Toxic Effects of Pesticides

David J. Dix, Ph.D.National Center for Computational Toxicology Office of Research & DevelopmentU.S. Environmental Protection AgencyResearch Triangle Park, NC 27711Email: dix.david@epa.govhttp://www.epa.gov/ncct/

Biochemical and Molecular ToxicologyUNC ENVR/TOXC 44205nov2009www.unc.edu/courses/2009fall/envr/442/001/

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Pesticides in the News

August 23, 2009Debating How Much Weed Killer Is Safe in Your Water GlassBy CHARLES DUHIGGFor decades, farmers, lawn care workers and professional green thumbs have relied on the popular weed killer atrazine to protect their crops, golf courses and manicured lawns. But atrazine often washes into water supplies and has become among the most common contaminants in American reservoirs and other sources of drinking water. Now, new research suggests that atrazine may be dangerous at lower concentrations than previously thought. Recent studies suggest that, even at concentrations meeting current federal standards, the chemical may be associated with birth defects, low birth weights and menstrual problems. Laboratory experiments suggest that when animals are exposed to brief doses of atrazinebefore birth, they may become more vulnerable to cancer later. An investigation by The New York Times has found that in some towns, atrazineconcentrations in drinking water have spiked, sometimes for longer than a month. But the reports produced by local water systems for residents often fail to reflect those higher concentrations.

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August 23, 2009

Pesticides in the News

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Pesticides in the News

October 7, 2009Regulators Plan to Study Risks of AtrazineBy CHARLES DUHIGGThe Environmental Protection Agency plans to conduct a new study about the potential health risks of atrazine, a widely used weedkiller that recent research suggests may be more dangerous to humans than previously thought.Atrazine — a herbicide often used on corn fields, golf courses and even lawns — has become one of the most common contaminants in American drinking water. For years, the E.P.A. has decided against acting on calls to ban the chemical from environmental activists and some scientists who argued that runoff was polluting ecosystems and harming animals. More recently, new studies have suggested that atrazine in drinking water is associated with birth defects, low birth weights and reproductive problems among humans, even at concentrations that meet current federal standards.The E.P.A. is expected to announce on Wednesday that it will conduct a new evaluation of the pesticide to assess any possible links between atrazine and cancer, as well as other health problems, such as premature births. The E.P.A. may determine that new restrictions are necessary.

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Pesticides in the Scientific Literature

PubMed, “atrazine AND birth defects”, 03nov2009

Agrichemicals in surface water and birth defects in the United States. Winchester PD, Huskins J, Ying J. Acta Paediatr. 2009 Apr;98(4):664-9. Epub 2009 Jan 22.PMID: 19183116

Effects of atrazine and endosulfan sulphate on the ecdysteroid system of Daphnia magna. Palma et al. Chemosphere. 2009 Feb;74(5):676-81. Epub 2008 Nov 29.PMID: 19042009

The herbicide atrazine activates endocrine gene networks via non-steroidal NR5A nuclear receptors in fish and mammalian cells. Suzawa M, Ingraham HA. PLoS One. 2008 May 7;3(5):e2117.PMID: 18461179

Atrazine-induced reproductive tract alterations after transplacental and/or lactationalexposure in male Long-Evans rats. Rayner JL, Enoch RR, Wolf DC, Fenton SE. Toxicol Appl Pharmacol. 2007 Feb 1;218(3):238-48. Epub 2006 Nov 23.PMID: 17204298

Characterization of atrazine-induced gonadal malformations in African clawed frogs (Xenopus laevis) and comparisons with effects of an androgen antagonist (cyproterone acetate) and exogenous estrogen (17beta-estradiol): Support for the demasculinization/feminization hypothesis. Hayes et al. Environ Health Perspect. 2006 Apr;114 Suppl 1:134-41.PMID: 16818259

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In Principles and Methods of Toxicology (5th ed), A Wallace Hayes (editior)Pgs 727-840, Informa Healthcare, NY, 2007

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Publisher : CRC | ISBN : 0748409106 | edition 2004 | PDF | 296 pages

Chapter 2 Why is a toxicant poisonous?

Seven routes to death.

Pesticides prevent, destroy, repel or mitigate any pestranging from insects, animals and weeds, to microorganisms such as fungi, molds, bacteria and viruses.

• insect killers (insecticides)

• mold and fungi killers (fungicides)

• weed killers (herbicides)

• slug pellets (molluscicides)

• plant growth regulators

• bird and animal repellents

• rat and mouse killers (rodenticides)

• antimicrobials

• inert ingredients

Pesticides help to manage and prevent pests that spread disease, that damage crops, buildings, and other property, and that are a public nuisance. 2

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Pesticidal Classes- Fungicides

• Acylalanines and Oxazolidinones• Benzimidazoles and Thiophanates• Carboxamides• Methoxyacrylate and Oximinoacetate (Strobilurins)• Organotin Compounds • Anilinopyrimidines• Phenylpyrroles• Dicarboximides• Demethylase Inhibitors• Inorganic Fungicides• Dithiocarbamates and Ethylenebisdithiocarbamates• Phthalimides• Chloronitriles

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Pesticidal Classes- Insecticides • Carbamates: AChE Inhibitors• Organophosphorus Insecticides: AChE Inhibitors • Cyclodiene Organochlorines: GABA Antagonists • Organochlorines: Sodium Channel Modulators • Pyrethroids: Sodium Channel Modulators • Nicotine and Neonicotinoids: Acetylcholine Receptor Agonists• Spinosyns: Acetylcholine Receptor Agonists• Avermectins and Milbemycin: Chloride Channel Activators • Juvenile Hormone Mimics and Selective Feeding Blocker • Phenyltetrazines/Aminotriazines: Larvacides/ Molt Disruptors • Delta-Endotoxins Derived from Bacillus thuringiensis• Benzoylureas: Chitin Synthesis Inhibitors • Diacyhydrazine: Ecdysone Agonists • Octopaminergic Agonists and Monoamine Oxidase Inhibitors • Respiratory Inhibitors and Uncouplers• Pheromones

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Pesticidal Classes- Herbicides • Acetyl–CoA Carboxylase Inhibitors • Aryloxyphenoxypropionates• Cyclohexanediones• Acetolactate Synthase Inhibitors• Sulfonylureas• Imidazolinones• Triazolopyrimidines• Pyrimidinylthiobenzoates• Inhibition of Photosynthetic Electron

Transport• Triazines and Triazinone• Uracils and Pyridazinones• Ureas• Nitriles• Benzothiadiazoles and Phenylpyridazine• Bipyridyliums• Protoporphyrinogen Oxidase Inhibitors

• Diphenyl Ethers• N-Phenylphthalimides, Thiadiazoles,

and Triazolinones• Oxadiazole and Pyrimidindione

Herbicides• Bleaching Herbicides• Pyridazinones• Triketones and Isoxazoles• Triazoles and Isoxazolidinones• Synthase Inhibitors• Dinitroaniline Microtubule Assembly

Inhibitors• Chloroacetamide Inhibitors of Very-

Long-Chain Fatty Acid Synthesis• Cellulose and Lipid Synthesis

Inhibitors• Synthetic Auxin Mimics (Phenoxy,

Benzoic, and Pyridine Acids)• Herbicides with Unknown

Mechanisms of Action

Complexities of the Nomenclature: Example

Stenersen, J. Chemical pesticides: Mode of Action and Toxicology. CRC Press 2004

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Compendium of Pesticide Common Names

http://www.alanwood.net/pesticides/

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Pesticide Mass

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Pesticide Toxicity

Toxicity = Hazard = Dose x Response

Environmental Toxicity = Human + Ecological

• acute• chronic• cancer• mutagenicity• reproductive• endocrine• neurotoxicity• immunotoxicity

• aquatic• avian• terrestial• honey bee• plant• worms• ecosystems

• Disturbance in energy production• Inhibition of photosynthesis• Free radical generation & SH-group reactivity• Interference with cell division• Inhibition of nucleic acid synthesis• Inhibition of enzymes:

Ergosterol synthesisAmino acid synthesisChitin synthesisCholinesterase

• Hormone-like and behavior-modifying agents

Modes of action of pesticides:

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Pesticide Exposures

• Routes– Oral (mouth and digestive system)– Dermal (skin)– Inhalation (nose and respiratory system)

• Pathways– Food and water– Residential and non-occupational

• External to Internal– ADME modifiers

• Aggregate– 1996 Food Quality Protection Act (FQPA)– multiple pathways and routes of exposure– http://www.epa.gov/opp00001/trac/science/aggregate.pdf

Risk = Hazard x Exposure

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Pesticide Regulation• Federal Insecticide, Fungicide, and Rodenticide Act

(FIFRA, 1947) administered by USDA• Federal Food, Drug, and Cosmetic Act (FFDCA, 1954)

established pesticide tolerances on food– Delaney Clause, forbade the use of carcinogens as food

additives• Food Quality Protection Act (FQPA, 1996) reauthorized

FFIFRA provisions– Tolerances reassessed as part of reregistrations– single, health-based standard– aggregate risk from all routes of non-occupational exposure– evaluating endocrine effects– extra tenfold uncertainty factor for children/in utero

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Vulnerability of Children

Greater exposure• On a caloric consumption:body-weight ratio

Children are 2.5x adults. Diet less varied (fruit and milk)

• Hand to mouth activity• Skin surface area per body weight is double

that of an adult• Rate of respiration

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Vulnerability of Children

Greater physiological susceptibility• Period of rapid development of nerve cells• Loss of organ function can be permanently

imprinted• Absorption and elimination of pesticides• Metabolizing enzymes not fully developed

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Pesticide Testing- US EPAHarmonized Test Guidelines

810 - Product Performance Test Guidelines830 - Product Properties Test Guidelines835 - Fate, Transport and Transformation Test Guidelines840 - Spray Drift Test Guidelines 850 - Ecological Effects Test Guidelines860 - Residue Chemistry Test Guidelines870 - Health Effects Test Guidelines

Test Guidelines/Acute Toxicity - Acute Oral Toxicity Up-And-Down-Procedure875 - Occupational and Residential Exposure Test Guidelines880 - Biochemicals Test Guidelines885 - Microbial Pesticide Test Guidelines890 - Endocrine Distruptor Screening Program Test Guidelines

http://www.epa.gov/opptsfrs/home/guidelin.htm

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Pesticide Testing-Health Effects Test Guidelines

Group A – Acute Toxicity Test Guidelines870.1000 - Acute Toxicity Testing--Background (December 2002) (PDF) (9 pp, 23K)

870.1100 - Acute Oral Toxicity (December 2002) (PDF) (10 pp, 27K)870.1200 - Acute Dermal Toxicity (August 1998) (PDF) (10 pp, 24K)870.1300 - Acute Inhalation Toxicity (August 1998) (PDF) (11 pp, 21K)870.2400 - Acute Eye Irritation (August 1998) (PDF) (8 pp, 22K)870.2500 - Acute Dermal Irritation (August 1998) (PDF) (8 pp, 23K)870.2600 - Skin Sensitization (March 2003) (PDF) (9 pp, 29K)

Group B – Subchronic Toxicity Test Guidelines870.3050 - Repeated Dose 28-Day Oral Toxicity Study in Rodents (July 2000) (PDF) (17 pp, 29K)

870.3100 - 90-Day Oral Toxicity in Rodents (August 1998) (PDF) (13 pp, 33K)870.3150 - 90-Day Oral Toxicity in Nonrodents (August 1998) (PDF) (12 pp, 30K)870.3200 - 21/28-Day Dermal Toxicity (August 1998) (PDF) (15 pp, 36K)870.3250 - 90-Day Dermal Toxicity (August 1998) (PDF) (14 pp, 34K)870.3465 - 90-Day Inhalation Toxicity (August 1998) (PDF) (17 pp, 40K)870.3550 - Reproduction/Developmental Toxicity Screening Test (July 2000) (PDF) (13 pp, 89K)870.3650 - Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000) (PDF) (17 pp, 89K)870.3700 - Prenatal Developmental Toxicity Study (August 1998) (PDF) (11 pp, 126K)870.3800 - Reproduction and Fertility Effects (August 1998) (PDF) (14 pp, 35K)

Group C – Chronic Toxicity Test Guidelines870.4100 - Chronic Toxicity (August 1998) (PDF) (18 pp, 44K)

870.4200 - Carcinogenicity (August 1998) (PDF) (17 pp, 41K)870.4300 - Combined Chronic Toxicity/Carcinogenicity (August 1998) (PDF) (20 pp, 49K)

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Pesticide Testing-Health Effects Test Guidelines (cont.)

Group D – Genetic Toxicity Test Guidelines870.5100 - Bacterial Reverse Mutation Test (August 1998) (PDF) (13 pp, 36K)

870.5140 - Gene Mutation in Aspergillus nidulans (August 1998) (PDF) (7 pp, 18K)870.5195 - Mouse Biochemical Specific Locus Test (August 1998) (PDF) (7 pp, 19K)870.5200 - Mouse Visible Specific Locus Test (August 1998) (PDF) (6 pp, 16K)870.5250 - Gene Mutation in Neurospora crassa (August 1998) (PDF) (6 pp, 17K)870.5275 - Sex-linked Recessive Lethal Test in Drosophila melanogaster (August 1998) (PDF) (6 pp, 16K)870.5300 - In vitro Mammalian Cell Gene Mutation Test (August 1998) (PDF) (14 pp, 37K)870.5375 - In vitro Mammalian Chromosome Aberration Test (August 1998) (PDF) (13 pp, 33K)870.5380 - Mammalian Spermatogonial Chromosomal Aberration Test (August 1998) (PDF) (11 pp, 28K)870.5385 - Mammalian Bone Marrow Chromosomal Aberration Test (August 1998) (PDF) (11 pp, 28K)870.5395 - Mammalian Erythrocyte Micronucleus Test (August 1998) (PDF) (12 pp, 31K)870.5450 - Rodent Dominant Lethal Assay (August 1998) (PDF) (6 pp, 15K)870.5460 - Rodent Heritable Translocation Assays (August 1998) (PDF) (7 pp, 17K)870.5500 - Bacterial DNA Damage or Repair Tests (August 1998) (PDF) (7 pp, 18K)870.5550 - Unscheduled DNA Synthesis in Mammalian Cells in Culture (August 1998) (PDF) (7 pp, 18K)870.5575 - Mitotic Gene Conversion in Saccharomyces cerevisiae (August 1998) (PDF) (6 pp, 16K)870.5900 - In vitro Sister Chromatid Exchange Assay (August 1998) (PDF) (7 pp, 18K)870.5915 - In vivo Sister Chromatid Exchange Assay (August 1998) (PDF) (6 pp, 15K)

Group E – Neurotoxicity Test Guidelines870.6100 - Acute and 28-Day Delayed Neurotoxicity of Organophosphorus Substances (August 1998) (PDF) (10 pp, 27K)

870.6200 - Neurotoxicity Screening Battery (August 1998) (PDF) (13 pp, 32K)870.6300 - Developmental Neurotoxicity Study (August 1998) (PDF) (14 pp, 35K)870.6500 - Schedule-Controlled Operant Behavior (August 1998) (PDF) (8 pp, 20K)870.6850 - Peripheral Nerve Function (August 1998) (PDF) (9 pp, 22K)870.6855 - Neurophysiology Sensory Evoked Potentials (August 1998) (PDF) (14 pp, 35K)

Group F – Special Studies Test Guidelines870.7200 - Companion Animal Safety (August 1998) (PDF) (10 pp, 23K)

870.7485 - Metabolism and Pharmacokinetics (August 1998) (PDF) (14 pp, 34K)870.7600 - Dermal Penetration (August 1998) (PDF) (14 pp, 37K)870.7800 - Immunotoxicity (August 1998) (PDF) (13 pp, 34K)

Group G – Health Effects Chemical-Specific Test Guidelines870.8355 - Combined Chronic Toxicity/Carcinogenicity Testing of Respirable Fibrous Particles (July 2001) (PDF) (17 pp, 181K)

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http://www.epa.gov/opp00001/reregistration/status.htm

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What is the acute and chronic Point-of-Departure for pesticide toxicity?

Reference Dose (RfD) = NOAEL x UF

26ToxRefDB website: http://www.epa.gov/ncct/toxrefdb/

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In Vivo Toxicity Data in ToxRefDB

Chronic/CancerMultigenationDevelopmental

Che

mic

als

30 years and more than $2B worth of data

Martin et al 2009a,bKnudsen et al 2009

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Chronic/Cancer Toxicity Profiling

http://www.ehponline.org/members/2008/0800074/0800074.pdf

Liver & ThyroidToxicants

Spleen & KidneyToxicants

29

Chronic Rat & Mouse Endpoints

30

Reproductive Toxicity Profiling

http://toxsci.oxfordjournals.org/cgi/reprint/kfp080

Systemic Toxicity&

Delayed SexualMaturation

DecreasedReproductivePerformance

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ReproductiveEndpoints

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SOURCE: Knudsen et al. (2009) Reproductive Toxicology (in press) DOI 10.1016/j.reprotox.2009.03.016Also see Abstract #16

in vivo endpoints (target, description)www.epa.gov/ncct/toxrefdb

ToxRefDB 387 chemicals, 751 prenatal studies,988 effects annotated (enhanced DevTox.org)

283 chemicals x 293 effects 19 targetsystems from rat ( ) and rabbit ( ) studies

Profiling developmental toxicity

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Is a pesticide carcinogenic?

• Genotoxic?• Non-genotoxic?• Human relevance?

34

Rat Liver Histopathologyfrom Chronic Bioassays

68

37

21

No PathologyProliferative LesionsPre-neoplastic LesionsNeoplastic Lesions

N = 248 Chemicals

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Chemicals Evaluated for Carcinogenic Potential by US EPA

http://www.epa.gov/pesticides/carlist/

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Is a pesticide an endocrine disruptor?

• Estrogenic• (anti) Androgenic• Thyrotoxic• Other

37

Panzica et al 2005

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http://www.epa.gov/endo/pubs/regaspects/testorders.htm

39

Reproductive and

Endocrine Organ

Toxicity Endpoints

from ToxRefDB

40

What is a hit?

41

HISTORY OF DDT1,1,1-trichloro-2,2-bis-(p-chlorophenyl) ethane

• WWII – DDT was used by the allies to suppress a typhus epidemic in Naples

• 1943-1944 DDT was applied directly to the head of humans to control lice

• Success with DDT hastened the development of aldrin, dieldrin, endrin, chlordane, benzene hexachloride etc.

DDT was discovered to be an insecticide in 1939 by Paul Muller. He was a scientist working for Geigy, a Swiss firm that was focused on the chemical development of agricultural insecticides. Products with DDT entered the Swiss market in 1941. Seven years later, in 1948, Muller received the Nobel Prize for medicine and physiology in recognition for the lives DDT saved.

DDT• DDT can take more than 15 years to break down• Found in animals far from where they were it is used • Bio-accumulates in fish and marine mammals. Found concentrations in

these animals are many thousands of times higher than levels in water

• DDT can be absorbed by some plants and by animals and humans whoeat those plants

• DDT is fat-soluble and is stored in adipose tissues of humans and animals

CURRENT STATUS:• No US registration, most uses cancelled in 1972, all uses by 1989• No US production, import, or export• DDE (metabolite of DDT) is regulated as a hazardous air pollutant

(Clear Air Act)• Priority toxic pollutant (Clean Water Act)

HUMAN EXPOSURE FROM:• Eating contaminated fish and shellfish • Eating imported food exposed to DDT • Infant exposed through breast milk • Eating products from crops grown in contaminated soil

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Insecticide advantages of DDT• Low volatility• Chemical stability• Lipid solubility• Slow rate of biotransformation and degradation

Disadvantages of DDT• Persistence in the environment• Bioconcentration• Biomagnification in food chain• Profound effects on wild life (“Silent Spring”)

Health Effects of DDT• Paresthesia of tongue, lips, and

face• Irritability, dizziness, vertigo,

tremor, and convulsions• Hypersusceptibility to external

stimuli (light, touch, and sound)

• Hypertrophy of hepatocytes• Hepatic tumors• No epidemiological evidence linking DDT

to carcinogenicity in humans• Low rate of absorption through the skin• Human health effects minor

44Klaassen, CD. CASARETT AND DOULL's Toxicology: The Basic Science of Poisons. McGraw-Hill 2001

45

Sites of DDT poisoning

Klaassen, CD. CASARETT AND DOULL's Toxicology: The Basic Science of Poisons. McGraw-Hill 2001

46

Is a pesticide a developmental neurotoxicant?

47

Inhibition of choline esterase or action potential

• Organochlorine Insecticides

• Organophosphate Insecticides

• Carbamates

• Pyrethroidinsecticides

• Botanical Insecticides

Klaassen, CD. CASARETT AND DOULL's Toxicology: The Basic Science of Poisons. McGraw-Hill 2001

• Most chemical insecticides act by poisoning the nervous system of the target organisms

• CNS of insects are highly developed and similar to that of the mammal

• Chemicals that act on the insect nervous system may have similar effects on higher forms of life

Stenersen, J. Chemical pesticides: Mode of Action and Toxicology. CRC Press 2004

49

General Modes of Action

Pesticides acting on the axon (impulse transmission):

• Interference with transport of, Na+, K+, Ca2+, or Cl- ions

Pesticides acting on synaptic transmission:

• Inhibition of specific enzyme activities:GABA-ergic (inhibitory) synapsesCholinergic synapses

• Contribution to the release or persistence of chemical transmitters at nerve endings

Stenersen J, Chemical Pesticides Mode of Action and Toxicology, CRC Press 2004

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PON1 polymorphism:PON1 – human serum paraoxonase, enzyme important for lipid metabolisms that is also involved in metabolism of organophosphate compounds

From: Hulla et al. Toxc. Sci. (1999)

PON1R192 PON1Q192

Rapid hydrolysis of paraoxon

Rapid hydrolysis of sarin, soman,

diazoxon

Two-dimensional enzyme analysis to characte-rize PON1 polymorphisms in human population: analyze hydrolysis of PON1 substrates, diazoxone vs. paraoxone (panel c)O PON1Q192/PON1Q192 (glutamine)■ PON1Q192/PON1R192 (glu/arg) PON1R192/PON1R192 (arginine)

51

Pyrethroid Insecticides

• Newest class of insecticides• New analogs will be (hopefully):

– More stable in light and air– Better persistence– Low mammalian toxicity

Soderlund et al. (2002)

Importance of Structure-Activity-Toxicity

Relationships

Soderlund et al. (2002)

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Pyrethroid Use

• Household sprays• Flea preparations for pets• Plant sprays for home• Plant sprays for greenhouses

• Similar to DDT• Not highly toxic in animals• Toxic ingredients

– Chrysanthemic acid– Pyrethric acid

Pyrethroid Poisoning

Figure 1. Nine neonicotinoidinsecticides and four nicotinoids.The neonicotinoids are nitromethylenes(C==CHNO2), nitroguanidines(C==NNO2), and cyanoamidines(C==NCN). Compounds with 6-chloro-3-pyridinylmethyl, 2-chloro-5-thiazolylmethyl, and 3-tetrahydro-furanmethyl moieties are referred to as chloropyridinyls (or chloronicotinyls),chlorothiazolyls (or thianicotinyls), and tefuryl, respectively. The nicotinoidsare naturally occurring [(−)-nicotine and (−)-epibatidine] and synthetics (ABT-594 and desnitroimidacloprid).

Tomizawa & Casida (2004)

Tomizawa & Casida (2004)

Tomizawa & Casida (2004)

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Developmental NeurotoxicityBasic Clin Pharmacol Toxicol. 2008 Feb;102(2):228-36.Pesticide toxicity and the developing brain.Eskenazi B, Rosas LG, Marks AR, Bradman A, Harley K, Holland N, Johnson C, Fenster

L, Barr DB.Center for Children's Environmental Health Research, School of Public Health, University

of California, Berkeley, CA 94704, USA. eskenazi@berkeley.eduOrganochlorine pesticides are used in some countries for malaria control and

organophosphate pesticides are widely used in agriculture and in homes. Previous literature documents children's exposure to these chemicals both in utero and during development. Animal studies suggest that many of these chemicals are neurodevelopmental toxicants even in moderate doses, but there are few studies in human beings. Associations of children's pesticide exposure with neurodevelopment from studies being conducted worldwide are summarized. In addition, we present the work of the CHAMACOS study, a longitudinal birth cohort study of Mexican-American children living in the Salinas Valley of California. In this study, we investigated the relationship of children's neurodevelopment with maternal dichlorodiphenyltrichloroethane and dichlorodiphenyldichloroethylene serum levels, as well as prenatal and child organophosphate urinary metabolite levels. We have examined the association with children's performance on the Brazelton Neonatal Assessment Scales and at 6, 12 and 24 months on the Bayley Scales of Infant Development (mental development and psychomotor development) and mothers report on the Child Behaviour Checklist. We observed a negative association of prenatal dichlorodiphenyltrichloroethane exposure and child mental development. We also observed adverse associations of prenatal but not postnatal organophosphate pesticide exposure with mental development and pervasive developmental disorder at 24 months.

58

59

Biotransformation of XenobioticsThe M in ADME (absorption, distribution, metabolism and excretion)

• Metabolism of xenobiotics in liver is defense against lipophiliccompounds accumulating in body

• Nuclear Receptors coordinate body’s defense against these compounds

• Biotransformation: Phase I, II, III– Phase I: functionalization reaction

• Cytochrome P450 Monooxygenase System• Microsomal• Major reaction is oxidative• Minor reaction is reductive• May result in metabolic activation

– Phase II: conjugation reaction• Glucuronidation, sulfation, acetylation• Cytoplasmic• Increases water solubility

– Phase III: transporter activity• Basolateral and bile canalicular expression• ATP-dependent export pumps

60

Importance of Biotransformation

ER radioligandbinding assay

ER cellular (HEK293) transactivationassay

Parent/Metabolite

-3 -2 -1 0 1 2-20

0

20

40

60

80

100

120

Methoxychlor (human ER)

HPTE (human ER)Methoxychlor (bovine ER)

HPTE (bovine ER)

BottomTopLogIC50HillSlopeIC50

HPTE (hER)2.448= 100.0-1.349-0.91970.04476

HPTE (bER)1.725= 100.0-1.697-0.98770.02009

Conc (log M)

% o

f Inh

ibiti

on

Parent/Metabolite

-3 -2 -1 0 1 2-20

0

20

40

60

80

100

BottomTopLogEC50HillSlopeEC50

Methoxychlor0.301741.860.57145.4663.727

HPTE-3.91235.70-0.71312.2580.1936

MethoxychlorHPTE

Conc (log M)

% o

f Con

trol

61

NR Regulation of Metabolic

Enzymes

Phase I Phase II Phase IIIXenobiotics AhR XRE CYP1A1(+) UGT1A1(+) ABCG2(+)

CYP1A2(+) UGT1A6(+)

CYP1B1(+)

Xenobiotics CAR DR-3, DR-4, DR-5 CYP2A6(+) UGT1A1(+) ABCC2(+)

Phenobarbital SR-6, ER-6 CYP2B1(+) ABCC3(+)

CYP2B6(+) ABCC4(+)

CYP2C9(+)

CYP2C19(+)

Xenobiotics SXR/PXR DR-3, DR-4, DR-5 CYP1A2(+) SULT2A1(+) ABCA1(+)

Steroids ER-6, ER-8 CYP2B6(+) UGT1A1(+) ABCB1(+)

CYP2C9(+) UGT1A3(+) ABCB11(+)

CYP2C19(+) UGT1A4(+) ABCC1(+)

CYP3A4(+) ABCC2(+)

CYP3A7(+) ABCC3(+)

CYP7A1(-) ABCG2(+)

CYP3A(+)

Bile Acids FXR IR-1 CYP7A1(-) UGT2B4(+) ABCB4(+)

DR-1 CYP8B1(-) SULT2A1(+) ABCB11(+)

ABCC2(+)

Oxysterols LXR DR-4 CYP2B6(-) ABCA1(+)

CYP3A4(-) ABCG1(+)

ABCG4(+)

ABCG5(+)

ABCG8(+)

Fatty acids PPAR DR-1 CYP4A1(+) UGT1A9(+) ABCA1(+)

Fibrates CYP4A3(+) UGT2B4(+) ABCC2(+)

CYP7A ABCD2(+)

ABCD3(+)

Fatty acids PPAR DR-1 CYP4A(+) UGT1A(+) ABCA1(+)

Carboprostacyclin

Eicosanoids PPAR DR-1 CYP4AB(+) UGT1A(+) ABCA1(+)

Thiazolidinediones ABCG2(+)

1,25(OH)- VDR DR-3 CYP2B6(+) SULT2A1(+) ABCC2(+)?vitamin D3 ER-6 CYP2C9(+)

IR-0 CYP3A4(+)

Glucocorticoids GR GRE CYP2C9(+)

CYP2B6(+)

Ligand NR Response ElementTarget Gene

Phase I Phase II Phase IIIoxidation conjugation transportersreduction glucuronic acidhydrolysis sulfationcyclization glutathione

decyclization

62

Cytochrome P450/CYP

• Heme-containing proteins

• RH+O2+H++NADPH → ROH+H2O+NADP+

Medscape

63

Phase II Metabolism• Glutathione S-transferases • Mercapturic acid biosynthesis • UDP-Glucuron(os)yltransferases• N-Acetyltransferases• Amino acid N-acyl transferases • Sulfotransferases

64

Phase III: Transporters

Bile Canaliculus

65

PXR Ligands

• Estimated that 50% of drugs are PXR ligands

• Many environmental chemicals are PXR ligands

• Most promiscuous of the NRs

• Large, flexible binding pocket

66

EnvironmentalChemicals

Molecularresponse

Cellularresponse

Tissueresponse

Cell fate

Proliferation

DeathApoptosisNecrosis

AdverseOutcome

Hyperplasia

Tumor

Cancer

Chemicals

PesticidesConazolesPyrethroids

ToxicsDE-71PCBsPhthalatesPFOA/PFOS

NR-sig Gene-reg. Transcription

CARPXRPPAR

cis-reg.trans-reg.

Xen. Met.Phase I

Phase II

Phase III

Molecular Response (Early)

NR activators stimulate intracellular processes that lead to hyperplasiaChronic stimulation increases the risk of neoplasms

Focus On a Mode of Action …