Pollution:

OutlineOutline

1) Endrocrine disruptors

2) Drug studies: DES, Hormone Replacement Therapy

3) Pesticides: Estrogen mimics and other actions

Pollution

Alterations of the natural environment producing conditions harmful to living organisms

Examples:Fossil FuelsOzone depleting compoundsMutagensEndocrine DisruptorsNeurotoxins

Types of Pollution

CO2, CH4, N2O Green House Effect Climate changeClimate change

CFCs Ozone depletion UV DamageUV DamageLots Mutagens DNA damage, CancerDNA damage, CancerLots Endocrine Disruptors Development, Development,

CancerCancerHeavy Metals some are Neurotoxins Neurological Neurological

ProblemsProblemsRadionuclides Mutagens DNA damage, CancerDNA damage, Cancer

CHEMICALS & THE ENVIRONMENT

11,000,000 Chemicals are known

100,000 Chemicals are produced deliberately

90,000 Registered Chemicals in the US

1200-1500 New Chemicals are registered in the US/year

Only ~50 Organic toxins with legally enforceable environmental standards in drinking water

http://www.epa.gov/safewater/mcl.html

Historical Note

Industrial Revolution-1950s :

• Conquest of Nature

• Age of Optimism

Technology can cure all our problems

Historical Note

At the same time, information on Health and Physiology was sometimes lacking

Los Angeles in 1972

Accumulation of Environmental disasters spawned environmental movements in the 1960s and 1970s

Discussed effects of pesticides on songbird populations throughout the United States, whose declining numbers yielded the “silent spring”

Silent Spring1962by Rachel Carson

Regulatory Measures

• Environmental Protection Agency created in 1970

– Clean Air Act of 1970

– Safe Drinking Water Act 1974

– Clean Water Act 1977

– Water Quality Act 1987

http://www.epa.gov/history/publications/formative5.htm

William D. RuckelshausFirst EPA Administrator

More Recent Update:

Our Stolen Future 1996

Theo Colborn et al.

Website on news and articles:http://www.ourstolenfuture.org/index.htm

What are endocrine What are endocrine disrupters?disrupters?

Many pollutants are endocrine Many pollutants are endocrine disruptersdisrupters

Steroid Hormones are lipid soluble, bind to cytoplasmic receptors and then enter the nucleus

Chemicals which interfere with endocrine system function

• Estrogenic• Androgenic• Anti-Estrogenic• Anti-Androgenic• Something more complex• Combination of effects depending on concentration

and interaction with other compounds

Endocrine Disruptors

Health Effects:• Nonpolar compounds that store in fat cells

• Might have no obvious effect until 2nd generation

• Disrupt Normal Sexual development Hermaphroditism, Sterility, abnormal testes, ovaries, etc.

• Cancer

Many pollutants are endocrine Many pollutants are endocrine disruptersdisrupters

Estradiol-17ßA natural estrogen hormone found in all vertebrates.

Many pollutants are estrogenic

Estrogens in Humans

• Estrone

• Estradiol

• Estriol

• Several others

Estrogens

• Estrone: produced during menopause

• Estradiol: predominant form in nonpregnant females

• Estriol: primary estrogen of pregnancy

• several others

Critical Functions of Estrogens

• Essential hormone for development and reproduction

• In humans, present in both men and women, but significantly higher levels in women of reproductive age.

• Promote the development of female secondary sex characteristics

• Stimulates endometrial growth during the menstrual cycle• Responsible for the maintenance of blood vessels and the skin• Reduces bone resorption, therefore increasing bone formation• Increases uterine growth

Detrimental effects• Estrogen and the ERs have been implicated in breast cancer,

ovarian cancer, colon cancer, prostate cancer, and endometrial cancer. This is true even when estrogen is administered with progestin (Chlebowski et al. JAMA 2010).

• Estrogen receptors are over-expressed in around 70% of breast cancer cases, referred to as "ER-positive". Two hypotheses have been proposed to explain why this causes tumorigenesis, and available evidence suggests that both mechanisms contribute:

• First, binding of estrogen to the ER stimulates proliferation of mammary cells, with the resulting increase in cell division and DNA replication, leading to mutations.

• Second, estrogen metabolism produces genotoxic waste.

• Both processes are thought to result in disruption of cell cycle, apoptosis and DNA repair, and, therefore, tumor formation.

Estrogen Effects• Creates proliferative endometrium • Breast cell stimulation (fibrocystic breasts*) • Increased body fat and weight gain* • Salt and fluid retention • Depression, anxiety, and headaches* • Cyclical migraines* • Poor sleep patterns* • Interferes with thyroid hormone function* • Impairs blood sugar control* • Increased risk of blood clots* • Little or no libido effect* • Loss of zinc and retention of copper* • Reduced oxygen levels in all cells* • Causes endometrial cancer*• Increased risk of breast cancer* • Increased risk of prostate cancer* • Restrains bone loss • Reduces vascular tone (dilates blood vessels) • Triggers autoimmune diseases* • Creates progesterone receptors • Increases risk of gall bladder disease*

* Indicates that these effects are caused by estrogen dominance, or an imbalance of estrogen caused by too little progesterone.

Tradeoffs during Ageing(Antagonistic Pleiotropy)

• Age-specific effects• Early Life

– Necessary for development (e.g. cell growth and proliferation of breast tissue)

– Necessary for functions related to reproduction (increase in fitness early in life)

• Later Life– Increases in Breast Cancer and other ailments later in life – Cell growth and proliferation early in life would promote

development of breast tissue, but then could cause cancer later…more cell divisions = more mutations

– In older women, estrogen use was linked to a 15% increase in occurrences of a range of diseases (studies by Andrea LaCroix)

Estrogen Receptor 1 (ER-alpha)

Estrogen Receptor 2 (ER-beta)

Estrogen Receptors

Estrogen Receptor-alpha;Ligand-binding domain complexed to estradiol

Testosterone and other hormones could also have detrimental effects, esp later in life; however, pollutants are often estrogenic because:

Estrogen receptors arefairly nonspecific

• Bind to multiple ligands,~12 estrogens (estradiol, estriol, estrone…etc.)

• They are ancestral receptors

So... many compounds will bind to them, such as pesticides, plastics, etc.

Estrogen Receptors

Androgenreceptor

Glucocorticoidreceptor

Progesteronereceptor

Mineralocorticoidreceptor

Eel ERHuman ER

Trout ERXenopus ER

Human ER

Sex steroid response probably occurred in the early Cambrian

Estrogen response evolved in jawless fish or tunicates (early chordates)

Baker, ME. 2001. Adrenal and sex steroid receptor evolution: environmental implications. Journal of Molecular Endocrinology 26:119–125

the most ancient of the adrenal and sexsteroid receptors

Estrogen Receptors

Androgenreceptor

Glucocorticoidreceptor

Progesteronereceptor

Mineralocorticoidreceptor

Eel ERHuman ER

Trout ERXenopus ER

Human ER

• More Ancestral receptors tend to be less specific…

• Specificity for particular ligands often evolves over time

the most ancient of the adrenal and sexsteroid receptors

• A gene encoding one receptor might duplicate and evolve specificity for different ligands

Steroid Hormones are lipid soluble, bind to cytoplasmic receptors and then enter the nucleus

Estrogen (ligand) + Estrogen Receptor (ER) form a complex that acts as a transcription factor, binding to DNA and regulating gene expression

• Different ligands differ in their affinity for alpha and beta isoforms; estradiol binds equally well to both receptors; estrone binds preferentially to the alpha receptor; estriol prefers the beta receptor

• Estrogen receptors form dimers– the receptors may form ERα (αα) or ERβ (ββ)

homodimers or ERαβ (αβ) heterodimers

• The effects of estrogens vary depending on the type of estrogen, the type of receptor, and the type of dimer

• The same ligand may be an agonist in some tissue (where coactivators predominate) while antagonistic in other tissues

Estrogen Receptor 1 (ER-alpha)

Estrogen Receptor 2 (ER-beta)

Estrogen Receptors

• Animation of estrogen binding to estrogen receptor, and the estrogen-receptor complex acting as a transcription factor:

• http://e.hormone.tulane.edu/learning/animations/hormone-docking-estrogen.html

• It is difficult to make simple statements on the effects of estrogens and estrogenic compounds

• The effects of estrogens depends on many factors, such as the type of estrogen, the type of receptor, the dimer that is formed between the receptors, the type of tissue, the age of the individual, etc…

• Estrogens also function differently in the presence of progestin

• So, it is hard to make simple statements about the effect of estrogens or endocrine disruptors… the pollutants (plastics, pesticides, etc.) that bind to estrogen receptors…

• Exogenous estrogenic compounds (plastics, pesticides, etc.) can have many different effects… often negative.

Estrogenic compounds

Estrogenic Compounds

What have we learned from the action of Drugs?

Estrogens used in Medicine:

• DES

• Hormone Replacement Therapy

DES Diethylstilbestrol

• First manufactured 1938

• During 1938-1971, U.S. physicians prescribed DES to pregnant women to prevent miscarriages and other pregnancy problems

• ~ 5-10 million pregnant women and children born of these pregnancies were exposed to DES

• Breast cancer

DES Daughters:• Clear-cell adenocarcinoma (CCA), a rare kind of vaginal & cervical cancer

• Pregnancy complications, including ectopic (tubal) pregnancy and pre-term (early) delivery

•Reproductive tract structural differences (such as T-shaped uterus)

• Infertility

DES Sons:•Non-cancerous epididymal cysts (growths on the testicles)

Women prescribed DES while pregnant are at modest increased risk for:

Importance of findings on DES

• Shattered optimism of the pre-1950s regarding the generally beneficial effects of drugs

• Instilled caution regarding chemicals in the environment

• Long term effects still unknown, DES “babies” are still on average in their mid-40s, early-50s

Hormone Replacement Therapy

• 1942 Premarin® is introduced into the U.S. market

• Influential Book: Feminine Forever in 1966Dr. Robert Wilson wrote that women taking estrogen

"will be much more pleasant to live with and will not become dull and unattractive"

• By 1995, 38% of postmenopausal women were participating in HRT

Premarin®

• A popular product used in HRT

• Derived from urine of pregnant horses

• Contains seventeen different types of estrogen, most of which are not native to human beings

• Contains primarily just one "human" estrogen, estrone (75-80%), plus equilin (6-15%), a form of estrogen found exclusively in horses

The longstanding Dogma on HRT: Fountain of Youth

• Protection against osteoporosis• Protection against heart disease• Enhanced memory, mental acuity• Supple skin, better appearance

Star Trek Episode:

“Mudd’s Women”Venus drug of Youth turns out to be a Placebo

But...

(1) First study with double-blind controls in 2002: estrogen and progestin trial study on 16,608 women ages 50 to 79 with an intact uterus during 1993-1998(Women’s Health Initiative. 2002. JAMA 288:321-328)

(2) Statistical meta-analysis of previous studies

(Nelson et al. 2002. JAMA 288:872-881)

Results(1) First Controlled Study in 2002

(Women’s Health Initiative. 2002. JAMA 288:321-328)

Estrogen and progestin trial study on 16,608 women ages 50 to 79 with an intact uterus during 1993-1998

Double blind study in which patients were administered either HRT or a placebo

On July 9, 2002, the NIH halted this trial after 5.2 years, concluding that the risks for the study group on combined HRT outweighed the benefit

RISKSHeart Disease: 29% higher

Stroke: 41% higher Breast Cancer: 26% higher

Blood clot: 113% higher

BenefitsColon Cancer: 37% lower

Bone Fractures: 24% lower

Women’s Health Initiative. 2002. JAMA 288:321-328

Hormone Replacement relative to Placebo

Problems with Previous Studies on HRT

• Most were poorly conducted (lacking controls), not account for socioeconomic status, exercise, etc...

• Correlational

• Not double blind

(2) Meta-analysis (reanalysis of previous data)(Nelson et al. 2002 JAMA 288:872-881)

• HRT did not reduce heart disease when controlled for socioeconomic status, exercise

• Increase in stroke, blood clots, breast cancer

• Reduction in bone fractures, colorectal cancer

• Prevention of dementia uncertain

Endocrine Disruptors in the Environment

• Persist in environment

• Spreads throughout the world: airborne, ocean currents

• Concentrates at higher trophic levels in food webs

PCBs and pesticide transport by air

Artic Ocean Currents

High concentrations of PCBs and DDT in the Arctic

Bioaccumulation

Breast milk of Inuit women from northern Quebec who eat marine mammals has been found to contain PCB concentrations 2-10 times higher than that of women in the southern part of the province.

High levels of PCBs have been found in polar bears, with concentrations of ~10 ppm in the Canadian Arctic and up to 100 ppm in the European Arctic.

Humans

Blue whale Sperm whale

Crabeater seal

Killerwhale Elephant

seal

Leopardseal

Adéliepenguins Petrel

Fish

Squid

Carnivorous plankton

Krill

Phytoplankton

Herbivorouszooplankton

Emperorpenguin

Antarctic Food Web

Toxins bioaccumulate at each trophic level

Phytoplankton 0.025 ppm

zooplankton 0.123 ppm

smelt 1.04 ppmLake Trout 4.83 ppm

Herring Gull Eggs 124 ppm

2004

PCBs and Dioxin are both banned, but persist in the environment and are in food products and plastics

2004

Most endocrine disruptors are nonpolar and do not get removed from urine

They tend to accumulate in fats cells of an animal and become concentrated…

Resulting in accumulation in milk (and even more in butter, cheese)

Sources

• Pharmaceuticals (DES, estrogens)• Agricultural Fields (Food)• Golf Courses and Yards• Factories (paper production), Industry• Plastics

DDT

DESKepone

PCB

4-nonylphenol

Endocrine Disruptors

Dioxin

Atrazine

Pesticides that act as Endocrine Disruptors

(we talked about some pesticides that act as neurotoxins when we

discussed CYP450s)

As recently as the 1950s, potent toxins used as pesticides or herbicides:

Lake Mendota: Arsenic, Copper Sulfateadministered by students without protective clothing to deter algal blooms

At the end of WWII, new chemical technology: synthetic organics

• A• acylalanine fungicides see acylamino acid fungicides• acylamino acid fungicides• aliphatic amide organothiophosphate insecticides• aliphatic nitrogen fungicides• aliphatic organothiophosphate insecticides• amide fungicides• amide herbicides• anilide fungicides• anilide herbicides• antiauxins• antibiotic acaricides• antibiotic fungicides• antibiotic insecticides• antibiotic nematicides• aromatic acid herbicides• aromatic fungicides• arsenical herbicides• arsenical insecticides• arylalanine herbicides• aryloxyphenoxypropionic herbicides• auxins• avermectin acaricides• avermectin insecticides• B• benzamide fungicides• benzanilide fungicides• benzimidazole fungicides• benzimidazole precursor fungicides• benzimidazolylcarbamate fungicides• benzofuranyl alkylsulfonate herbicides• benzofuranyl methylcarbamate insecticides• benzoic acid herbicides• benzothiazole fungicides• benzothiopyran organothiophosphate insecticides• benzotriazine organothiophosphate insecticides• benzoylcyclohexanedione herbicides• bipyridylium herbicides see quaternary ammonium

herbicides• botanical insecticides• botanical rodenticides• bridged diphenyl acaricides• bridged diphenyl fungicides• C• carbamate acaricides• carbamate fungicides• carbamate herbicides• carbamate insecticides• carbamate nematicides• carbanilate fungicides• carbanilate herbicides• chitin synthesis inhibitors• chloroacetanilide herbicides• chloropyridine herbicides see pyridine herbicides• chlorotriazine herbicides• conazole fungicides• copper fungicides• coumarin rodenticides• cyclic dithiocarbamate fungicides• cyclodiene insecticides• cyclohexene oxime herbicides• cyclopropylisoxazole herbicides• cytokinins

Ddefoliantsdiacylhydrazine insecticides see moulting hormone agonistsdicarboximide fungicidesdicarboximide herbicidesdichlorophenyl dicarboximide fungicidesdimethylcarbamate insecticidesdinitroaniline herbicidesdinitrophenol acaricidesdinitrophenol fungicidesdinitrophenol herbicidesdinitrophenol insecticidesdiphenyl ether herbicidesdithiocarbamate fungicidesdithiocarbamate herbicidesEethylene releasersethylthiotriazine herbicidesFfluorine insecticidesformamidine acaricidesformamidine insecticidesfumigant insecticidesfuramide fungicidesfuranilide fungicidesGgibberellinsgrowth inhibitorsgrowth retardantsgrowth stimulatorsHhalogenated aliphatic herbicidesheterocyclic organothiophosphate insecticidesIimidazole fungicidesimidazolinone herbicidesindandione rodenticidesinorganic fungicidesinorganic herbicidesinorganic insecticidesinorganic mercury fungicidesinorganic rodenticidesinsect growth regulatorsisoindole organothiophosphate insecticidesisoxazole organothiophosphate insecticidesJjuvenile hormone mimicsjuvenile hormonesMmercury fungicidesmethoxytriazine herbicidesmethylthiotriazine herbicidesmilbemycin acaricidesmilbemycin insecticidesmite growth regulatorsmorphactinsmorpholine fungicidesmoulting hormone agonistsmoulting hormonesmoulting inhibitorsNnereistoxin analogue insecticidesnicotinoid insecticidesnitrile herbicidesnitroguanidine insecticidesnitromethylene insecticidesnitrophenyl ether herbicides

Oorganochlorine acaricidesorganochlorine insecticidesorganochlorine rodenticidesorganomercury fungicidesorganophosphate acaricidesorganophosphate insecticidesorganophosphate nematicidesorganophosphorus acaricidesorganophosphorus fungicidesorganophosphorus herbicidesorganophosphorus insecticidesorganophosphorus nematicidesorganophosphorus rodenticidesorganothiophosphate insecticidesorganothiophosphate nematicidesorganothiophosphate acaricidesorganotin acaricidesorganotin fungicidesoxadiazine insecticidesoxathiin fungicidesoxazole fungicidesoxime carbamate acaricidesoxime carbamate insecticidesoxime carbamate nematicidesoxime organothiophosphate insecticidesPphenoxy herbicidesphenoxyacetic herbicidesphenoxybutyric herbicidesphenoxypropionic herbicidesphenyl ethylphosphonothioate insecticidesphenyl methylcarbamate insecticidesphenyl organothiophosphate insecticidesphenyl phenylphosphonothioate insecticidesphenylenediamine herbicidesphenylsulfamide acaricidesphenylsulfamide fungicidesphenylurea herbicidesphosphonate acaricidesphosphonate insecticidesphosphonothioate insecticidesphosphoramidate insecticidesphosphoramidothioate insecticidesphosphoramidothioate acaricidesphosphorodiamide acaricidesphosphorodiamide insecticidesphthalic acid herbicidesphthalimide acaricidesphthalimide fungidesphthalimide insecticidespicolinic acid herbicidespolymeric dithiocarbamate fungicidespolysulfide fungicidesprecocenespyrazole acaricidespyrazole insecticidespyrazolopyrimidine organothiophosphate insecticidespyrazolyloxyacetophenone herbicidespyrazolylphenyl herbicidespyrethroid acaricidespyrethroid ester acaricidespyrethroid ester insecticidespyrethroid ether acaricidespyrethroid ether insecticidespyrethroid insecticidespyridazine herbicidespyridazinone herbicides

pyridine fungicidespyridine herbicidespyridine organothiophosphate insecticidespyridylmethylamine insecticidespyrimidinamine acaricides

pyrimidinamine insecticidespyrimidinamine rodenticidespyrimidine fungicidespyrimidine organothiophosphate insecticidespyrimidinediamine herbicidespyrimidinyloxybenzoic acid herbicidespyrimidinylsulfonylurea herbicidespyrrole acaricidespyrrole fungicidespyrrole insecticidesQquaternary ammonium herbicidesquinoline fungicidesquinolinecarboxylic acid herbicidesquinone fungicidesquinoxaline acaricidesquinoxaline fungicidesquinoxaline organothiophosphate insecticidesSstrobin fungicidessulfite ester acaricidessulfonanilide fungicidessulfonanilide herbicidessulfonylurea herbicidesTtetronic acid acaricidestetronic acid insecticidesthiadiazole organothiophosphate insecticidesthiadiazolylurea herbicidesthiazole fungicidesthiocarbamate acaricidesthiocarbamate fungicidesthiocarbamate herbicidesthiocarbonate herbicidesthiophene fungicidesthiourea acaricidesthiourea herbicidesthiourea rodenticidestriazine fungicidestriazine herbicidestriazinone herbicidestriazinylsulfonylurea herbicidestriazole fungicidestriazole herbicidestriazole organothiophosphate insecticidestriazolone herbicidestriazolopyrimidine herbicidesUuracil herbicidesurea fungicidesurea herbicidesurea insecticidesurea rodenticidesVvalinamide fungicidesXxylylalanine fungicides see acylamino acid fungicides

Classes of Pesticides

Mechanism of Action (on the pests) of some Major Classes

• Chlorinated hydrocarbons (DDT, Lindane, dioxin): Accumulate in fatty tissue, causing chronic disease

• Organophosphates (Malathion): Inhibit acetylcholinesterase

• Carbamates (NHRCOOR’): Inhibit acetylcholinesterase

• Pyrethroids (modeled after natural products): neurotoxin

• Growth regulators: Block juvenile hormone receptors (Methoprene), block chitin synthesis, formation of cuticle

• Triazines (Atrazine): Inhibit photosynthesis

• Phenoxy herbicides: Mimic plant hormone auxin, causing abnormal growth

Mechanism of Action (on the pests) of some Major Classes

• Chlorinated hydrocarbons (DDT, lindane, dioxin): Accumulate in fatty tissue, causing chronic disease

• Organophosphates (malathion): Inhibit acetylcholinesterase

• Carbamates (NHRCOOR’): Inhibit acetylcholinesterase

• Pyrethroids (modeled on natural products): Neurotoxin

• Growth regulators: Block juvenile hormone receptors (Methoprene), block chitin synthesis, formation of cuticle

• Triazines (Atrazine): Inhibit photosynthesis

• Phenoxy herbicides: Mimic plant hormone auxin, causing abnormal growth

Chlorinated Hydrocarbons

• Chlorinated Ethane Derivatives• DDT, DDE• Methoxyclor

• Chlorinated Cyclodienes• Aldrin, Dieldrin, Endrin• Heptachlor• Chlordane• Toxaphene

• Hexachlorocyclohexanes• Lindane

Last time I talked a bit about neurotoxins (in terms of evolution of pesticide resistance).

This time: Endocrine Disruptors

Various Chemicals and their breakdown products can have estrogenic or other endocrine effects

DDT

DES Kepone

PCB

4-nonylphenol

Endocrine Disruptors

Dioxin

Atrazine

Agent OrangeAgent Orange

In 2004 Vietnamese activists took the chemical companies that produced Agent Orange to court in the US.

But an American Federal District Judge dismissed the case on the grounds that use of the defoliant did not violate international law at the time.

During the Vietnam War, the US sprayed 80 million litres of toxins in Vietman, including 45 million litres of Agent Orange over a 10th of Vietnam. Agent Orange laced with Dioxins (binds aggressively to receptors)

PCBs

• Polychloronated biphenyls (phenyl refers to the benzene rings; pheno is the old name for benzene)

• 209 different PCBs have varying levels of estrogenic activity

• Once widely used in electrical transformers and cooling systems and as pesticides, but are now restricted (commercially produced in North America from 1929 to 1977)

C

C

C C

C

C

C

C C

C

CC

H

Cl

H

H

H

H H Cl

H Cl

generic chemical structure

Dichloro-diphenyl-trichloroethane; a synthetic pesticide

DDT is banned in Europe and the US (in 1972), but is still used in developing countries to control malaria.

Neurotoxin (blocks Na channels)Estrogen MimicCarcinogen??? DDE, the principle breakdown product of DDTAnti-androgenic

DDT (one type of PCB)

Bald Eagle

• Nearly went extinct

• Due to thin egg shells caused by effects of DDT

• In the approximately twenty six years following the banning of DDT, the number of bald eagle pairs increased by eight fold, from approximately 791 pairs in 1974 to 5,948 nesting pairs in 1998.

DDT was banned for use as a pesticide in the U.S. by the EPA in 1972, but still persists in environment; breakdown products are also endocrine disruptors, but with different effects

Atrazine

2-chloro-4-ethlytlamino-6-isopropylamine-1,3,5-triazine

“The most commonly used herbicide in the U.S. and probably the world”

Atrazine

Given its molecular structure, would it bind to estrogen

receptors?

Why or why not?

Atrazine

No

Atrazine is estrogenic, but in a very different way

What types of molecules bind to estrogen receptors?

Estrogenic Compounds

The Estrogen Receptor Relative Binding Affinities of 188 Natural and Xenochemicals: Structural Diversity of LigandsToxicoogical Sciences (2000) 54: 138-153Robert M. Blair et al. AbstractWe have utilized a validated (standardized) estrogen receptor (ER) competitive-binding assay to determine the ER affinity for a large, structurally diverse group of chemicals. Uteri from ovariectomized Sprague-Dawley rats were the ER source for the competitive-binding assay. Initially, test chemicals were screened at high concentrations to determine whether a chemical competed with [3H]-estradiol for the ER. Test chemicals that exhibited affinity for the ER in the first tier were subsequently assayed using a wide range of concentrations to characterize the binding curve and to determine each chemical's IC50 and relative binding affinity (RBA) values. Overall, we assayed 188 chemicals, covering a 1 × 106-fold range of RBAs from several different chemical or use categories, including steroidal estrogens, synthetic estrogens, antiestrogens, other miscellaneous steroids, alkylphenols, diphenyl derivatives, organochlorines, pesticides, alkylhydroxybenzoate preservatives (parabens), phthalates, benzophenone compounds, and a number of other miscellaneous chemicals. Of the 188 chemicals tested, 100 bound to the ER while 88 were non-binders. Included in the 100 chemicals that bound to the ER were 4-benzyloxyphenol, 2,4-dihydroxybenzophenone, and 2,2′-methylenebis(4-chlorophenol), compounds that have not been shown previously to bind the ER. It was also evident that certain structural features, such as an overall ring structure, were important for ER binding. The current study provides the most structurally diverse ER RBA data set with the widest range of RBA values published to date.

Estrogen binding affinities for 188 chemicals

• A ring structure is of primary importanceWith the exception of 4 chemicals (kepone, norethynodrel, 5a-androstane-3a, 17b-diol, and 5a-androstane-3b,17b-diol)

• Phenol ring (Hydroxyl group on the phenyl [=benzene]): Thought to act as a hydrogen bond donor and acceptor in the ER-binding site

• Longer side chains

• In general, chemicals with 2 ring structures separated by 2 carbon atoms (steroidal and synthetic estrogens and diphenyl ethanes) have higher affinities than chemicals with a single ring structure or 2 rings separated by one carbon atom (but the latter compounds might still bind, perhaps not as strongly).

Atrazine

2-chloro-4-ethlytlamino-6-isopropylamine-1,3,5-triazine

“The most commonly used herbicide in the U.S. and probably the world”

• Recommended application levels: 2,500,000-29,300,000 parts/billion (ppb)

• Midwestern agricultural areas (tailwater pits): 2,300 ppb

• Midwestern streams: 224 ppb

• Atrazine in agricultural runoff: 40 ppb

• Groundwater: 21 ppb

• Allowable level for drinking water: 3 ppb

• Hayes et al: 0.01-200 ppb affects sexual development of Xenopus laevis

Atrazine

Weed Killer Found to Sexually Deform FrogsNew York Times April 17, 2002

“the most important paper in environmental toxicology in decades”Dr. Stanley Dodson, University of Wisconsin

Hayes et al. 2002. Proceedings of the National Academy of Sciences. 99: 5476-5480

Atrazine

?

• What experiments were performed in this study?

• What were the Results?

• What is the Significance?

Results

• Atrazine (> 0.1 ppb) induces hermaphroditism and demasculinzed the larynges of exposed males

• Adult males suffered a 10-fold decrease in testosterone levels when exposed to 25 ppb

Control Male Control Female

Control Male

Control Female

An Atrazine TreatedHermaphrodite (1 ppb)

Effect of 46-day exposure to atrazine on testosterone levels in sexually mature males of Xenopus laevis (frogs)

Abbreviated Pathway of Estradiol Synthesis

Hypothesis: Atrazine induces aromatase to convert testosterone

into estrogen?

Dodson et al. 1999. Environmental Toxicology and Chemistry. 18: 1568–1573

Results

• Exposure of Daphnia to atrazine during embryogenesis resulted in a shift in sex determination toward males.

• The shift was detectable at 0.5 ppb (nominal concentration) and pronounced at 10 ppb or more

Significance of these studies

• Atrazine blocks photosynthesis, so the assumption was that it would have no effect on animals

• Yet, Atrazine has physiological effects at levels lower than concentrations present in the environment

Testing for Toxicity• Does the compound affect development?

– Does it act as an antagonist or agonist with hormone receptors?

• How does it affect the second generation?– Is the compound lipophilic?

– Does it bioaccumulate?

• What are the breakdown compounds and what are their effects?– What is the half-life of the compound and the breakdown

compounds?

How to avoid endrocrine disruptors?

• Eat Organic: esp. eggs and dairy, meat , berries, spinach(Madison area organic farms – many have delivery service -

http://www.csacoalition.org/category/all-farms/)

• Avoid contact between plastics and foodavoid canned foods, plastic containers

• Avoid imported produce, especially from countries where the US sells banned pesticides

• Examine ingredients of your food, lotions, cosmetics– Examine the molecular structure of the compounds. – Even if a compound has not been studied, you might be able

to predict its ER receptor affinity

Take Home Message• Much of the medical literature is plagued with

erroneous conclusions based on poor experimental design and faulty statistics

• A lot of chemicals produced can be very harmful… many are produced by combustion, which produces benzene rings bind to estrogen receptors

• Pesticides are likely to be more harmful to us than the organisms we are trying to kill (as smaller organisms can evolve much more rapidly due to large population size and short generation times)

Which of the following is LEAST likely to act as an endocrine disruptor by binding to estrogen receptors?

Which of the following is most likely to be FALSE regarding Atrazine?

(a) Atrazine reduces the concentration of testosterone in adult frogs(b) Atrazine is thought to induce the activity of aromatase(c) Atrazine concentrations in the environment, such as allowable levels in drinking water, is higher than the concentrations which had a physiological effect in frogs (in the study by Tyrone Hayes)(d) Atrazine is a type of endocrine disruptor that resembles the structure of estrogens(e) Atrazine blocks photosynthesis in plants

Which of the following would prevent the action of a pesticide before it reaches its target?

(a) Point mutation in a voltage-gated ion channel in response to DDT(b) Amino acid substitution at the active site of acetylcholinesterase in response to organophosphates(c) Duplications of DME alleles in response to PCBs(d) Duplications of Rdl alleles in response to cyclodienes