1ES/RP 531Fundamentals of Environmental ToxicologyLecture 7Hazard ID--Types of StudiesEndocrine System ToxicityPart 1: Overview of Endocrine SystemInstructor: Allan [email protected] 2005Types of Experiments forAssessing Hazard In vitro In vivo EpidemiologicalFor risk assessment, the in vitro and in vivo experimentsgive us a NOEL (or NOAEL) and LOEL (LOAEL).In Vitro Experiments Use of biological material isolated from thewhole organism but kept functional Cell cultures Tissue cultures Enzymes Receptors Measure specific response in relationship toincreasing dose or concentration of toxicantIn Vitro Testing Can estimate an LD50, LC50, ED50, EC50 ifmeasuring a specific endpoint like death orcell numbers, etc. If measuring enzyme function or receptorbinding and function can measure Km (Michaelis Menton affinity constant);concentration at which velocity is 0.5 max Kd (Equilibrium constant for binding) I50 (Concentration inhibiting activity by 50%) Can estimate a NOAEL or NOAECMCF-7 Cell Line:In-Vitro Assay forPotency ofEstrogenic Activityof BenzophenoneDerivativesE2 = estradiol (estrogen)OR1R2In-Vitro Assays for Potency of Estrogenic & AntiandrogenicActivity of Sunscreen Additives 0.300.104’-OH2,4-OH 3.20 >1002’-OH2-OH26.50 31.34’-OH2,3,4-OH11.908.13HH 1.530.302’,4’-OH2,4-OH 4.780.144’-OH4-OHNA<0.001(estradiol)E2AntiandrogenicEC50 (µM)Estrogenic EC50(µM)R2R1from Suzuki et al. 2005Tox. Appl Pharmacol. 203:9OR1R2Benzophenone Derivatives2In Vivo Testing Use whole animals Determine a NOAEL or NOAEC Measure death or sub-lethal responses Use a range of doses Dose range limited to control and 1 - 3 doses Doses tend to be high relative to environmentalexposuresHershberger Assay: In-Vivo Test (with rats) forAnti-Androgenic ActivityEpidemiological Studies Objective: Relate the incidence of a diseaseor condition with exposure to some agent(microbiological, chemical) or activity(lifesyle, behavior, product use, place ofresidence, etc.) Epidemiological studies of chemical effectsare normally applied to chronic exposuresand maladies classified as chronic toxicity Controversial when applied to chemicals Confounding factors (variables that may affect orinfluence the outcome of the results but notcontrollable) For example, if wanted to examine the effect of lowresidues of insecticide on a population of song birds in anarea with urbanization, the confounding factor would behabitat fragmentation Epi studies can only make associations, notconclusions about causation For human applications, doses hardly evermeasured Epi studies good for pathogen causeddiseases (follow Koch’s postulates)Epidemiological StudiesExample Results from an Ecological Epi StudyGeneral Mechanismsof Toxic Action Interference with normal receptor-ligand interactions Can result in neurotoxicity, endocrine toxicity,developmental & reproductive toxicity Interference with membrane functions Interference with cellular energy production Binding to biomolecules Perturbations in calcium homeostasis Toxicity from selective cell loss Nonlethal genetic alterations in somatic cells3Endocrine System Toxicity Over the last 10 years, new hypothesis born(but there were earlier “warnings” Numerous chemicals, both synthetic and naturallyoccurring can affect the normal functioning of theendocrine system Contaminants called endocrine disrupters National Academy of Sciences (NRC) prefers the termshormonally active agents (HAAs) because mechanismof action of the various syndromes seen in wildlife arenot entirely clear as to cause and not necessarilycaused through endocrine system actionEndocrine System Toxicity Effects on endocrine system observed atdoses that are sub-lethal and not associatedwith cellular toxicity Adverse effects observed: Physiological Developmental Reproductive Morphological Behavioral Individuals shown affected, but unclear iflarger scale effects can be shownPopularization ofEndocrine System Effects Theo Colborn et al (1996) Our Stolen Future Premise: Synthetic chemicalsreleased into the environment (andsome natural ones, too) mimichormones of the endocrine system(mainly estroen and testosterone),and because of their wide rangingeffects, essentially threaten humanintelligence, fertility, and survival Wildlife is a sentinel for future effectson humansScientific Roots The idea that synthetic chemicals couldmimic estrogen dates back over 50 years Phytoestrogens in clover affect sheep (‘40s) Organochlorine pesticides affect uterine wetweight (an estrogenic effect) (‘50’s) DES (diethylstilbesterol and effects on malechildren) “Chemically Induced Alterations in SexualDevelopment--the Human/WildlifeConnection” --1992 conference in Racine, WIConsensus Statement from theRacine, WI Conference “A large number of man-make chemicals thathave been released into the environment, aswell as a few natural ones, have the potentialto disrupt the endocrine system of animals,including humans” Note the the evidence for effects in humans wasbased solely on the experience of women usingthe drug DES; Conference attendees believes wildlifepopulations were already affectedHOOHdiethylstilbestrolClClClCl4,4',5,5'-tetrachlorobiphenylOOHO ClClCl2,3,7,8-tetrachlordibenzodioxin (TCDD)Presence of chlorine atoms is not a prerequisite forpotential to affect endocrine physiology(PCB congener)(polyhalogenated benzodioxins)4National Academy of ScienceNational Research Council Rept.Hormonally Active Agents in theEnvironment (1999) Link to effects on humans is weak Except for known effects of the drug,DES Possible that wildlife poulations in someinstances and in some environments haveshown effects from hormone mimicexposures Hypothesis has more plausibility thanthat of effects on humansHormonally Active Agents(Endocrine Disrupters) The new paradigm in hazard id essentially links alladverse effects (rightly or wrongly) to adverse effectson the endocrine system Endocrine system, nervous system, and immunesystem linked with another (like a computer network) This biological “internet” either controls orintegrates all growth and physiological functions Communicate via chemical transmitters Hormones, neurotransmitters, cytokinesWhat Is the Endocrine System? One of three interdependent