Biomarkers
Indicators (measures) of exposure to a chemical or
class of chemicals
Not generally indicators of effects
Vitellogenin (phospholipoglycoprotein) is an egg yolk
precursor produced in the liver of females during egg
production. Males have the ability to produce egg yolk
protein but generally never receive an internal signal to do
so.
The production of vitellogenin in male fish has been used
as a biomarker of exposure to chemicals in the
environment that produce estrogenic responses
(xenoestrogens).
Estrogen (estradiol) and its metabolites (listed
in order of their potency) estrone, and estriol
have all been shown to be capable of inducing
the production of vitellogenin in male fish.
Ethynyl estradiol is a synthetic estrogen that
is used in the Pill. It too has been shown to be
capable of inducing the production of
vitellogenin in male fish.
Should we care that male fish have elevated levels of VTG,
and if so why?
Death rate < Birth rate
Ova
Sperm
Individual
Born
Lives
Reproduces
Dies
?
Population
Increases
Population
Decreases
Population Persists
Total Fertility
2.1
Death rate > Birth rate
Two systems control all physiological processes
The nervous system exerts point-to-point
control through nerves, similar to sending
conventional messages by telephone (hard
wired).
The endocrine system broadcasts its
hormonal messages to essentially all cells by
secretion into blood and extracellular fluid. Like
a radio broadcast it requires a receiver (cells
must have a receptor for the hormone being
broadcast).
These two system often act together
Endocrinology is the study of hormones, their
receptors and the intracellular signaling pathways
they invoke.
Distinct endocrine organs are scattered throughout
the body.
Hypothalamus
Pituitary
Parathyroid glands
Testes
Thyroid glands
Pancreas
Adrenal glands
Ovaries
In addition to the classical endocrine organs,
many other cells secrete hormones, through what is
sometimes called the “diffuse” endocrine system.
All pathophysiological events are influenced
by the endocrine milieu
All “large” physiologic effects are mediated
by multiple hormones acting in concert
There are many hormones known and little
doubt that others remain to be discovered.
What exactly are hormones and how are they different from
“non-hormones”?
Hormones are chemical messengers secreted into blood or
extracellular fluid by one cell that affect the functioning of other
cells.
Most hormones circulate in the blood, coming into contact
with essentially all cells. However, a given hormone usually
affects only a limited number of cells, which are called target
cells. Target cells respond to the hormones because they have
receptors for the hormone.
Hormone receptors are found either exposed on the
surface of the cell or within the cell depending on the type of
hormone. In very basic terms binding of hormone to receptor
triggers a cascade of reactions within the cell that affects
function.
Two important terms are used to refer to molecules that
bind to the hormone-binding sites of receptors:
Agonists are molecules that bind the receptor and induce
all the post-receptor events that lead to a biologic event.
In other words they act like the “normal” hormone.
Antagonists are molecules that bind the receptor and
block binding of the agonist, but fail to trigger
intracellular signaling events. (Antagonists are like
certain types of bureaucrats – they don’t themselves
perform useful work, but block the activities of those
that do have the capacity to contribute.) Hormone
antagonists are widely used as drugs (e.g. statins).
Atorvastatin (Lipitor)
Fluvastatin (Lescol)
Lovastatin (Mevacor)
Pravastatin (Pravachol)
Simvastatin (Zocor)
Rosuvastatin (Crestor)
The statin drugs inhibit HMG CoA reductase in the liver and
prevent the formation of cholesterol.
Lipitor, followed closely by Zocor was the most frequently
prescribed drug in the United States.
All have the same MOA and probably act additively
Normal response
Timing
Abnormal Response No Response
Dormant receptor
Receptor Blocked
turned on by mimic
Normal receptor over
stimulated by mimic
Endocrine System regulates biological processes


Growth and function of reproductive system (androgen
[testosterone], estrogen [estradiol], and related compounds
from gonads)
Control of blood sugar (pancreatic insulin)

Regulation of metabolism (adrenal cortisol and thyroid
thyroxin)

Development of nervous system including brain (estrogen and
thyroid hormones)

Overall development from conception to old age = homeostasis
Hormone Classes

Steroids : derived from cholesterol
 androgens and estrogens and cortisol

Amines : synthesized from amino acids
 give rise to adrenaline and noradrenaline

Peptides and proteins : a.a. chains
 growth hormone

Eicosanoids - 20 carbon fatty acid derivatives
 prostoglandins = adenyl cyclase activator,
cellular energetics
Receptor Types

Cell membrane : peptide hormones

Cytoplasmic : steroids

Nuclear receptors : thyroid hormones

a cell may contain as many as 10,000
receptors for a single hormone

~ 50-100 genes may be controlled by a single
hormone
Specific Case Example
Fish Response to
Wastewater Effluent Estrogenicity
Estrogen Mimics

Intended disruptors
 DES (diethylstilbestrol)

ethynylestradiol
phytoestrogens (soy protein, soy milk)
 Paper mills, Canada

Estrogen Mimics – death by “progress”

Unintended disruptors
 nonyl/octylphenol
 commercial and domestic detergents

phthalate & Bisphenol A
 plastics : bottles and liners

DDT (DDE)

PCBs
Estrogen Mimics : promiscuous receptor
Estradiol
Intended disruptors
Ethynylestradiol
DES
Estrogen Mimics : promiscuous receptor
Estradiol
Unintended disruptors
-C9H19
Bisphenol A
Polycarbonate plastic
DDT
DEHP
Di(2-ethylhexyl)
phthalate
Nonylphenol
Vitellogenin Biomarker : fish model
Endogenous / Exogenous Estrogens
Vitellogenin
Liver
Gonad
Vitellogenin – egg yolk precursor produced in the liver
Pilot Study Methods

Adult male fathead minnows (n=5/exposure)

1-3 Weeks of exposure to 100% WWTP

RHW control in lab

Plasma VTG content via ELISA

Likelihood Ratio Test
 ND left censored at 3,000 ng/ml
Pilot Study Results
Number of male fish with detectable VTG levels / site with 5 fish
5
4
3
2
1
0
Control
1 Week
2 Weeks
3 Weeks
Non-detects were left censored at 3,000 ng/ml = DL.
Pilot Study Results
Mean plasma vitellogenin concentrations (ng/ml)
30000
*
25000
*
20000
15000
10000
5000
0
Control
1 Week
2 Weeks
3 Weeks
*Statistical significance from the controls (=0.05)
Comparison to Similar Studies
Mean plasma vitellogenin concentrations (ng/ml) in fish
exposed to wastewater effluent receiving systems.
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
Background
Exposed
TX FHM
UK TRT
UK FLO
MI FHM
Conclusions



Induction of vitellogenesis (biomarker) in male
fish indicates the presence of estrogenic
components in the wastewater effluent tested.
Vitellogenin concentrations increased and were
more frequent with increased exposure duration.
Vitellogenin concentrations in male fish
decreased when UNT and TWU were not in
session.
Additional Research

Assessment of the efficacy of a constructed
wetland to reduce or remove wastewater
effluent estrogenicity using the vitellogenin
biomarker in fathead minnows (Pimephales
promelas Rafinesque, 1820).


wetland vegetative coverage and degradation
activity
suspect chemical constituents
 estradiol and ethynylestradiol vs
 DEHP, DDT, Bisphenol A, Nonylphenol
Wetland Exposure Design
Replicate minnow traps
(seven male fish per trap)
2
4
Out
3
1
In
Fish Measurements








Vitellogenin Content of Plasma
GSI – Gonado Somatic Index testes wt/somatic
(total body) wt x 100
HSI – Hepato Somatic Index liver wt/total body
wt x 100
Hematocrit (%packed blood cells)
Secondary Sex Characters (turbercles, fat pad, stripes)
Length
Weight
Gonadal Differentiation
Chemical Analysis
Estradiol
Ethynylestradiol
-C9H19
Bisphenol A
DDT
DEHP
Nonylphenol
Wetland Characterization


vegetation types and density
depth and width of channels

retention time as estimated by input flow

relative to chemical constituents & fish response
Fish health as measured by condition factor (K)
and hematocrit values were significantly
reduced at wetland sites 1 and 2. A negative
trend was observed between VTG concentration
and condition factor among wetland sites. A
positive trend was observed between condition
factor and hematocrit value.
Gonadosomatic index was significantly reduced
at site 1 in the wetland while the hepatosomatic
index was significantly increased.
Tubercle number, fatpad thickness, and stripe
density were all reduced at site 1.
Vitellogenin concentrations in fish from site 1
were significantly elevated compared to
control and other wetland sites.
The constructed wetland significantly reduced
VTG levels in fish and increased measures of
fish well-being.
As population continues to increase and pressures on
available freshwater in our area increase use of “reclaimed
water” will become more and more important. One of the
ways to reclaim the water may be to run Trinity River water
through a constructed wet land and then reintroduce it into
our reservoirs. What negatives can you see in this?
Vitellogenin Gene Expression in Fathead
Minnows Exposed to EE2 in a Whole Lake
Dosing Experiment
Greg Toth
*Lazorchak,
1
JM1, Flick, R1, Lattier, D.L.1,
U.S. EPA, National Exposure Research Laboratory, Cincinnati, OH
Kidd, K2, Palace, V2, Evans, B2 , Blanchfield, P2, Mills, K2 , Hodge, T2,.
2
Canadian Division of Fisheries and Oceans, Freshwater Institute, Winnipeg, Manitoba,
Smith, ME3, Wiechman, B3,,
3
Sobran Inc., c/o U.S. EPA, Cincinnati, OH.,
3
Why look at
17a-ethynylestradiol
(EE2)
effective component of birth control pills
 potent estrogen mimic
 70-80 % degraded in sewage treatment
 found at significant and effective
concentrations downstream of municipal
wastewater treatment plants

K. Kidd
Why Work with the Canadian
DFO
Fathead Endpoints

Vitellogenin (spring, mid-summer, fall) V. Palace

Liver, kidney and gonad development, GSI and LSI (spring &
fall) B. Evans

Secondary sex characteristics (mid-summer) P. Blanchfield
Male reproductive behaviour (mid-summer) P. Blanchfield
Nest size and egg development (mid-summer) P. Blanchfield


Population size structure, growth, abundance (spring and
fall) K. Mills
K. Kidd

Located in northwestern Ontario approximately 250 km east of Winnipeg
and 50 km east-southeast of Kenora.
K. Kidd
Lake 260 – Ethynylestradiol (EE2) Addition
Lake
• 34 ha in surface area
• Max. depth 14 m
• Outflow into lake that is
30 times larger
• Contains well-defined
populations of lake
trout, white sucker,
fathead minnow and
pearl dace
• Long-term records on
plankton and water quality
Dosed during stratification
Study Design
recovery?
effects on individuals & populations
ethynylestradiol additions
baseline data
1999
2000
2001
2002 2003
2004
reference lake data
2005
2006
Additions of EE2 to Lake 260, 2001-2003
• EE2 added 3 times a week for 5 months
• 100-450 mg added/day to maintain
constant concentration (4.5% loss/day)
• Season mean of 6.1 (2.9) and 5.0 (1.8)
ng/L in surface waters (SPE solid phase
extraction and RIA measure hormone levels in
blood without a bioassay)
water sampling
K. Kidd
Mean EE2 Concentrations in Lake 260
14
12
2002
2001
EE2 (ng/L)
10
8
6
4
2
0
31 4-J 11 18 25 2-J 9-J 16 23 30 6-A 13 20 27 4-S 10 17 24 1-O 8-O
-M un -Ju -Ju -Ju ul ul -Ju -Ju -Ju ug -Au -Au -Au ep -Se -Se -Se c c
ay
n n n
l l l
g g g
p p p t t
additions started
Concentrations of EE2 in Stratified Lake 260
Epilimnion EE2 = 6.1+/- 1.3 ng/L
Metalimnion 1.9 +/- 0.93 ng/L
Hypolimnion 1.7 +/- 0.61 ng/L
U.S. EPA MERB 2001 Objectives

Evaluate exposure of indigenous male fathead minnows in
Lake 260 to ethynylestradiol using vitellogenin gene
expression

Evaluate short-term exposure of male fathead minnows
from Lake 114 deployed in Lake 260

Evaluate exposure of Cincinnati male fathead minnows to
Lake 260 water at the US EPA facility in Cincinnati

Evaluate exposure of Cincinnati fathead minnow fry to
sediment from Lake 260
Approach - Indigenous Male
Fathead Minnows

Male Fathead Minnows were collected from Lake 260
after 7 weeks, 9 weeks and 12 weeks of dosing.

Male minnows were collected from Lake 114 at the same
timepoints.

Livers were collected.

RT-PCR was performed on samples and vitellogenin
expression quantified relative to 18s ribosomal RNA
expression.
2001 Semi-quantitative PCR
Gel Based
2001 Results of Indigenous Male
Fathead Minnows
Vitellogenin Expression
FHM Adult Livers - 17 Cycles
0.8
Pixel Density
Vg/(Vg + 18S)
1
0.6
Average
0.4
+ 1 StD
0.2
- 1 StD
0
-0.2
La
ke
11
4
July 9
La
ke
26
0
k
La
e
4
11
M
k
La
e
0
26
July 25
M
La
ke
4
11
F
L
e
ak
11
4
Sept
La
ke
26
0
EPA MERB Approach - Deployment Study

Fish were collected in Lake 114 using minnow
traps two days before deployment.

On day of deployment males were separated from
females and placed in cages in Lakes 114 and 260.

Minnows were retrieved from cages on days 1, 3,
7 and 13.

Livers were collected and RT-PCR performed.
2001 Results of 13-day Deployment Study
Vitellogenin Expression
FHM Adult Livers - 17 Cycles
1.2
0.8
Pixel Density
Average
+ 1 SD
- 1 SD
0.6
0.4
0.2
0
13
7
D
D
ay
ay
7
0
0
La
ke
26
26
ke
La
La
ke
11
4
D
ay
3
D
ay
3
0
26
ke
La
La
ke
11
4
D
ay
1
ay
D
0
26
ke
La
ke
11
4
D
ay
1
-0.2
La
Vg/(Vg + 18S)
1
EPA MERB Approach - Grab Samples

Water was collected from Lakes 114 and 260.
Samples were shipped to Cincinnati.

Male Cincinnati fathead minnows were exposed to
water samples for 48 hours with water renewal
after 24 hours.

Male fathead minnows were also exposed to 5
ng/L ethynylestradiol.

RT-PCR was performed on RNA from liver
samples.
2001 Comparison of Exposure of Minnows to
Lakes 114 and 260 Grab Samples
Vitellogenin Expression
FHM Adult Livers - 17 Cycles
3
2
Avg
1.5
+1 Std
1
-1 Std
0.5
0
26
La
ke
E
/L
ng
5
ke
La
0
E2
4
11
ne
Li
b
La
M
S
O
-0.5
D
Pixel Density
Vg/(Vg+18S)
2.5
Vitellogenin Expression by QPCR*
Study Number 1-11 (In-House ELA)
FHM Adult Male Livers
0.9
Vg / 18S
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
DMSO
Labline
Lake 114
Treatment Group
5 ng/L EE2
Lake 260**
EPA MERB Approach - Sediment Study

Sediment samples were collected and shipped to Cincinnati.

Sediment was combined with two volumes of water, shaken
for one hour, and centrifuged. Liquid phase (elutriate) was
used for exposures.

Fry were exposed to elutriate for five days, with elutriate
renewed daily.

Fry were homogenized and RT-PCR was performed on the
samples.
2001 Results of Fathead Minnow Fry
Exposed to Sediment Samples
Vitellogenin Expression
Pixel Density
Vg/(Vg + 18S)
FHM Fry - 32 Cycles
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
-0.2
H
M
Average
+ 1 St D
-1 St D
RW
D
SO
M
5
ng
/L
2
EE
La
ke
11
4
L
e
ak
2
60
#1
L
e
ak
2
60
#2
L
e
ak
2
60
#3
L
e
ak
2
60
#4
L
e
ak
2
60
#5
2002 Objectives Vitellogenin expression
Evaluate exposure of indigenous male fathead minnows
in Lake 260 to ethynylestradiol using vitellogenin gene
Expression before and 3 times throughout the dosing.
Evaluate exposure of Cincinnati male fathead minnows
to Lake 260 water at the US EPA facility in Cincinnati
Evaluate
exposure of Cincinnati fathead minnow fry to
sediment from Lake 260
2002 Vitellogenin expression
Analyzed by quantitative real-time PCR
Extrapolation to standard curve
Use of reference
Quantitation
relative fluorescence
0.35
0.3
0.25
K. Kidd
0.2
0.15
0.1
0.05
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39
cycle number
K. Kidd
27
-M
ay
26
19
-J
0
un
26
90
Ju
l2
60
1O
ct
26
0
ay
1
3Ju 14
n
11
54
Ju
n
19
11
-J
4
un
21
11
-J
4
un
27
11
-S
4
ep
11
4
27
-M
02
2
44
2
44
ep
1- 442
O
ct
44
2
24
-S
27
5-
ay
14
-M
Ju
l3
5-
Vg/18s
Indigenous Fathead minnows: males 2002 Results
2.5
2
1.5
1
0.5
0
ay
1O
ct
26
0
26
0
26
0
ay
19
-J
un
27
-M
44
24
2
-S
ep
44
2
1O
ct
44
2
27
-M
27
-M
16
ay
,1
11
94
M
ay
11
27
4
-S
ep
11
4
Vg/18S
Fathead minnows: females 2002
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
EPA Observations: 2002 fathead minnows
Females
in lake 260 continued to have higher
vitellogenin gene expression into October, compared to
Lake 114 and Lake 442 females
A
very impressive lack of vitellogenin gene expression
in all males from all control lakes
Males
in lake 260 exhibited high vitellogenin gene
expression after dosing, with expression remaining high
at least until October 1
Histologically Male Fathead Minnow With
Ovipositor Collected From Lake 260 In
Summer Of 2003
(photo by C. Podemski).
Vitellogenin (mg/g whole body homogenate)
0
10000
8000
6000
4000
2000
Fall 1999
Spr 2000
Summ 2000
Fall 2000
Spr 2001
Summ 2001
Fall 2001
Fall 1999
Spr 2000
Summ 2000
Fall 2000
Spr 2001
Summ 2001
Fall 2001
12000
11000
10000
9000
8000
2
Fall 1999
Spr 2000
Summ 2000
Fall 2000
Spr 2001
Summ 2001
Fall 2001
DFO Vitellogenin in Fathead Minnow
Pre-EE2 Post EE2
Males
0
Lake 260
Lake 114
Lake 442
1000
Females
DFO Histopath Results Lake 260 Fathead
Minnow Testes – Spring
1999
2000
2001
100 mm
2002
100 mm
spermatogonia
spermatocytes
100%
fibrosis, no tubules
spermatozoa
K. Kidd
DFO Male Fathead Minnow Behaviour –
Nest Defense Results
23
Nesting aggression
chases per min (+ 1s.e.)
7
6
5
20
4
31
3
21
19
8
8
2
11
9
1
0
K. Kidd
EE2
additions
start
n/a n/a
n/a
1999 2000 2001 2002
1999 2000 2001 2002
1999 2000 2001 2002
Lake 302
Lake 260
Lake 375
Summary - Fathead Minnow
•Spring 2001 - EE2 additions began
- Vg gene expression induction in deployed 114 fish in 260 in 24-hrs
–significant vitellogenin plasma induction after 7 weeks
•Fall 2001 (4 months)
–proteinaceous accumulation in kidney
–liver cell size increased
•Spring 2002 (12 months)
–disorganized testes, immature ovaries
–decreased spawning aggression, fewer & less-developed eggs
–reduction in 2o sex characteristics
– No fish population impacts observed
•Fall 2002 (17 months) - reproductive failure, few age 0 fish
•Spring 2003 (2 years) - only age 2 fish remaining
–one male found, females with large ovipositors
• 2006 (3yrs post additions) – Fathead population recovered
Endocrine Disruption Legislation




Food Quality Protection Act
TOSCA – Toxic Substances Control Act
FFIRA – Federal Insecticide, Fungicide, and
Rodenticide Act
Safe Drinking Water Act Ammend.

develop a screening program, using appropriate validated test
systems and other scientifically relevant information, to determine
whether certain substances may have effects in humans that are
similar to an effect produced by a naturally occurring estrogen, or
other such endocrine effect as the Administrator may designate

by August 1998 and implement by Aug 1999 (EPA extended the
comment period on “Proposed Chemical Selection Approach for
Initial Round of Screening, to April 1, 2003).
Once the chemicals to be tested are chosen Tier 1 screening
tests will be performed followed by Tier 2 testing where
warranted.
Tier 1 will be comprised of a battery of screening assays that
would identify substances that have the potential to interact
with the estrogen, androgen, or thyroid hormone systems.
The purpose of the Tier 2 assays is to determine whether the
substance may cause endocrine-mediated effects via or
involving estrogen, androgen, or thyroid hormone systems,
determine the consequences to the organism of the activities
observed in Tier 1, and establish the relationship between
doses of an endocrine-active substance administered in a test
and the effects observed.
They is little doubt that random blood samples taken from
adults will contain as many as 200 chemicals that did not exist
a century ago.
EDSTAC Developed

Endocrine Disruptor Screening and Testing
Advisory Committee

Composed of scientists and representatives from :

EPA and other federal agencies, state
agencies, industry, water providers, worker
protection groups, environmental groups,
environmental justice groups, public health
groups and research scientists
Endocrine Disruptor Definitions

An exogenous agent which interferes with the
synthesis, secretion, transport, binding, action,
or elimination of natural hormones in the body
which are responsible for the maintenance or
homeostasis, reproduction, development or
behavior.

Kavlock, 1996 = Too open ended for regulation
Endocrine Disruptor Definitions

An exogenous agent that changes endocrine
function and causes adverse effects at the level
of the organism, its progeny, and/or (sub)
populations of organisms.

EDSTAC 1997

also includes androgens and thyroid hormones
EDSTAC Dilemma


Examine estrogen,
androgen, and thyroid
(EAT)
Evaluate endocrine
disrupting properties of
both chemical substances
and common mixtures


The universe of chemicals
to be prioritized for
screening and testing
number >87,000 plus
mixtures.
Chemicals include
pesticides (carriers)
commodity chemicals,
food additives, nutritional
supplements, naturally
occurring, non-steroidal
estrogens phytoestrogens);
soy
Estimated Number of Chemicals
Number of chemicals: 5,000,000
Chemicals in commerce: 80,000
Industrial chemicals: 72,000
New Chemicals: 2,000/year (1,000 in US)
Pesticides: 600 (21,000 products)
Food Additives: 8,700
Cosmetic ingredients: 7,500 (40,000 products)
Human pharmaceuticals: 3,300
EPA estimates for the world, 1995.
TOSCA – The law does not require routine testing of
chemicals, and critics contend required tests only provide
limited information about new chemicals. To approve a
new chemical for commerce, EPA chemists compare its
structure to a list of similar compounds. If no red flags go
up, off to the market it goes. The EPA has 90 days to
review a chemical, though approval typically comes
earlier because the agency has accumulated enough
chemistry data to fast track large categories of
compounds.
The EPA receives 108 applications on average per month
from companies seeking to introduce new chemicals on
the market – 32,559 since 1979. With the application
comes “all available data” on production volume, use
and environmental releases but not a word on toxicity
unless the manufacturer happens to have some data.
Since 1979 EPA has forced restrictions on just nine
applications.
Federal Law in place since 1979 (TOSCA) directs
regulators to assess the hazards of chemicals in
commerce and control those of greatest concern. NAS,
GAO, Congressional Office of Technology Assessment,
EPA have all concluded that TOSCA falls short of its
objective.
Europe in 2006 is set to switch to a chemical policy that
requires chemicals to be evaluated for safety before going
on the market (precautionary principle). Called REACH –
Registration, Evaluation, and Authorization of Chemicals
– the policy promises to revolutionize the way European
regulators look at chemicals. They’re basically saying no
data, no market, and the industry is up in arms about it.
More Info

Our Stolen Future, Colborn, Dumanoski, Myers, 1996.

Generations at Risk, Reproductive Health and the
Environment, Schettler, Solomon, Valenti, Huddle, 1999.

http://website.lineone.net/~mwarhurst/chemicals.html

http://www.tmc.tulane.edu/ecme/eehome/

http://www.epa.gov/scipoly/oscpendo/index.htm