Znt. J. Cancer: 23, 691-696 (1979)
CELL-MEDIATED MUTAGENESIS I N CULTURED CHINESE
HAMSTER CELLS BY POLYCYCLIC HYDROCARBONS :
MUTAGENICITY AND DNA REACTION RELATED TO
CARCINOGENICITY IN A SERIES OF COMPOUNDS
Caroline B. WIGLEYls2, Robert F. NEWBOLD,
Jacqueline AMOS,and Peter BROOKES
Chemical Carcinogenesis Division, Institute of Cancer Research, Pollards Wood Research Station, Nightingales
Lane, Chalfont St. Giles, Bucks. HP8 4SP,England
Three polycyclic hydrocarbons, benz(a)anthracene. 3-methylcholanthrene and 7,12-dImethylbenr(a)anthracene, have been studied in a cellmediated mutagenesis system using B H K 21 cells
to metabolize the hydrocarbons and V-79 cells as
targets for detecting induced cytotoxicity and
mutation. I n large-scale experiments, the D N A of
V-79 cells was analyzed by column chromatography
to determine the nature and true extent of reaction
of hydrocarbons with deoxyribonucleosides. Products with D N A formed by the two carcinogenic
compounds were qualitatively very similar to those
reported to occur in vivo and in primary cell cultures.
Binding indices were calculated from the tritium
content of DNA-hydrocarbon products, related to
overall metabolism, for these two compounds
together with benzo(a)pyrene and 'I-methylbenz(a)anthracene using data from a previous study. These
values reflected differences in carcinogenic potency
between the compounds. Induced mutation frequencies were related to the extent of D N A reaction
with each compound. A t equivalent extents of D N A
reaction with hydrocarbon products, levels of
induced mutation were not significantly different.
We have previously described (Newbold et al.,
1977) a system for studying the biological effects of
chemical interactions between carcinogenic polycyclic
hydrocarbons (CPH) and the DNA of mammalian
cells in culture, in which both biological response
(mutation to drug resistance) and DNA reaction
could be quantitated in the same population of cells.
This was a modification of the cell-mediated
mutagenesis (CMM) system devised by Huberman
and Sachs ( I 974). We used lethally X-irradiated
BHK 21 cells as a feeder layer to metabolize hydrocarbons to reactive derivatives which are transferred
to overlying target V-79/4 cells, probably via direct
or proximate contact between the two cell types
(Kuroki and Drevon, 1978). Toxicity and mutation
resulting from exposure of V-79 cells to CPH
metabolites were then assayed after differential
trypsinization and cell counting techniques to
separate target cells from BHK feeder cells.
Similar experiments carried out on a large scale
permitted analysis of DNA from V-79 cells after
exposure to tritiated CPH. Using two hydrocarbons
of different carcinogenic potencies, benzo(a)pyrene
(BP) and 7-methylbenz(a)anthracene (7-MBA), we
were able to show that chromatographic profiles of
hydrocarbon-DNA products were identical in both
cases to those reported to occur in primary rodent
embryo cell cultures (Baird and Brookes, 1973;
Baird et a/., 1975) but different from those generated
in vitro when rat liver microsomes were used as the
source of metabolizing enzymes (King et al., 1975).
In the case of BP, the major hydrocarbon-DNA
product isolated from V-79 cells was shown to
co-chromatograph with the deoxyribonucleoside
product formed after reaction between the bay
region diol-epoxide and DNA.
We attempted to quantitate the number of
mutants induced for a given amount of hydrocarbonDNA binding in the major product peak of each
compound (referred to as true DNA reaction).
When variable rates of cell division were accounted
for, both BP and 7-MBA proved to be equally
mutagenic per ,umole/mole DNA phosphorus of
true product reaction. This suggested that the
difference in carcinogenic potency between the two
compounds was reflected by the extent of reaction
with DNA rather than the nature of that reaction.
We have now extended our original study to
include three further hydrocarbons, the weak or
non-carcinogenic benz(u) anthracene @A) and two
potent carcinogens, 3-methylcholanthrene (3-MC)
and 7,12-dimethylbenz(a)anthracene (DMBA). With
7-MBA and BP, investigated in the previous study
(Newbold et al., 1977) these five compounds span a
range of carcinogenic potencies in laboratory
animals. In principle, this study is directed towards
answering the same questions as those posed by
Brookes and Lawley (1964), Duncan et al., (1969),
and Huberman and Sachs (1977), who all attempted
to explain differences in carcinogenic activity
between a number of CPH. We have looked at all
the parameters used in their studies (metabolism,
toxicity, DNA binding and mutation) in the same
experimental system and, where possible, in the
same experiment.
MATERIAL AND METHODS
Chemicals
Unlabelled polycyclic hydrocarbons, 8-azaguanine
and DNA degradation enzymes were obtained from
Received : January 3, 1919 and in revised form March
9, 1979.
To whom reprint requests should be sent.
Present address: Department of Cellular Pathology,
Imperial Cancer Research Fund, Lincoln's Inn Fields,
London W.C.2.
692
WIGLEY ET AL.
the Sigma Chemical Co. (Kingston-upon-Thames,
Surrey, England). Generally tritiated hydrocarbons
were obtained from the Radiochemical Centre
(Amersham, Bucks., England). Radioactive compounds were stored as stock solutions in benzene in
the dark at room temperature. Immediately before
use, aliquots were taken and the benzene evaporated
off with nitrogen. The solid residue was redissolved
in a DMSO solution of unlabelled compound to
reduce the specific activity to l-5mCi/mg and give
total hydrocarbon concentrations of 200 pg/mI
(BA, 3-MC) or 40 pg/ml (DMBA). Precautions were
taken to keep all solutions in the dark but, with
DMBA, these precautions were especially rigid at
all stages of preparation and cell culture treatment
to minimize photo-oxidation (Baird and Dipple,
1977).
Cell culture and mutation assays
FIGURE
1 - The time course of metabolism of taH]hydrocarbons to water-soluble derivatives by BHK cells
during 48-h exposure of mixed cultures, expressed as pg
metabolized per 175 ,ma flask. Initial amounts of hydrocarbon were 50 pg BA, 3-MC and 10 p g DMBA per
BA; (o), 3-MC; (A), DMBA.
flask. (o),
All the techniques used in these experiments have
been described previously (Newbold et al., 1977).
In brief, lethally X-irradiated BHK 21 clone 13 cells
were plated in 175 cm2 culture flasks (Nunclon) at
a density of 2 x lo7 cells/flask in Dulbecco's modification of Eagle's minimal essential medium (DMEM)
with 10% foetal calf serum (Gibco Biocult Ltd.,
Paisley, Scotland). Eighteen hours later, 3 x loe
V79/4 cells, harvested from log phase cultures, were
plated on to the confluent BHK in 50 ml of fresh
medium. Two hours later, the mixed cultures were
treated with 0.25 ml (0.5 %) of dimethylsulphoxide
(DMSO) or DMSO containing tritiated hydrocarbon
to give the required final concentration in medium.
TABLE I
SUMMARY OF DATA ON THE TOXICITY, MUTAGENICITY A N D EXTENT OF D N A BINDING IN V-1914
CELLS OF 5 POLYCYCLIC HYDROCARBONS, ASSAYED IN A BHK CELL-MEDIATED SYSTEM
DMSO
Control
A. Carcinogenic potency (Iball index)
B. Increase in cell number/48 h
7-MBA
BP
3-MC
DMBA'
-
ND
45
75
80
151
x11.2
x11.4
x9.4
x3.9
~4.2
~2.3
C. Percentage plating efficiency
97.2
107.9
64.5
21.6
38.0
42.9
D. Mutation frequency (Az') per survivor x lo6
1.7
1.2
73
227
169
208
~
E. Metabolism to water-soluble derivatives/48 h
-
4.25
2.95
3.65
2.35
0.69
-
0.76
-
5.7
0.7
4.4
5.4
-
3.9
2.5
5.3
4.0
-
-
6.6
15.2
10.5
9.2
I. Binding index (H/E)
-
-
2.2
4.2
4.5
13.3
J. Az' mutants/106survivors/mole/pmole P
corrected DNA reaction (D/H)
-
0
11.1
14.9
16.1
22.6
(n mole/ml)
Observed hydrocarbon binding to DNA
(prnole/mole P)
F. i) Total
G. ii) Products
H. Hydrocarbon product binding to DNA
corrected for cell division during treatment
(Cix B) &mole/mole P)
IbaH (1939). - Absolute values. - * Maximum value at optimum expression time. - ' Initial concentration in medium was 0.2 p&l,
for all other compounds this was l.Opg/ml.
693
HYDROCARBON MUTAGENESIS AND DNA REACTION
(Newbold et al., 1977). A sample of DNA degraded
to deoxyribonucleosides was assayed for total
radioactivity and UV absorption at 260 nm so that
the overall extent of hydrocarbon-DNA binding
could be calculated. The remainder was then
analysed by LH20 Sephadex column chromatography
as before, to separate unchanged deoxyribonucleosides from products with covalently bound hydrocarbon metabolite.
RESULTS
Metabolism of hydrocarbons by mixed cultures
FIGURES
2 - Sephadex LH20 column elution profiles
of D N A isolated from V-79 cells after exposure to
t3H]-3-MC in mixed cultures with BHK feeder cells.
D N A (1.9 mg) was degraded to deoxyribonucleosides
prior to chromatographic analysis. UV absorbance
(hatched line) at 260 nm and radioactivity (solid line)
were monitored in 5-ml eluent fractions. The vertical
arrow indicates the elution volume of marker, p-nitrobenzylpyridine.
Forty-eight hours later, V-79 cells were harvested as
a pure population by differential trypsinization and
replating techniques described previously. Contamination by BHK was monitored at each stage of
the purification by differential counting in a
haemocytometer. The bulk of the purified V-79
population (BHK contamination <2 %) was stored
at-20" C for subsequent DNA isolation and
analysis.
A sample of V-79 cells was assayed for cytotoxicity
and mutation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus; mutation at
this locus confers resistancs to 8-azaguanine ( A z r ) .
Two hundred V79 cells were plated per 5-cm dish
for cytotoxicity assay and 5 x lo4 cells per 9-cm
dish for assay of mutation. Care was taken to ensure
maximum recovery of mutants (Newbold et al.,
1975) and expression time curves were constructed
in every experiment so that mutation frequencies
could be calculated at the optimum time of selection
with 8-azaguanine (0.2 mM) Mutation frequency per
lo6 survivors was calculated as follows: mean
number of mutant colonies per plate x 2 x 200/mean
number of colonies per survival plate. We routinely
obtain better recovery of mutants using this in situ
method than we do with a replating technique. A
similar observation was made by Barrett and Ts'o
(1978). Theoretical reasons for this were discussed
by Newbold et al., (1975).
Analysis of hydrocarbon metabolism and D N A
binding
Metabolism was assayed at various times during
the 48-h treatment period by sampling medium and
measuring the extraction into cyclohexane of
remaining parent hydrocarbon which leaves the
more water-soluble metabolites in the aqueous
phase. This procedure has been described elsewhere
(Duncan and Brookes, 1970). Isolation and degradation of DNA from the purified V-79 cell fraction
were performed exactly as described previously
Figure 1 shows the time-course of metabolism to
water-soluble derivatives of three hydrocarbons, BA,
3-MC and DMBA. The extent of metabolism at times
between 0 and 48 h is expressed as the amount (pg)
of hydrocarbon metabolized per 175 cm2 flask at
that time. This allows direct comparison of amounts
metabolized to be made between BA and 3-MC,
initially at 1.0 ,ug/ml and DMBA at 0.2 pglrnl. This
concentration of DMBA was chosen to induce a
similar degree of cytotoxicity t o that obtained with
3-MC.
BA was metabolized efficiently with only 2 % of the
total 50 pg remaining after 48 h. 3-MC in contrast
was metabolized relatively slowly so that 38 % of the
initial concentration was still extractable into
cyclohexane after 48 h. Metabolism continued to
increase linearly, however, so that only 22% of the
radioactivity remained in the organic phase at
76 h. This suggests that the observed slow rate of
metabolism is not entirely due to the production of
metabolites which extract into cyclohexane with the
parent hydrocarbon. Thin-layer chromatographic
analysis of cyclohexane extracts demonstrated that
a maximum of 18 % of the radioactivity extractable
at 48 h could be due to metabolites (M. R. Osborne,
pxsonal communication). Maximal metabolism of
the low concentration of DMBA was reached by
about 24 h. Since at 48 h, 12% of the radioactivity
was still extractable into cyclohexane, much of this
may have been due to metabolites of low watersolubility (Diamond et al., 1968). Values of the
fi
Ib
FIGURE
3 - Sephadex LH20 column elution profiles
of D N A (1.0 mg) isolated from V-79 cells after exposure
to t3H]-DMBA in mixed cultures with BHK feeder cells.
Experimental procedure and symbols as in Figure 2.
694
l AL.
WIGLEY E
--
amount of hydrocarbon metabolized to watersoluble derivatives in 48 h were taken as measures of
the effective dose of hydrocarbon, and used in the
calculations of binding index shown in Table I
(Duncan et al., 1969).
Chromatographic analysis of the hydrocarbon-DNA
reaction
DNA was isolated by the phenol method (Parish
and Kirby, 1967) from purified V-79 fractions
harvested from large-scale mixed cultures after
48 h exposure to the tritiated hydrocarbons BA,
3-MC or DMBA. Contamination of the V-79 cell
fraction by BHK feeder cells was always less than
2 % in the present experiments and did not affect
DNA binding data (Newbold et al., 1977). After
degradation to deoxyribonucleosides, DNA concentration and the total amount of tritium were
measured to obtain overall DNA binding values.
These are given in Table I, together with equivalent
values obtained in the previous study for BP and
7-MBA. It can be seen that these results do not
reflect the carcinogenic potencies of the hydrocarbons, which increase from left to right across the
Table. Values of carcinogenic potency in skinpainting experiments (Iball index) are quoted from
lball (1939). BA was thought to be non-carcinogenic
but has since been shown to be weakly active.
Although variations in values of Iball index can be
found in the literature, the rank order of potencies
shown in Table I is consistent. All data shown for
each compound in Table I were obtained from the
same experiment. This was representative of the
three or four repeat experiments performed in
each case.
Degraded DNA from V-79 cells treated with 3-MC
or DMBA was then fractionated by LH20 column
chromatography as in the previous study. The total
binding observed for BA-treated cells was too low
for this DNA to be analysed further. The value of
0.76 ,umole/mole P probably represents an overestimate of BA binding, since this was calculated
from radioactive counts in DNA which were only
just above the background level. Chromatographic
profiles, shown in Figures 2 and 3, were constructed
for 3-MC and DMBA respectively. In both cases
these were qualitatively very similar to those published for DNA from mouse skin (Phillips et al.,
1978) or mouse embryo cells in vitro (Dipple and
Nebzydoski, 1978; King et al., 1977) after treatment
with the same compounds. In the case of 3-MC,
major peaks eluted at about 400 ml and 510 ml, with
a minor peak at 545 ml. The DMBA profile showed
a major peak at about 520 ml elution volume and
smaller peaks at 440, 465 and 550 ml.
From these LH 20 profiles, it was possible to
recalculate binding values in terms of tritium in
DNA-hydrocarbon products. Tritium eluting ahead
of unchanged deoxyribonucleosides, the X peak in
Figures 2 and 3, was included in calculations of
product binding. There is evidence from the studies
of Dipple and Nebzydoski (1978) with DMBA, that
this material contains hydrocarbon derivatives and
Phillips et a/. (1978) have shown that it probably
consists of nucleotide-hydrocarbon adducts resistant
to complete enzyme hydrolysis. The recalculated
figures therefore exclude tritium associated with UV
absorbance characteristic of unchanged bases which,
to a greater or lesser extent, contributes to an
overestimate of DNA-hydrocarbon binding. Table I
shows binding values for 3-MC and DMBA obtained
from the profiles shown in Figures 2 and 3. Equivalent values for BP and 7-MBA were calculated from
data obtained in the earlier study. Revised hydrocarbon binding values quoted in that study were
estimated from the major peak in the product profile
(e.g., the peak co-chromatographing with diolepoxide-[14C] DNA in the case of BP). Since the
elution profiles obtained with 3-MC and DMBA
were more complex than those published for the
other two compounds, it was necessary to include the
whole of the late-eluting product region, as well as X
peak material, in calculating true DNA reaction for
each compound. The adjusted values for BP and
7-MBA are given in Table I. It can be seen that
values of product binding reflect the carcinogenic
potency of the compounds more closely than the
same values calculated from the total tritium content
of the DNA.
When binding indices were calculated from product binding and total metabolism over 48 h,
corrected for cell division during the treatment
period (Table I), ihe values correlated well with the
carcinogenic potency of the four compounds
analysed. This was not the case when the total
tritium content of the DNA was used to calculate
binding index.
Cytotoxicity and mutagenesis as a function of DNA
binding
A sample of V-79 cells from the population used
to isolate DNA for chromatographic analysis was
assayed for cytotoxicity (percentage plating efficiency) and mutation to azaguanine resistance
(mutant colonies per lo5 survivors) after 48 h
incubation with tritiated BA, 3-MC or DMBA.
The results obtained are given in Table I. BA was
non-toxic and non-mutagenic relative to control
cells treated with 0.5% DMSO solvent alone.
However, both 3-MC and DMBA were cytotoxic
and induced high mutation frequencies at optimum
expression times (62 and 72 h after plating, respectively).
Induced mutation was then related to the amount
of DNA-hydrocarbon binding, excluding tritium in
unchanged deoxyribonucleosides. When these results
were corrected for the amount of the cell replication
dming the treatment time (Newbold et al., 1977),
numbers of mutants induced per unit true DNA
reaction could be estimated; these are given in
Table I. All four carcinogenic compounds proved
almost equally mutagenic for a given amount of
DNA reaction, there being at most a two-fold
difference between the highest and lowest values.
DISCUSSION
In a previous paper we attempted to quantitate
mutation in mammalian cells in terms of DNA
reaction for two carcinogenic polycyclic hydro-
HYDROCARBON MUTAGENESIS AND DNA REACTION
carbons. We reported that both compounds proved
to be equally mutagenic for a given amount of true
DNA reaction. In the present study we have
extended this analysis to include three additional
hydrocarbons in an attempt to draw more general
conclusions which might be applicable t o the mode
of action of this class of carcinogen.
The LH20 chromatographic profiles obtained
for 3-MC and DMBA, as well as the profile published earlier for BP (Newbold et al., 1977) were in
good agreement with those published by Phillips
et al. (1978) for fractionated DNA from mouse skin
after topical application of the same compounds.
All three compounds are potent carcinogens when
applied to mouse skin and the degree of potency is
related to the extent of reaction with DNA, but
not RNA or protein, from mouse skin (Brookes
and Lawley, 1964). The nature of the reaction of
CPH metabolites with V-79 cell DNA in the experiments described here has thus been shown to
closely reflect the situation in vivo where carcinomas
are induced. Mutation in V-79 cells resulting from
this DNA binding is therefore a relevant parameter
in measurements of the biological activity of CPH
in an in vitro system.
Values of total DNA binding based on the specific
activity of degraded DNA (Table I) did not correlate
well with carcinogenicity, although the noncarcinogenic BA showed a much lower value than
the carcinogenic compounds. If binding values were
recalculated on the basis of product binding alone,
omitting tritium incorporated into unchanged bases,
the weak carcinogen 7-MBA was shown to bind to
DNA to a much lesser extent than the three potent
carcinogens. The biochemical events which lead to
this tritium incorporation are not known, but the
extent of uptake appeared to be related to the
amount of cell replication during the treatment time.
Hydrocarbon binding was related to the extent
of metabolism to water-soluble derivatives and
binding indices derived which were corrected for
variable cell division during the treatment time
(Newbold et al., 1977). Calculations based on
product binding rather than total tritium content of
DNA (Table I) showed a good correlation between
binding index and biological potency within the
range of carcinogens. This suggests that this parameter (true DNA reaction related to overall extent
of metabolism) may be a good indicator of carcinogenic potential and could reflect a facet of the
mechanism of action of CPH.
Finally, induction of mutation was related to the
extent of product binding for each compound at the
end of the treatment period (Table I). Essentially,
695
within the limits of the experimental methods used,
all the compounds were approximately equally
mutagenic for a given extent of true DNA reaction.
The two-fold difference in induced mutation between
the weakest and most potent carcinogens in the
series was insufficient to explain the difference in
their biological activities. In any experiments
attempting to quantitate mutation, the role of DNA
repair should be considered. Although no correction
has been .made in these experiments for repair
during the treatment time, it is likely that within
the range of closely related compounds, used at
concentrations allowing comparable cell survival,
factors due to repair will be similar for each compound.
Differences in biological activity between CPH
may therefore be most easily explained in terms of
the direction and rate of cellular metabolism and the
effective doses of particular reactive metabolites
which reach target macromolecules. These conclusions will be tested as derivatives of the parent
hydrocarbons responsible for product binding are
identified, synthesized and assayed for mutagenesis.
It has already been shown that the bay region diol
epoxide of BP, the metabolite responsible for most
of the DNA binding in rodent embryo cells, is
itself a particularly efficient mutagen in mammalian
cells (Newbold and Brookes, 1976). Evidence is
accumulating that equivalent diol epoxides of other
carcinogenic hydrocarbons are major species involved in generating the observed pattern of hydrocarbon-DNA products in primary rodent embryo cell
cultures (Dipple and Nebzydoski, 1978; Ivanovic
et al., 1978; King et al., 1977; Vigny et al., 1977).
Evidence was presented by Swaisland et al. (1974)
that the low extent of reaction of tritiated BA with
DNA from hamster embryo cells was due to a
non-bay-region diol epoxide. There has been a
need, however, to investigate and quantitate fully
the biological properties of parent hydrocarbons in
mammalian cell culture systems, since their carcinogenic activity in vivo may depend on additional
factors besides the generation of mutagenic bayregion diol epoxides.
ACKNOWLEDGEMENTS
The authors would like to thank Mrs. Mary
White for excellent technical assistance.
The work was supported by NIH (USA) contract
No. N01-CP-33367 and in part by grants t o the
Institute of Cancer Research from the Medical
Research Council and the Cancer Research Campaign.
MUTAGENESE A MEDIATION CELLULAIRE INDU ITE
PAR DES HYDROCARBURES POLYCYCLIQUES DANS DES CLJLTURES DE CELLULES
DE HAMSTER CHINOIS: MUTAGfiNICITE ET R E A F I O N AVEC L’ADN
EN RELATION AVEC LA CANCEROGENICITE
Trois hydrocarbures polycycliques - benz(a)anthracene, 3-methylcholanthrene et 7,12-dimCthylbenz(a)anthracene ont BtC Btudi6s dam un systeme de mutaghkse cellulaire utilisant les cellules BHK pour le metabohme des hydrocarbures et
les cellules V-79 pour montrer l’induction de la cytotoxicitB et de mutations. L’ADN des cellules V-79 a Ctt analysC par
chromatographie sur colonne pour mettre en Cvidence le type et I’Btendue r e e k de la rCaction des hydrocarbures avec les
d6soxyribonuclCosides.Les deux substances canctrigenes on1 form6 avec 1’ADN des complexes qualitativement tres semblables,
696
WIGLEY ET AL.
B ceux demontrts in vivo ou en cultures cellulaires primaires. Les indices d’association pour ces deux substances, et pour le
benzo(a)pyrene et le 7-mtthylbenz(a)anthracene (avec des donnees provenant d’une etude prealable), ont ete calcults d’apres
le contenu triti6 des complexes ADN-hydrocarbures par rapport a u metabolisme global. L’efficacit6 canc6rigene de ces produits
se reflete dans les valeurs de ces indices. La frequence des mutations induites est associee ?
l’etendue
i
de la reaction ADNhydrocarbure, et les niveaux d’induction de mutations n’ont pas present6 de difference significative lorsque ces reactions
etaient kquivalentes.
REFERENCES
BAIRD,W. M., and BROOKES,
P., Isolation of the hydrocarbon
deoxyribonucleoside products from the D N A of mouse
embryo cells treated in culture with 7-methylbenz(a)anthrac~ne-~H
.
Cancer
Res., 33, 2378-2385 (19731.
BAIRD,W. M., and DIPPLE,A., Photosensitivity of DNAbound 7,12-dimethylbenz(a)anthracene. Int. J. Cancer, 20,
427-431 (1977).
BAIRD,W. M., HARVEY,
R. G., and BRODKES,
P., Comparison
of the cellular DNA-bound products of benzo(a)pyrene with
the products formed by the reaction of benzo(a)pyrene4,5-oxide with DNA. Cancer Res., 35, 54-57 (1975).
BARRETT,C., and Ts’o, P. 0. P., Relationship between
somatic mutation and neoplastic transformation. Proc. nut.
Acad. Sci. (Wash.), 75, 3297-3301 (1978).
BROOKES,
P., and LAWLEY,
P. D., Evidence for the binding
of polynuclear aromatic hydrocarbons to the nucleic acids
of mouse skin: relation between carcinogenic power of
hydrocarbons and their binding to deoxyribonucleic acid.
Nature (Lond.), 202, 781-784 (1964).
DIAMOND,L., SARDET,C., and ROTHBLAT,G. H., The
metabolism of 7,12-dimethyIbenz(a)anthracene in cell
cultures. Int. J . Cancer, 3, 838-849 (1968).
DIPPLE,A., and NEBZYDOSKI,
J. A., Evidence for the involvement of a diol-epoxidein the bindingof 7,12-dimethylbenz(a)anthracene to DNA in cells in culture. Chem.-Biol. Interact.,
20, 17-26 (1978).
DUNCAN,
M. E., and BROOKES,
P., The relation of metabolism
to macromolecular binding o f the carcinogen benzo(a)pyrene,
by mouse embryo cells in culture. Int. J. Cancer, 6, 496-505
(I 970).
DUNCAN,M., BROOKES,
P., and DIPPLE,A., Metabolism
and binding to cellular macromolecules of a series of hydrocarbons by mouse embryo cells in culture. Int. J . Cancer, 4,
813-819 (1969).
HUBERMAN,
E., and SACHS,L., Cell-mediated mutagenesis of
mammalian cells with chemical carcinogens. f n t . J. Cancer,
13, 326-333 (1974).
E., and SACHS,L.,DNA binding and its relationHUBERMAN,
ship to carcinogenesis by different polycyclic hydrocarbons.
Int. J. Cancer, 19, 122-127 (1977).
J., The relative potency of carcinogenic compounds.
IBALL,
Amer. J. Cancer, 35, 188-190 (1939).
IVANOVIC,
V., GEACINTOV,
N. E., JEFFREY,
A. M., Fu, P. P.,
I. B., Cell and microsome
HARVEY,
R. G., and WEINSTEIN,
mediated binding of 7,12-dimethylbenz(a)anthracene t o
D N A studied by fluorescence spectroscopy. Cancer Lett.,
4, 131-140 (1978).
KING, H. W. S., OSBORNE.
M. R., and BROOKES,P., The
metabolism and D N A binding of 3-methylcholanthrene.
Int. J. Cancer, 20, 564-571 (1977).
M. H., and BROOKES,
P., The
KING,H. W. S., THOMPSON,
benzo(d)pyrene deoxyribonucleoside products isolated from
D N A after metabolism of benzo(a)pyrene by rat liver
microsomes in the presence of DNA. Cancer Res., 34, 12631269 (1975).
KUROKI,T., DREVON,C., Direct or proximate contact
between cells and metabolic activation systems is required
for mutagenesis. Nature (Lond.), 271, 368-370 (1978).
NEWBOLIJ,R. F., and BROOKES,
P., Exceptional mutagenicity
of a benzo(a)pyrene diol epoxide in cultured mammalian
cells. Nature (Lond.), 261, 53-54 (1976).
NEWBOLD,
R. F., BROOKES,
P., ARLETT,C. F.,BRIDGES,
B. A,,
and DEAN,B., The effect of variable serum factors and
clonal morphology on the ability t o detect hypoxanthineguanine phosphoribosyl transferase (HPRT) deficient
variants in cultured Chinese hamster cells. Mutation Res.,
30, 143-148 (1975).
NEWBOLD,
R. F., WIGLEY,C. B., THOMPSON,
M. H., and
BROOKES,
P., Cell-mediated mutagenesis in cultured Chinese
hamster cells by carcinogenic polycyclic hydrocarbons :
nature and extent of the associated hydrocarbon-DNA
reaction. Muration Res., 43, 101-116 (1977).
PARISH, J. H., and KIRBY,R. S . , An extension of the naphthalene disulphonate method for mammalian nucleic acids.
Biochem. Biophys. Acta., 142, 273-275 (1967).
PHILLIPS,
D. H., GROVER,
P. L., and SIMS,P., The covalent
binding of polycyclic hydrocarbons t o D N A in the skin of
mice of different strains. Inr. J. Cancer, 22, 487-494 (1978).
SWAISLAND,
A. J., HEWER,A., PAL, K., KEYSELL,G . R.,
BOOTH,J., GROVER,P. L., and SIMS,P., Polycyclic hydrocarbon epoxides: the involvement of 8,9-dihydro-8,9dihydroxybenz(a)anthracene 10,l I-oxide in reactions with
the D N A of benz(a)anthracene-treated hamster embryo
cells. FEBS Leu., 47, 34-38 (1974).
M., COULOMB,
H., TIERNEY,B.,
VIGNY,P., DUQUESNE,
GROVER,
P. L.,and SIMS,P., Fluorescence spectral studies on
the metabolic activation of 3-methylcholanthrene and
7,12-din~ethylbenz(a)anthraceiiein mouse skin. FEBS Lett.,
82, 278-282 (1977).