Endocrine disruptors: effects on wildlife and human health

1
Endocrine disruptors effects on wildlife and
human health
Pacific tree frog, Rancho Santa Margarita, CA
2
What is an Endocrine Disruptor ?
Endocrine Disrupting Chemicals (EDCs) are

• Environmental chemicals (typically persistent
  pollutants e.g. organochlorines)
• Perturb normal endocrine hormonal signaling
• Disrupt development or physiology
• In many cases, EDCs target the steroid nuclear
  receptor family of transcription factors (e.g.
  environmental estrogens activating the estrogen
  receptor)
• Mimic natural receptor ligands

3
The Nuclear Hormone Receptor Superfamily
A/B
C
D
E
F
DNA
LIGAND
N
C
Classical receptors (from biochemistry)GR corti
solMR aldosteroneAR testosteronePR
progesteroneER a,ß estradiolVDR 1,25-(OH)
2 vit D3TR a,ß triiodothyronine (thyroid
hormone)EcR 20-OH ecdysone (insect molting
hormone)

• NR ligands are
• Small
• Lipophilic (steroids or fatty acid derivatives)
• Signaling molecules (hormones)

Nuclear Receptor Genes in Different Species C.
elegans (nematode worm) 250 nuclear
receptors D. Melanogaster (fruitfly) 20
nuclear receptors H. sapiens (humans) 48
genes Arabidopsis (plants) no family
members
4
Some Steroidal and Lipophilic Hormones
Cholesterol (essential membrane lipid)
Estradiol (female sex characteristics, menstrual
cycle)
Testosterone (male sex characteristics, anabolic
steroid)
Progesterone (pregnancy)
Cortisol (stress hormone)
Vitamin D (bone homeostasis)
Retinoic Acid (vitamin A acid) (morphogenesis,
skin)
5
Estrogenic
Bisphenol A (polycarbonate plastics)
Polychlorinated biphenyls (e.g. PCB-110,
industrial chemical)
Polybrominated diiphenylethers (e.g. PBDE-85,
flame retardants)
Nonylphenol (from detergents and agrichemical
polymers)
Diethylhexylphthalate (plasticiser)
DDT (pesticide)
Anti-estrogenic/ NR agonist
Anti-androgenic
Tributyltin (ship paints, fungicide, PVC)
Vinclozolin (fungicide, wine, transgenerational
epigenetic effects)
6
Crystal Structure of a Typical Nuclear Receptor
Complex
RXR-PPARg bound to 9-cis RA and rosiglitazone in
complex with DNA hormone response element
Nuclear receptor heterodimer (bluered) binding
to DNA helix (silvergreen). Receptor ligands are
shown bound in yellow
7
Endocrine Disruption via Nuclear Receptors
Inhibitor
Activator
RNA polymerase II
Target Gene
Hormone Response Element (HRE)
Wrong time or place
8
Organotins as Endocrine Disruptors

• Marine ship paints Trialkytins are potent
  biocidal antifouling agents for molluscs
• Widely used during 1960-1970s
• Regulated but not completely phased out (date
  varies by country worldwide ban by 2008 ?)
• Fungicide on high value food crops
• e.g. Brestan (triphenyltin acetate) for potatoes,
  rice, celery, pecans
• Wood preservative
• Catalysts for organic synthesis

• Heat stabilizers in manufacture of
  polyolefin plastics (PVC) up to 3 w/w
• Bioaccumulative and NOT biodegradable

9
Organotins and Imposex
2
1

• Imposex in molluscs (female to male sex
  characteristics)
• Direct inhibitory effect on aromatase (CYP19)
  enzymatic activity
• Also appears to block storage of testosterone as
  esters
• Alters shell development in bivalve molluscs

1 National Environmental Research Institute,
Aarhus University, Denmark 2 Hagger JA et al.
EHP 2006, 114, S-1
10
Organotin Mechanism for Induction of Imposex
Aromatase
x
Testosterone
Estradiol
Male sex hormone
Female sex hormone
Animals Masculinized
11
Imposex is causally linked to environmental
organotin levels

• Prevalence of imposex can be quite high
• Closely associated with shipping and boating in
  most locations

12
Endocrine Disrupting Properties of Organotins

• Vertebrates
• Spermatotoxic
• Sperm lack flagella or have impaired motility
  (fish, rat)
• Sex reversal in fish (Danio rerio and Japanese
  flounder)
• Increased males
• Masculinization of genetically female flounder
  (25-30)
• Mild effects on mammalian sex characteristics
• Inhibits aromatase in cultured ovarian granulosa
  cells
• Reduced seminal vesicle weights in male rats
• Immunotoxic
• induces neutrophil apoptosis
• Inhibit cytotoxic function of NK cells
• Hepatotoxic (liver toxicity)

Effects of early exposure can persist in adulthood
13
Organotin Effects in Frogs
fat body
fat body
Vehicle (DMSO)
TBT
testis
adipocytes
kidney
t
t
X. laevis tadpoles exposed to low levels of
organotins exhibit ectopic fat cell production
compared to controls.
k
k
14
Organotin Action on Aromatase is NOT the Complete
Story.

• Organotins can also bind and activate nuclear
  receptors (RXR and PPARg) at nanomolar levels
  (ppb). These NRs are critical for adipogenesis
  (fat cell production) and lipid regulation.
• Animal exposure models suggest a link to the
  obesity epidemic and metabolic syndrome diseases
  such as diabetes, hypertension and cardiovascular
  disease)
• Environmental Obesogens a new class of endocrine
  disruptors
• pollutant chemicals that disturb lipid and fat
  cell regulation to promote obesity

Endocrine-disrupting organotin compounds are
potent inducers of adipogenesis in vertebrates.
Grün F, Watanabe H, Zamanian Z, Maeda L, Arima K,
Cubacha R, Gardiner DM, Kanno J, Iguchi T,
Blumberg B.Mol Endocrinol. 2006 Sep20(9)2141-55.
So is the environment making us fat ?
15
Obesity Epidemic

• Cause
• Caloric Intake (diet) ? Caloric Expenditure
  (metabolic rate, growth and exercise)
• Excess 100 calories/day 28 pounds of fat/year
• Definition Body Mass Index (BMI) gt30

BMI 704.5 x Weight (lbs)/(Height
(in))2 Overweight 25.0-29.9 Obese
gt30.0 Overweight 6ft man 185-220 lbs 5ft 6
woman 155-185 lbs
60 million Americans

• Complications
• Increased risk of diabetes
• Increased risk of hypertension
• Increased risk of heart disease,
  artherosclerosis
• Stroke
• Gallbladder disease
• Increased risk of breast, prostrate and colon
  cancer
• Decreased lifespan/quality of life
• 120 billion/year healthcare and economic costs

Metabolic syndrome
300,000 excess deaths
16
Atrazine Effects on Amphibians

• Atrazine is most common herbicide employed in
  US gt60,000,000 lbs/yr.
• Typically farm application rate is 2-30 ppm.
• EPA allows short term exposure up to 200 ppb 3
  ppb in drinking water
• Agricultural tailwater pits 2,000 ppb
  contaminated midwestern streams gt 100 ppb
  rainwater in farm land reaches 40 ppb.
• Prof. Tyrone Hayes (UC Berkeley) tested effects
  of atrazine on frog tadpoles between 0.1-25 ppb.
• Found mixed gonads in 20 animals at 0.1 ppb
• Above 1 ppb males become demasculinized
• Appears to enhance aromatase action ? lower
  testosterone levels
• Also linked to low sperm counts in men and
  chromosomal damage
• Possible link to decreased amphibian populations
  ?

17
(No Transcript)
18
Atrazine Effects on Frog Gonad Formation/Hormone
Levels
Hayes, TB, A Collins, M Lee, M Mendoza, N
Noriega, AA Stuart, and A Vonk. 2002.
Hermaphroditic, demasculinized frogs after
exposure to the herbicide, atrazine, at low
ecologically relevant doses. Proceedings of the
National Academy of Sciences (US) 995476-5480.
19
Gender-bender' fish problem widens Wednesday, 6
September, 2000 By BBC News Online's Helen
Briggs The entire male fish population of some
European rivers show feminising effects from
so-called "gender-bending" chemicals, according
to new research.Freshwater fish in five out of
seven northern European countries surveyed so far
showed some signs of exposure to the chemicals,
which mimic female hormones and are present in
sewage effluents. "We are finding this problem
right across northern Europe, it is clearly
widespread," Professor Alan Pickering of the
Natural Environment Research Council's Centre for
Ecology and Hydrology, UK, said. He was
addressing the British Association's Festival of
Science in London.Male and female"It seems to
relate to a mixture of chemicals both industrial
and also some of the natural excretory products
from the human body. "Symptoms of exposure to
the chemicals, known as endocrine-disrupters,
ranged from relatively minor effects to, in the
worst cases, fish developing both male and female
reproductive organs.In some places, such as the
River Aire, Yorkshire, UK, 100 of male fish were
found to show evidence of feminisation. Professor
Pickering said it was still not clear whether
the chemicals were having a direct impact on the
ability of fish to reproduce normally. But in one
UK river at least, he said, detrimental effects
had been established.
Frequency of wild and hatchery salmon exhibiting
signs of sex reversal
20
Waste Water Effluent Feminizes Fish
Male Head Fat Pads (male 2o sex characteristic)
Vittelogenin Egg Protein
Estrogen Receptor Levels
Nonylphenol (from detergents and agrichemical
polymers)
21
Lake Apopka DDE Contamination and Alligators

• 4th largest lake in Florida. About a 100 years
  of human alteration to water system and
  environment
• In 1980 Tower Chemical (pesticide company)
  improperly disposed of large amounts of DDE and
  other toxic chemicals.1981 EPA declares area a
  Superfund site but despite efforts spill
  contaminated waterways and aquifer.
• Irrigation flooding of adjacent farmland also
  introduces phosphorus laden farm runoff ?
  eutrophication
• Significant impact on wildlife bird and fish
  kills and reproductive problems for alligators
• Alligator sex is determined by temperature of
  eggs in nest but also sensitive to environmental
  estrogens
• At 6 months of age, female alligators from Lake
  Apopka had plasma estrogen concentrations
  gt2-times greater than normal females. Also noted
  abnormal ovaries, and an increased mortality
  rate.
• The plasma testosterone levels in male juvenile
  alligators gt3-fold lower than control males in
  Lake Woodruff. Lake Apopka males also had poorly
  organized testes and abnormally small phalli.

Prof. Louis Guillette
22
Trends in Reproductive Abnormalties

• Trend for earlier age of onset of puberty
• normal girls 9-14 years of age
• normal boys between 10-17
• Average age of onset lt0.5 yr but extreme cases
  of lt8 yrs of age
• Link to improved nutrition, obesity and EDCs

Sperm count
Age of Onset (boys)
Age of Onset (girls)
Elisabeth Carlsen and others, "Evidence for
decreasing quality of semen during past 50
years," BRITISH MEDICAL JOURNAL Vol. 305 (1992),
pgs. 609-613.
Peak Growth Rate (boys)
Peak Growth Rate (girls)

• Decreasing sperm counts
• (110 million ? 60 million)
• Increased infertility

23
Observations with links to EDCs

• Premature breast bud development in Puerto
  Rican girls exposed to phthalate esters
• Phthalates linked to obesity (waist
  circumference). Ubiquitous in US population.
• Premature puberty in Michigan girls exposed to
  PBDEs
• Significant exposure to bisphenol A (estrogenic)
  from food plastics and baby bottles. Bisphenol A
  is present in gt90 of US population
• Vinclozolin is a fungicide used in viticulture
  (wine making) and on fruits (e.g. strawberries)
  since 1975.
• Vinclozolin exposure in rodents during
  development leads to epigenetic changes
  maintained over 3 generations affecting sperm
  count and fertility i.e. a single exposure
  affects all offspring of subsequent generations.
  (Anway, MD, AS Cupp , M Uzumcu, and MK Skinner.
  2005. Epigenetic Transgenerational Actions of
  Endocrine Disruptors and Male Fertility. Science
  3081466-1469)

24
Malformed frogs in North America Endocrine
disruption of vitamin A signaling during limb
formation or trematode parasitic infection ?
Duplicated paired limbs
Split hindlimbs
Supernumerarylimbs
25
Hypothesis 1 Infection by Parasites
Newt infected with trematode cysts

• Trematodes are parasitic worms
• Life-cycle includes different hosts (amphibians,
  birds, snails)
• Larvae encyst in amphibians
• Cysts cause disturbance of critical regions
  controlling limb development
• Published work supports this cause for many
  cases The effect of trematode infection on
  amphibian limb development and survivorship.
  Johnson PT, Lunde KB, Ritchie EG, Launer AE.
  Science. 1999 Apr 30284(5415)802-4.

but can find malformed amphibians with no
infections ?
26
Bocks Lake, Crow Wing County, Minnesota (July
2000)
Frog Minnow Die-off
Mink frog with bony triangle gt 70 metamorphs
deformed

• CWB lake 30 acres
• 1 family house adjacent dairy farm
• Phenotypes
• Dead fish and frogs
• Malformed metamorphic frogs (bony triangles,
  duplications) similar to retinoic acid exposure
• Male adults do not call or initiate amplexus
  (mating hold) feminized ?

27
Hypothesis 2 Are there endocrine disrupting
chemicals present in lake water that can activate
nuclear hormone receptors ?
Collect water samples, fractionate ? test for
ability to activate the Retinoic Acid Receptor
(RAR)
28
Minnesota Lake Water Fractions NR Activation
Assay
Fractionate and test lake water samples for
receptor activation

• Conclusion
• Chemicals are present that behave as predicted
  at the molecular level. Same activity found in
  Minnesota and California
• Lead components
• Chlorinated herbicide or pharmaceutical (for
  dairy cattle/horses)
• Inert agrichemical filler ingredient (alkylphenol
  polymer ?)

29
A Plague of Frogs, William Souder Hyperion, 1st
edition (2000)
Pacific tree frog, Rancho Santa Margarita, CA
30
Conclusions

• Endocrine disruption of wildlife populations by
  chemical pollutants are an ever growing threat
• Effects may not be readily apparent since acute
  toxicity and lethality are not always evident
• Instead chemicals that interfere with
  reproductive functions (e.g. altered sex steroid
  hormone levels) can signifcantly impact fertility
  ? reproductive success ? leading to a decline in
  population numbers
• EDCs have been shown to affect courtship and
  mating behaviors, egg/sperm quality, morphology
  of genitalia, fecundity (reproductive capacity),
  growth, immunity and disease resistance,
  development and morphogenesis (body pattern
  formation)
• Most worrisome are emerging threats from
  epigenetic changes, since problems persist into
  future generations who were never directly exposed