EFFECT OF DOSE AND DOSE RATE ON BENZO[a]PYRENE
INDUCED MUTATIONAL SPECTRA
IN HUMAN CELLS
by
JIA CHEN
B.S., Beijing (Peking) University, 1986
M.A., College of William and Mary, 1989
Submitted in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF SCIENCE
in
CIVIL & ENVIRONMENTAL ENGINEERING AND TOXICOLOGY
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
May 1994
© Massachusetts Institute of Technology, 1994
All right reserved
Signature of
A
h
-uLI I
J
%r
Department of Civil and Environmental Engineering
May 13, 1994
Certified
by
,
-
William G. Thilly
Thesis Advisor
Accepted
by
(.
Joseph M. Sussman
Chairman, Departmental Committee on Graduate Student
|
OF TFC#PftY
.Eaft2IftlEs-
This doctoral thesis has been examined by a committee of the Department of Civil
and Environmental Engineering as follows:
/~
Professor William Thilly.
Professor Harold Hemond
Professor Howard Liber
i,
..
,"
IO 1
/-
I
Harvard School of Public Health
EFFECT OF DOSE AND DOSE RATE ON BENZO[a]PYRENE
INDUCED MUTATIONAL SPECTRA
IN HUMAN CELLS
by
Jia Chen
Submitted to the Department of Civil and Environmental Engineering
in Partial Fulfillment of
the Requirement of the Degree of
Doctor of Science in Civil & Environmental Engineering and Toxicology
It is possible to find the causes of genetic change from human exposure to
environmental agents by comparing the mutational spectrum of an individual (or a
population) to that of the suspected mutagen. However, humans are chronically exposed
to low levels of environmental mutagens, yet all of the mutational data available
nowadays have been obtained using high-dose, short-term protocols. Since the possibility
exists that different mutational mechanisms might operate under different conditions, it is
important to evaluate whether the high-dose, short-term treatment protocols accurately
mimic chronic exposures.
Herein the effect of dose and dose rate on Benzo[a]pyrene induced mutations in
human cells has been studied. A human lymphoblast cell line (AHH-1) which
constitutively expresses cytochrome P450IA1 in amounts similar to those found in human
lung epithelia was used. To assure the statistical quality of the mutational spectra, a 4
liter human cell culture system which allows exposure to large number of cells (> 4 x
109) was designed. The combination of large-scale human cell cultures and hi-fi
PCR/DGGE enable us to study BP-induced human mutational spectra at a concentrations
within the range of human exposure. Low-melting domain of hprt exon 3 induced spectra
have been studied in duplicate cultures at each of the following conditions: 0.02 gM x 20
days, 0.5 gM x 6 days, 0.5 gM x 20 days, and 30 gM x 28 hours. The results
demonstrate that the BP mutational spectra is markedly dose and dose rate dependent.
This study suggests that different mutagenic pathway may be operative depending on
dose rate. It is thus clear that the mutational spectra should not be used to define
expectations for mutational spectra in human tissue. Rather the more difficult
experiments of low-dose, long-term mutation studies are justified in terms of seeking
better approximation to human mutational pathway.
Thesis Advisor:
Title:
William G. Thilly
Professor of Civil & Environmental Engineering and Toxicology
ACKNOWLEDGEMENTS
I would like to express my appreciation to my thesis advisor, Prof. Bill Thilly, whom
I bumped into six years ago in the hallway of Bldg. 16. I was struck by Bill's confidence,
enthusiasm, broad interest, and his ability to manipulate numbers (i.e. from 2x to 10Y). As a
student in Oceanography at that time, I was eager to start a new career because of my
propensity to sea sickness. Pursuing my degree under his guidance, I had an unique
opportunity to experience life as both a toxicologist and an environmental engineer.
I would like to acknowledge the other members of my thesis committee, Professors.
Harold Hemond and Howard Liber, for their insights, recommendations and guidance.
I would like to thank Gengxi Hu for enduring my endless barrage of questions; John
Hanekamp and Lucy Ling for introducing me to the lab techniques; Konstantin Khrapko
and Paulo Andre for their constructive suggestions about my research; Riitta Mustonen,
Lata Shirname-More and Joyce Morrill, for their encouragement and friendship; and John
Durant for his generosity and friendship. I am deeply indebted to other members of the
"fabulous five" (Katz, MIT Thesis, 1993), Hilary, Tracy, Dave and Peter. Their friendship,
moral support and humor made the notoriously harsh life at MIT bearable.
I would like to thank all my colleagues at the 6th floor of E18 for their continued
support over the years. Special thanks to Rita DeMeo, Cindy Slick-Flannery, Jackie
Goodluck-Griffith, Paula Lee, Xiaocheng Li, Aoy Tomita, Rita Cha, Shihong Wang, Emba
Selvaraj and Al Atkinson. I am blessed to have such friends.
My family has been a constant source of inspiration, encouragement, and support over
the years. None of my achievements would have been possible without them. Finally, I
would like to express my deep appreciation to Xiaohong, whose love, patience and
understanding helped me endure. His genius in math made all the numbers and equations
personable to me.
TABLE OF CONTENTS
Title page
1
Acknowledgments
4
Table of contents
5
List of figures
7
List of tables
9
List of abbreviations
10
I. Introduction
12
II. Background
14
2.1. Mutational spectrometry
14
2.2. Definition of terminology
20
2.3. Experimental approaches to study low-dose mutations
21
2.4. BP as a model mutagen
24
2.4.1. Transport of BP
27
2.4.2. Metabolic activation of BP
27
2.4.3. DNA binding of BP and PAH
31
2.4.4. Repair of BP induced DNA adducts
36
2.4.5. BP induced mutations in human cells
36
2.4.6. Dose-dependence of BP mutagenesis in human cells
39
III. Experimental Design
44
3.1. A statistical model to estimate variance in long-term,
low-dose mutation assay
44
3.2. Design an experiment to study low dose rate mutations
46
3.3. Large-scale human cell cultures
51
IV. Methodology
58
4.1. Cell line
58
4.2. Cell maintenance
63
4.3. Benzo[a]pyrene treatment
64
4.4. Determination of survival and mutant fraction
65
4.5. Reducing background mutant fraction
69
4.6. Molecular analysis of point mutations on low-melting domain of hprt exon 3
70
V. Results and discussion
77
5.1. Selection of dose and dose rate
77
5.2. Toxicity and mutagenicity of BP in large cultures
83
5.3. Statistical analysis of variance in large-scale human cell cultures
90
5.4. Dose dependence of BP mutational spectra
98
5.4.1. Definition of mutational hotspot
98
5.4.2. Low-melting domain of hprt exon 3 as the target sequence
100
5.3.3. BP mutational spectra under various dose and dose rate
101
5.4.4. Characteristics of BP mutational spectra
107
5.5. Significance toward human study
115
5.5.1. A model to estimate BP exposure in human lungs
117
5.5.2. Estimation of BP concentrations in human lungs
119
5.6. How do mutants disappear?
121
5.6.1. General considerations on BP mutagenesis
121
5.6.2. Hypotheses for disappearing mutants
122
A. Selective pressure or neutrality of 6TG r phenotype
124
B. Gene amplification
125
C. Original Heterogeneity
133
D. BP Induced Heterogeneity
144
VI. Summary and conclusions
151
VII. Suggestions for future research
154
References
156
LIST OF FIGURES
Figure
Number
2.1
Title
Determination of mutational spectra from a treated bulk
culture using PCR/DGGE
16
2.2
Comparison of mutational spectrum obtained by PCR/DGGE method
18
2.3
Shapes of dose-response curves for chemically induced mutations
22
2.4
Proposed pathway to BP mutagenesis in human cells
25
2.5
Metabolic fate of BP
29
2.6
Stereochemistry of BP activation
33
2.7
BPDE spectra in human cells
37
2.8
Summary of long-term, low-dose BP mutagenesis in AHH-1 cells
40
2.9
Comparison of long-term, low-dose with short-term, high-dose
BP-induced mutation in AHH-1 cells
42
3.1
Effect of number of cells treated on dispersion of mutation rate
49
3.2
Schematic design of large scale human cell cultures
52
3.3
Protocol for long-term, large-scale human mutation assay
56
4.1
Concentration dependence of BP induction of AHH activity
59
4.2
Time course of AHH activity induction
61
4.3
Determining survival by growth curve extrapolation
66
4.4
Protocol for molecular analysis of 6TGr mutant population
71
4.5
Layout and sequences of primers used for the analysis of 6TG r
mutations in the low-melting region of hprt exon 3
73
Dose and dose rate dependence of BP mutagenesis in small
stationary AHH-1 cultures
79
Selection of doses and duration for large-scale human cell
cultures
81
5.3
Mode of exposure with selected dose rates
84
5.4
Growth curves of spontaneous, 0.02 pM and 0.5 g±M BP treated
cultures
86
5.1
5.2
Figure
Number
Title
Pagc
5.5
Growth curves for 30 pLM BP treated cultures
88
5.6
Summary of BP induced mutant fraction in large-scale AHH-1 ceel
91
5.7
A proposed model of the hprt catalytic domains
102
5.8
DGGE analysis of BP induced mutations in the low-melting
domain of hprt exon 3 under various dose and dose rate
104
5.9
Display of BP mutational hotspot under various dose and dose rate
110
5.10
Test for neutrality of 6TGr phenotype
126
5.11
Illustration of "onionskin" model for gene amplification
130
5.12
Test for gene amplification
134
5.13
Hypothesis of original cell heterogeneity
138
5.14
Short-term toxicity and mutagenicity of BP to AHH-1 cells
140
5.15
Short-term toxicity of BP in "disturbed" and "disturbed"
AHH- 1 cells
142
5.16
Hypothesis for BP induced heterogeneity
145
5.17
Short-term toxicity of BP on 6TGr cells induced by BP treatment
148
LIST OF TABLES
Table
Number
Title
Pag
2.1
DNA adducts of BP and PAH from various sources
35
3.1
Experimental conditions for large-scale design
48
5.1
Summary of the large-scale cell culture experiment
93
5.2
Experimental conditions used in large-scale cell cultures
95
5.3
Variance of mutant fraction in large-scale human cell cultures
96
5.4
Variance of mutation rate in large-scale human cell cultures
97
5.5
Comparison of mutant rate under various doses
99
5.6
Summary of BP hotspots in the low-melting domain
of hprt exon 3
107
5.7
Contingency table analysis of clone by clone BPDE induced
mutations under different doses
116
5.8
Human non-occupational and occupational exposure to BP
120
5.9
Analysis for selective pressure on 6TGr phenotype
128
5.10
Analysis for gene amplification
136
LIST OF ABBREVIATIONS
AHH
aryl hydrocarbon hydrolase
BP
benzo[a]pyrene
bp
base pair or base position
d
day(s)
DGGE
denaturing gradient gel electrophoresis
DMSO
dimethyl sulfoxide
EMS
ethylmethanesulfonate
H
helical structures
HAT
2x10 -4 M hypoxanthine + 8x 10- 7M aminopterin + 3.5x10 -5 M thymine
hi-fi
high-fidelity
hprt
hypoxanthine-guanine phosphoribosyl transferase
J
treatment time point
Jay
average of the treatment time points
j
days of treatment
k
days of phenotypic expression
mo
the number of mutant at time zero
Am
the number of mutant generated each day
MF
mutant fraction
MFo
mutant fraction at time zero
MFO
mixed function oxidase
MNNG
methylnitronitrosoguanidine
MNU
methylnitrosurea
NO
the number of cells at time zero
Nt
the number of cells there would be at a particular time
4NQO
4-nitroquinoline-N-oxide
ni
the number of observation at each time point
PAGE
polyacrylamide gel electrophoresis
PAH
polyaromatic hydrocarbon
PCR
polymerase chain reaction
PE
plating efficiency
r
mutation rate
S
spontaneous culture(s)
ss
single strand
SD
standard deviation
t
growth factor
6TG
6-thioguanine
6TG r
6-thioguanine resistant
TH
3.5 x 10-5 M thymine + 2 x 10-4 M hypoxanthine
Var
variance
WT
wild type
P3
beta strand
'
doubling time
a
variance at treatment time point calculated by Eq. A
I. INTRODUCTION
The central premise of genetic toxicology applied to humans is that:
environmental mutagens to which human are exposed cause a significant portion of
their genetic changes. One line of scientific argument hypothesized that it will be
possible to estimate the genetic risks inherent in human exposure to environmental agents
by comparing the mutational spectrum of an individual (or a population) to that of the
suspected mutagen. Efforts have been made to assess the genetic risk of particular
mutagens by using single-cell and whole-animal bioassays, both of which use dose levels
much higher than human would likely encounter. Attempts to extend these investigations
to low-dose ranges common to human experience are limited by the time, cost and
statistical precision. Quantitative assessment of genetic risk has primarily relied on
mathematical extrapolation of risk from high dose animal or cell culture study to the
anticipated human scenario which in general is chronic exposure to low levels of
environmental mutagens. However, the possibility exists that different mutational
mechanisms might operate under different conditions, it is important to evaluate whether
the high-dose, short-term treatment protocols widely used in risk assessment accurately
mimic chronic exposures in humans.
Our lab endeavored to mimic human exposure phenomena and genetic changes by
developing a human lymphoblast mutation assay. To develop a system for the study of
the induction of gene mutations which closely approximates human, a gene-locus
mutation assay using diploid human lymphoblast lines was designed in our lab (Thilly et
al., 1980). Crespi and Thilly (1982) improved the system by isolating a human
lympoblast line, AHH-1, competent for xenobiotic metabolism. Danheiser et al. (1989)
performed the first study to assess the mutagenic effects of long-term, low-level exposure
to a chemical (benzo[a]pyrene) requiring activation to its mutagenic counterparts in
human cells. They demonstrated the lowest detectable concentration of benzo[a]pyrene
(0.02 gM), common to human experience, for statistically significant mutations in human
cells. In addition, nonlinearity of the dose-dependence of mutagenicity was observed at
high dose ranges.
In this study, I extend the work of Danheiser in mimicking human exposure
scenario in cell cultures and studying the mutational spectra resulted from such exposure.
This is the first time anyone has studied the long-term, low-dose mutational spectra in
human cells or any living organism as well as the dose effects. It is a crucial study before
mutational spectra is used to diagnose whether human suffers any genetic damage related
to exposure of suspected mutagens.
The dose-dependence of DNA base sequence changes in the coding region of the
hprt gene in human lymphoblast cells (AHH-1) has been examined using benzo[a]pyrene
(BP) as a model mutagen. To achieve that, an experimental system was designed in such
a way that the distribution of BP-induced mutations in a human cell population could be
accurately analyzed when cells were treated with BP at a concentration common to
human experience (0.02 jM).
This study demonstrates BP induces mutations by molecular pathways that are
dose-dependent. It suggests that short-term, high- or toxic-dose protocols commonly
used in regulatory decision making can not predict health risk of human experiencing
chronic exposure to low levels of environmental mutagens.
I. BACKGROUND
2.1. MUTATIONAL SPECTROMETRY
When a homogenous cell population is exposed to a mutagen under certain
exposure conditions, there exists a specific reproducible pattern of genetic changes: the
mutational spectrum. This fact was discovered by Benzer and Freese (1958) who
showed that the position and frequency of mutations in a genome of the T4 bacterial virus
was reproducible among independent experiments and unique for untreated and chemically
treated virus populations. Since their observation over 30 years ago, the study of
mutational spectrometry has extended to bacteria, fungi, rodent, and human cells.
Mutational spectrometry has become a powerful tool for exploration of the molecular
biology of mutagenesis, of structure and function relationships of proteins and nucleic
acids, and perhaps most importantly, of the quantitative relationships between exposure to
environmental mutagens and genetic disease (reviewed by Southam and Thilly, 1994).
Two general approaches have been widely used in the field mutational
spectrometry: use of endogenous genes such as hprt or shuttle vectors carrying E. coli
supF tRNA gene or the E. coli lacI gene. Shuttle vector system involves the mutagen
treatment of naked plasmid DNA containing a reporter gene. The plasmid must contain a
selectable sequence. After treatment in vivo with a mutagen, the plasmid is transfected
into a mammalian cell where it is acted upon by repair enzyme and replicated. The
plasmid is isolated from the mammalian cells and transfected into bacteria for selection of
mutant phenotype. The major advantage of shuttle vector system is allowing the host cell
machinery to work on mutagen treated DNA, so the repair mechanism of the host cell can
be studied. But it is often the case that much higher proportion of damage is achieved
when plasmids are treated outside of the cell compared to a living cell. As a result, one
plasmid often contains several mutations. Endogenous genes, however, are treated and
selected in situ, and thus more relevant to mutagenic process in vivo.
In addition, two modes of phenotypic selection have been applied: clone by clone
and en masse selection. In the former case, the descendants of each mutation are isolated
as a separate colony and their sequences are analyzed on a clone by clone basis. Such
practice is always tedious and seldom generates sample size large enough to analyze
mutational spectrum with statistical significance. In the latter, one uses cell cultures in
which descendants of induced mutations are selected and analyzed en masse. By
increasing culture size, one may obtain enough to mutants to study their mutational
spectra.
Since 1980's, our lab has innovated the means for elucidating mutational spectra
(Thilly, 1985; Cariello and Thilly, 1986). The development of denaturing gradient gel
electrophoresis (DGGE) (Fischer and Lerman, 1983), which discriminate DNA with single
base difference, was coupled with the polymerase chain reaction (PCR) (Mullis and
Faloona, 1987), which allows rapid reproduction of specific target DNA sequences. Such
technique was further improved by optimizing the fidelity of the polymerase (Keohavong
et al., 1990; Ling et al., 1991) so that induced mutations should not be obscured in
polymerase induced "noise".
PCR/DGGE technique (Fig. 2.1) is a powerful tool for determining the spectra
from selected population of cells (Thilly, 1985; Cariello and Thilly, 1986; Cariello et al.,
1988; Keohavong and Thilly, 1992). By treating a bulk human cell culture en masse with
a suspected mutagen, one can induce at least 10,000 independent survival independent
mutants, which insures the statistical quality of the mutational spectrum. Of course, only
mutations exceeding the sensitivity of the assay, now about 0.2% of total mutants, are
detected.
Fig. 2.2 represents mutational spectra data from some of the studies using
PCR/DGGE method. These mutational spectra were observed in hprt exon 3. It is a clear
Fig. 2.1
Determination of mutational spectra from a treated bulk culture
using PCR/DGGE
(Adapted from Southam and Thilly, 1994)
DGGEIPCR Method
r--ý
~C7
Surviving mutants and
wild type caB
Tratw/mutarmpt
to give looo0000
urvivtn
mutants
Generatiomnfor
phantypic cprmion
and selection
culture
Isolate DNA
Sequare
seuec
w/al
HPRT mutants
..... -ap
Boil and
rearneal with
ecs wild typ
load on DGG
atant heteroduplexes
Recover from gel
- Wild
type homoduplex
and sequence
stational spectrum
bp posw
Fig. 2.2
Comparison of mutational spectrum obtained by PCR/DGGE method
The kinds, positions and frequencies of 6TGr mutations in hprt exon 3, represented by
base positions 135-318. The frequencies of mutations relative to total 6TG mutations are
represented by the vertical bar. The kinds of the predominant hotspots were indicated on
the top of each spectrum, and those for other hotspots were indicated on the top of the
vertical bars.
(Adapted from Southam and Thilly, 1994)
Mutational Spectra in axon 3 of hprt using
DGGEIPCR method
Spe-fma-e
SpWrma COUr.15
i2I"
a
km ir
00021o
In
mi0lwm
IPDE Spectrun
tunve.
o
20
3"00
200
200
300
Dowsigearpsittll
t•,i
X-ray $prmanIkt,
c-T
-IT
SI'
I4
I"
130
200
220
2
2W0
200
300
0
10 230
240
I20
Im eN
asr elten
MN psaosm
omit"en
JMNNcpja,,
r."se14
I
ii
II
~·IlU
· ·
ISO
W10
200
. I .
220
.
.
240
20
20
· I·
300
·
140
10
ISO
20
lms petr pglltt
.
.
.
220
240
20
.I
.
.
.
20
300
230
300
bIC-91e• posittn•
(r.tro
4Specurman
2
ICA-191 Specutrut
Mwulew.
1990)
I
U'
.40
30o.
2 0·
ri
I I,.
c0
t20
220
240
rv
NW rclm
230
230
300
4o
0
3W
In
200
220
240
km sew OpWIl.a
20
.
demonstration that mutational spectrum is indeed unique to each mutagen. This implies
that one can use mutational spectrum can be used as a "fingerprint" of each mutagen to
study causative effect, if any, of a mutagen on human genetic integrity.
2.2. DEFINING DOSE AND DOSE RATE
The "dose" implies the amount of mutagen administered or being exposed to over
a period of time (i.e. the whole life span or length of the experiment). The "dose rate" is
the temporal pattern of the total dose, so the dose can be calculated by integration of dose
rate over time.
The human situation is exposure to mutagens occurs at very low levels over long
period of time. A principal question facing environmental scientists concerns how to
measure mutations induced by low level of mutagens. As analyzed in Liber et al. (1985),
cellular exposure to mutagens occurs via four modes:
(I) High dose rate x short time, yielding a high dose; typical in cell and animal
assays;
(II) High dose rate x long time, yielding a very high dose; usually marked by
extreme toxicity;
(III) Low dose rate x short time, yielding a low dose;
(IV) Low dose rate x long time, yielding a high dose.
As one can see, Modes I and II only reflect extreme circumstances outside of
human experience, so such protocols may be unrealistic in terms of mimicking human
exposure. In order to devise experiments which useful in predicting human health effects
which result from living and working in the presence of low levels of environmental
pollutants, then our interests must focus on low dose rates, in which exposure occurs
intermittently or continuously (Mode IV).
2.3. EXPERIMENTAL APPROACHES TO STUDYING LOW-DOSE
MUTATIONS
Long-term, low-dose exposure to chemicals, a situation more likely to be
encountered by man, can be mimicked in vitro. Low dose rate x short time protocol can
be repeated many times with a defined time interval separating the exposure. The
background mutant fraction (which is always non-zero), and the population size
determine the sensitivity of the mutation assay. As stock cultures age, mutants arising
spontaneously accumulate and the background mutant fraction rises. As a result, the
sensitivity of the mutation assay decreases. To study low-dose mutations we require that
the treated and concurrent control mutation rates demonstrate a statistically significant
difference. One thus needs to treat large number of cells with a mutagen for a long period
of time. Such an approach is experimentally difficult and costly.
The common strategy used to overcome such limitations is to use doses that are
much higher than those encountered in the environment. The results are then
extrapolated to the dose levels of interest, usually using dose-response relationships with
little biological evidence. Thus the establishment of a reasonable dose-response
relationship becomes crucial in the process.
Four most common dose-response relationships are illustrated in Fig. 2.3. Type (I)
is a linear function of the mutagen concentration as seen with methylnitronitrosoguanidine
(MNNG) in human cells (Penman and Thilly, 1976) and hamster cells (Schwartz and
Samson, 1983). It may be the case of direct-acting mutagen without enzymatic induction
of DNA repair. Linearity is a common phenomenon among many other mutagens at low
dose levels (i.e. Nakamura and Okada, 1983), but departure from linearity is frequently
observed at high dose rates. Penman (1980) showed that the dose response of
ethylmethanesulfonate (EMS) was linear at low doses and switched to another linear
relatioship with an higher slope (concave upward); and 4-nitroquinoline-N-
Fig. 2.3
Shapes of dose-response curves for chemically induced mutations
Graphs illustrate four basic curve shapes observed in cell culture using mutant fraction as
genetic endpoint.
I:
Linear;
II:
Plateau;
III: Concave-up;
IV: Combination of II & III.
w
z
2
rcc
I\I
w
z
DOSE
oxide (4NQO) switched from a linear relationship at low doses to a nonlinear relationship
at high doses (plateau). Nakamura and Okada (1981) studied mutational dose-rate effects
of gamma rays in mouse lymphoma cells. Their data indicated a quadratic curve at the
high dose rate, but a linear curve with a lower slope at the low dose rate. Nevertheless,
similar treatment yielded a linear quadratic equation in both low and high dose rate in
Chinese hamster cells (Thacker and Stretch, 1983). Type (II) is a plateau with increasing
mutagen concentration. Such a relationship may be explained by saturation in transport
and/or metabolism of the mutagen, and/or the induction of repair enzymes. ICR-191
(DeLuca et al., 1977) and aflatoxin B1 (Kaden et al., 1987) demonstrated type (II)
response. Type (III) is concave up as mutagen concentration rises as seen in MNU or
methylnitrosourethane (Thilly et al., 1976; Penman et al., 1979). This response may be
the result of saturation of DNA repair. Type (IV) is a combination of type (II) and (m).
ENU-induced mutations in mouse spermatogonia falls into this category (Russell et al.,
1982). The mutant fraction shows a significant departure from linearity at both high and
low dose. This type of response may be the result of induced DNA repair at low dose and
saturation of mutagen transport and metabolism at high dose.
2.4. BP AS A MODEL MUTAGEN
Benzo[a]pyrene, a member of the polyaromatic (or polycyclic) hydrocarbon
(PAH) family, is an ubiquitous environmental pollutant. It is released into the
atmosphere, along with other PAH, during the combustion of fossil fuels and vegetation,
and is present in tobacco smoke and food. The exposure to BP is virtually unavoidable
(Baum, 1978; Weinstein, 1988).
Mutagenesis of BP is a cascade process which may be modeled as a series of steps
(Fig. 2.4), and each process can be dose-dependent. BP is transported into cells and
metabolized into electrophilic forms that are reactive with nucleophilic sites on DNA,
Fig. 2.4
Proposed pathway to BP mutagenesis in human cells
BP
Transport
Metabolism/Activation
DNA Binding
Potentially mutagenic Lesion
Misrepair
Correct Repair
\ýk
No Repair
Misrep lication
Replication
N
Wild Type
Nonreplication
4
.
Mismatch Repair
Ir
Mmmaaom
Wild Type
Cell Death
forming DNA adducts (Miller and Miller, 1981; Singer and Grunberger, 1983; Lawley,
1989). DNA binding of carcinogen and the damage caused by this are thought to constitute
the initial step in the complex multistage phenomenon of chemical carcinogenesis (Lawley,
1989). Accordingly, studies of factors, such as dose effects, that influence mutagenesis in
human cells should enhance our understanding of cancer etiology.
2.4.1. Transport of BP
The first step of BP mutagenesis is the transport of BP into the cell. Modes of
transport include passive diffusion and active transport (Timbrell, 1991). Most chemicals
cross cell membranes by simple diffusion. Small hydrophilic compounds diffuse across
lipid membranes through aqueous channels, whereas larger organic molecules diffuse
across hydrophobic, lipid domain. Passive diffusion is a process driven by the
concentration gradient across the cell membrane. The rate of diffusion is proportional to
the concentration gradient, and thus is a dose-dependent process.
BP was also shown to be transported across the membrane by lipoprotein (HansonPainton et al., 1983). It was shown that BP bound to the protein carrier was preferentially
transferred to the microsomes. The carrier protein fraction transferred levels of BP manyfold in excess of its BP-binding capacity. In addition, the carrier fraction if capable of
both transferring BP to the microsomes and accepting oxidized BP from microsomes. It is
not hard to imagine that such active transport process can be saturated at high doses.
2.4.2. Metabolic Activation of BP
BP is metabolized to exert its mutagenic function onto DNA. The primary BPmetabolizing enzyme system is the cytochrome P450IAl-dependent enzyme called aryl
hydrocarbon hydroxylase (AHH). The oxidative metabolism of BP is catalyzed by AHH
that is part of the endoplasmic reticulum and consist of a terminal cytochrome (P450) and
an electron transport chain (Sipes and Gandolfi, 1986). A highly significant positive
correlation between the lung microsomal AHH activity and formation of BPDE-DNA
adducts was reported (Alexandrov et al., 1992). This result suggests that in human lung
BPDE-DNA adducts are mediated predominantly by P450IA1 as rate-limiting step.
The level of P4501A1 is found in all human tissues examined, including liver
(Selkirk et al., 1975), placenta (Nebert et al., 1969), Bronchus (Cohen et al., 1976), Lung
(Prough et al., 1979), Colon (Autrup et al., 1978), Kidney (Prough et al., 1979),
lymphocytes (Busbee et al., 1972; Kellermann et al., 1973), monocytes (Bast et al., 1974;
1976) and lung macrophages (Bast et al., 1973), bladder (Selkirk et al., 1983) and
esophagus (Harris et al., 1979), and both increases and decreases in activity have been
reported. The inducibility of AHH (P450IA1) activity to exposure of PAH is controlled
by a single Ah locus, which encodes the cytosolic Ah receptor involved in the regulation
of P450 genes (Gonzales et al., 1986).
The induction of P450IA1 (AHH) activity has been associated with the
appearance of human cancer. In a study carried out in lung cancer patients (Anttila et al.,
1991), P4501A1 activity was detected in all but one cancer patients, but in none of the
controls. Law (1990) also reported about half of the studies found higher P450IA1
inducibility in lung cancer cases than controls.
A summary of the metabolic pathways of BP metabolism is shown in Fig. 2.5.
The mechanism and stereoselectively of BP metabolism was reviewed by Gelboin (1980).
Five phenols have been identified as metabolites: 1-OH; 3-OH, 6-OH, 7-OH, and 9-OH.
The phenols can be converted to quinones like 1,6-, 3,6-, and 6,12-quinones. Three
dihydrodiols: 4,5-, 7,8-, and 9,10-dihydrodiols are produced from corresponding arene
oxides. The primary epoxides can be conjugated to glutathione S-conjugates and the
Fig. 2.5
Metabolic fate of BP
(Adapted from IARC, 1983).
METATOLIC FATE OF BENZO[a]PYRENE
1
12
9
8
7
6
P4
:-
GSH conjugates
Arene Oxides
4,57,89,10-
GSH Conjugates
5
P450
Phenols
A
-
1- 73- 9-
-
Ouinones
1,63,6
6,12-
Epoxide Hydrolase
Glucuronides
&
-------
Sulphate Esters
Glucuronides
Dibydrodiols
4,57,8-
9,10-
9-OH-4,5-diol
6-OH-7,8-diol
1-(3-)OH-9,10-diol
IP450
Tetraols
GSH Conjugates -~
7,8-diol-9,10-epoxide
9,10-di01-7,8-epoxide
Sulphate Esters
phenols and diols can be conjugated to water-soluble compounds by either sulfate
conjugation or glucuronide conjugation. The 7,8-diol is converted to four stereoisomers
of 7,8-diol-9,10-epoxides illustrated in Fig. 2.6. The diol epoxides are highly reactive
and react with macromolecules such as DNA or protein. The stereochemistry of the diol
epoxides plays important roles in BP toxicity and mutagenicity. Anti-(+)-BP-7,8-diol9,10-epoxide (BPDE) is considered the ultimate mutagen and has been shown to be
mutagenic in most of the experimental system.
Metabolite profile differs among human tissues. Three major classes of metabolite
were detected in human system using liver microsomes. They include dihydrodiols
(9,10-, 7,8- and 4,5-dihydrodiols), phenols and quinones. The major metabolite produced
by human peripheral blood lymphocytes were phenols. Quinones, trans-7,8-dihydrodiol
BP, and one unidentified metabolites were also reported (Vaught et al., 1978). The
metabolite profiles in human lymphocytes also depends on the incubation time with BP
(Selkirt et al., 1975). During a 30 minute incubation no trans dihydrodiols were
observed, the major metabolites were phenols and quinones. During a 24 hour
incubation, human lymphocytes produce 4,5-, 7,8-, and 9,10- trans dihydrodiols,
quinones and phenols.
2.4.3. DNA Binding of BP and PAH
DNA adduct levels measured in human tissues can be compared with the known
level of exposure to allow assessment of potential individual risk (Perera, 1988; Van
Zeeland, 1988). The levels of DNA adducts may reflect the dose of environmental
mutagens, it is thus important biomakers which can provide information about the
exposure to mutagens and the effect of genotoxic compound.
Extensive work has been done on DNA binding of total PAH in human tissues, but
detection of BP-DNA adduct in human tissues had not been successful till recently. This
is because antibody designed to recognize BP-DNA adduct in immunoassay shows
various level of cross-reactivity with other PAH, while
32P-postlabeling
of adducts gives
unknown spots of unidentified nature. In this section, DNA bindings of both BP and
PAH in humans are reviewed.
The binding of BP and PAH is highly complex because of the stereochemistry of
the BP and PAH metabolites (Fig. 2.6). Most of the data on BP or PAH adduct concern
BPDE. The major BP adduct is formed by binding to the exocyclic N2-atom of guanine
(Baird et al., 1975; Osborne et al., 1981; Cooper et al., 1983), but other binding sites have
been identified as well. The minor products of BPDE include C-8, N-1, N3, N-7 and 0 6
atom of guanine (Cooper, 1983).
DNA adducts in exposed individual can be quantified by radiolabeling (i.e. 32p.
postlabeling), chromatographic (i.e. HPLC; GC-MS), immunological (ELISA), or
physicochemical (i.e. SFS) methods. All the techniques are sufficiently sensitive and
specific for the detection of environmental and occupational exposure (Bartsch et al.,
1988).
Table 2.1 summarizes the level of BP and PAH adduct in person exposed from
various sources. For BP-DNA adduct, a new HPLC-fluorometric assay was used.
Human occupational data for PAH were obtained by immunoassay analysis (ELISA,
USERIA) using antibodies against BPDE-DNA, while total binding of aromatic DNA
adducts was measured by
32P-postlabeling.
Synchronous fluorescence spectrometry was
applied to detect aromatic DNA adducts in peripheral blood lymphocytes of workers in
foundries and production of coke. As one can see from the table, the adduct levels in the
exposed populations are in general higher than the controls.
Fig. 2.6
Stereochemistry of BP activation
Formation of BP metabolites responsible for the mutagenicity and carcinogenicity of the
parent hydrocarbon. Solid arrows indicate major metabolic pathways.
Adapted from Levin et al. (1980).
STEREOCHEMISTRY OF BP ACTIVATION
o
1112 1
9
7
6
7.
6
j2
o~i
0
OH
OH
OH
OH
OH
0
0H
5
BP
OH
OHO
OH
OH
VII
I: (+)-BP-7R,8S-OXIDE
II: (-)-BP-7R,8R-DIHYDRODIOL
m: (anti)(+)-BP-7R,8S-DIOL-9S,10OR-EPOXIDE-2
IV: (syn)(-)-BP-7R,8S-DIAL-9R,10S EPOXIDE-1
V: (-)-BP-7S,8R-OXIDE
VI: (+)-BP-7S,8S-DIHYDRODIOL
VII: (syn)(+)-BP-7S,8R-DIOL-9R, 10OS-EPOXIDE-1
VIII: (anti)(-)-BP-7S,8R-DIOL-9R,10S-EPOXIDE-2
Viii
Table 2.1. DNA Adducts of BP and PAH from Various Exposure
Occupation
Tissue' Adduct (108 Nucleotides) Method*
Reference
Exposed
Controls
WBC
62-533
4-10
HPLC/FI
Rojas et aL., 1994
LP
0.6-9.9
-
HPLC/FI
Alexandrov et al., 1992
L
31
13
PP
Savela & Hemminki, 1991
G
9.6
7.6
PP
Savela & Hemminki, 1991
WBC
0.1-9.6
0.1-0.3
PP
Herbert er al., 1990
BCC
1.3-80
-
M
Shamsuddin er al., 1985
WBC
17.6
4.4-8.2
PP
Hemminki er al., 1990a
WBC
15.3
2.3-13
IM
Hemminki et al., 1990b
L
57
-
IM
Haugen er al., 1986
Benzoralpyvrene
smoking
PAH
Smoking
Roofer
Coke plant
Foundry
Fire fighters
L
13-1143
-
IM
Harris er al., 1985
L
20-66
-
SFS
Haugen et al., 1986
WBC
0.8
0.2
PP
Phillips et al., 1988
BCC
26
2.2
IM
Perera et al., 1988
BCC
1.3-80
-
lM
Shamsuddin et al., 1985
WBC
62
38
IM
Liou et al., 1989
* WBC: white blood cells; L: lymphocytes; LP: lung parenchyma; G: granulocytes; BCC:
buffy coat cells.
** HPLC/Fl: HPLC-Fluorometric assay; PP: 3 2P-postlabeling, IM: immunoassay; SFS:
synchronous fluoresence spectrophotometry.
2.4.4. Repair of BP Induced DNA Adducts
The damage induced by PAH, and particularly the formation and repair of BP
adducts, have been studied in mammalian cells (Maher et al., 1977; Cerutti et al., 1978;
Busbee et al., 1984, McCormick and Maher, 1978, 1985; Chen et al., 1992). BP-DNA
adducts can be repaired through excision pathways. In a study with XP12DE cells, the
kinetics of the removal of DNA adducts formed by BPDE, cytotoxicity and the formation
of mutagenic lesions were about the same. Residues were removed by repair enzymes of
the normal cells over a period of four days. There was no loss of adducts in excisiondeficient XP12BE cells (McCormick and Maher, 1985).
The repair of BP-DNA adducts occurs preferentially on transcribed strand of the
gene (Chen et al., 1990, 1991, 1992; Andersson et al., 1992). A cell cycle-dependent
strand bias was observed in repair-proficient human cells (Chen et al., 1990), but not in
excision repair-deficient human cells (Chen et al., 1991). The preferential repair of BPDNA adducts also occurs on an activated gene compared to an inactivated genes (Chen et
al., 1992).
2.4.5. BP Induced Mutations in Human Cells
Most of the studies on BP-induced human cells involve BPDE, the genetically
active form of BP. It is because few of the human cell lines used today have the ability to
activate BP. The BPDE mutational spectra were obtained by using either shuttle vectors
containing BPDE adduct (Yang et al., 1987) or selectable genes (Keohavong and Thilly;
1992; Yang et al., 1991; Chen et al., 1990; Carothers and Grunberger, 1990). Fig. 2.7 is a
display of BP spectra in hprt exon 3. One has to bear in mind that most of the studies,
except the one by Keohavong and Thilly (1992), used clone by clone analysis (see Section
Fig. 2.7
BPDE spectra in human cells
Mutational spectra of BPDE were obtained on the low-melting domain of hprt exon 3.
The horizontal axis represents the target sequence from position 215-318. The frequency
of each mutation is represented either as number of observations (panel A-C) or as
percentage of total 6TGr mutations. The kind of the predominant hotspots were indicated
on the top of each spectrum and those for other hotspots were indicated on the top of the
vertical bars.
Panel A: Human fibroblasts, S phase (Chen et al., 1990);
Panel B: Human fibroblasts, G1 phase (Chen et al., 1990);
Panel C: Human fibroblasts, synchronous (Yang et al., 1991);
Panel D: Human lymphoblasts, asynchronous (Keohavong and Thilly, 1992).
BPDE SPECTRA IN HUMAN CELLS
FIBROBLASTS
G -> T
A
S phase
N = 24
B
G -> T
FIBROBLASTS
Glphase
N = 24
I
C
I!
G -> T
FIBROBLASTS
N = 34
A
A
D
G -> T
LYMPHOBLASTS
cc 3-
N =
18,200
0
135
155
175
195
215
<
235
Base Position
255
275
295
315
2.1), so the sequence specificity of the mutation was not statistically significant. Yet it is
obvious from Fig. 2.7 that BPDE induces predominantly G ->T transversion mutations
based on the fact that the repair of the BP adduct occurred preferentially on transcribed
strand (see Section 2.5.4).
2.4.6. Dose-Dependenceof BP Mutagenesis in Human Cells
Effects of dose and dose rate on BP mutagenicity has been studied in human
lymphoblast cells (Danheiser, 1985; Danheiser and Thilly, 1989). AHH-1 cells were
treated with BP up to 20 days at concentrations between 0.02-1 rM, and dosedependence of BP mutagenicity was observed (Fig. 2.8 & 2.9). When AHH-1 cells were
exposed to BP between 0.1 and 1 jtM, the induced rate of mutation was high and constant
from 0 to 5 days, after which the rate dropped sharply and remained steady until 20 days.
Exposure to low concentrations of BP (0.02 jiM) resulted in a low but constant mutation
rate throughout 20 days of treatment (Fig. 2.8). For a given integral dose (BP
concentration x time of exposure), long-term, low-dose exposure induced 4 times as
many mutations as short-term, high-dose exposure (Fig. 2.9). Thus, it is impossible to
extrapolate from a short-term, high-dose mode of BP exposure to a long-term, low-dose
mode.
The striking feature of Danheiser's findings is the saturation effect after 5 days
exposure to BP at 0.1 tM or higher. There are several steps along the BP mutagenesis
pathway (Fig. 2.4) at which this saturation response might be affected, such as the
blockage of BP transport into the cells; saturation of BP metabolism; or induction of
DNA repair. Danheiser's results suggest that the DNA binding rate has not been a factor
in the saturation effect. The event leading to the observed drop of mutation rate with time
is occurring after DNA binding, either by enhanced adduct removal or induced DNA
repair.
.39
Fig. 2.8
Summary of long-term, low-dose BP mutagenesis in AHH- 1 cells
Weighted least squares lines for 0, 0.02, 0.1, 0.5, and 1 gM BP were fit to the mean
mutant fraction of 3 independent experiments (performed in duplicate or triplicate) over a
treatment period of 20 days. Numbers on the line indicate mutation rates (mutation per
cell per day).
(Adapted from Danheriser, 1985).
40
-1 I IKA
6r0
50
z
0
40
t--
K.
rr
30
LL
z
~-20
10
I
I
|
•
-
0
10
15
TIME OF TREATMENT (DAYS)
20
Fig. 2.9
Comparison of long-term x low-dose with short-term x high-dose
BP-induced mutations in AHH-1 cells
(Adapted from Danheiser, 1985).
___
CD
,61
-dose
O
I-5
04
U-
z
3
02
LU
0
1-dose
zl
0
5
BP CONCENTRATION X TIME (uM X DAYS)
4,2
10
III. EXPERIMENTAL DESIGN
3.1. A STATISTICAL MODEL TO ESTIMATE VARIANCE IN LONG-TERM,
LOW-DOSE MUTATION ASSAY
A long-term, low-dose protocols used in this experiment permits evaluation of BP
mutagenesis in the context similar to human experience. However, imprecision of such
protocol arising from random error and systematic bias limits our ability to obtain
mutational spectra with desired statistical precision and to distinguish mutational
distribution from different dose conditions. As one might imagine, random error is
increased by extending the treatment period, by decreasing the number of mutants
generated each day (low mutation rate) and by increasing the number of cycles of growth
and dilution (during treatment and phenotypic lag). Oller et al. (1989a) developed a
statistical model to analyze the variance of long-term, low-dose protocol, which allows
the design of experimental protocol that can provide any desired degree of dispersion
around the estimated mutant fraction and mutation rate. This model separates the
physical steps of mutation assay and calculate the dispersion expected from each step.
The dispersion is expressed in terms of statistical variance. These variances from each
steps of the assay are summed up to predict the variance expected from the long-term,
low-dose protocol.
Experimental conditions are designated as follow: At the beginning of the
experiment (to), No cells and mo mutants were present. The background mutant fraction
(MFo) at the beginning was determined by plating, so mo was calculated by No x MFo.
The growth factor t was around 2 since AHH-1 cells doubled every day. Each day, Am
mutants were generated. It could be quantified from the mutation rate. For the
spontaneous cultures Am represented the spontaneous mutants generated each day and for
treated cultures Am represented the sum of spontaneous plus induced mutants generated
each day. During the j day treatment followed by k day phenotypic expression, there
were j + k -1 dilution steps with a dilution factor of 1/t. The repeated sampling and
dilution steps contributed to the variance of mutant fraction. At last, cells were plated into
microtiter plates to determine their colony forming ability in the presence and absence of
selective conditions.
Variance of Mutant Fraction
The experimental protocol used in large-scale human cell cultures consists four
physical steps: 1) cell treatment with BP; 2) cell dilution during treatment; 3)
phenotypic expression of the mutations after treatment; 4) plating for mutant fraction.
Each independent step was the source of random error. The total variance of the protocol
could be obtained by summing the variance of each step:
Var(MF)dilution during j days of treatment
(1-1/t)*[(j*Am/t*2)+mo]*(j- 1)/N 0 2
Var(MF)treatment for j days
(j*Am/t+m0)/N 0 2
Var(MF)dilution during k days of phenotypic lag
(1-1/t)*(m0+j*Am/t)*k/d*N0 2
Var (MF)plating
[(1-d)/d]*(mo+j*Am/t)/N0 2
+[(j*Am/t+mo)/N 0]2*( [1-(xs/ns)]/xs*[ln(xs/ns)] 2+[1-(xodno)]/xo*[ln(xo/no)]2
-----------------------------------------------------------------------= Total Var(MF)
(Eq. A)
Variance of Mutation Rate
The variance of the mutation rate can be approximated as follows:
a 2 (mutation rate) =
X[nj-(J-Jav) 2 -aj2]/[nj.(J-Jav)2] 2
nj: number of observation at each time point;
(Eq.B)
J: treatment time point;
Jay: average of the treatment time points;
aj: variance at treatment time point calculated by Eq. A.
Goals for quantitative studies of BP-induced mutations are to discriminate between
1) zero mutation rate with untreated cultures and low spontaneous mutation rate; 2)
treated cultures mutating at low mutation rate and untreated cultures; 3) cultures with
different treatment conditions.
3.2. DESING AN EXPERIMENT TO STUDY LOW DOSE RATE MUTATIONS
The determining factor for designing a mutation assay is the ability to discriminate
between a treated culture mutating with the same mutations rate as the untreated culture
or a treated culture that is mutating at a low mutation rate such a twice the spontaneous
rate (doubling dose of a chemical). Thus, to study mutagenicity of a chemical, one has to
design an experiment which allows us to study mutations at a rate of 10-7 mutation per
cell per day (or generation). To compare the mutation rates we could calculate the 95%
confidence interval for the difference of the mutation rate based on Eq. B. If that
confidence interval excludes zero, we can reject the hypothesis of equal mutation rate.
Oller et al. (1989a) demonstrated an analysis of variance in a long-term, low-dose
study. Using total hprt mutagenicity as an end-point, they indicated that 500 ml human
cell cultures enables them to discriminate a treated culture that is mutating at a low
mutation rate (3 x 10-7) such as twice the spontaneous rate (1.5 x 10-7).
The end-point of this study, however, is a mutational hotspot comprised of mutant
population carrying the same mutations. Such hotspot may contain 1%of total hprt
mutants (discussed in Section 5.4.1). So we have to design an experiment to study the
mutations arise at a rate of 10-9. We now consider a culture with a hotspot mutation rate
of 1.0 x 10- 9 mutation per cell per day. In order to distinguish spontaneous rate (1.0 x 107) from zero, 1.0 x 10-9 ± (1.96 x SDspontaneous) has to exclude zero. As a result, the
dispersion around the estimated mutation rate has to be less than 50% (SDspontaneous <
0.5 x 10-9 ).
From the model discussed in Section 3.1, one can estimate the variance of mutation
rate by Eq. B. Herein, I calculated the variance of mutation rate using the conditions
reported in Table 3.1. These conditions were based on a long-term, low-dose experiment
performed by Danheiser et al. (1989), but at a larger scale. The major difference is that
studying mutational hotspot doesn't involve microtiter plating. The mutant cells are
obtained by en masse selection with a selective agent. The variances of this process is
likely to be small, since large number of mutants are selected. As a result, our statistical
model only includes chemical treatment, cell dilution during treatment, and culture
sampling and growth after treatment. One can see from the Eq. B that variance of
mutation rate is markedly affected by the number of treated cells. Fig. 3.1 illustrates the
dispersion around the mutation rate expressed as SDmutation rate/mutation rate decreases
as the number of cells treated increases, for a given mutation rate. In order to achieve
SDmutation rate/mutation rate < 0.5 at a mutation rate of 1 x 10-9, one has to treat 3 x 109
cells, equivalent of 3 liters of human cell culture. To ensure the statistical quality, a 4
liter culture system was used in this study.
Now, let's examine whether a 4 liter culture allows us to distringuish mutational
spectra between untreated culture and culture treated with a doubling dose. As a
numerical example to illustrate this point, let's consider spontaneous and mutation rates
for a mutational hotspot to be 1.0 x 10-9 and 2.0 x 10-9 mutation per cell per day,
respectively (Table 3.1). So the difference between them (Ar) is 1.0 x
Table 3.1. Experimental Conditions for Large-Scale Design
untreated culture
Volume (ml)
treated culture
4000
4000
# cells treated (No)
2.4 x 109
2.4 x10 9
Doubling time (hr)
24
24
Growth factor (t)
2
2
Mutation rate (r)
# mutants/day (Am)
Bkg. mutant fraction (MFo)
1x 10-9
2 x 10-9
2.4
4.8
1.0 x 10-8
1.0 x 10-8
# mutants at to (mo)
24
24
Days of treatment (j)
20
20
4000
4000
d factor
1
1
days of phenotypic
expression (k)
6
6
Volume after treatment (ml)
Fig. 3.1
Effect of number of cell treated on dispersion of mutation rate
Effect of the number of cells treated (No) on the percentage dispersion around the
estimated mutation rate. The statistical model was applied to protocol described in Table
3.1. Calculations were made for the following mutation rate: (o) 1 x 10-9 mutations per
cell per day; (A)2 x 10.9 mutations per cell per day.
vo
0.8-
e3
0.6aCu
N
0.4-
*am
0.2·I
I
I
I
1
I
1
I
1
I
1
I
1
I
1
I
1
1
I
1
1
12
16
Number of cells treated x 109 (NO)
20
10-9 . The variance of mutation rate (4 liter culture, NO = 4 x 109) are 2.1 x 10-19 and 1.5
x 10-19 (Fig. 3.1). The variance of the difference of the mutation rate is then equal to 3.7
x 10- 19 (the sum of the two). The confidence interval for the difference in mutation rate
becomes 1.0 x 10-9±(1.96 x 6.0 x 10-10). The lower bound of the interval is -1.8 x 10-10
which is extremely close to zero. This indicates a 4 liter culture system is the minimum
requirement to study mutational spectrum induced by a doubling dose.
3.3. LARGE-SCALE HUMAN CELL CULTURES
Device for Lare Cultures
Large scale human cell culture was designed using modified 7 liter spinner flasks.
A schematic is shown in Fig. 3.2. One sidearm was fitted with a silicone stopper with an
air outlet, an air inlet and a sampling tube. Oxygen was provided as filtered air pumped
through a sparger at a rate of 500 ml per min (see below for discussion). The culture was
sampled daily through the sampling tube. The other sidearm of the flask was fitted with a
medium inlet port and cell outlet port. A portion of the cell culture was pumped out
(depending upon the cell density) and fresh medium of equal volume was replenished
through the medium inlet port, so a constant volume was maintained. Sterile connections
were made using C-flex splicable tubing (Cole-Parmer, Chicago, IL) spliced with a sterile
connection device (Haemonetics Corp., Braintree, MA). Temperature was maintained at
37'C by placing cultures in water jacketed incubators. The working volume was between
2 and 4 liters. Cells were grown in RPMI 1640 media supplemented with 5%donor
horse serum.
Fig. 3.2
Schematic design of large scale human cell cultures
Cell cultures were carried out in modified spinner flask. The working volume was
between 2 and 4 liters. Cultures were maintained on a stirring plate in 37'C dry
incubator. No C02 was enriched into the incubator.
(Graph is not drawn in scale).
S"Vent
Sar
Air
m In
I Out
Oxven Reauirement
It is worth mentioning that oxygen is an essential requirement for the normal growth
of mammalian cells, but in excess or shortage of oxygen may inhibit the growth and
metabolism of the cells. Oller et al. (1989b) studied the growth of another human
lymphoblast cell line (TK6) at various oxygen concentrations. No adverse effect of
oxygen on TK6 cell survival was seen for concentrations ranging from 60 jLM to 410 jiM.
Such range of dissolved oxygen has a pronounced effect on the design of large-scale
mammalian cell culture devices.
The oxygen transfer rate in a fermentor is described by:
OTR = KLa (Cgas - Cliq),
where
OTR = oxygen transfer rate in gmol 1 -Ih- 1;
KLa = mass transfer coefficient in h-l;
Cgas = gas-phase oxygen (equilibrium) concentration in gM;
Cliq = liquid-phase oxygen concentration in gM.
Air is 21% oxygen. It was shown (Schumpe et al., 1978) that the concentration of
oxygen in medium in equilibrium with dry air at 37'C is approximately 200 JIM. It was
also shown by Oiler et al. (1989b) that the oxygen concentration in medium dropped to
50 gM prior to dilution. In addition, they indicated KLa of 2.5 h-1 for oxygen transfer in
a system similar to this study. The oxygen transfer rate attainable using air in the system
is thus 370 inmol -1h-1.
Does this oxygen transfer rate sustain AHH-1 cells grow at 1 x 106 cells per ml?
AHH-1 cell line has an oxygen consumption rate of 0.1 pmole cell-h
-1
(personal
communication with Dr. W.G. Thilly). The concentration of AHH-1 cell culture is
around 1 x 106 cells per ml, the oxygen consumption then becomes 100 jimol 1-h-1 . This
indicates the oxygen transfer rate of the system (370 pmol l-lh-1 ) is enough to sustain the
growth of AHH-1 cells in culture.
General Protocol for Long-term. Low-dose Study
The experimental protocol is illustrated in Fig. 3.3. The cultures were diluted daily
to 5 to 6 x 105 cells per ml. BP was added daily to the culture with fresh media
containing BP at desired concentration (see Section 5.1). At the end of treatment period,
cells were centrifuged (1,000 g x 10 min) and resuspended in fresh media and carried
over for 6 days for the phenotypic expression of hprr mutants. 6TG was then added to
each culture to the final concentration of 1 gig per ml. Cells were grown with the
presence of 6TG for 3 days, and then spun down from 6TG and resuspended in fresh
media. After around 15 days, 6TGr cells grew back and were harvested for the molecular
analysis. Throughout the experiment, all cells removed from the culture during daily
dilution were spun (1,000g x 10 min) and frozen at -135 0 C for later analyses.
Fig. 3.3
Protocol for long-term, large-scale human mutation assay
AHH- 1 cells were HAT treated for 3 days, followed by a 2 day recovery period in
TH. HAT treated cells were then scale up and continually exposed to BP from 0 to 20
days, during which time samples were taken for mutant fraction determination and
molecular analysis. At sampling, BP was removed from the cultures and 6 days were
allowed for phenotypic expression. To determine mutant fraction, the cells were plated in
the presence and absence of 6TG. Plates were incubated at 37"C in 5%C02 humidified
incubators and scored 14 days later. Cells for molecular analysis were grown in 6TG (1
gg/ml) for 3 days after phenotypic expression and resuspended into fresh medium.
Exponential growth resumed 15 days later. Cells were then harvested for molecular
analysis. Throughout the experiment, all cells removed from cultures during daily
dilution were frozen and stored at -135"C.
TIMES (days)
I
I
HAT
t
I
I
I
LL
2
etc.
etc.
Remove BP
Phenotypic Expression
(6 days)
Plate
(w/ & w/o 6TG)
6TG Selection
(14-18 days)
Score Plates
Determine MF
Harvest Mutant Cells
DNA Isolation
Mutational Spectrum
hifi-PCR/DGGE
IV. MATERIALS AND METHODS
4.1. CELL LINE
A human lymphoblast cell line, AHH-1, was developed in our lab and licensed to
the Gentest Corp. (Woburn, MA). This cell line was isolated and characterized by Dr. C.
L. Crespi (Crespi, Ph. D thesis, MIT, 1982; Crespi and Thilly, 1984).
AHH-1 cell line was isolated by from a subclone of the RPMI-1788 human
lymphoblast line with some BP-oxygenating ability (Freedman et al. 1979a, b; Moore and
Minowada, 1969). RPMI-1788 cells contained high basal and induced level of P4501A1
(AHH) activity. This activity could be induced about 3.5-fold by pretreatment with 10
p.M BP for 48 hours. Because the RPMI-1788 cell line was contaminated with
mycoplasma, mycoplasma-free subclones were isolated, and the cell were subject to
antibiotic treatment. One of the mycoplasma-free clone was found to be hypersensitive to
BP-induced growth inhibition and was designated AHH-1. This cell line has been
routinely tested for mycoplasma contamination and all tests have been negative.
AHH-1 cell line is competent in oxidative xenobiotic metabolism, thus it is
sensitive not only to directly active mutagens (i.e. EMS, ICR-191) but also mutagens
which require metabolic activation (i.e. BP, aflatoxin B i). It expresses high basal levels
and inducible benzo[a]pyrene hydroxylase (AHH) activity and 7-ethoxyresofurin
deethylase activity, but minimum level of epoxide hydrolase. These activities can be
induced by pretreatment with various PAH such as BP or 8-naphthoflavone. A
significant induction of AHH activity was observed after a 24 hr pretreatment of 0.01 gM
BP with a plateau of 13.8-fold increase in AHH activity after treatment of AHH-1 cells
with 10 g.M BP for 24 hrs (Fig. 4.1). The time course of AHH activity was also
determined (Fig. 4.2). Significant induction occurred after a 1 hr delay with exposure to
10 .rMBP. AHH activity increased linearly with time, reached a maximum at 24 hrs, and
Fig. 4.1
Concentration dependence of BP induction of AHH activity
Cells were treated with different concentrations of BP for 24 hours. Cells were
washed and AHH activity measured. Each point represents the mean of 4 independent
determinations (2 independent experiments). Confidence limits represent on standard
deviation.
(Graph adapted from Crespi, Ph.D. Thesis, MIT, 1982)
maowrrmwNC
OanMNN
Fig. 4.2
Time course of AHH activity induction
Relative AHH activity is plotted vs. time (0 - 72 hours) after induction of AHH-1
cells with 10 pLM.
(Graph adapted from Crespi, Ph.D. Thesis, MIT, 1982).
LO
OhO
COURS AFTER
CUCTION
remained fairly stable up to 72 hrs. Phenol production (indicative of AHH activity) was
found to increase linearly with induced hprrmutations (Crespi, Ph.D. Thesis, MIT,
1982).
Many aspects of AHH-1 cell biotransformation resembles that of human peripheral
lymphocyte. AHH- 1 cells produces primarily phenolic metabolites of BP with minor
amounts of quinones and dihydrodiols. However, AHH-1 cells don't produce any known
amount trans-dihydrodiol-BP metabolite. BP-adduct analysis showed that BP-4,5-oxide
accounts for 7% of the adduct and 9-hydroxy-4,5-oxide-BP accounts for about 2%. The
majority of adducts consist of uncharacterized species (Crespi, Ph.D. Thesis, MIT, 1982).
AHH-1 cells, in addition to being competent for xenobiotic metabolism, are
phagocytic (Crespi et al., 1985). This characteristic can be used for studying the effects
of xenobiotics present in particulate, since materials carried by particulate are extracted
under near-physiological conditions.
4.2. CELL MAINTENANCE
AHH-1 cell line is anchorage independent, so it grows in both stationary and stirred
cultures with a doubling time of around 24 hrs. AHH- 1 cells were grown in RPMI 1640
media (JRH Biosciences, Lenexa, KS) supplemented with 5%donor horse serum (JRH
Biosciences, Lenexa, KS). It should be noted that response of AHH- 1 cells to inducers of
AHH activity is strongly influenced by unknown serum factors. Screening of serum
batches is necessary in order to sustain the cells' expression of cytochrome P450 enzyme
(Drs. Crespi and Penman, personal communication). The cultures were diluted daily to 56 X 105 cells per ml and maintained in 37"C incubator with a 5%C02 atmosphere. Care
was exercised that the cell concentration never exceeded 1.2 x 106 cells per ml. In
stationary cultures, cells were maintained in 175 cm 2 T-flasks. They tend to grow as
loose aggregates which were dissociated by agitation before cells were counted on a
model B Coulter counter (Coulter Electronics, Hialeah, Fl.). In stirred cultures, cells
were grown in large spinner flasks (7 liter capacity). The volumes were kept at 2 to 4
liters as required to assure statistical reproducibility (See section 3.2). Media conditions
were the same as stationary cultures.
4.3. BENZO[a]PYRENE TREATMENT
Consumption of BP by AHH-1 cells can be estimated by AHH activity. Fig. 4.1
indicates that the maximum consumption of 1.2 pmole BP/10 6 cells/min is achieved after
treating cells with 3 pLM BP for 24 hours. Taken into the account that concentration of
AHH-1 cell culture is around 106 cells per ml, this consumption rate can be converted to
1.7 gmole 1-Id- 1 or 1.7 ptM per day. So the removal of BP via incubation in media with
cells can only be considered negligible when dose rate is high (i.e. 10 pM). At low dose
rate (i.e. < lg.M), all (or majority) of the BP is consumed by cells before the next dosing.
The exposure scheme of BP is then considered continuous at high dose rate but
intermittent at low dose rate.
BP was dissolved in a known amount of DMSO. Stock solutions of required
concentrations was prepared by serial dilution, and then divided into aliquots for daily
uses and stored at -70°C in the dark. At the beginning of the experiment, fresh BP was
added to AHH-1 cultures at the desired concentrations and then replenished daily by
adding fresh media supplemented with BP at studied concentrations. Considering a 2
liter culture treated with 0.5 tM BP treated culture for example, one dilutes the culture
2x daily. One liter culture is removed and replenished with 1 liter fresh media containing
250 gg BP (1 LM), so the final concentration is 0.5 pM assuming most of BP the
previous day has been removed by cells. DMSO concentrations never exceeded 0.1%
(v/v) in the culture. The spontaneous cultures contains DMSO only. To end treatment,
the cells were centrifuged and resuspended in fresh media. Cultures were carried over for
6 days with daily dilution for the phenotypic expression of hprt mutants.
4.4. DETERMINATION OF SURVIVAL AND MUTANT FRACTION
Two methods were used in this experiment to determine the survival fraction of the
cells after acute BP treatment. The first method involves the daily monitoring of cell
growth after the treatment (DeLuca er al., 1983). Growth curves were plotted in loglinear fashion. Given the assumption that surviving cells grow exponentially and no
growth lag occurs, the survival fraction of cells after treatment can be determined by
back-extrapolation of growth curves (Fig. 4.3). The extrapolation of the exponential
phase of the curve back to time zero reveals the number of live cells (YI) following
treatment. Percent survival is determined by dividing Yj by the number of cells originally
counted at time zero (YO). Were there to be a growth lag or of slower growth, this
method would tend to overestimate toxicity.
The other method involves comparison of colony-forming ability of treated vs.
untreated cultures (DeLuca et al., 1983). It is done by seeding a known number of cells
(e.g. 2 cells per well) into microtitre wells immediately following treatment. Relative
survival of cultures is determined by comparing the colony-forming efficiencies of the
untreated to treated cultures.
Mutant fractions after BP treatment were estimated by comparing the colony
forming ability of the cells in the presence and absence of selective conditions (Furth et
al., 1981). Cells were plated into 96-well microtiter plates. Based on the Poisson
distribution, the average number of colony forming units per well (g.) can be calculated as
long as some positive and some negative wells are observed. The probability of
observing a negative well is P(O) = e-9L where gt = -In (fraction of observed negative
Fig. 4.3
Determining survival by growth curve extrapolation
The survival fraction of cells after treatment can be determined by backextrapolation of growth curves. Cell counts are monitored daily. The extrapolation of the
exponential phase of the curve back to time zero reveals the number of live cells (Y
1 )
following treatment. Percent survival is determined by dividing YI by the number of cells
originally counted at time zero (YO): S = YI/Yo
e
E
3·
c,
u,
CD
0
Y
to
Time
wells). The plating efficiency (PE) of a culture is then defined as g divided by the
average number of cells plated per well.
-In[# wells with no colonie
# wells scored
PE PE
=
# cells/well
The survival of cells after treatment was calculated by the ratio of PE between
treated and untreated cultures.
When the cells are plated in the presence of selective agent that allows growth only
for the surviving mutants, the mutant fraction was calculated as:
MF =
[# wills with no colonies]
-In
# wells scored
J
#cells/well X PE
The number of cells per well put down to determine PE was 2 cell per well whereas
the number for MF was 20,000 cells per well.
The number of cells per well put down to determine PE (2 cells per well) is much
lower than the number of cells per well put down to determine MF (20,000 cells per
well). Thus, the means of determining the mutant fraction is biased on the assumption
that the cloning efficiency of cells at low density is the same as high density. Such bias
was tested by Kersch and Thilly (1987) by adding an experimentally distinguishable
population to the cell cultures as an internal standard. They demonstrated that the
calculated mutant fractions using the conventional low-density PE method were
significantly higher than the mutant fraction calculated using the internal-standard
method. In addition, growth-curve extrapolation yielded the higher estimate of survival
than low-density PE.
4.5. REDUCING BACKGROUND MUTANT FRACTION
The mutagenic effect of a particular mutagenic treatment is measured as the
difference between the mutant fractions of treated and untreated cultures. As spontaneous
mutants accumulate, there is a non-zero background mutant fraction. The magnitude of
the background mutant fraction is a function of spontaneous mutation rate and time. The
estimation of an induced mutant fraction becomes more imprecise as the treated
approaches the untreated mutant fraction. The magnitude of the background mutant
fraction limits the sensitivity of any assay (Oller et al., 1989a).
In order to eliminate any mutants which arose prior to BP treatment, cells were
grown under conditions which select against pre-existing mutants. This deliberate
reduction in the background mutant fraction increases the sensitivity of the mutation
assay. Exponentially growing AHH-1 cells were first grown for 3 days in a HAT
medium containing 2 x
4
10A
M hypoxanthine, 8 x 10-7 M aminopterin and 3.5 x 10-5 M
thymidine (all from Sigma Chemicals, St. Louis, MO). Aminopterin blocks de novo
synthesis of purines. Only cells able to incorporate the thymidine and hypoxanthine via
salvage pathways can grow in the presence of HAT. Cells without hprt activity, however,
do not have a hypoxanthine salvage pathway and are killed and diluted out by the growth
of hprt+ cells in HAT.
After treatment with HAT, cells were centrifuged, resuspended in medium
containing TH (3.5 x 10-5 M thymidine and 2 x 104 M hypoxanthine) and maintained for
two days. Such time is required for the synthesis of new tetrahydrofolate reductase,
since the old enzyme is depleted by forming a strong bond with aminopterin. Cultures
were then scaled up and treated with test substance.
4.6. MOLECULAR ANALYSIS OF POINT MUTATIONS ON LOW-MELTING
DOMAIN OF hprt EXON3
The hprt locus is the most frequently studied gene study of mammalian
mutagenesis. The gene is X-linked (Szybalski, 1958) and expressed constitutively in all
tissues (Krenisky 1969; Kelley and Wyngarrden 1983). Hereditary deficiency of hprt in
people leads to a severe condition known as Lesch-Nyhan syndrome with symptoms
including mental retardation and self mutilation (Seegmiller et al., 1967). In vitro, the
absence of hprt enzyme activity confers no selective disadvantage to human B cells in
culture (Thilly et al. 1976). It has been suggested however that hprr mutants in vivo are
at a selective disadvantage and do not persist for long periods. Albertini and DeMars
(1974) suggested not only selection against hprr mutants in the lymphocyte population in
vivo, but increasing selection against the mutants with age.
The hprt gene codes for a total of 218 amino acids. It is around 42 kb and contains 9
exons. Exon 3 is the largest exon of the gene and encompassing 28% of the coding
region. The exon is split into a 80 bp high melting domain and 104 bp low melting
domain. Exon 3 is the only hprt exon with a stable high melting region situated within
the exon boundaries, and the low melting region has the appropriate length and number of
mutable sites. As a result, the low melting domain of exon 3 is chosen to be the target
sequence for mutational spectra in this study (Thilly, 1985).
The protocol for mutation analysis is outlined in Fig. 4.4. DNA from the 6TGr cells
was isolated with anion exchange columns (Qiagen, Studio City, CA) and then PCR
amplified using high fidelity enzyme pfu (Strategene, La Jolla, CA). 20mer primers P1
and P2, immediately flanking the exon were used (Fig. 4.5). PCR products were purified
using polyacrylamide gel electrophoresis (PAGE). DNA was recovered using D-gel
(Epigene Corp., Baltimore, MD) electroelution and then end-labeled with y-32P (NEN-Du
Pont, Wilminton, DE) using T4 polynucleotide kinase (New England Biolabs, Beverly,
Fig. 4.4
Protocol for molecular analysis of 6TGr mutant population
PROTOCOL FOR MOLECULAR ANALYSIS OF MUTATIONS
U
DNA Isolation
?3
5
10 cell pellet
Genomic DNA
PCR
Page Purify the
Desired Band
32
P Endlabelling DNA
_
S*
WT
•Mutant
Boil and Reaneal
Sequencing
*
S
*
. Heteroduplex 1
- Heteroduplex 2
Mutant
PCR
SWT
Exercized from DGGE
i------i
I~I
] Het 1&2
Mutant
WT
Fig. 4.5
Layout and sequences of primers used for the analysis of 6TG mutations in the lowmelting region of hprt exon 3
20mer primer P1 and P2 hybridized to the flanking intron sequences of exon 3 and
were used to amplified the exon from genomic DNA. P4 adjacent of primer P2 was used
for DNA sequencing.
Melting Map of Hprt Exon 3 Low Melting Domain
76
727068666462I
60
I , I , I
1
150
-
-
t
It I I I f t I I
t1
.
a,
.,
a I
,
300
250
200
Base Pair Position
EXON 3 CODING 184 bp
350
r-
P4
P2
P4
(Sequencing)
P1
P1
PRIMER SEQUENCES
P1
5' CAT ATA TTA AAT ATA CTC AC 3'
P2
5' TCC TGA TTT TAT TTC TGT AG 3'
P4
5' GAC TGA ACG TCT TGC TCG AG 3'
MA). In order to form wild type (WT)-mutant heteroduplexes for DGGE analysis, DNA
samples were boiled for 3 minutes, and then allowed to reanneal at 650C for 2 hours.
Denaturing gradient gel electrophoresis (DGGE) was used to separate mutant DNA
sequence from the WT. DGGE uses a linear denaturant gradient to mimic the
temperature gradient. The mutant-wild type heteroduplex is less stable than wild type so
that the double strands of heteroduplexes are denatured at lower temperature. As a result,
mutant DNA migrates at different position compared to the wild type. The 50%
denaturant stock consisted 3.5 M urea, 20% formamide (v/v), 6.5% acrylamide (37.5:1
acrylamide:bisacrylamide) and 1X TAE. 0% stock contained only 6.5% acrylamide and
LX TAE. DGGE gels were 1 mm thick and run at 150 V for 12 to 18 hours submerged
in 400 C circulating lx TAE buffer. After electrophoresis had been completed, gels were
dried and exposed to X-ray film and PhosphorImager (Molecular Dynamics, Sunnyvale,
CA) screen.
The fraction of a particular mutation within the population was quantified by
comparing the intensity of altered bands to the WT bands. It should be mentioned that
this approach takes into account only point mutations in the target sequence. Mutants
with large deletions are neglected. Intensity of bands were measured with storage
phosphor technology (PhosphorImager, Molecular Dynamics, Sunnyvale, CA).
In order to discover the underlying DNA alteration, mutant bands separated on a
DGGE gel was excised from the gel and purified. Purification involved reamplification
of the band, PAGE purification and analysis on second DGGE. Such process might have
to repeat couple of times before the band was pure enough to be sequenced. Amplified
fragments were then sequenced with a modification of the Sanger method (Sanger et al.
1977) suggested by Dr. Gengxi Hu (1994). In brief, 1011 copies of PAGE-purified DNA
were mixed in 10 pM solution containing 1013 copies of primer P4, 50 gCi a- 35S-dATP
(1000 Ci per mmole), 20 mM tris.HC1, pH 7.5, and 4 mM MgC12. 2.5 pl of the mixture
was aliquot to each of the four 2.5 pl A, G, C and T termination solutions, all of which
contain 3 mM each dATP, dGTP, dCTP, dTTP and one of the following: 0.7 PM ddATP,
0.9 mM ddGTP, 0.9 mM ddCTP and 0.7 mM ddTTP, respectively. The four termination
mixtures were then heated at 95"C for 1 min; chilled to room temperature for 1 min; and
incubated at 37'C. Two units of Sequenase II (United State Biochemical, Cleveland, OH)
were added into each of the four mixture. The reaction were incubated at 37'C for 5 min;
chased by 2 unit Sequenase II in 40 mM dATP, 10 mM each dGTP, dCTP, dTTP at 37'C
for another 5 min; and then mixed with 4 Wi 95% formamide, 20 mM EDTA, 0.05%
bromophenol blue, 0.05% xylene FF. The mixtures were denatured by being heated at
95'C for 3 min and run on a 6% sequencing gel.
V. RESULTS AND DISCUSSION
5.1. SELECTION OF DOSE AND DOSE RATE
It is worth emphasizing the difference between "dose" and "dose rate". As defined
in Section 2.1, "dose rate" (concentration) is the temporal pattern of the "dose"
(concentration x time). Defining a "low dose" and "low dose rate" of a mutagen is quite
arbitrary. In this study, "low dose rate" refers to situations common to human experience
(discussed in Section 5.5). In such case, total "dose" is not necessarily low since the
duration of exposure can be long.
Most biological data available nowadays fail to reach low dose rate because of the
technical (and possible financial) limitation. Crespi (Ph.D. Thesis, MIT, 1982) found that
BP induced statistically significant mutations at 0.3 gM for an exposure time of 24 hours
(when compared to the experimental negative control) or 1 gM for 24 hours (when
compared to the historical distribution of negative controls). Danheiser et al. (1989) first
demonstrated the mutation rate for 0.02 gM BP was equal to the spontaneous rate in
AHH-1 cells. This constitutes the lowest detectable concentration for statistically
significant long-term BP-induced mutations in AHH-1 cells. In this study, an experiment
was designed to study BP-induced mutational spectrum under the lowest detectable BP
concentration, and compare it to that under a higher dose. Concentrations of 0.02 pM BP
and 0.5 gM BP were chosen to be tested on the long-term dose effect on BP mutational
spectra in AHH- 1 cells. In addition to 0.02 pM and 0.5 gM BP, both of which are shown
to be nontoxic, a toxic dose of 30 gM was also studied.
Initial Exoeriment (Small Scale)
A small scale experiment was carried out to assess the mutagenicity of BP under the
selected doses. AHH-1 cells were grown in 150 ml stationary cultures and treated with
0.02 pM and 0.5 p.M BP every day for up to 20 days. Each condition involved three
independent cultures. At different time point, BP was removed from cultures and 6 day
were allowed for phenotypic expression. Mutant fraction at hprt locus was then
determined. Spontaneous cultures were treated with 100 pt DMSO only.
For both 0.02 p.M and 0.5 prM treated cultures, there was no apparent toxicity.
Mutagenicity results are demonstrated in Fig. 5.1. The spontaneous mutation rate of
AHH-1 cells was 4.6 x 10-7 mutation per cell per day. Treatment with 0.02 prM BP
resulted in the observed mutation rate of 10 x 10-7. Interesting observation was made that
for 0.5 prM BP, the rate of BP-induced mutation was high and constant (2.9 x 10-6 )
between days 0 and 7, at about which time it dropped off and remained at a reduced level
(1.2 x 10-6) for at least 20 days exposure. This reduced mutation rate is similar to the
observed mutation rate with 0.02 lpM BP. These results confirm the findings by
Danheiser (M.S. Thesis, MIT, 1985).
Selecting Dosing Scheme
The selection of doses for mutational spectra study is illustrated in Fig. 5.2.
Concentrations of BP include 0.02 pM (nontoxic), 0.5 p.M (nontoxic) and 30 pM (toxic).
Duration ranges from short term (< 6 days) to long term (20 days). To study the effect of
dose and dose rate on BP mutagenesis, we compared the BP mutational spectra of 0.02
p.M vs. 0.5 p.M, toxic dose rate vs. nontoxic dose rate, and long term vs. short term.
Now, let's analyze the mode of exposure with the selected dose rate. From Fig. 4.1,
one can calculated the removal rate of BP in AHH- 1 cultures. For example, at anticipated
0.04 p.M BP, the rate of BP consumption is 0.4 pmole/10 6 cells/min, equivalent to 0.58
p.M per day. Such removal is more than an order of magnitude higher than the supply
(0.04 pgM per day) that assumes no removal of BP. The anticipated 0.04 pM treatment is
thus intermittent (or pulse) with actual concentration of 0.02 pM. On the first treatment
day of this study, cultures of low dose rate were dosed with 0.04 pM BP, instead
Fig. 5.1
Dose and dose rate dependence of BP mutagenesis
in small (150 ml) stationary AHH-1 culture
Cultures were treated in triplicate with 0, 0.02 and 0.5 .M BP for up to 24 days.
Error bars represent one population standard deviation of the mean. The lines represent
the best fit lines calculated by the method of least squares. 0 LM BP cultures were treated
with DMSO (<0.1% v/v). 6TGr mutant fraction was determined after treatment of
following days:
0 gM: days 0, 2, 5, 8, 14, 24;
0.02 gM: days 0, 2, 6, 8, 12, 18, 20;
0.5 gM: days 0, 2, 6, 8, 12, 18, 20.
mutant fraction
0
(n
O
0,
(N)
U,
5;
Fig. 5.2
Selection of doses and duration for large scale human cell cultures
The selection of doses and duration was based on the results from data in Fig. 4.1.
Both low dose and high dose are subtoxic. Selection of the toxic dose was based on
Crespi's data (Ph.D. Thesis, MIT, 1982). Duration ranges from 28 hrs to 20 days. The
number in the bracket indicates the total dose (M*-d).
Dose rate (MU)
Duration (d)
Total Dose (UM-d)
0
20
0
0.02
20
0.4
0.5
6
3
0.5
20
10
30
1.2
36
c
0
LLL
L-
0.5 uM x 20 d
5
10
15
Time (days)
20
of 0.02 i±M. Because the BP treatment was done assuming the removal of BP was
negligible, so at the second day of treatment, the culture was diluted 2 x with medium
containing 0.04 gM BP. Since the BP added the day before was removed by cells, the
concentration of BP in the culture becomes 0.02 .gMinstead of anticipated 0.04 pM from
day 2 to day 20 as illustrated in Fig. 5.3.
In the case of anticipated 1 p.M BP, removal rate is a 1.1 pgM per day which is close
to the daily supply rate. As discussed above, from day 2 to day 20, the BP concentration
may be close to 0.5 pM. The exact BP concentration in the culture is difficult to estimate.
The best way is to measure the concentration directly in the culture. Nevertheless the
mode of 1 p.M exposure can be considered as semi-continuous (or semi-intermittent) with
a equilibrium concentration close to 0.5 pgM. An diagram illustrating the mode of
exposure with the selected dose conditions is presented in Fig. 5.3.
5.2. TOXICITY AND MUTAGENICITY OF BP IN LARGE CULTURES
Ten large scale cultures were completed. They include duplicate cultures of each
following conditions: 0 .gMx 20 days (Spontaneous), 0.02 p.M x 20 days (dose: 0.4
gtM-d), 0.5 gpM x 6 days (dose: 3 gM.d), 0.5 pgM x 20 days (dose: 10 gM*d), and 30 pM x
28 hours (dose: 36 M*-d).
Fig. 5.4 illustrates the growth of AHH-1 cells under various BP concentrations. BP
demonstrated no toxic effects on AHH- 1 cells with the concentrations of 0.02 .rMand 0.5
pM over a 20 day exposure. Cultures with no treatment and these subtoxic concentrations
grew exponentially with doubling times of 21.8 to 23.5 hours. Cultures treated with 30
p.M BP, however, slowed down after the treatment (Fig. 5.5). Back-extrapolation of the
growth curve revealed survival fractions of 82% and 76% for two independent cultures
and doubling times of 25.6 and 27.3 hours, respectively.
Fig. 5.3
Mode of exposure with selected dose rates
AL
0.04
0.02 uM x 20 d
-
-0
C
o
0.02
>
0c.
C
0
I
I
I
I
I
Days
0.5 uM x 20 d
r-
.0
E-
o
0,_
o
0.5
E-
a.
m
Days
I
I
I
I
Fig. 5.4
Growth curves of spontaneous, 0.02 gM and 0.5 gM BP treated cultures
Cell counts were recorded each day for the duration of the experiment. The
doubling time for each culture was determined from regression analysis of the growth
curve according to the equation:
t/
Nt = No2V
NO: the number of cells at time 0;
Nt: the number of cells there would have been on the last day of BP exposure;
t: days of treatment;
r: doubling time of the experiment.
Spontaneous:
t = 23.2 ± 0.4 h;
0.02 p.M treated:
t = 23.5 ± 0.5 h;
0.5 p.M treated:
t = 21.8 ± 1.7 h.
1 013
,,
1
1 09
1 07
1 05
100
200
300
400
500
100
200
300
400
500
1 012
~1 010
o
1 08
1 06
0
101 5
1011
1 09
10
1 07
1 05
0
100
200
300
time (h)
87
400
500
600
Fig. 5.5
Growth curves of 30 gM BP treated cultures
Cells were treated with 30 gM BP for 28 hrs. After the treatment, cells were
centrifuged and resuspended into fresh media (time 0). Cell concentration was measured
daily. Toxicity was assessed by back-extrapolation of the growth curve. The best fit
lines in the graph were calculated by least squares.
Cell survival:
culture 1: S = 0.82, T = 25.6 ± 0.4 h;
culture 2: S = 0.76, t = 27.8 ± 0.6 h.
8
IU
7
10
o
10 s
5
1n
-50
•
IV
0
50
100
150
8
10 7
106
10
-50
0
50
time (h)
100
150
Mutant fraction results are demonstrated in Fig. 5.6. The mutation rate is 7.5 x 10-8
mutation per cell per day for spontaneous and 8.4 x 10-7 for 0.02 pM BP treated cultures.
The tenfold increase in mutation rate resulted by 0.02 p.M BP treatment is much higher
than anticipated twofold increase based on results from stationary cultures (Fig. 5.1).
This is primarily due to the unexpected decrease in spontaneous mutation rate in large
stirred cultures (4.6 x 10-7 in stationary to 7.1 x 10-8 in stirred). The reason for such
decline is not known. One speculation is that high mutation rates in stationary cultures
were results of oxygen depletion since cells tended to aggregate at the bottom of the Tflasks. A micro-environment of anoxia might exist around cell aggregate which enhanced
the spontaneous mutation rate.
Treatment of 0.5 gM BP for 6 days resulted in a mutation rate of 2.3 x 10-6 which
then was reduced to 8.7 x 10-7 by further treatment of 0.5 gM BP for additional 14 days
(Fig. 5.6). The later rate was virtually the same with that of 0.02 pM treated culture (8.4
x 10-7).
The number of independent surviving mutants was calculated simply by the
product of mutation rate and the number of cells treated. It was shown that all the BP
treated cultures demonstrated elevated mutant fraction compared to untreated cultures,
and contained over 20,000 independent surviving mutants at the end of treatment (Table
5.1). The two spontaneous cultures, however, yielded 6302 and 3532 hprr independent
mutants due to unexpected low spontaneous mutation rate.
5.3. STATISTICAL ANALYSIS OF VARIANCE IN LARGE-SCALE HUMAN
CELL CULTURES
Herein, I reported the statistical results on the large-scale human cell cultures based
on the model by Oiler et al. (1989a). This model was also used to design the large-scale
Fig. 5.6
Summary of BP induced mutant fraction in large-scale AHH-1 cultures
Each point represents the mean of the mutant fractions of two independent cultures.
Numbers on the line represent mutation rate (mutation per cell per day):
Spontaneous:
7.1 x 10-8;
0.02 gM x 20 d:
8.4 x 10-7;
0.5 pM x 6 d:
2.3 x 106 ;
0.5 gM x 20 d:
8.6 x 10-7;
30 gM x 28 h:
7.5 x 106.
3e-5
1-
2e-5
F
d
I
e-5
r\
7.1E-8
DAYS
Table 5.1. Summary of the Large-Scale Cell Culture Experiment
BP
Duration
Volume
Mutant
Mutation
# Independent
(gM)
(days)
(liters)
Fraction
Rate
Mutant
1
0.00
20
4
2.90 x 10-6
9.1 x 10-8
6,300
2
0.00
20
4
2.10 x 10-6
5.1 x 10-8
3,530
0.02gtMx20d 1
0.02
20
4
1.83 x 10- 5
8.6 x 10-7
59,600
2
0.02
20
4
1.75 x 10- 5
8.2 x 10-7
56,900
1
0.5
6
4
1.45 x 10- 5
2.1 x 10-6
21,800
2
0.5
6
4
1.71 x 10-5
2.5 x 10-6
26,300
1
0.5
20
2
2.95 x 10-5
1.1 x 10-6
37,600
2
0.5
20
2
2.62 x 10- 5
8.5 x 10-7
42,100
1
30.0
1.2
4
1.01 x 10-5
7.4 x 10-6
25,200
2
30.0
1.2
4
1.03 x 10-5
7.6 x 10- 6
23,900
Cultures
Spontaneous
0.5gLMx6d
0.5pMx20d
30gMx28h
experiment (Section 3.2). Experimental conditions used in the experiment are
summarized in Table 5.2.
Variance ofMutant Fraction
Table 5.3 summarizes the variance from each physical step and the total variance of
the experimental protocol based on Eq. B and experimental conditions presented in Table
5.2. The relative error on the mutant fraction is expressed by the ratio of standard
deviation and mutation fraction (SDMF/MF). From the analysis, it is obvious that plating
is the major source of variance. The dispersion of the protocol is accounted for nearly
entirely (>94%) by the random error due to fluctuation in mutant colony number in
plating. Spontaneous cultures have the highest dispersion (SDMF/MF=18.5%) because of
their low mutant fractions.
Variance of Mutation Rate
Variance of mutation rate are summarized in Table 5.4. Again the spontaneous
cultures demonstrated the highest dispersion (SDmutation rate/mutation rate = 27.3%). For
the other three conditions, the dispersion is quite small.
An important goal for quantitative studies of mutations is the desire to discriminate
between 1) zero mutation rate and low spontaneous mutation rate; 2) treated cultures
mutating at low mutation rate and untreated cultures; 3) cultures with different treatment
conditions. To compare the mutation rate we could calculate the 95% confidence interval
for the difference of the mutation rate. If that confidence interval excludes zero, we can
reject the hypothesis of equal mutation rate.
As a numerical example to illustrate this point, let's consider spontaneous and 0.02
gM x 20 d mutation rates of 7.1 x 10-8 and 8.4 x 10-7 mutation per cell per day,
respectively (Table 5.4). So the difference between them (Ar) is 7.7 x 10-7 . The variance
of mutation rate are 4.2 x 10-16 and 2.6 x 10-15 (Table 5.4). The variance of the difference
Table 5.2. Experimental Conditions Used in Large-scale Cell Cultures
S
D.02pMx20d
0.5jM x 6d
D.5pM x 20d
5)0M x 28 h
4000
4000
4000
2000
4000
# cells treated (No)
2.4 x10 9
2.4 x 109
2.4 x10 9
1.2 x 109
2.4 x 109
Doubling time (hr)
23.6
23.5
21.8
21.8
26.5
Growth factor (t)
2
2
2
2
2
Mutation rate (r)
7.10 x10-8
8.41 x10-7
2.32 x 10-6
8.60 x 10-7
7.50 x 10-6
340
4037
11136
2064
36000
1.0 x 10-6
1.0 x 10-6
1.2 x 10-6
1.6 x 10- 5
1.2 x 10-6
# mutants at to (mo)
2400
2400
2880
18840
2880
Days of treatment (j)
20
20
6
14
4000
4000
2000
2000
4000
d factor
1
1
0.5
1
1
days of phenotypic
expression (k)
6
6
6
6
6
# wells plated w/o 6TG (no)
768
768
768
768
768
# wells plated w/ 6TG (ns)
1536
1536
1536
1536
1536
# negative wells w/o 6TG(xo)
285
282
279
378
311
# negative wells w/ 6TG (xs)
1502
1271
1311
1311
1421
Volume (ml)
# mutants/day (Am)
Bkg. mutant fraction (MFo)
Volume after treatment (ml)
1.2
Table 5.3. Variance of Mutant Fraction in Large-Scale Human Cell Cultures
S
0.02 pM
x20d
0.5 pM
x6d
0.5p M 30 jM
x20d x28 h
Var(treatment) (xlO-15 )
1.04
7.46
6.25
23.2
0.24
Var(dilution during treatment) (x10 -15 )
6.94
37.4
8.44
118
4.25
Var(dilution after treatment) (xl0-15)
3.13
3.36
37.5
69.7
12.8
Var(plating) (xl0-13)
1.98
19.3
15.1
55.4
1.63
7Variance (x10 -12)
0.21
1.98
1.56
5.75
0.18
Standard Deviation (x10 -6)
0.46
1.40
1.25
2.38
0.42
Mutant fraction (x10-6)
2.50
17.9
15.8
27.9
10.2
SDMF/MF (%)
18.5
7.82
7.91
8.53
4.20
Table 5.4. Variance of Mutation Rate in Large-Scale Human Cell Cultures
S
0.02 IiM 0.5 pM
x 20 d
x6d
0.5 p.M
x 20 d
30 pM
x 28 h
Mutation rate (x10-7)
0.75
8.45
23.0
8.71
75.0
Varmutation rate (x10-15)
0.42
2.64
22.6
14.8
13.9
SDmutation rate (x10-8)
2.05
5.14
15.0
12.2
11.8
SDmutation rate/mutation rate (%)
27.3
6.08
6.52
14.0
1.57
of the mutation rate is then equal to 3.1 x 10-15 (the sum of the two). The confidence
interval for the difference in mutation rate becomes 7.7 x 10-7±(1.96 x 5.5 x 10-8). The
lower bound of the interval is 6.6 x 10-7 (> 0), which indicates the protocol enables us to
distinguish spontaneous mutation rate and that induced by exposure to 0.02 prM BP for 20
days. As shown in Table 5.5, there are clear differences between mutation rate of:
spontaneous and zero; spontaneous and 0.02 pM x 20 d; 0.02 gM x 20 d and 0.5 W.M x 6
d; yet the mutation rate of 0.5 pgM x 20 d and 0.02 pM x 20 d is virtually the same.
5.4. DOSE DEPENDENCE OF BP MUTATIONAL SPETRA
5.4.1. Definition of Mutational Hotspot
A mutational spectrum is the frequency of all mutants of a defined DNA sequence
in a defined cell population. In a global sense, the mutational spectrum of a cell
population is an ensemble of mutations from all possible genetic events: base pair
substitution, recombination, large and small deletions, aneuploidy and heritable DNA
modifications. This study is restricted to point mutations, i.e. base pair substitution and
small deletion and additions.
There is no precise definition of a hotspot as related to mutational spectra. Hotspot
is usually considered as a mutant sequence that occurs at higher frequency than expected
by chance with 95% confidence. Yet many researcher have arbitrarily considered a
hotspot as a mutant represented in the sample as a fraction greater than 5%or 10%.
Obviously, as the length of the target sequence increases, the number of possible
mutations increases and the fractional contribution of a particular mutation decreases.
Therefore, defining a hotspot is subject to the target sequence one is studying.
For the sake of argument let us agree that there are 3 kinds of base substitutions and
insertion or deletion possible at each base pair. Let us also agree to ignore mutations
Table 5.5. Comparison of Mutation Rate (r) under Various Doses
S
vs.
Zero
Ar (x10 - 7 )
X Variance
(x10-15 )
SD (x10 - 7 )
Ar ± 1.98 x SD (x10 -7 )
Distinguishable?
S
0.02p.Mx20d 0.5p.Mx6d 0.5gMx20d
V
vs.
vs.
vs.
vs.
0.02gMx20d 0.5gMx6d 0.5gMx20d
0.02pMx20d
0.75
7.70
14.6
14.3
0.26
0.42
3.06
25.2
37.4
17.44
0.21
0.55
1.59
1.93
1.32
14.6±3.1
14.3±3.8
0.26±2.6
yes
yes
no
0.75±0.42 7.70±1.09
yes
yes
involving more than one base pair substitution even though such events do occur.
Therefore, there are 5 kinds of mutations can occur at any base pair. Considering hprt
gene which contains 654 base pairs, one expects 3270 possible mutations. Chance
implies that all these possible mutations arise with equal probability, so the probability of
a hprt mutation, if occurs randomly, equals 0.03% (1/3270). If a mutant sequence is
observed at significantly higher frequency than 0.03%, it is then considered a hotspot.
In this study, a hotspot is defined as a mutant sequence which occurs at frequency
higher than 1%. It can also be represented by 1%of 6TGr population which is composed
of cells with possible mutations distributing over the entire hprt region. Are hotspots
shared by 1%6TGr cells significant within a hprt mutational spectrum? As discussed
above, a random distribution of mutations would give a baseline of 0.03%. It should be
taken into account that not all mutations are selected with 6TG. Let us assume that only
half of the hprt mutations yield 6TGr phenotype, the baseline then becomes 0.06%. A
1%hotspot is therefore 16.7x over the background.
Analyzed by PCR-DGGE, a hotspot can be expressed by the relative intensity of the
mutant band to the wild-type band. Multiplying the percentage of an mutant bands by the
original number of mutants after mutagen treatment gives the number of mutants sharing
a particular hotspot. The number of "original" mutant is crucial to the reproducibility of a
hotspot. Hotspot originating from 100 mutants would have a precision of ±20%
according to Poisson distribution. Low numbers of "original" mutants, however, can
cause large variation on the relative fractions of the mutational hotspot, and in certain
case absence of a hotspot.
5.4.2 Low-melting domain of hprt Exon 3 as the Target Sequence
Computerized database containing information on mutations at the hprt locus has
been established (Cariello and Skopek, 1993). This database contains over 1000 mutants
100
from both human cell cultures and T-cells from human peripheral blood. It was reported
that mutations occurred more frequently than predicted in hprt exon 3 (p = 0.013). Fig.
5.7 shows the observed mutated sites (Cariello and Skopek, 1993) in a proposed structure
of the hprt catalytic domain (Busetta, 1988). Low-melting domain of hprt exon 3 (bp 214
- 318) consists codon 71 to 105. This region is composed of a helical structure (HI), a B
sheet and a hairpin connecting the two. A clustering of mutated amino acids was
observed in H 1 region which is the putative PRPP binding site. The density of mutable
amino acids was considerably reduced in the other portions of the predicted structure.
The amino acids which participate in the PRPP binding may be very critical to the
functioning of the hprt protein, so substitutions in this region may have a high probability
of producing 6TG r phenotype. Cariello and Skopek (1993) reported there were DNA
base pair substitution hotspots around bp 200 where low-melting region of exon 3 is
located. Mutations induced by BPDE (Keohavong and Thilly, 1992), UV (Keohavong et
al., 1991), H20 2 (Oiler and Thilly, 1992), K2Cr 20 7 (Chen and Thilly, 1994) were
detected in this region. In addition, hprt exon 3 is the largest exon in hprt gene and the
only one with naturally occurred high- and low-melting domain which is suitable to
directly use DGGE for mutational spectra study. For the reasons discussed above, lowmelting domain of hprt exon 3 was chosen as the target sequence for this study.
5.4.3. BP MutationalSpectra under Various Dose and Dose Rate
Fig. 5.8 demonstrates the DGGE analysis of the cultures treated with different dose
and dose rate. Dark bands above the WT indicate mutational hotspots. Several features
are worth mentioning. First of all, BP treatment resulted in mutations in the target
sequence, since some mutant bands were observed only in BP treated cultures but not in
spontaneous cultures and PCR controls. Second, results from duplicates of treated
cultures were very reproducible in BP treated cultures. Such reproducibility can only be
101
Fig. 5.7
A proposed model of the hprt catalytic domains
The approximate location of amino acid substitutions is given by the carets (^).
Helical structures are noted by an H, and 8 strands are noted by a B. The amino acid
number of the start and finish of each 8 strand and helix strand is given. Note that the
model is not to scale. The proposed location of the PRPP and purine binding sites are
shown. Highly conserved residues thought to function in purine binding are given in one
letter amino acid code. The initiation codon is Met 0.
(Graph taken from Cariello and Skopek, 1993)
102
103
Fig. 5.8
DGGE analysis of BP induced mutations in the low-melting domain of
hprt exon 3 under various dose and dose rate
The sequence was amplified 106 times with primers P1 and P2. The resulting 204bp fragment was end-labeled with 32P. Samples were boiled, reannealed and migrated
through 8% polyacrylamide gel containing a linear 60-75% denaturant gradient. The gel
was run at 40'C for 16 hrs, dried and autoradiographed. Small arrows indicate the
position of bands containing hotspot alterations. The identity of the alterations was
determined by sequencing and is given in Table 5.6. The heteroduplex of a particular
hotspot are designated with the same letter in lower case (i.e. al and a2) followed by
either the number 1 (heteroduplex closer to the wild type) or 2 (heteroduplex further from
wild type). If two heteroduplexes focus at the same position, they are designated with
one single letter in lower case (i.e. e or d). The hotspot bearing a particular mutations is
designated by the same letter in upper case (i.e. hotspot A).
Lane 1:
PCR control: pfu amplification of untreated unselected culture;
Lane 2 & 3:
Spontaneous: 0 pM BP x 20 days, 0 gM*d;
Lane 4 & 5:
0.02 gM BP x 20 days, 0.4 .gM.d;
Lane 6 & 7:
0.5 .M BP x 6 days, 3 gM*d;
Lane 8 & 9:
0.5 gM BP x 20 days, 10M*d;
Lane 10 & 11: 30 gM BP x 28 hours, 36 gM*d.
104
MUTATIONAL SPECTRA:
EFFECT OF DOSE RATE
BENZO[a]PYRENE
AHH-1 CELLS
1
2
3
4
5
6
7
8
9
10
11
w
uJ
xuI
.. I
0
cc
ui
I-.
uI
3I-.
z
I--
WT
ss
MUTATIONAL SPECTRA:
AHH-1 CELLS
1
2
3
4
5
6
EFFECT OF DOSE RATE
BENZO[a]PYRENE
7
8
9
10
11
W
A
A A
I
1
oE
C4
1
=
<C4..
c -4
,n
0
o
x
0
nA,
U,
0<0
0
rei:Too0crý cn
14
*c1-
-
C"
U ~c
C
N
o
u
C,
z
c
0
0
N~
A
I
o
N
tIM
.0
'0
0\ o0W
Cl
Cl
'I~
o,
o
'0
c'0
Un
U
0
U
U,
(-4 n
c
C% C14C
\O
(N
N
0
c ,
Cl
Clf
U,
U
0
8
0
Ue,
C3
o
t
U
E
0<
U,
U,
QQ
< uO
U
U,~
U
'0
U,
U
U
0
s-
U:
U
S.
'0
U
U,
S
U
I-
U
U,
'-
U
0
0
U
7.
L)
107
-
30 IM BP x 28 h
AHH-1 cells were treated with a toxic dose of BP (30 pM) for a short time (28
hours). Only 82% and 76% of the cells survived the treatment in two independent
cultures (Fig. 5.5). The major mutational hotspot (3.4% of hprr cells) is a transversion
mutation, G -> T at bp 229 (bands al and a2 in lanes 10 and 11). It is the same mutation
(mutation A) as in 0.02 .M x 20 d cultures, but the intensity of the mutant bands differs
dramatically in two cases. This hotspot comprised 3.4% of the 6TGr population in 30 JLM
x 28 h cultures but only 1.5% in 0.02 pM x 20 d cultures.
Other minor bands were also observed in 30 gpM x 28 h cultures. They represent
mutation F, TG -> CC at bp 233-234 (band f), and mutation G, G -> T at bp 237 (bands
gl and g2). Mutation F results in Leu -> Pro substitution. Mutation G, however, replaced
one Leu codon by another. Such "silent" mutation were not suppose to be detected by
6TG selection. Possible mechanisms are discussed later.
There are large variations regarding to the intensity of bands f, gl and g2 in two 30
pgM x 28 h cultures. Such variations are expected when the number of independent
mutants representing each band becomes small. As shown in Table 5.1, 24,600 (average)
independent mutants survived 30 gM BP treatment. Each of the minor bands represent
about 0.2% of the 6TG r population, which means 50 mutants in each bands. This gives
the precision of 30%, agreeable to what was observed.
0.5 tM x 6 d
Four mutant bands (cl, c2, d, e in lanes 6 and 7) are present in cultures treated with
0.5 p•M BP for 6 days. Mutation C (bands cl and c2) is A -> T substitution at bp 261
resulting in Arg -> Ser. Band d actually contains two heteroduplexes from the same
mutation D focused extremely close on the DGGE gel. It represents a 7 bp deletion
between bp 307 and 313 (mutation D). This mutation results in deletion of two highly
conserved residues thought to be functioning in purine binding (Busetta, 1988). Such
108
mutation may completely knockout the hprt protein function. Two heteroduplexes of
mutation A -> T at bp 247 (mutation E)migrated to the exact position on DGGE gel, so
only one band was observed (band e). This mutation substitutes Lys with Tyr at codon
82, which encode an amino acid in a helical region (H1) in hprt catalytic domains.
LM BP x 20 d
0.5
It is worth mentioning that 0.5 gM x 20 d cultures were the extension of the 0.5 rM
x 6 d cultures. At the sixth day of 0.5 gM BP treatment, cultures were splitten into two.
One continued the 1 gM BP treatment (0.5 gM x 20 d). The other was spun out of BP
and carried out six day phenotypic expression in fresh media (0.5 pM x 6 d). An
interesting phenomenon was observed in 0.5 gM x 20 d cultures. The only mutational
hotspot present is A ->T at bp 247 (mutation E). The other two mutational hotspots
(mutation C and D)in 0.5 gM x 6 d cultures unexpectingly disappeared (intensity <
0.2%). Several hypotheses were proposed to explain this surprising result. They are
discussed in detail in the next section.
In summary, BP induced mutational spectrum is dependent upon the concentration
and duration of the treatment. Dose and dose rate not only affect the intensity of
mutational hotspots, but also the distribution of mutations (Fig. 5.9).
5.4.4. Characteristics of BP Mutational Spectra
BP was mutagenic to human cells at doses as low as 0.02 gM. Within a 104-bp
target sequence, nine distinct mutational hotspots were observed. Among them, one
generated stop codon (hotspot E), two caused frameshift mutations (hotspots B3 and D),
five substituted one amino acid by another (hotspots A, B 1, B2, C, F) and one appeared to
be a "silent mutation" (hotspot G). All the single base pair substitutions were transversion
109
Fig. 5.9
Display of BP mutational hotspot under various dose and dose rate
Kinds, positions, and frequencies of 6TG mutations induced by BP with different
doses and duration in the low-melting domain of hprt exon 3. Base positions 215-318
represent our target sequence. The frequencies of mutations relative to total 6TG
mutations are indicated by the height of the vertical bars. The kinds of mutations are
indicated on the top of vertical bars. Designation of hotspots is indicated by a capital
letter in the bracket. The positions of hotspots are as follows:
0.02 pM x 20 d:
bp 229, 238, 256 or 257, 295;
0.5 gM x 6 d:
bp 247, 261, 307-313;
0.5 pM x 20 d:
bp 247;
30 gM x 28 h:
bp 229, 233-234, 237.
110
0.02RM x 20d
t
I--
A
A
I[A]
0.5j±M x 6d
A3
r[B1
AI
E]
I
[B3]
*
[C]
[B2]
[D
0.5gM x 20d
E
30uM x 28hr
IrA
A
[A]
215
230
G]
,
270
250
290
Base Pair Position
111
310
318
mutations. Six out of seven single base pair changes occurred in single nucleotide repeats
(i.e. -(A)n-, -(T)n-, -(G)n-, -(C)n-; n 2 2).
In the study by Chen et al. (1990), it was found that BPDE induced mainly G -> T
transversions in hprr gene of diploid human fibroblasts, with the mutated G located on the
non-transcribed strand, and it was suggested that human fibroblasts preferentially repair
BPDE adducts on the transcribed strand. To further elucidate the specificity of BPDE
mutagenesis in vivo, Andersson et al. (1992) found that in human T-lymphocytes all
mutated GC base pairs in BPDE-induced spectrum were oriented so that the G was
located on the nontranscribed strand. Assuming that the premutagenic lesion in these
cases was covalent binding of the BPDE to guanine and that BPDE bound randomly to
both strands, the strand specificity of the BPDE-induced mutations indicates that
preferential excision repair of BPDE adducts on the transcribed strand occurs in the hprt
gene in human T-lymphocytes. In addition, Vrieling et al. (1991) reported that there are
difference in the rate and efficiency of removal of UV-induced pyrimidine dimers and
photoproducts from the transcribed and non-transcribed strands of the hprt gene in
cultured hamster cells. Mellon et al. (1987) also showed that in human cells nucleotide
excision repair significantly faster in the transcribed strand than in the non-transcribed
strand of dhfr gene. If such preferential repair of BP adducts from the transcribed strand
occurs in AHH- 1 cells, a greater proportion of the premutagenic lesions is expected to
remain in the non-transcribed strand. Results from this study indicated purine (G or A)
was the target base for BP in AHH-1, since majority (5/6) of the single base pair changes
involved G or A on the nontranscribed strand. It has also been shown that > 95% of the
DNA adducts involved guanine when mammalian cells were treated with BPDE (Osborne
et al., 1981; Straub et al., 1977) and G -> T transversion dominated BPDE mutational
spectra (Mazur and Glickman, 1988; Carothers and Grunberger, 1990; Yang et al., 1991).
Kakefuda and Yamamoto (1978a, b) indicated however, that although guanine might be
quantitatively the most important base reacting with BP diol epoxide, the adenine also
112
reacted with the BP diol epoxide and the latter interaction might be responsible for the
local unwinding-induced denaturation of the DNA. Several reports (Kakefuda and
Yamamoto, 1978b; Meehan, et al., 1977) demonstrated that the BP diol epoxides bound
to adenine as well as the guanine of DNA. Preferential modification of G and A by BP in
this study seems agreeable with these findings.
BP metabolism in AHH-1 cells resembles that reported for human lymphocytes.
Human lymphocytes produce BP phenols, quinones, and a limited array of transdihydrodiols (Selkirk et al., 1975; Vaught et al., 1978). AHH-1 cells produced
predominantly phenols and quinones (Crespi, Ph.D. Thesis, MIT, 1982). It is worth
emphasizing that no trans-dihydrodiol BP metabolites were observed in both cases, so
BPDE which have been reported to be the major pathways to DNA binding in vivo were
unlikely. The majority of the BP-DNA adduct profile in AHH-1 cells was
uncharacterized. Even though the possibility of BPDE binding to DNA was ruled out,
preferential damages on purine bases suggested BP metabolites generated by AHH-1 cells
were mechanistically similar to BPDE and targeted guanine and adenine sites.
It is also interesting that hotspot A (G -> T at bp 229) in this study was observed in
BPDE treated human fibroblasts (Yang et al., 1991). This hotspot was detected in 6%
(2/34) of 6TGr clones treated with 0.15 giM BPDE for 1 hr. Since BPDE was not
considered to be produced by AHH-1 cell line, hotspot A detected in this study might
result from BP metabolite structurally or mechanistically similar to BPDE.
Mutational hotspot B3 (lbp deletion at bp 256 or 257) was also observed
spontaneous cultures of human lymphoblastoid (TK6) cells (Oller and Thilly, 1992).
Because this mutation was observed in triplicate cultures, it was considered a true
spontaneous hotspot and comprised 1.2% (average of three cultures) of 6TG r mutants in
spontaneous cultures. In this study, mutant rate of 0.02 ptM treated culture was only
tenfold higher compared to spontaneous cultures. Detection of hotspot B3 is thus not
surprising. Possibility also exists that BP treatment enhanced this particular hotspot.
113
Mutation E (A -> T at bp 247) is also a chromium(VI) hotspot (Chen and Thilly, 1994)
and detected in human population in vivo (Cole and Skopek, 1993).
It is a surprise that hotspot G appeared to be "silent". Such a hotspot was not
expected in a selected mutational spectrum. Since the hotspot has arisen in two
independent cultures, it is regarded as a true hotspot. Several mechanisms might be
involved: 1) This mutation might be a "silent partner" mechanistically linked to an
intragenic expressed mutation outside the target sequence (Timms et al., 1992); 2) There
might be preferential codon usage of CUG over CUU (or CIT on DNA), both coding for
leucine, by AHH-1 cells (Grantham et al., 1980; Grosjean and Fier, 1982; Shields, 1990).
"Silent" mutations were also observed in BPDE treated human fibroblast cells (Yang et
al., 1991) and K2Cr20 7 treated human lymphoblast cells (Chen and Thilly, 1994). In
Yang's study, the "silent" mutation contained a second base substitution in other part of
the gene.
It is an interesting phenomenon that the long-term, low-dose rate (0.02 gM x 20 d =
0.4 igMd) and acute, high-dose rate (30 p.M x 28 h = 36 .M.*d) spectra shared the same
major hotspot A, even though they are not identical. The common hotspot is reflecting
mechanistic similarities between the two populations. As discussed in Section 5.1, 0.02
gM BP treatment is intermittent or pulse, since of the removal of BP is far grater than the
supply. Each 0.02 pgM treatment can be considered acute or as single dose, like the
treatment with 30 IiM BP. So the hotspot A is associated with acute (or single dose)
treatment. In such case, the dosing period is so short that DNA repair enzyme may not
have been fully induced. Cultures treated with 0.5 gM BP follows semi-continuous
mode. The mutagenic mechanisms associated it may be completely different from
intermittent exposure.
Finally and most importantly, this study is the first to demonstrate that mutational
spectrum can indeed be dose and dose rate dependent. Wei et al. (1991) studied the
effects of dose vs. spectra in hamster cells. They claimed a significant difference when
114
mutations occurring at G:C base pairs were compared to those at A:T base pairs.
Unfortunately, their study failed to generate sample size large enough to differentiate any
of the differences regarding to kinds and sequence specificity of mutations that might
have been seen between treatment types (Table 5.7). The dose dependence of BP
mutational spectra observed in this study may be reflection of the dose dependence of
processes like BP metabolism, DNA binding and DNA repair. Human lymphocytes
produced different metabolite profiles depending on the incubation time with BP (Selkirk
et al., 1975). During a 30 minute incubation no trans dihydrodiols were observed, the
major metabolites were phenols and quinones. During a 24 hr incubation however,
human lymphocytes produced 4,5-, 7,8-, and 9,10- transdihydrodiols, quinones and
phenols and seven unidentified metabolites. Strand specificity discussed above can also
be affected by dose. There were less stand bias for UV-induced mutations at the Chinese
hamster hprt gene after irradiation with relatively high dose (Vrieling et al., 1989)
compared to a lower dose (Vrieling et al., 1991). It was suggested at high dose, cell cycle
progression was considerably delayed, more time for repair was available and
photoproducts could be removed from both strands insignificant amounts resulting in a
much less pronounced strand bias for mutation induction. To pin-point the exact
mechanism of dose dependence of BP mutational spectrum is beyond the scope of this
study. These are just examples among many possibilities.
5.5. SIGNIFICANCE TOWARD HUMAN STUDY
This study is the first to study a mutational spectrum at a dose rate close to real
human situation (discussed below). Such task was once thought impossible because of its
technical and financial difficulties. Herein, a large-scale cell culture experiment was
designed so one can treat large amount of human cells with low levels of chemicals for a
long period of time. Statistical analysis shows that such protocol induces enough
115
Table 5.7. Contingency Table Analysis of Clone by Clone BPDE Induced
Mutations under Different Doses
Treatment
G -> T
G -> A
0.04 lVM BPDE x 1 hr
28 (24.2)
0.48 pM BPDE x 1 hr
19 (22.8)
47
Total
G -> C
Others
Total
1 (2.1)
7 (6.7)
1(4.1)
37
3 (4.9)
6 (6.3)
7 (3.9)
35
4
13
8
72
* Data from Wei et al. (1992)
X2 = 7.25 < 7.82; No statistical difference
3 degrees of freedom 95% confidence limit
Chi squared values are calculated using the fomula:
2 columns rows
x2 =
j=1
2
row total x column total
all observed
[nij- E(nij)] 2
i=1
E(nij )
116
independent mutations to study the frequency and sequence specificity of chemically
induced point mutations on a selectable gene with statistical significance.
5.5.1. A Model to Estimate BP Exposure in Human Lungs
Let's first find out what dose range of BP that human is experiencing from nonoccupational and occupational sources. A model was proposed to estimate such
exposure. Given the fact that lung is the target organ for BP in human, I herein try to
estimate the dose range in human lungs when exposed to BP from various sources.
Let's first consider the dimension of the alveolar component of the lung. A thin
film of fluid wetting the alveolar walls is thought to aid in the initial absorption of toxins
from the alveolar air (Guthrie, 1980). This film is estimated to be about 0.75 micron
thick (Menzel and McClellan, 1980). If the alveolar respiratory surface area is 70 m2
(Guyton, 1977), then we can estimate the volume of this fluid to be about 50 ml.
PAH in the environment are primarily associated with the carbonaceous particulate
that are formed simultaneously during combustion and cigarettes smoke (Perera, 1981).
To assess health risk associated with human exposure to BP, it thus is necessary to
evaluate the bioavailability of BP associated with particulate. In the study by Bevan and
Ruggio (1991), they demonstrated that > 70% BP was deposited in lungs when SpragueDawley rats were intratracheal instilled with BP-containing particulate. The absorption of
BP into lungs is a fast process. Merely 1 hr after instillation, 77% of BP was detected in
lungs. Another striking feature is that BP is very stable in lungs, about 50% BP remained
in lungs 3 days after instillation.
A mathematical model is proposed to estimate the BP concentration in human
lungs. The following hypotheses are made based on the results discussed above: 1) The
absorption of BP from the particulate by human lungs is instantaneous; 2) The
elimination of BP from human lungs follows first order kinetics with rate constant ke; 3)
117
Equilibrium concentration of BP (C.) is estimated when time (t) ->
0o;
4) Bioavailability
(B) of BP is 70% and the volume of alveolar fluid (V) is 50 ml.
The time function of BP concentration in alveolar fluid can then be expressed as:
S= -keC + A
(Eq. D)
dt
where:
C:
BP concentration in the alveolar fluid;
k:
elimination constant;
A:
Alveolar concentration from daily exposure to the sources;
so:
A = (daily intake) x (bioavailability) = -(daily intake;
(volume of alveolar fluid)
V
In the rodent experiment (Bevan and Ruggio, 1991), rats were treated with one dose
of BP. A was then zero because the exposure was not continuous. About 50% of
radioactivity remained in lungs at 3 days following instillation. So the Eq. D became:
In ~ = -ket where t = 3 d, and Ct/Co = 0.5/0.7 = 0.71;
Co
ke is then equal to 0.11 d- 1. ke also provides information on how fast the target
organ reaches the equilibrium (or saturated) concentration once it starts experiencing
chemical insult. In this case, it takes 6 days (ln2/ke) for the lung to reach 0.5 C..
The equilibrium concentration of BP in alveolar fluid can be estimated by solving
Eq. D at t -> oo:
C.=A = A
ke
(Eq. F)
0.11
118
Now, let's use human exposure to urban air as an example to illustrate the model. In
urban area, the concentration of BP in the air was reported as high as 74 ng/m 3
(Hoffmann and Wynder, 1976). Thus, breathing 10 m3 air per day (based on a minute
volume of 7,500 ml/min; Menzel and McClellan, 1980) would deposit 0.5 gg BP per day
into the lung (B=0.7) so the alveolar concentration from daily exposure to the urban air
(A) equals to 0.04 gM. Using Eq. F, one can then estimate that the equilibrium
concentration of BP in human lung (C.) is 0.36 gM
5.52. Estimation ofBP Concentrations in Human Lungs
BP exists in mainstream and sidestream smoke from cigarettes and smoke-polluted
environments. Based on Eq. 7, calculated concentrations of BP in human alveolar fluid
from various sources of exposure displayed in Table 5.8. "A" in column 4 represents
dose rate of BP. C. indicates the concentration of BP in human lungs when reaches
equilibrium. One cigarettes contains 20-40 ng of BP (Baker, 1980). Smoking ten
cigarettes per day would result in daily exposure of 0.01 - 0.02 pM BP in alveolar fluid.
In a smoke-polluted environment, BP concentration has been shown to range from 2.8760 ng/m 3 (Elliot and Rowe, 1975; Galuskinova, 1964; Perry, 1973). Thus, breathing 10
m3 air per day results in daily exposure of BP up to 0.4 gM.
Occupational exposure to BP occurs in coal-tar production and coke plants,
aluminum production plants, road and roof-tarring operations, and around municipal
incinerators. Historically, industrial workers have experienced the highest level of
exposure to BP. BP concentrations ranging from 800 - 23,100 ng/m 3 have been reported
to occur in the workroom air of coke oven operation in the UK (Davies et al., 1986). For
the workers working in such environment, breathing 3.3 m3 air per eight-hour-working
day could result in 0.14 jM - 4.3 jiM daily exposure to BP.
119
r:
t<
r-
1C
CN
oo
00
0%
U.
6
00
.o
0\
0N
\.
00
- a-
T-4
r"-
1-)
0
0
N3
0,,k~
P-
00
6
S-
0
0
0
1:4
oc
o
c
C4
(N
-4
I--a
o
a.)
V
V
c
C/5
0o·
6C)
C)
0
C#.)
0
0
0
6o
C.T)
Pt
(N
o0
0
0o
oa
o
0
Cclý
Cc
T.
Cci
o660%
I
6
a
z
CN
00
0-L
r:
0
Sc
E:
C
00
C%
(N
00
00
00
r(=
0
00
O
21
o
o0
cli
N
t~
00
zS
U
U
U,
E
a.)
U,
0
U,
S0
S
g
a~
c0
ci
C4.
Ql
Sai
;0
0
to
v
>
120
cz
to
a
z
From the estimation, one can see that the dose conditions used in this study is within
the range of human experience. 0.02 gM treatment falls into the range of nonoccupational exposure and 0.5 gpM into occupational exposure. In extreme cases, the
occupational exposure can be toxic to tissue cells. Results from this study indicates that
different mutational mechanisms may be in operation under these difference schemes of
exposure so different genetic effects may be expected as well. Thus, cell and animal
experiments carried out at exposure higher than common to human experience cannot be
rationally considered as reflecting human mutational pathways.
5.6. HOW DO MUTANTS DISAPPEAR?
The most striking feature of dose-dependence of BP induced mutational spectrum is
the transient mutations associated with 0.5 gM BP treatment. In this section, processes
likely to be responsible for the disappearance of these mutants are discussed in detail.
5.6.1 General Considerationson BP Mutagenesis
BP mutagenesis involves absorption, metabolism, DNA binding, and DNA repair.
These process can be explained by a mathematical model (Liber et al., 1985) based upon
the "target theory": that there exists within the cell a target for mutagenic damage (hprt
gene); that mutation is related to a hit (a result of the adduct and its subsequent fate, as
opposed to a triggering of general cell response); and that the target region has the same
chance of being hit as any non-target region (other part of the genome). Additionally, this
model assumes that hits (adducts) can be modified by repair enzymes with limited
capacity. It is important to mention the model discussed by Liber et al. (1985) implies a
homogeneous cell population and each cell has the same probability of being mutated.
121
Even though the model permits multiple repair systems with their different capacities and
fidelity factors, it requires each cell has identical composition of these repair enzymes.
An alternative model for BP mutagenesis involves heterogeneous cell populations.
A cell population may contain two or more subpopulations, prior or as results of mutagen
treatment, with different responses to the toxic and mutagenic effects of a toxic mutagen.
In this case, the model described by Liber et al. (1985) is still applicable to each
homogeneous subpopulation. The overall population can be viewed as a summation of
the behavior of its parts. Heterogeneity can be of many origins and characteristics. It can
be referred to the sensitivity to toxicity and mutagenicity of a chemical. It can also be
referred to the difference in enzyme composition or activity, repair capacities and cell
cycles. Heterogeneity can be originated from nonclonal origin of a cell population or
aging of the culture. It can also be induced by chemicals or radiation.
5.6.2 Hypotheses For Disappearing Mutants
In order to discover the mechanism of transient mutants, one first has to know how
mutants arise. Molecular analyses of mutations at the hprt locus in human T-lymphocytes
and human lymphoblast cell cultures have identified a wide spectrum of alterations,
including total and partial genomic deletions, point mutations causing coding and splicing
errors as well as microdeletions and insertions in the hprt coding region (Recio et al.,
1990; Rossi et al., 1990, 1992; Hou et al., 1993; Steingrimsdottir et al., 1992; Cariello and
Skopek, 1993; Cole and Skopek, 1993). Isolation of hprt mutants depends on their ability
to grow in media containing toxic purine analog 6TG, which is lethal to wild type cells
expressing hprt activity. 6TG r mutants have been found to be mostly deficient in activity
of the purine salvage hprt enzyme which is considered nonessential for cell survival.
6TG selection is not a systems without its pitfall. First of all, not all mutants lead to
6TG r cells. Only mutants result in significant change in hprt activity can be observed, so
122
the mutational spectrum obtained from these mutants is actually biased. In certain cases,
6TG r cells doesn't mean hprr mutants. Cells with normal hprt activity can escape 6TG
selection (Brown and Thacker, 1984) by faulty transportation of 6TG across the cell
membrane (Crawford et al., 1990) or slow turnover (Hou et al., 1993). Wild-type cells
escaping the toxic effect of the selection medium may interfere the analysis of mutational
spectrum and reduce the ability to detect mutational hotspots. In addition, the length of
phenotypic expression is also a likely source of uncertainty. The progeny of cells
surviving environmental mutagenic exposure has demonstrated "delayed reproductive
death" (Chang and Little, 1991) associated with a prolonged expression of mutations at
the hprt locus (Little et al., 1990). These processes can prevent mutational spectra from
representing the true mutational event both qualitatively and quantitatively. Finally,
chemical selection can induce gene amplification in mammalian cells (reviewed by
Schimke, 1984a), which causes over-representing of certain mutational event in
mutational spectrum.
Let's now consider the possibilities of the disappearance of 6TGr mutants. It is
worth emphasizing the mutant bands observed in DGGE gel represent real mutant cells not
premutagenic lesions which can be removed by DNA repair or diminished by stopping
DNA replication. So the disappearance of mutant bands is a post-mutagenic event.
Mutant cells carrying certain mutations can gain selective growth advantage and give
more prominent mutant bands than other mutants with normal growth rate. One the other
hand, the growth of mutant cells can be selectively repressed. These lead to either
overestimation or underestimation of the mutational hotspot. Another possibility arises in
considering mutants identified solely at the DNA sequence level in which phenotypic
selection is avoided. If a mutational event is accompanied by a gene amplification event
encompassing the mutant sequence, then the mutational signal would over-represent the
mutational event to the extent of the degree of gene amplification. Usually, gene
amplification is not a stable trait. Amplified sequences, especially those exist
123
extrachromosomally, tend to dilute out with cell division. Transient mutants can also be
explained by heterogeneity hypothesis. Different subpopulations may exist in the
population that certain mutants (not others) were selectively killed off or slowed down.
Such heterogeneity may exist in the original cell population or induced by chemical
treatment.
In summary, many possibilities exist for the disappearance of a mutational hotspot.
Finding out the exact mechanism of this phenomenon is beyond the scope of this study.
Herein four likely hypotheses were proposed and tested individually:
A. Selective advantage or disadvantage of 6TG r mutants;
B. Gene amplification / deamplification;
C. Original heterogeneity;
D. BP induced heterogeneity.
A. Selective Pressure or Neutrality of Mutant Phenotype
It has frequently been suggested that hprr mutants in vivo are at a selective
disadvantage and do not persist for long periods. Albertini and DeMars (1974)
demonstrated the frequency of 8-azaguanine resistant lymphocytes in 3 known LeschNyhan heterozygous girls was only 5-10% and was zero in a 40 year old heterozygous
mother. This was taken to imply not only selection against hprr mutants in the lymphocyte
population, but increasing selection against the mutants with age. Selective advantage or
disadvantage of mutant phenotype has not been reported in cultured human cells.
Quantitative analysis of the 0.5 gM x 6 d and 0.5 gM x 20 d mutational spectra
(Fig. 5.8) revealed an at least twentyfold decrease of mutant C and D (the transient
hotspots). These mutants might have selective pressure against them with continuous
exposure of BP and were diluted out. It is also possible that mutant C and D acquired
124
growth advantage during phenotypic expression or 6TG selection, so that the mutational
events leading to mutation C and D were over-represented.
Fig. 5.10 provided clear answers for these speculations. Lane 2 represents a 10 day
growback of 6TGr cells from 0.5 AM x 6 d cultures. Compared to lane 5 (original 0.5 AM
x 6 d 6TGr cells), relative intensities of hotspots C, D and E demonstrate no significant
difference between the growback and original 6TGr cells (Table 5.9). For example, the
hotspot D represents 3.8% 6TG r cells in the original 0.5 p.M x 6 d culture, and 6.0% 6TGr
cells in 10 day grow-back cultures. This observation indicated no selective pressure
favoring or against all three hotspots observed in 1 AM x 6 d cultures under normal
growth conditions (no BP and 6TG).
Further more, the possible selective pressure imposed by 6TG was also tested. Lanes
3 and 4 in Fig. 5.10 demonstrate 3 day growth of 0.5 A.M x 6 d 6TG r cells with presence of
6TG. 6TG was added to 1 gpg per ml to 6TGr cells from 0.5 AtM x 6 d treatment. No
selective pressure against or favoring any mutational hotspots were observed. The relative
intensities of hotspots with and without 6TG selection are presented in Table 5.9. For
hotspot D, it represents 3.8% of 6TG cells in the original 0.5 A.M x 6 d cultures and 5.2%
6TG cells when 6TG was added back for additional three days.
In summary, no significant growth advantage or disadvantage of any mutational
hotspot were acquired during processes of normal growth, phenotypic expression and
6TG selection.
B. Gene Amplification
The increase in gene copy number of one or a few genes, called gene amplification,
is a mechanism leading to increased expression of normal or altered gene products and is
thought to participate in mutagenesis and carcinogenesis (Zur Hausen and
125
Fig. 5.10
Test for neutrality of 6TGr phenotype
The sequence was amplified 106 times with primers P1 and P2 and end-labeled
with
32p.
Samples were boiled, reannealed and migrated through 8%polyacrylamide gel
containing a linear 60-75% denaturant gradient. The gel was run for 16 hrs at 40'C, dried
and autoradiographed. Small arrows indicate the position of mutant bands designated in
Fig. 5.8.
Lane 1:
0.5 gM x 20 d, 6TGr cells;
Lane 2:
0.5 pM x 6 d, 6TG r cells, 10 day growth in fresh media;
Lane 3 & 4:
0.5 gM x 6 d, 6TG r cells, 3 day growth in 1 gg/ml 6TG;
Lane 5 & 6:
0.5 gM x 6 d, 6TG r cells.
126
TEST FOR SELECTIVE PRESSURE
1
2
3
4
5
6
dC2
d
aw-e
~
·
~.·; ~Ci
m %-WT
TEST FOR SELECTIVE PRESSURE
1
2
3
4
5
6
O- C2
4- C,
"-- WT
Schlehofer, 1987). Amplification of certain genes has been shown to result in cells
overcoming growth constraint of the selecting agent by an increased production of the gene
product (Schimke, 1984b; Stark and Wahl, 1984). Amplified oncogenes were found in a
variety of human tumors (Shwab and Amler, 1990) and are commonly believed to play
important roles in allowing the host cells to escape growth control and becoming mobile
and invasive, or escape immune surveillance. Gene amplification occurs physiologically in
normal cell regulation and differentiation (Long and Dawid, 1980; Spradling and
Mahowald, 1980) and is also the result of genotoxic insult. In particular, various types of
radiation (Liicke-Huhle et al., 1986; Ehrfeld et al., 1986; Liicke-Huhle and Herrlich, 1986,
1986) and chemical carcinogen (Lavi, 1982; Pool et al., 1989), including BP, efficiently
induce gene amplification in mammalian cells. An at least twentyfold amplification of hprt
gene was detected in a mouse neuroblastoma cell line (Brennand et al., 1982).
The "onionskin model" (Fig. 5.11) illustrated by Stark and Wahl (1984) explains the
process of gene amplification. This process involves unscheduled DNA synthesis plus
recombination. Two unusual cytological phenomena have been associated with amplified
DNA: first, the presence of small extrachromosomal linear duplexes or small acentric
chromosomes which because they often appear in pairs, are called double minutes (DM);
and second, the appearance of long chromosome segments with little evidence of Gbanding, referred to as homogeneously staining regions (HSR).
Gene amplification is a dose-dependent and transient process (reviewed by LiickeHuhle, 1989). Increasing doses of radiation induce higher enhancement of DNA
amplification. The number of amplified copies decreased by day 4 or 7 after radiation as
well as after exposure to chemicals (Lavi, 1982). This time course, however, is
independent of the dose of carcinogen and might be explained by the fact that amplified
sequences, not offering any growth advantage to the host, get lost during cell divisions.
In this study, disappearance of certain mutational hotspots (but not the others)
indicates hotspots were induced by different pathways. The transient mutants C and D may
129
Fig. 5.11
Illustration of "onionskin" model for gene amplification
Bidirectional replication at an origin generates a bubble that can undergo further
rounds of unscheduled DNA replication, resulting in a nested set of partially replicated
duplexes. Note that there are only two contiguous chromosomal strands. It is possible
for linear duplex DNA to become detached from the structure if two replication forks can
approach one another very closely (pathway 3). Recombination within the same duplex
could generate extrachromosomal circles (pathway 2), while multiple recombination
among different duplexes could resolve the structure into an intrachromosomal linear
array (pathway 1).
(Adapted from Stark and Wahl, 1984)
130
e
a
b
c
d
a
b
c
d/,
f
g
h
f
g
h
REPLICATION
UNSCHEDULED REPLICATION
UNSCHEDULED
REPLICATION
MITOTICALLY
UNSTABLE
.3
-5
abc dcd e def g/d e fgh
/,
/,
d e
/'I
o d ef
I
d
c
RECOMBINATION -
e
131
d
i~
be results of gene amplification and deamplification. BP induced amplification of DNA
sequences encompassing hprt exon 3. Mutants C and D arose predominantly in the
amplified sequences which later were deamplified. It is very unlikely that mutants C and
D were first induced in hprt exon 3 and selectively amplified, since preferential gene
amplification of mutant sequences with one base pair difference is hard to comprehend
based on the "onionskin model".
Let's assume the intensity of mutant bands carrying mutation Z is Y% of the WT
bands. If there is no DNA amplification, mutant Z represents 0.5Y% of 6TG r population
(0.5x is because mutant bands are heteroduplexes). If there is however an X-fold DNA
amplification, mutant Z only represents (0.5Y/X)% of 6TG r population while the relative
intensity of the mutant bands is unchanged. Let's consider hotspot D, Y is 7.6 so mutant
D represents 3.8% 6TG r cell population in case of no amplification. If gene amplification
is the reason for disappearance of mutant D, X has to be much greater than 40. This is
because the mutant heteroduplexes should have been visible (0.2% WT bands) on DGGE
gel if the amplification was merely fortyfold. So an experiment was designed to
distinguish zero and fortyfold amplification. The fraction of 6TG r cells carrying mutation
D would be 0.19% if DNA amplification (fortyfold) hypothesis was true.
These two situations can be distinguished by an experimental design as follows: 0.5
gM x 6 d 6TG r cells were divided into many small subpopulations containing small
number of mutant D, so the distribution of mutant D obeyed Poisson distribution. 0.5 gM
x 6 d 6TGr cells were plated into 96 well microwell plates at a concentration of 1000 cells
per well. Considering 50% PE, each well actually contained 500 living cells. Cells were
allowed to grow in microwell plates for 5 days. Six colonies were randomly picked and
expanded in T-flasks. DNA was isolated from these cells, amplified and analyzed by
DGGE. If no amplification occurred, the average number of cells carrying mutation D
among 500 cells plated would be 19 (500 x 3.8%). Since the fraction of mutant cells is
small, we expect a Poisson distribution regarding to the chance of a colony containing a
132
mutant cell. The probability of not observing mutation D would virtually be zero (e-19)
based on Poisson distribution. Analyzing six colonies, we should detect this mutation in
every single case. In case of fortyfold amplification, however, the expected number of
cells carrying mutation D would be 0.5. The chance of no observable mutation would be
60% (e-0 .5). As a result, 4 out of 6 colonies analyzed would be absent of mutation D.
Fig. 5.12 demonstrates the results of analysis of 6 colonies originated from 500
living cells. Mutant bands containing mutation D were detected in all colonies. The
intensities of these bands varies from 4.78 to 13.9% of WT (Table 5.10). This threefold
variation is agreeable to what is expected with average of 19 according to Poisson
distribution. The experimental results indicated no evidence of DNA amplification.
It is worth mentioning that there is uncertainty regarding to the PE at 1000 cells per
well. Possibility exists that PE is close to 100% instead of 50% assumed base on the PE
data from 2 cells per well. In case of fortyfold gene amplification and 100% PE, the
expected number of cells carrying mutation D among 1000 plated cells would be 1. The
chance of no observable mutation would be 37% (e-1 ). As a result, colonies analyzed
without mutation D would be 2 out of 6, which is not significantly different from 0 out of
6. To exclude gene amplification hypothesis, one needs to analyzed 12 colonies, 4 of
which would contain no mutation D.
C. Original Heterogeneity
AHH-1 cells used in this study was obtained from Gentest Corp. (Woburn, MA).
This cell line was first isolated in our laboratory by Dr. Crespi (Ph. D. Thesis, MIT, 1982)
from RPMI-1788 cells. It was originated from a clone which showed hypersensitive to
BP induced growth inhibition, which was found to be a stable trait. The AHH-1 cell line
has been routinely tested for mycoplasma contamination and all tests are negative. The
133
Fig. 5.12
Test for gene amplification
The sequence was amplified 106 times with primers P1 and P2 and end-labeled
with
32 P.
Samples were boiled, reannealed and migrated through 8%polyacrylamide gel
containing a linear 60-75% denaturant gradient. The gel was run for 16 hrs at 40'C, dried
and autoradiographed. Small arrows indicate the position of mutant bands designated in
Fig. 5.8.
Lane 1:
0.5 p.M x 20 d, 6TGr cells;
Lane 2:
0.5 ptM x 6 d, 6TGr cells;
Lane 3-8:
six colonies each containing 500 0.5 pgM x 6d, 6TGr cells.
134
TEST FOR GENE AMPLIFICATION
1
2
3
4
5
6
7
8
-
CPW
~
aml
C2
-d
e
C1
al"'"
-~WVT
TEST FOR GENE AMPLIFICATION
1
2
3
4
5
6
7
8
"*a
00 d
0 C,
"W-WT
original sample from Gentest Corp. and all stock cultures in this lab were absent from any
antibiotics to prevent mycoplasma contamination.
Since the isolation of the AHH- 1 cell line, it has been recloned occasionally to
ensure the homogeneity of the population. Possibility exists that heterogeneity arise
during the maintenance of the cell line. The heterogeneity may due to the difference in
cell cycle or cell physiology including metabolism and repair capacity, etc..
Hypothesis of original heterogeneity is illustrated in Fig. 5.13. Before BP was
added, there were two populations of cells with inherently different sensitivity to BP.
Mutation C and D were induced in sensitive population which later were killed off by BP
treatment. Mutation E, on the other hand, arose from resistant or both. Toxicity data
from Dr. Crespi (Ph.D. Thesis, MIT, 1982) seems suggesting the existence of resistant
and sensitive subpopulations (Fig. 5.14). When AHH-1 cells were treated with BP for 24
hrs, there was apparent toxicity when BP concentration was less than 3 gM. There was
an obvious saturation of toxicity however when the BP concentration exceeded 3 pLM
while the mutagenicity of BP kept increase. It seems that cells behaved as sensitive
population at low BP concentrations and as resistant population at high BP concentrations
regarding to BP toxicity indicating cells survived 30 p.M BP treatment came solely from
resistant population.
In order to test this hypothesis, killing curves were analyzed in both "undisturbed"
population, cells never treated with BP, and "disturbed" population, cells survived
treatment of 30 g.M BP for 24 hours (Fig. 5.15). If the hypothesis was true, one would
observe no (or reduced) killing in surviving cells which were thought to be BP resistant.
Instead, two very similar killing curves were obtained. When surviving cells were
removed from BP and allowed to resume exponential growth in fresh media, the
sensitivity to BP toxicity was restored. This result suggests homogeneity of the
"undisturbed" AHH-1 population. Heterogeneity observed in "disturbed" population was
thus attributed to cell responding to cytotoxic insult by BP.
137
Fig. 5.13
Hypothesis of original cell heterogeneity
The original AHH-1 population used in this study contains two subpopulations,
sensitive (S) and resistant (R) population, regarding to BP toxicity. The transient mutants
(hotspots C & D) arose solely in the sensitive subpopulation which was killed off by BP
treatment. The persistent mutants (hotspot E) might arose in both or resistant population
only. As a result, one observes mutational hotspots from both S and R populations after
AHH-1 being treated with 0.5 gM BP for 6 days. After 20 days treatment with 0.5 A.M
BP, one observes mutational hotspots solely from R population.
138
Herterogeneity of the Cell Population
_S
00-
0.5gM BP
0.5gM BP
6 days
O Homogeneous
r-
14 days
Population
Sensitive Polulation
E]Resistant Population
*
Persistent Mutants
0 Transient Mutants
139
Herterogeneity of the Cell Population
s
0.5gM BP
rwn
6 days
]
O
0.5gM BP
14 days
Homogeneous Population
Sensitive Polulation
Resistant Population
Persistent Mutants
Transient Mutants
139
z
0
-C
t(
0
z
0
U.I--
zCL
%
'
0
(0
PT
(
u
9.
6
8
BP CONCENTRATION, PIM
141
10
Fig. 5.15
Short-term toxicity of BP in "undisturbed" and "disturbed" AHH-1 cells
Each points represents two independent cultures. "Undisturbed" cells were HAT
treated and never exposed to BP. "Disturbed" cells were those survived 30 gM BP x 24
hr treatment. They were allowed to resume exponential growth in fresh media. Both
populations were treated with 1, 3, 5, 10 and 30 gM BP for 24 hrs. Error bars represent
one population standard deviation of the mean.
142
1-
BP T oxicity
"undisturbed" vs. "disturbed"
-
ai
Disturbed
Undisturbed
0.3
I
-5
0
1
2
10
15 20
Dose (uM)
143
25
I
I
30
35
D. BP Induced Heterogeneity
Heterogeneity of the cell population can be induced by treating with certain
chemicals or radiation (Oakberg, 1978; Cattanach et al., 1989; Russel and Rinchik, 1993).
Most of the study was carried out by treating mouse spermatogonial stem cell system
with split dose of radiation or combination of chemical and radiation. A common
observation was differential cell killing accompanying by augmentation of mutation
response. It was believed that the first (priming) dose induces changes in the composition
of the spermatogonial population so that there is a predominance of mutagen-sensitive
cells 24 hrs after mutagen treatment (Russell, 1962). Cattanach et al. (1989) concluded
that the first exposure constitutes a cytotoxic insult, and that the responsive cells presents
24 hrs later are the formerly resistant component of the stem-cell population that had
survived the priming treatment and had been triggered into a sensitive phase. There had
been controversial evidence regarding to where mutations originated. Oakberg (1978)
suggested that most of the mutations recovered in 24 hr fractionation experiments were
derived from the first dose, with the second dose serving to elevate the overall yield by
selectively killing those survivors of the first treatment and did not carry mutations.
Other findings that augmentation can be obtained following a priming treatment with an
ineffective dose of radiation (Russell, 1964) or chemical (Cattanach et al., 1989; Ehling
and Neuhduser-Klaus, 1987) have pointed to the conclusion that the increased overall
mutation yield is due to mutation induction by the second (challenging) dose. In addition,
Russell and Rinchik (1993) demonstrated the nuclear state of "sensitized" stem-cell
spermatogonia were different from that of previously "undisturbed" stem cells.
Hypothesis of BP induced heterogeneity is outlined in Fig. 5.16. At the beginning,
cell population was homogeneous. After BP was added to the culture, cells
144
Fig. 5.16
Hypothesis for BP induced heterogeneity
The original AHH-1 population used in this study was homogenous. Cell
population splitten into sensitive (S) and resistant (R) fractions in response to BP
treatment. The transient mutants (hotspots C & D) arose solely in the sensitive
subpopulation which was killed off by BP treatment. The persistent mutants (hotspot E)
might arose in both or resistant population only. As a result, one observes mutational
hotspots from both S and R populations after AHH-1 being treated with 0.5 jM BP for 6
days. After 20 days treatment with 0.5 gtM BP, one observes mutational hotspots solely
from R population.
145
BP Induced Heterogeneity
0: QQ
008
Homo
0.5gM BP
0.5gM BP
6 days
14 days
D3 Homogeneous Population
D Sensitive Polulation
Resistant Population
Persistent Mutants
Transient Mutants
146
BP Induced Heterogeneity
F71]
O
0.51±M BP
0.5p.M BP
6 days
14 days
Homogeneous Population
E Sensitive Polulation
*
Resistant Population
*
Persistent Mutants
*
Transient Mutants
146
Fig. 5.17
Short-term toxicity of BP on 6TG r cells induced by BP treatment
Each point represents two independent cultures. (o) indicates toxicity on 6TGr cells
from 0.5 p.M x 6 d cultures; (A)indicates toxicity on 6TGr cells from 0.5 gM x 20 d
cultures. AHH-1 cells were treated with 1, 3, 5 , 10, 20 and 40 pM BP for 24 hrs. Error
bars represent one population standard deviation of the mean.
148
BP Toxicity
4m
1
0.3
0.
(o) 0.5 gM
0.5 [LM
0.3
I
-10
_
I
10
I
20
BP (uM)
149
6 d, 6TG(r)
20 d, 6TG(r)
I
30
40
50
Quantitatively, the observed difference in BP toxicity of 6TGr cells from 0.5 gM x
6 d and 0.5 g.M x 20 d cultures can not explain the "disappearance" of mutations C and D.
As observed in Fig. 5.17, 0.5 g.M x 20 d treatment only reduced the fraction of resistant
population by half (25% -> 10%). Taken into the account of increase in MF from 15.8 x
10-5 in 0.5 gM x 6 d to 27.9 x 10-5 in 0.5 g.M x 20 d cultures, the intensity of mutant
bands carrying mutations C and D should be decreased 4 x, which should still be visible
on DGGE gels. Complete disappearance (relative intensity of < 0.2% WT band) of these
mutant bands suggested the number of these mutants was reduced at least fortyfold. It
suggests there might be more than one sensitive populations in 0.5 pgM x 6 d 6TGr cells.
Each sensitive population had its own characteristic killing curves. Mutations C and D
came from one of the populations which is hypersensitive to BP and killed off prior to
other sensitive populations by 0.5 IpM x 20 d treatment.
In summary, results of this study indicate BP-induced heterogeneity. However, the
exact nature of such heterogeneity remains unknown. More experiments need to be done
to solve the mystery of BP-induced transient mutants.
150
VL SUMMARY AND CONCLUSIONS
This study has investigated the effect of dose and dose rate on BP induced
mutational spectra selected by 6TG in human lymphoblast cells (AHH-1). The
mutational spectra was determined in the low-melting domain of hprt exon 3 using a
combination of hi-fi PCR and DGGE. Statistical significance of the mutational spectra
was achieved by designing large-scale human cell cultures and analyzing large number of
independent surviving mutants. In this way predominant types of mutation could be
assigned to populations involving over 100 mutants per hotspot. Comparison was carried
out among AHH-1 cells treated with various doses (0.02 - 30 gM) of BP for various
length of time (28 h - 20 d). Mechanisms of the dose-dependence of BP mutational
spectra were also studied. The conclusions derived from these studies are summarized
below.
1) Combination of large-scale human cell culture and hi-fi PCR/DGGE is a
powerful technique to study mutational spectra under low dose and dose rate.
This study is the first to demonstrate the feasibility of using large-scale human cell
culture and hi-fi PCR/DGGE to study the chemical-induced point mutations under dose
and dose rate close to human experience. Such technique assures the statistical quality
(precision and reproducibility) of the mutational spectra. The low dose rate (0.02 p.M)
investigated in this study falls into the dose range common to human exposure. The
induced mutation rate from 0.02 giM BP treatment is tenfold as high as the spontaneous
mutation rate. Yet the combination of large-scale human cell cultures and hi-fi
PCR/DGGE has the potential to study even lower doses which induce the same
mutational rate as the spontaneous rate (doubling doses).
151
2) BP induced mutational spectra are dose and dose rate dependent.
This study demonstrates BP mutational spectra are distinct from each other under
different dose conditions. Cultures treated with 0.02 gM BP for 20 days yielded 4
hotspots (A: G -> T at bp 229; Bi: G -> T at bp 238; B2: T-> G at bp 295; and B3: - A at
bp 256 or 257). Cultures treated with 30 jgM BP (a toxic dose) for 28 hours (a short term)
yielded the same hotspot A as the in cultures treated with 0.02 jiM (a nontoxic) for 20
days (a long term), which suggested mechanistic similarities between the two conditions.
Two extra hotspots were also observed in 30 gM BP treated cultures: F: TG -> CC at bp
233-234, and G: G -> T at bp 237. Cultures treated with 0.5 gM BP yielded interesting
yet unexpected results. After 6 days of treatment, three mutational hotspots (C: A -> T at
bp 261; D: - AAGAGCT at bp 307-313; and E: A -> T at bp 247) were observed. After
treating the same culture for 14 extra days, hotspots C and D mysteriously disappeared
and E was the only hotspot present in the culture. The possible mechanisms involved in
these observations were investigated.
3) Treatment of BP results in heterogeneity in AHH-1 cells.
Several hypothesis were proposed to solve the mystery of disappearing mutants,
including possibilities of selective pressure for/against 6TG r mutants; gene amplification;
original population heterogeneity; and BP induced heterogeneity. Results indicated no
growth advantage or disadvantage of 6TGr mutants in neither selective (with 6TG) nor
normal (without 6TG) conditions. No evidence of gene amplification was observed.
Possibility of heterogeneity in the original AHH-1 cell population was ruled out by
obtaining similar killing curves in both "undisturbed" and "disturbed" populations. On
the other hand, 6TG r populations for 0.5 gM x 6 d and 0.5 gM x 20 days demonstrated
different sensitivity toward BP toxicity, suggesting the existence of heterogeneity after
0.5 jiM BP treatment. Since the possible heterogeneity of AHH-1 population was ruled
152
out, the observed heterogeneity in these two populations was likely to be results of BP
treatment. To find out the nature of BP induced heterogeneity is beyond the scope of this
study.
In summary, this study is the first to demonstrate the dose-dependency of a human
mutational spectra with statistical significance. It is also the first to illustrate a
experimental design to allow the study of human mutational spectra resulted from a
exposure scheme close to real human situation. Results from this study indicate that
people exposed to BP from non-occupational and occupational sources many inherent
different mutations. This finding demonstrates that cell and animal experiments carried
out at exposure higher than common to human experience cannot be rationally considered
as reflecting human mutational pathways.
153
VII. SUGGESTIONS FOR FUTURE RESEARCH
Results in this study suggests the BP-induced mutational spectra are dose and dose
rate dependent. They can be attributed to dose-dependency of BP transport across the
membrane, metabolism of BP, DNA repair, and/or BP induced heterogeneity in AHH- 1
cells. One could continue this study to find out the mechanisms of the dose-dependency
of BP mutational spectra. The following topics are of particular interest to pursue:
1) Find out whether the phenomenon of "disappearing mutant" also occurs in the
cultures treated with 0.02 gM BP.
In cultures treated with 0.5 gM BP, three hotspot were present after 6 days of
treatment. Continuously treating the same culture for 14 more days, two of the hotspots
disappeared. It is interesting to see whether the same phenomenon would occur at lower
dose or dose rate (cultures treated with 0.02 gM BP). Since every cell in this experiment
has been frozen, one can simply resume the culture treated with 0.02 gM BP for 6 days
and continue the procedure in Fig. 3.3. Caution needs to be taken that not all the cells
would survive when a frozen sample thaws. Usually, when to thaw a sample containing
108 cells in one ml freezing solution (RMPI 1640 + 10% donor horse serum + 10%
DMSO), around 50% cells survive (data not shown). Based on the results in Table 5.1,
one has to pool the two independent cultures together to obtain about 10,000 independent
6TGr mutants.
2) Find out the mechanisms and the nature of "disappearing mutant".
Results of this study suggests that the transient mutants arose from a sensitive
subpopulation induced by BP treatment. However, the exact mechanisms of such
heterogeneity have not been fully understood. The best way is to clone the "disappearing
mutant" so one can analyze its biochemical characteristics. Such work is going to be
154
tedious, since each of the two transient mutant population comprises less than 4% 6TGr
cells. One thus has to isolate and analyze large number of colonies to get the desired
clone. After obtaining such colony, one can investigate its sensitivity toward BP toxicity,
AHH activity, BP metabolite, DNA binding and repair capacity. These will shed light on
the exact mechanism of transient mutants.
155
REFERENCES
Albertini, R.J. and R. DeMar (1974) Mosaicism of peripheral blood lymphocyte
population in females heterozygous for the Lesch-Nyhan mutation, Biochemical Genet.,
11: 397-411.
Albertini, R.J., J.P. O'Neill, J.A. Nicklas, N.H. Heintz and P.C. Kelleher (1985)
Alterations of the HPRT gene in human in vivo-derived 6-thioguanine resistant T
lymphocytes, Nature (London), 316: 369-371.
Alexandrov, K., M. Rojas, O. Geneste, M. Castegnaro, A.M. Camus, S. Petruzzelli, C.
Giuntini and H. Bartsch (1992) An improved fluorometric assay for dosimetry of
benzo[a]pyrene diol-epoxide-DNA adducts in smokers' lung: comparisons with total
bulky adducts and aryl hydrocarbon hydroxylase activity, Cancer Res., 62: 6248-6253.
Andersson, B., S. Filt and B. Lambert (1992) Strand specificity for mutations induced by
(+)-anti BPDE in the hprt gene in human T-lymphocytes, Mutat. Res., 269: 129-140.
Anttila, S, H. Vainio, E. Hietanen, A.M. Camus, C. Malaveille, G. Brun, K. HusgafvelPursiainen, L. Heikkila, A. Karjalainen and H. Bartsch (1992) Immunohistochemical
detection of pulmonary cytochrome P450IA and metabolic activities associated with
P450IA1 and P450IA2 isozymes in lung cancer patients, Environ. Health Perspect., 98:
179-182.
Baird, W.M., R.G. Harvey and P. Brookes (1975) Comparision of the cellular DNAbound products of benzo[a]pyrene with the products formed by the reaction of
benzo[a]pyrene-4,5-oxide with DNA, Cancer Res., 35: 54-57.
Bartsch, H., K. Kemminki and O'Neill (eds.) (1988) Methods for Detecting DNA
Damaging Agents in Humans: Applications in Cancer Epidemiology and Prevention,
IARC Scil Pub. No. 89, IARC, Lyon, pp. 9-51.
Bast, R.C.Jr., B.W. Shears, H.J.Rapp and H.V. Gelboin (1973) Aryl hydrocarbon
hydroxylase in guinea pig peritoneal macrohages: Benz[a]anthracene-induced increase of
enzyme activity in vitro and in cell culture, J. Natl. Cancer Inst., 51: 675-678.
Bast, R.C.Jr., J.P.Whitlock, Jr., H. Miller, H.J. Rapp and H.V. Gelboin (1974) Aryl
hydrocarbon hydroxylase in human peripheral blood monocytes, Nature, 250: 664-665.
Bast, R.C.Jr., T. Okuda, E. Plotkin, R. Tarone, J.H. RApp and H.V. Gelboin (1976)
Development of an assay for aryl hydrocarbon hydroxylase in human peripheral blood
monocytes, Cancer Res. 36: 1967-1974.
Baum, E.J. (1978) Occurence and survaillance of polycyclic aromatic hydrocarbons. In:
Geloin, H.V. adn Ts'o POP (eds.), Polycyclic Hydrocarbons and Cancer, vol.2, Academic
Press, New York 1978, pp. 45-70.
Benzer, S. and E. Freese (1958) Induction of spectra mutations with 5-bromouracil, Proc.
Natl. Acad. Sci. USA, 44: 112-119.
Bevan, D.R. and D.M. Ruggio (1991) Bioavailability in vivo of benzo[a]pyrene adsorbed
to diesel particulate, Toxicology and Insdustrial Health, 7: 125-139.
156
Brennand, J., A. C. Chinault, D. S. Konecki, D.W. Melton and C.T. Caskey (1982)
Cloned cDNA sequences of the hypoxanthine/guanine phosphoribosyltransferase gene
from a mouse neuroblastoma cell line found to have amplified genomic sequences, Proc.
Natl. Acad. Sci. USA, 79: 1950-1954.
Brown, R. and J.Thacker (1984) The nature of mutants induced by ionising radiation in
cultured hamster cells. I. Isolation and initial characterisation of spontaneous, ionising
radiation-induced, and ethyl methanesulphonate-induced mutants resistant to 6thioguanine, Mutat. Res. 129: 269-281.
Busbee, D.L., C.R. Shaw and E.T. Cantrell (1972) Aryl hydrocarbon hydroxylase
induction in human leukocytes, Science 197: 315-316.
Busbee, D.L., C.O. Joe, J.O. Norman and P.W. Rankin (1984) Inhibition of DNA
synthesis by an electrophilic metabolite of benzo[a]pyrene. Proc. Natl. Acad. Sci. USA,
81: 5300-5304.
Busetta, B. (1988) The use of folding patterns in the search of protein structural
similarities; a three-dimensional model of phosphoribisyl transferases. Biochem.
Biophys. Acta., 957: 21-23.
Cariello, N.F. and W.G. Thilly (1986) Use of gradient denaturing gels to determine
mutatioanl spectrum in human cells, Mechanisms of DNA Damage and Repair:
Implications for Carcinogenesis and Risk Assessment. New York, Plenum Press, pp.
439-452.
Cariello, N.F., J.K. Scott (1988) Resolution of a missense mutant in human genomic
DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro
DNA amplification: HPRT munich, Am. J. Human Gen., 42: 726-734.
Cariello, N. F., Keohavong, P., Kat, A. G., & Thilly, W. G. (1990). Molecular analysis of
complex human cell population: Mutational spectra of MNNG and ICR-191. Mutation
Research, 231: 165-176.
Cariello, N.F. and T.R. Skopek (1993) Analysis of mutations occuring at the human hprt
locus.
Carothers, A.M. and D. Grunberger (1990) DNA base changes in benzo[a]pyrene diol
epoxide-induced dihydrofolate reductase mutants of Chinese hamster ovary cells,
Carcinogenesis, 11: 189-192.
Cattanach, B.M., J. Peters and C.Rasberry (1989) Induction of specific locus mutations
in mouse spermatogonial stem cells by combined chemical X-ray treatments, Mutat.
Res., 212: 91-101.
Cerutti, P., K. Shinohara, M.-L. Ide and J.Remsen (1978) Fromation and repair of
benzo[a]pyrene-induced DNA damage in mammalian cells. In: Gelboin H.V. and P.O.P.
Ts'o (eds.) Polycyclic Hydrocarbons and Cancer, Vol. 2, Academic Press, New York
1978, pp. 203-212.
Chang, W.P. and J. B. Little (1991) Delayed reproductive death in X-irradiated Chinese
hamster ovary cells, Int. J. Radiat. Biol., 60: 483-496.
157
Chen, J. and W.G. Thilly (1994) Mutational spectrum of chromium(VI) in human cells,
Mutat. Res., 323: 21-27.
Chen, R.-H., V.M. Maher and J.J. McCormick (1990) Effect of excision repair by diploid
human fibroblasts on the kinds and locations of mutations induced by (±)-78,8adihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in the coding region of the
HPRT gene, Proc. Natl. Acad. Sci. USA, 87: 8680-8684.
Chen, R.-H., V.M. Maher and J.J. McCormick (1991) Lack of a cell cycle-dependence
strand bias for mutations induced in the HPRT gene by (±)-78,8a-dihydroxy-9a,10aepoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in excision repair-deficient human cells,
Cancer Res., 51: 2587-2592.
Chen, R.-H., V.M. Maher, J. Brouwer, P. van de Putte and J.J. McCormick (1992)
Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in
HPRT gene of diploid human fibroblasts, Proc. Natl. Acad. Sci. USA, 89: 5413-5417.
Cohen, G.M., S.M. Haws, B.P. Moore and J.W. Bridge (1976) Benzo[a]pyrene-3-yl
hydrogen sulfate, a major ethyl acetate-extractable metabolite of B[a]P in human,
hamster, and rat lung culures. Biochem. Pharmacol., 25: 2560-2571.
Cole, J. and T.R. Skopek (1993) Mutant frequency, mutation rate and mutational spectra
in the human population in vivo, Mutat. Res.
Cooper, C.S., P.L. Grover and P. Sims (1983) The metabolism and activation of
benzo[a]pyrene. In: Bridges, J.W. and L.F. Chasseaud (eds.), Progress in Drug
Metabolism, Vol. 7, Wiley & Sons Ltd., pp. 295-396.
Crawford, C.R., C.Y.C. Ng and J.A. Belt (1990) Isolation and characterization of an
L1210 cell line retaining the sodium-dependent carrier if as its sole nucleoside transport
activity, J. Biol. Chem., 265: 13730-13734.
Crespi, C.L. (1982) Xenobiotic metabolism and mutation in diploid human lymphoblasts.
MIT Thesis.
Crespi, C.L. and W.G. Thilly (1984) Assay for gene mutation in a human lymphoblast
line, AHH-1, competent for xenobiotic metabolism. Mutat. Res. 128: 221-230.
Crespi, C.L., H.L. Liber, T.D. Behymer, R.A. Hites and W.G. Thilly (1985) A human cell
line sensitive to mutation by particle-borne chemicals. Mutat. Res., 157: 71-75.
Danheiser, S. L. (1985). Long-term, low-dose benzo(a)pyrene-induced mutation in
human lymphoblasts competent in xenobiotic metabolism. MIT Master Thesis.
Danheiser, S.L., H.L. Liber, and W.G. Thilly (1989). Long-term, low-dose
benzo(a)pyrene-induced mutation in human lymphoblasts competent in xrnobiotic
metabolism. Mutation Research, 210: 143-147.
Davies, G. M. and A. Hodkinson (1986). Measurement and Analysis of Occupational
Exposures to Coke Oven Emissions. Ann. Occup. Hyg., 30: 51-62.
158
DeLuca, J. G., D.A. Kaden, J. Krolewski, T.R. Skopek and W.G. Thilly (1977).
Comparative mutagenicity of ICR-191 to S. typhimurium and diploid human
lymphoblasts. Mutation Research, 46: 11-18.
DeLuca, J.G., L. Weistein and W.G. Thilly (1983) Ultraviolet light-induced mutation of
diploid human lymphoblasts. Mutat. Res., 107: 347-370.
Ehling, U.H. and A. Neuhauser-Kltlus (1988) Induction of specific-locus and dominantlethal mutations by cyclophosphamide and combined cyclophosphamide-radiation
treatment in male mice, Mutat. Res., 199: 21-30.
Ehrfeld, A., F. Planas Bohne, and C. Liicke-Huhle (1986) Amplification of oncogenes
and integrated SV40 sequences in mammalian cells by the decay of incorported iodine125. Rad. Res., 108: 43-51.
Fischer, S. and L.S. Lerman (1983). DNA fragments Differing by Single Base-pair
Substitutions Separated in Denaturing Gradient Gels: correspondence with Melting
Theory. Proc. Natl. Acad. Sci. USA, 80: 1579-1583.
Freedman, H.J., H.L. Gurtoo, J.Minowada, B. Paigen and J.B. Vaught (1979a) Aryl
hydrocarbon hydroxylase in a stable human B-lymphocyte cell line, RPMI-1788, cultured
in the absence of mitogens. Cancer Res. 39: 4605-4611.
Freedman, H.J., N.B. Parker, A.J.Marinello, H.L. Gurtoo and J. Minowada (1979b)
Induction, inhibition, and biological properties of aryl hydrocarbon hydroxylase in a
stable B-lymphocyte cell line, RPMI 1788. Cancer Res. 39: 4612-4619.
Furth, E.E., W.G. Thilly, B.W. Penman, H.L. Liber and W.M. Rand (1981) Quantitative
assay for mutation in diploid human lymphoblasts using microtiter plates. Anal.
Biochem., 110: 1-8.
Gelboin, H.V. (1980) Benzo[a]pyrene metabolism, activation, and carcinogenesis: role
and regulation of mixed-function oxidases and related enzymes, Physiological Reviews,
60: 1107-1166.
Gonzales, F.J. (1989) The molecular biology of cytochrome P450s, Pharmacol. Rev., 40:
244-288.
Grantham, R., C. Gautier, M. Gouy, R. Mercier and A. Pave (1980) Codon catalog usage
and the genome hypothesis, Nucleic Acids Res., 11: r49-r62.
Grosjean, H. and W. Fiers (1982) Preferentialr codon usage in prokaryotic genes: the
optimal codon-anticodon interaction energy and the selective codon usage in efficiently
expressed genes, Gene, 18: 199-209.
Grosovsky, A.J. and J.B. Little (1985). Evidence for linear response for the induction of
mutations in human cells by X-ray exposure below 10 rads., Proc. Natl. Acad. Sci. USA,
2092-2095.
Guthrie, F. E. (1980). Absorption and Distribution. In E. Hodgson & F. E. Guthrie (Eds.),
Introduction to Biochemical Toxicology (pp. 23, 24). New York: Elsevier.
Guyton, A. C. (1977). Basic Human Physiology. Philadelphia: W.B. Saunders.
159
Hanson-Painton, O., M.J. Griffin and J. Tang (1981) Involvement of a cytosolic carrier
protein fraction in the microsomal metabolism of benzo[a]pyrene in rat liver, Cancer
Res., 43: 4198-4206.
Harris, C.C., H. Autrup and G.D. Stoner (1979) Metabolism of B[a]P, Nnitrosodimethylamine, and N-nitrosopyrrolidine and identification of the major
carcinogen-DNA adducts formed in cultured human esophagus, Cancer Res., 39: 44014406.
Harris, C.C., K. Vahakangas, M.J. Newman, G.E. Trivers, A. Shamsuddin, N. Sinopoli,
D.L. Mann and W.E. Wright (1985) Detection of benzo[a]pyrene diol epoxide-DNA
adducts in peripheral blood lymphocytes and antibodies in serum form coke oven
workders, Proc. Natl. Acad. Sci. USA, 82: 6672-6673.
Haugen, A., G. Becher, G. Benestad, K. Vahakangas, G.E. Trivers, N.J. Newman and
C.C. harris (1986) Determination of polycyclic aromatic hydrocarbons in urine,
benzo[a]pyrene diol epoxide-DNA adducts in lymphocyte DNA, and antibodies to the
adducts in sera from coke oven workers exposed to measured amounts of polycyclic
aromatic hydrocarbons in the work atmosphere, Cancer Re., 46:4178-4183.
Hayashi, N. and T. Tsutsui (1993) Cell cycle dependence of cytotoxicity and
glastogenicity induced by treatment of synchromized human diploid fibroblasts with
sodium fluoride, Mutat. Res., 290: 293-320.
Hemminki, K, E. Grzybowska, M. Chorazy, K. Twardowska-Saucha, Sroczynski, K.
Putman, K. Randerath, D.H. Philips and A. Hewer (1990a)
Hemminki, K, E. Grzybowska, M. Chorazy, K. Twardowska-Saucha, Sroczynski, K.
Putman, K. Randerath, D.H. Philips and A. Hewer, R,M, Santella and F.P. Perera (1990b)
DNA adducts in human related to occupational and environmental exposure to aromatic
compounds. In: Vainio, H., M.Sorsa and A.J. McMichael (eds.) Complex Mixture and
Cancer Risk, IARC Sci. Publ. No. 104, IARC, Lyon, pp. 181-192.
Herbert, R., M. Marcus, M.S. Wolff, F.P. Ferera, L. Andrew, J.H. Godbold, M. Rivera,
M. Stenfandis, X.Q. Lu, P.J. Landrigan and R.M. Santella (1990) Detection of adducts of
deoxyribonucleic acid in white blood cells of roofers by 32p-postlabeling, Scand. J.
Work. Environ. Health, 16: 135-143.
Hou, S.-M., A.-M. Steen, S. Filt and B. Andersson (1993) Molecular spectrum of
background mutations at the hprt locus in human T-lymphocytes, Mutagenesis 8: 43-49.
IARC Monographs on the Evaluation of the Carconogenic Risk of Chemicals to Humans:
Benzo[a]pyrene. Lyon: IARC. 1983.
Kaden, D.R., K.M. Call, P.-M. Leong, E.A. Komives and W.G. Thilly (1987) Killing and
mutation of human lymphoblast cells by Aflatoxin B1: Evidence for an inducible repair
response, Cancer Res., 47: 1993-2001.
Kakefuda, T. and H. Yamamoto (1978) Modification of DNA by the benzo[a]pyrene
metabolite diol epoxide r-t, t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene,
Proc. Natl. Acad. Sci. USA, 75: 415-419.
160
Kakefuda, T. and H. Yamamoto (1978) Modification of DNA by benzo[a]pyrene diol
epoxide I. In: Polycyclic hydrocarbons and cancer, ed. H. V. Gelboin and P.O.P. Ts's.
New York: Academic, Vol. 2: 293-304.
Kellermann, G., E. Cantrell and C.R. Shaw (1973) Variation in extent of aryl
hydrocarbon hydroxylase inductionin cultured human lymphocytes, Cancer Res., 33:
1654-1656.
Kelley, W.N. and J.B. Wyngaarden (1983) Clinical syndromes associated with
hypoxanthine-guanine phosphoribosyltransferase deficiency. In: The metabolic basis of
inherited disease. (eds. J.B. Stanbury, J.B. Wyngaarden, D.S. Frederickson, J.L.
Goldstein, M.S. Brown), McGraw-Hill, New York, pp. 1115-1143.
Keohavong, P., V.F. Liu and W.G. Thilly (1989) Analysis of point mutations induced by
ultraviolet light in human cells, Mutat. Res., 249: 147-159.
Keohavong, P. and W.G. Thilly (1989). Fidelity of DNA polymerase in DNA
amplification. Proc. Natl. Acad. Sci. USA, 86: 9253- 99257.
Keohavong, P. and W.G. Thilly (1992). Mutational spectrometry: A general approach for
hot-spot mutations in selectable genes. Proc. Natl. Acad. Sci. USA, 89: 4623- 4627.
Kersch, R.L. and W.G. Thilly (1987) Internal standards for survival: increasing the
accuracy for human cell mutation assays. Mutat. Res., 182: 83-97.
Krenitsky, T.A., R. Papioannou and G.B. Elion (1969) Human hypoxanthine
phosphoribosyltransferase: purification, properties and specificity. J. Biol. Chem. 244:
1263-1277.
Law, M.R. (1990) Genetic predispostition to lung cancer, Br. J. Cancer, 61: 195-206.
Lavi, S. (1982) Carcinogen-mediated amplification of specific DNA sequences. J. Cell.
Biochem., 18: 149-156.
Lawley, P.D. (1989) Mutagens as carcinogens: development of current concepts, Mutat.
Res., 23: 3-25.
Levin, W., M.K. Buening, A.W. Wood (1980) An enantiomeric interaction in the
metabolism and tumorigenicity of (+)- and (-)-benzo[a]pyrene 7,8-oxide, J. Biol. Chem.
255: 9067-9074.
Liber, H. L., Danheiser, S. L., & Thilly, W. G. (1985). Mutation in single-cell systems
induced by low-level mutagen exposure. Basic Life Science, 33: 169- 204.
Ling, L.L., P. Keohavong, C. Dias and W.G. Thilly (1991) Optimization of the
polymerase chain reaction with regard to fidelity: Modified T7, Taq, and Vent DNA
polymerases, PCR Meth. Appl., 1: 63-69.
Liou, S.-H., D. Jacobson-Dram, M.C. Poirier, D. Nguyen, P.T. Strickland and M.S.
Tockman (1989) Biological monitoring of fire fighters: sister chromatid exchange and
polycyclic aromatic hydrocarbon - DNA adducts in peripheral blood cells, Cancer Res.,
49: 4929-4935.
161
Little, J.B., L. Gorgojo and H. Vetrovs (1990) Delayed appearance of lethal and specific
gene mutations in irradiated mammalian cells, Int. J. Radiat. Oncol. Biol. Phys., 19: 14251429.
Long, E.O. and I.B. Dawid (1980) Repeated genes in eukaryotes. Annu. Rev. Biochem.,
49: 727-746.
Liicke-Huhle, C (1989) Review: Gene amplification - a cellular response to genotoxic
stress, Molecular Toxicology, 2: 237-253.
Liicke-Huhle, C. and P. Herrlich (1986) Gene amplification in mammalian cells after
exposure to ionizing radiation and UV. In: Radiation Carcinogenesis and
DNAAlterations, eds. F.J. Bums, A.C. Upton, and G. Silini, pp. 405-411. Amsterdam:
Plenum Press.
Liicke-Huhle, C. and P. Herrlich (1987) Alpha-radiation-induced amplification of
integrated SV40 sequences is mediated by a trans-acting mechanism. Int. J. Cancer, 39:
94-98.
Maher, V.M., J.J. McCormick, P.L. Grover and P. Sims (1977) Effect of DNA repair on
the cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon derivatives in
normal and xeroderma pigmentosum human fibroblasts, Mutat. Res., 43: 117-138.
Mazur, M. and B.W. Glickman (1988) Sequence specificity of mutations induced by
benzo[a]pyrene-7,8-diol-9,10-epoxide at endogenous aprt gene in CHO cells, Somat.
Cell Mol. Genet., 14: 393-400.
McCormick, J.J. and V.M. Maher (1978) Effect of DNA repair on the cytotoxicity and
mutagenicity of polycyclic hydrocarbon metabolites in human cells. In: Gelboin, H.V.
and P.O.P. Ts'o (eds.) Polycyclic hydrocarbons and Cancer, Vol. 2, Academic Press,
New York, 1978, pp. 221-232.
McCormick, J.J. and V.M. Maher (1985) Role of DNA lesion and DNA repair in
mutagenesis and transformation of human cells, In: Harris, C. and H.N. Autrup (eds)
Human Carcinogenesis, Academic Press, pp. 401-429.
Meehan, T., K. Straub and M. Calvin (1977) Benzo[a]pyrene diol epoxide covalently
binds to deoxyguanosine and deoxyadenosine in DNA, Nature, 269: 735-737.
Mellon, I., G. Spivak and P.C. Hanawalt (1987) Selective removal of transcriptionblocking DNA damage from the transcribed strand of the mammalian DHFR gene, Cell,
51: 241-249.
Menzel, D. B., & McClellan, R. 0. (1980). Toxic Responses of the Respiratory System.
In J. Doull, C. D. Klassen, & M. O. Amdur (Eds.), Toxicology: the Basic Science of
Poisons (pp. 249, 251). New York: Macmillan.
Miller, E.C. and J.A. Miller (1981) Searches for ultimate chemical carcinogens and their
reactions with cellular macromolecules, Cancer, 47: 2327-2345.
Moore, G.E. and J. Minowada (1969) Human hematopoietic cell lines: a progress report.
In Vitro, 4: 100-114.
162
Mullis, K.B. and F.A. Faloona (1987) Specific synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction, Method in Enzymology, 155: 335-350.
Nakamura, N. and S. Okada (1981) Dose-rate effects of gamma-ray-induced mutations in
cultures mammalian cells, Mutat. Res., 83: 127.
Nakamura, N. and S. Okada (1983) Mutations resistant to bromodeoxyuridine in mouse
lymphoma cells selected by repeated exposure to EMS. Characteristics of phenotypic
instability and reversion to HAT resistance by 5-azacytidine, Mutat. Res., 111: 353.
Nebert, D.W., J. Winker and H.V. Gelboin (1969) Aryl hydrocarbon hydroxylase activity
in human placenta from cigarette-smoking and nonsmoking women, Cancer Res., 29:
1763-1769.
Oakberg, E.F. (1978) Differential spermatogonial stem-cell survival and mutation
frequency, Mutat. Res., 327-340.
Oller, A. R., Rastogi, P., Morgenthaler, S., and Thilly, W. G. (1989a). A Statistical Model
to Estimate Variance in Long term-Low Dose Mutation Assays: Testing of the Model in a
Human Lymphoblastoid Mutation Assay. Mutat. Res., 216: 149-161.
Oiler, A.R., C.W. Buser, M.A. Tyo and W.G. Thilly (1989b) Growth of mammalian cells
at high oxygen concentrations, J. Cell Sciences, 94: 43-49.
Oiler, A.R. and W.G. Thilly (1992) Mutational spectra in human B-cells. Spontaneous,
oxygen and hydrogen peroxide-induced mutations at the hprt gene, J. Mol. Biol., 228:
812-826.
Osborne, M.R., S. Jacobs, R.G. Harvey and P. Brookes (1981) Minor products from the
reaction of (+) and (-) benzo[a]pyrene-anti-diolepoxide with DNA, Carcinogenesis, 2:
553-558.
Penman, B. W. (1980) Mutation of Human Cells Exposed to Low Concentrations of
Chemical Mutagens for Long Periods of Time. Ph.D Thesis, massachusetts Institute of
Technology.
Penman, B. W., Hoppe, H., and Thilly, W. G. (1979). Concentration-dependent mutation
by alkylating agents in human lymphoblasts and Salmonella typhimurium: N-methyl-Nnitrosourethane and 8-propiolactone, J. Natl. Cancer Inst., 63(4): 903-907.
Penman, B. W., and Thilly, W. G. (1976). Concentration-dependent mutation of diploid
human lymphoblasts by methylnitronitrosoguanidine: the importance of phenotypic lag.
Somat. Cell Genet., 2(4): 325-330.
Perera, F. (1981) Carcinogenicity of airborne fine particulate benzo[a]pyrene: An
appraisal of the evidence and the need for control, Environ. Health Perspect., 42: 163185.
Perera, F. (1988) The significance of DNA and protein adducts in human biomonitoring
studies, Mutat. Res., 205: 255-269.
Pool, B.L., A.O. Yalkinoglu, P. Klein and J.R. Schlehofer (1989) DNA amplification in
genetic toxicoloty. Mutat. Res., 213: 61-72.
163
Prough, R.A., V.W. Patrizi, R.T. Okita, R.S.S. Masters and S.W. Jakobsson (1979)
Characteristics of benzo[a]pyrene metabolism by kidney, liver, and lung microsomal
fractions from rodents and humans, CancerRes., 39: 1119-1206.
Recio, L., J. Cochrane, D. Simpson and T.R. Skopek (1990) DNA sequence analysis of in
vivo hprt mutations in human T-lymphocytes, Mutagenesis, 5: 505-510.
Rossi, A.M., A.D. Thijssen, A.D. Tates, H. Vrieling, A.T. Natarajan, P.H.M. Lohman and
A.A. van Zeeland (1990) Mutations affecting RNA splicing in man are detected more
frequently in somatic than in germ cells, Mutat. Res., 244: 353-357.
Rossi, A.M., A.D. Tates, A.A. van Zeeland and H. Vrieling (1992) Molecular analysis of
mutations affecting hprt mRNA splicing in human T-lymphocytes in vivo, Environ. Mol.
Mutagen., 19: 7-13.
Russell, W. L., Hunsicker, P. R., Raymer, G. D., Steele, M. H., Stelzner, K. F., &
Thompson, H. M. (1982). Dose-response curve for ethylmitrosourea-induced specificlocus mutations in mouse spermatogonia. Proc. Natl. Acad. Sci. USA, 79: 3589-3591.
Russell, L.B. and E.M. Rinchik (1993) Structural differences between specific-locus
mutations induced by different exposure regimes in mouse spermatogonial stem cells,
Mutat. Res., 288: 187-195.
Sanger, F., S. Nicklen and A. R. Coulson (1977) DNA sequencing with chain-terminating
inhibitors. Proc. Natl. Acad. Sci. USA. 74: 5463-5467.
Savela, K. and K. Hemminki (1991) DNA adducts in lymphocytes and granulocytes of
smoker and nonsmokers detected by 32 P-postlabelling assay, Carcinogenesis, 12:
Schimke, R.T. (1984a) Gene Amplification in cultured animal cells, Cell, 37: 705-713.
Schimke, R.T. (1984b) Gene amplification, drug resistance, and cancer. Cancer Res., 44:
1735-1742.
Schwab, M. and L.C. Amler (1990) Amplification of cellular oncogenes. A predictor of
clinical outcome in human cancer. Genes Chromosomes Cancer 1: 181-193.
Schwartz, J. L., & Samson, L. (1983). Mutation Induction in Chinese Hamster Ovary
Cells after Chronic Pretreatment with MNNG. Mutat. Res., 119: 393-400.
Seegmiller, J.E., F.M. Rosenbloom and W.N. Kelley (1967) Enzyme defect associated
with a sex-linked human neurological disorder and excessive purine synthesis. Science
155: 1682-1684.
Selkirk, J.K., R.G. Croy, J.P. Whitlock and H.V. Gelboin (1975) In vitro metabolism of
benzo[a]pyrene by human liver microsomes and lymphocytes, Cancer Res., 35: 36513655.
Selkirt, J.K., A. Nikbakht and G.D. Stoner (1983) Comparative metabolism and
macromolecular binding of benzo[a]pyrene in explant cultures of human bladder, skin,
bronchus, and esophagus of eight individuals, Cancer Lett., 18: 11-19.
164
Shamsuddin, A.K.M., N.T. Sinopoli, K. Hemminki, R.R. Boesch and C.C. Harris (1985)
Detection of benzo[a]pyrene: DNA adducts in human white blood cells, Cancer Res., 45:
66-68.
Shields, D.C. (1990) Switches in species-specific codon preference: the influence of
mutation biases, J. Mol. Evol., 31: 71-80.
Singer, B. and D. Grunberger (eds.) (1983) Molecular Biology of Mutagens and
Carcinogens, Plenum Press, New York, 1983, 347 pp.
Sipes, I.G. and A.J. Gandolfi (1986) Biotransformation of toxicants, In: Klaassen, C.D.,
M.O. Amdur and J. Doull (eds.) Toxicology: The basic science ofpoisons, Macmillan
Publishing Company, New York.
Southam, P. and W.G. Thilly (1994) Human mutational spectra, in preparation.
Spradling, A.C. and A.P. Mahowald (1980) Amplification of genes for chorion proteins
during oogenesis in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA, 7: 1669-1100.
Stark, G.R. and G. M. Wahl (1984) Gene amplification, Ann. Rev. Biochem., 53: 447491.
Steingrimsdottir, H., G. Rowley, G. Dorado, J. Cole and A.R. Lehmann (1992) Mutations
which alter splicing in the human hypoxanthine-guanine phosphoribosyl transferase gene,
Nucleic Acids Res., 20: 1201-1208.
Straub, K.M., T. Meeham, A.L. Burlingame and M. Calvin (1977) Identification of the
major adducts formed by reaction of benzo[a]pyrene diol epoxide with DNA in vitro,
Proc. Natl. Acad. Sci. USA, 74: 5285-5289.
Szybalski, W. (1958) Resistance to 8-azaguanine as a selectable marker for a human cell
line, Microbiol. Genet. Bull., 16: 30.
Thacker, J. and A. Stretch (1983) Recovery from letahal and mutagenic damage during
postirradiation holding and low dose-rate irradiations of cultured hamster cells, Radiat.
Res., 96: 380.
Thilly, W. G., DeLuca, J. G., Hoppe, H., & Penman, B. W. (1976). Mutation of human
lymphoblasts by methylnitrosourea. Chem. Biol. Interact., 15: 33-50.
Thilly, W.G. (1985) Potential use of gradient denaturing gel electrophoresis in obtaining
mutational spectra from human cells. Carcinogenesis: The role of chemicals and
radiation in the etiology of cancer, New York, Raven Press, pp. 511-528.
Timbrell, J. A. (1991). Principlesof BiochemicalToxicology. London: Talor & Francis
Ltd.
Timms, A.R., H. Steingrimsdottir, A.R. Lehmann and B.A. Bridges (1992) Mutant
Sequences in the rpsL gene of Escherichia coli B/r: mechanistic implications for
spontaneous and ultraviolet light mutagenesis, Mol. Gen. Genet., 232: 89-96.
Toxicological Profile: Benzo[a]pyrene, U.S. Department of Health & Human Service,
1990.
165
Van Zeeland (1988) Molecular dosimetry of alkylating agents: quantitative comparison of
genetic effects on the basis of DNA adduct formation, Mutagenesis, 3; 179-191.
Vaught, J.B., H.L. Gurtoo, B. Paigen, J. Minowada, and P. Sartori (1978) Comparison of
benzo[a]pyrene metabolism by human peripheral blood lymphocytes and monocytes,
Cancer Lett., 5: 261-268.
Vrieling, H., M.L. van Rooijen, N.A. Groen, M.Z. Zdzienicka, J.W.I.M. Simons, P.H.M.
Lohman and A.A. van Zeeland (1989) Mol. Cell. Biol., 9: 1277-1283.
Vrieling, H., J. Venema, M.-L. van Rooyen, A. van Hoffen, P. Menichini, M.Z.
Zdzienicka, J.W.I.M. Simons, L.H.F. Mullenders and A.A. van Zeeland (1991) Strand
specificity for UV-induced DNA repair and mutations in the Chinese hamster HPRT
gene, Nucleic Acids Res., 19: 2411-2415.
Wei, S.-J.C., R.L. Chang, C.-Q. Wong, N. Bhachech, X.X. Cui, E. Henning, H. Yagi,
J.M. Sayer, D.M. Jerina, B.D. Preston and A.H. Conney (1991) Dose-dependent
differences in the profile of mutations induced by an ultimate carcinogen from
benso[a]pyrene, Proc. Natl. Acad. Sci. USA, 88: 11227-11230.
Weinstein, I.B. (1988) The origins of human cancer: Molecular mechanisms of
carcinogenesis and their implications for cancer prevention and treatment, Cancer Res.,
48: 4135-4143.
Yang, J.-L, R.-H. Chen, V.M. Maher and J.J. McCormick (1991) Kinds and location of
mutations induced by (±)-78,8a-dihydroxy-9a, 10a-epoxy-7,8,9. 10tetrahydrobenzo[a]pyrene in the coding region of the hypoxanthine (guanine)
phosphoribosyltransferase gene in diploid human fibroblasts, Carcinogenesis, 12: 71-75.
Zur Hausen, H. and J.R. Schlehofer (1987) The role of DNA amplification in
carcinogenesis. In: Accomplishments in Oncology, vol. 2, no. 1. Philadelphia:
Lippincott.
166