Preliminary Year 2000 Gene Expression Findings

1
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).
2
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.
3
Should we care that male fish have elevated
levels of VTG?
4
Death rate lt Birth rate
Population Increases
Ova
Population Persists Total Fertility 2.1
?
Population
Decreases
Sperm
Death rate gt Birth rate
5
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
6
Endocrinology is the study of hormones, their
receptors and the intracellular signaling
pathways they invoke. Distinct endocrine organs
are scattered throughout the body. Hypothalamus Pi
tuitary Thyroid glands Parathyroid
glands Pancreas Adrenal glands Testes Ovaries
In addition to the classical endocrine organs,
many other cells secrete hormones, through what
is sometimes called the diffuse endocrine
system.
7

• 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.

8
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.
9
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 dont 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).
10
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
11
Normal response Abnormal Response No
Response
Dormant receptor Receptor Blocked
turned on by
mimic
Normal receptor over
stimulated by mimic
Timing
Timing
Timing
12
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

13
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

14
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

15
Specific Case Example
Fish Response to Wastewater Effluent
Estrogenicity
16
Estrogen Mimics

• Intended disruptors
• DES (diethylstilbestrol)
• ethynylestradiol
• phytoestrogens (soy protein, soy milk)

17
Estrogen Mimics

• Unintended disruptors
• nonyl/octylphenol
• commercial and domestic detergents
• phthalate Bisphenol A
• plastics bottles and liners
• DDT (DDE)
• PCBs

18
Estrogen Mimics promiscuous receptor
Estradiol
Intended disruptors
DES
Ethynylestradiol
19
Estrogen Mimics promiscuous receptor
20
Vitellogenin Biomarker fish model
Endogenous / Exogenous Estrogens
Vitellogenin
Liver
Vitellogenin egg yolk precursor produced in the
liver
21
Pilot Study Methods

• Adult male fathead minnows (n5/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

22
Pilot Study Results
Number of male fish with detectable VTG levels /
site with 5 fish
Non-detects were left censored at 3,000 ng/ml
DL.
23
Pilot Study ResultsMean plasma vitellogenin
concentrations (ng/ml)


Statistical significance from the controls
(?0.05)
24
Comparison to Similar StudiesMean plasma
vitellogenin concentrations (ng/ml) in fish
exposed to wastewater effluent receiving systems.
25
Conclusions

• Induction of vitellogenesis 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.

26
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

27
Wetland Exposure Design
Replicate minnow traps (seven male fish per trap)
28
Fish Measurements

• Vitellogenin Content of Plasma
• GSI Gonado Somatic Index testes wt/somatic 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

29
Chemical Analysis
Ethynylestradiol
-C9H19
DEHP
DDT
Bisphenol A
Nonylphenol
30
Wetland Characterization

• vegetation types and density
• depth and width of channels
• retention time as estimated by input flow
• relative to chemical constituents fish response

31
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.
32
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 increased use
of reclaimed water will become more and more
important. One of the ways to reclaim the water
is to run Trinity River water through a
constructed wet land and then reintroduce it into
our reservoirs.
33
Vitellogenin Gene Expression in Fathead Minnows
Exposed to EE2 in a Whole Lake Dosing Experiment
Greg Toth Lazorchak, JM1, Flick, R1, Lattier,
D.L.1, 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.,
34
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
35
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
36
Located in northwestern Ontario approximately 250
km east of Winnipeg and 50 km east-southeast of
Kenora.
K. Kidd
37
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40
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

41
Study Design
recovery?
effects on individuals populations
ethynylestradiol additions
baseline data
2004
2000
2001
2002
2003
2006
1999
2005
reference lake data
42
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 and RIA)

water sampling
K. Kidd
43
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44
Concentrations of EE2 in Stratified Lake 260
45
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

46
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.

47
2001 Semi-quantitative PCRGel Based
48
2001 Results of Indigenous Male Fathead Minnows
49
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.

50
2001 Results of 13-day Deployment Study
51
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.

52
2001 Comparison of Exposure of Minnows to Lakes
114 and 260 Grab Samples
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54
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.

55
2001 Results of Fathead Minnow Fry Exposed to
Sediment Samples
56
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

57
2002 Vitellogenin expression
Analyzed by quantitative real-time PCR
Extrapolation to standard curve Use of reference
Quantitation
K. Kidd
K. Kidd
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• 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

61
Histologically Male Fathead Minnow With
Ovipositor Collected From Lake 260 In Summer Of
2003
(photo by C. Podemski).
62
DFO Vitellogenin in Fathead Minnow
Pre-EE2
Post EE2
Males
Vitellogenin (mg/g whole body homogenate)
Females
63
DFO Histopath Results Lake 260 Fathead Minnow
Testes Spring
2002
1999 2000 2001
100 mm
100 mm
spermatogonia spermatocytes spermatozoa
100 fibrosis, no tubules
K. Kidd
64
DFO Male Fathead Minnow Behaviour Nest Defense
Results
23
EE2 additions start
20
Nesting aggression chases per min ( 1s.e.)
31
21
19
8
8
11
9
n/a
n/a
n/a
2001
2002
2001
2002
1999
2001
2002
1999
2000
2000
1999
2000
K. Kidd
Lake 375
Lake 302
Lake 260
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66
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

67
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).

68
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.
69
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

70
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

71
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

72
EDSTAC Dilemma

• The universe of chemicals to be prioritized for
  screening and testing number gt87,000 plus
  mixtures.
• Chemicals include pesticides (carriers) commodity
  chemicals, food additives, nutritional
  supplements, naturally occurring, non-steroidal
  estrogens phytoestrogens) soy

• Examine estrogen, androgen, and thyroid (EAT)
• Evaluate endocrine disrupting properties of both
  chemical substances and common mixtures

73
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.
74
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.
75
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.
76
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.
Theyre basically saying no data, no market, and
the industry is up in arms about it.
77
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.ht
  ml
• http//www.tmc.tulane.edu/ecme/eehome/
• http//www.epa.gov/scipoly/oscpendo/index.htm