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ESRP 531 Fundamentals of Environmental Toxicology Fall 2005 September 26 2005 Lecture 9 Endocrine System Effects Part 111 Case Studies 1 Case of the Less than Manly Alligators A One of the most publicized cases of endocrine disruption at possibly the population levels comes from research on alligators conducted at the University Of Florida in the lab of L Guillette review of research in Guillette L J Jr et al 2000 Alligators and Endocrine Disrupting Contaminants A Current Perspective American Zoologisi Vol 40 No 3 pp 438452 1 The earliest work leading to the hypothesis that the alligator endocrine system was being affected by contaminants in water was published in 1994 Guillette et al 1994 Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida Environ Health Perspectives 102680 a This study suggests that at high enough environmental levels feminization of alligator eggs can occur this study is an example of eco epidemiology but over the many years that Guillette et al have been studying the phenomenon they have used a combination of experimental lab studies and field monitoring measurements B The Contaminated Environment and Affected Population 1 Guillette et al found in Lake Apopka FL declining alligator populations and evidence of an elevated estrogen to testosterone levels compared to Lake Woodruff a pristine area that is part of a National Wildlife Refuge Figure 1 Juvenile Alligators per km of Shoreline in Florida Lakes 30 25 Lake Griffin 20 Hl 15 K 10 5 Lake Apopka 0 1 1 1 1 1 1 1 1980 1981 1982 1983 1984 1985 1986 1987 Year Figure 1 Population abundance of juvenile alligators in two Lakes in central Florida Lake Griffin is another reference lake as is Lake Woodruff where alligator populations seemed normal in abundance Guillette et al 1994 2 Lake Apopka which is in central Florida is adjacent to a EPA Superfund site listing due to a pesticide spill in 1980 from the Tower chemical Company and receives agricultural drainage ESRP531 Lecture 9 CaseStudydoc Page 1 of 11 ESRP 531 Fundamentals of Environmental Toxicology Fall 2005 a The lake also receives treated sewage outfall from the city of Winter Garden Note the case of the surfactant degradation product nonylphenol below and its effects on fish b The pesticide spill involved dicofol an insecticidally active ingredient contaminated with DDT and its isomers Note the similarity in structure The DDT is a byproduct of the manufacturing process and in the environment it rapidly oxidizes to DDE 03 39c Ec13 H U C Cl Cl HO Cl Cl p p DDT Dicofol c1 ltc13 ltc12 lt3HC12 c c H H C1 c1 c1 c1 c1 op397DDT DDE DDD Figure 2 DDT occurs is several isomeric forms note op DDT In the environment DDT is rapidly oxidized to DDE Under reducing conditions which is characteristic of low oxygen waters especially in the sediment layer DDT is reduced to DDD Dicofol which is used as an acaricide rather than an insecticide is more readily biodegradable than DDT by virtue of being hydroxylated at the bridge carbon atom 3 In the 1990 s alligator populations began to rebound in Lake Apopka but not to the former levels before the pesticide spill a The rebound was hypothesized to be due to juvenile alligator recruitment ie greater survival to the juvenile stage as a result of an increase in clutch survivability 4 Other studies had shown that alligator eggs from Lake Apopka contained comparatively high levels of several DDTr an indication of DDT plus metabolites and isomers residues as well as other chlorinated hydrocarbon pesticides Figure 3 a Heinz et al 1991 Contaminants in American alligator eggs from Lake Apopka Lake Griffin and Lake Okeechobee Florida Environ Monitoring amp Assessment 16 277 reported concentrations of DDE ranging from 32 81 ppm wet weight basis b Bear in mind that alligator eggs from other reference sites which were supposedly not as contaminated also had measurable pesticide residues but there seemed to be no effect on juvenile alligator abundance ESRP531 Lecture 9 CaseStudydoc Page 2 of 11 ESRP 531 Fundamentals of Envlronmemal Toxlcology Fall 2005 Plasma Concentration Hgml I Dlel ml Endrm I Mllex oxycmotdane D transNouachlnr olp eDDT D Total PCBS plp rDDE Flgure 3 C mnan n tn luvenlle allrgator collected In Florlda Note that transenonachlor and oxychlordane are contarntnants m the now banned Er mluclde chlordane structure of chlord ne shown b w They are also envlronmental breakdown produc awa at Flond r tate wth trern s ter te problems and the envlronmentally persrstent chlordane wasused lustabouteverywhere stnce the 195039s Graph taken from Gurllette et al 2000 Cl Cl Cl C or C 0 Cl Cl Cl Cl Cl C 0 Cl Cl Cl Cl 0 Cl Cl Cl or Cl C C mirex dieldrin chlordane c pp 7 D er contarntnants reflected use ln the 1 r e ple the rnostcornrnonly used terrntucrde pnor of regl uon b e 199039s waschlordanel dleldrln and a related compound heptachlor a class known as chlonnated c lenesmade good lnse a plled to sol buLLhey are hlghly recalcrtrant and tend to be Very hydmphoblc They re um N The mostreValenLContammantm alllgator sera from Lake Apopka was 39 southeastern U s to suspenslon whlch also contalned yclodlenes cucldes espec LO lotransfo mane versally banne U N Lreaty and are classlfled as POPS PerslsLenLOrganlc Pollutants ESRP531 Lecture 9 CaseSmdy doc Page3 of 11 ESRP 531 Fundamentals of Environmental Toxicology Fall 2005 3 Another compound found in alligator sera was uniquely used in the southeast U S to control fire ants Mirex a 107carbon box structure is also highly chlorinated and classified as a POP C Benchmarks of Toxicity Guillette et al observed morphological changes in male reproductive tissue that suggested feminization of male gonads One of the morphological benchmarks was a comparatively reduced penis size Figure 4 a A graph of the relationship between alligator body mass and SVL showed no differences in populations lines were nearly coincidental Thus weight of the alligator was not a confounding factor that could explain differences in genitalia relative to size 20 7 7 i 7 7 N t O Apopka 3 D Wna l llquot Penis Tip Length mm m 30 an so 60 70 so 90 SVL cm Figure 4 The relationship of penis tip length to snout7vent length SVL in juvenile al igators One would expect that as SVL gets bigger the penis would get proportionally bigger also Note that the penis size is proportionally smaller in the Lake Apopka alligators compared to the Lake Woodruff population Graph is from Guillette 2000 3 Another benchmark of toxicity was the level of sex hormones found in the alligator blood a The potential feminization when males have female7like characteristics was an observation consistent with elevated levels of estrogen in males ESRP531 Lecture 9 CaseStudydoc Page 4 of 11 ESRP 531 Fundamentals of Environmental Toxicology Fall 2005 from Lake Apopka compared to control lakes ie Lake Woodruff the normal sex hormone ratio for males should be 11 Figure 5 b Thus one of the first hypothesis was that the lake contaminants which were found not only in the alligator eggs but also in the blood were mimicking estrogen 1 2 I Lake Apopka Lake Wood ruff Ratio ET Males Females Figure 5 Hormone ratios in alligators collected from Lake Apopka and compared to the reference site Lake Woodruff Lake Apopka had about a 391 y 4 estrogentestosterone level but normal for males should be 11 Guillette et al 1994 4 Other benchmarks of toxicity come from laboratory studies Guillette et al physiology of reptiles note that this phenomenon also occurs in fish to control sex determination in laboratory experiments a When alligators are exposed to cool temperatures 26 730 C females predominate Above 30 C the proportion of males increases and at 32 nearly all eggs turn out to be male Alligator eggs were experimentally exposed to different concentrations of pp39rDDE and op39rDDE as well as estradiol the natural estrogen as a quot Thee 39 L J 7 roduci 7 tempeiat e Doses of 01 and 03 mgkg egg mass did not affect the sex ratio all males hatched from the eggs However at doses of 1 3 and 10 mgkg egg mass the ratio of male to females was 11 ie 50 females hatched at a male produci g temperature 39 4 b with turtles and other compounds including DDE Sex reversal was observed following in ESRP531 Lecture 9 CaseStudydoc Page 5 of 11 ESRP 531 Fundamentals of Environmental Toxicology Fall 2005 ovo exposure to PCB hydroxylated metabolites nonachlor chlordane and d However Guillette et al have found a male biased sex ratio on Lake Apopka contaminated as well as on the comparatively uncontaminated Lake Woodruff Thus the ecological finding is not predicted by the results of laboratory experiments nor consistent with the reduced levels of testosterone relative to estrogen seen in plasma of Lake Apopka alligators D Given the mixture of pesticide residue contaminants found in alligators from all P1 TU ESRP531 Lecture 9 CaseStudydoc lakes but especially the higher levels found in alligators from Lake Apopka one would logically hypothesize some negative correlation between phallus morphology ie size and tissue contaminants 1 Although phallus size is smaller in alligators from Lake Apopka then in alligators from other lakes and testosterone levels are also lower Guillette et al 1999 were unable to show a relationship between phallus size and serum contaminant levels in juvenile alligators However the lower testosterone levels in juvenile alligators is correlated with smaller phallus size a Guillette explained their seemingly incongruent findings ie incongruent to the idea that the contaminants are causing endocrine disruption and therefore a correlation between residues and effect should be expected by concluding that current serum levels are essentially irrelevant Instead the observed diminished phallus sizes and testosterone levels of Lake Apopka alligators could be due to exposures during embryonic development to DDE or various other contaminants In another study Guillette concluded that other pesticide contaminants namely EDB ethane dibromide an agricultural fumigant with fungicidal and herbicidal properties could also be a factor in the shrinking alligator genitalia and populations 1 Furthermore and perhaps more importantly Guillette et al 2000 have emphasized that hormone mimicry may be only one of several other mechanisms for causing endocrine disruption In hypothesizing a mechanism for the male biased sex ratio mentioned above Guillette et al reported that mixture of DDE and trans nonachlor a component of the chlordane termiticide formulation did not cause sex reversal in treated alligator eggs but trans nonachlor alone did cause reversal a Interestingly pp DDE does show an affinity for the alligator estrogen receptor but unlike rats and humans wherein DDE shows an affinity for the androgen receptor there is no data on alligators b Thus Guillette hypothesize that pp DDE is a mixed function ligand hoving hormonal and anti hormonal action depending on the environment ie temperature hormonal milieu in which it resides Guillette 2000 Guillette et al have also examined whether the thyroid gland or thyroid hormones are different in Lake Apopka alligators then in alligators from other Florida lakes Crain A D et al 1998 Sexisteroid and thyroid hormone concentrations in juvenile alligators alligator mississippiensis from contaminated and reference lakes in florida USA Environmental Toxicology and Chemistry 17 34467452 Hewitt E A D A Crain M P Gunderson and L J Guillette Jr 2002 Thyroid staus in juevnile alligators alligator N Page 6 of 11 ESRP 531 Fundamentals of Environmental Toxicology Fall 2005 mississippiensis from contaminated and reference sites on lake Okeechobee florida USA Chemosphere 47 112971135 1 Crain et al 1998 found that whereas there were no differences in plasma E2 concentrations among animals of three lakes Lake Apopka Lake Woodruff and Lake Okeechobee male alligators from the contaminated lakes Lake Apopka and Lake Okeechobee had significantly lower plasma T concentrations compared to males from the reference lake Lake Woodruff 2 In another study Hewitt et al 2002 examined the thyroid gland and T4 hormone levels in alligators from three sites at Lake Okeechobee of varying degrees of contamination a They found significant differences in one histological benchmark epithelial cell widths b Thyroxine T4 concentrations were elevated in alligators from the site considered of intermediate contamination G In summary the Guillette et al studies represent a combination of in vitro in vivo and ecoepidemiological studies 1 Lake Apopka alligators and perhaps alligators from some other contaminated lakes eg Lake Okeechobee exhibit unusual male genitalia morphology which seems associated with comparatively low testosterone levels 2 Although it has been hypothesized that contamination by waste pesticides or pesticide residue runoff is causing these abnormalities tissue residue monitoring is not positively correlated with genitalia size nor with testosterone levels 3 Newer evidence suggests that the thyroid gland and hormones are exhibiting abnormal physiology in alligators from Lake Apopka and Lake Okeechobee 4 Finally current sera levels of pesticide residues may be irrelevant because the prevailing hypothesis is that exposure of the embryo may be responsible for morphological and hormonal abnormalities as a result of the organizational effects of hormonally active agents 11 The Case of the Sexually Confused Fish A Several research groups have shown that male fish caged near sewage outfalls have the unusual female characteristic of vitellogenin in the blood Jobling S and J P Sumpter 1993 Detergent components in sewage effluent are weakly oestrogenic to fish An in vitro study using rainbow trout Oncorhynchus mykiss hepatocytes Aquatic Toxicology 2723617372 1 Vitellogenin is an egg protein produced by females in the presence of male fish ready to mate a Both females and males are genetically competent to produce vitellogenin but the biochemical process is not turned on in males however males do have a very tiny amount of vitellogenin in the plasma compared to females 2 The working hypothesis that explains this odd benchmark of toxicity is the presence of certain contaminants in the treated sewage outfalls The final degradation product of the surfactant group known as alkylphenol polyethoxylates APEs has been hypothesized as being the main endocrine disrupting chemical The product is nonylphenol ESRP531 Lecture 9 CaseStudydoc Page 7 of 11 ESRP 531 Fundamentals of Environmental Toxicology Fall 2005 ngCgOOH ngcgOCHZCOOH nonylphenol nonylphenoxycarboxylic acid NP1EC H17C8 O OH ngcg O CHZCHZOCHZCHZOH octylphenol nonylphenoldiethoxylate NP2EO Figure 6 Breakdown products of the surfactant group known as alkylphenol polyethoxylates B Toxicity of APEs 1 Acute oral LD50 rats range from 1420 to gt28000 mgkg dermal in rabbits range from gt2000 mgkg to gt 10000 mgkg 2 Negative in carcinogenicity tests not mutagenic 3 3 Ecotoxicity a Freshwater fish LCSO 13 1000 mgL b Crustaceans LC50 29 10000 mgL Endocrine Disrupting Effects 1 At least two groups of researchers one in the US and one in the UK have detected elevated levels of vitellogenin in male fish that have been captured near municipal sewage outfalls a Folmar L C et al 1996 Vitellogenin induction and reduced serum testosterone concentrations in feral male carp Cyprinus carpio captured near a major metropolitan sewage treatment plant 0 b UK Harries et al 1996 A survey of estrogenic activity in United Kingdom inland waters Environ Toxicol amp Chem 15 1993 2002 c What s the big deal about vitellogenin detection in male serum of fish 1 Vitellogenin is a lipophosphoprotein normally synthesized in the female liver under the control of estradiol it is transported by the blood to the ovary where it is taken up into the oocytes during yolk formation a In maturing female fish it is found in the plasma in large amounts up to 100 mgL often constituting over 50 of blood protein 2 Males have very little estradiol thus normally you would not expect to see vitellogenin also why would a male produce it when they have no eggs in which to develop yolk proteins however there does seem to be a detectable background level of about 10 4 g L as described in Harries et al 1996 3 Exposure of males to estrogen has been associated with the production of vitellogenin ESRP531 Lecture 9 CaseStudydoc Page 8 of 11 ESRP 531 ESRP53l Lecture 9 CaseStudy doc 4 Fundamentals of Environmental Toxicology Fall 2005 Thus the presence of vitellogenin in the plasma of a male fish is a very sensitive biomarker of exposure to an estrogenic cherm a Sumpter J P and Jobling S 1995 Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment Environmental Health Perspectives 103 supplement 7 1737178 d Doseiresponse studies and single dose testing of NP OP NPlEC and NPZEO have been studied by Jobling et al 1996 EE2 ethynylestradiol a potent synthetic estrogen was used as a positive control N W U can ETHYNYLESTRADIOL EEZ The potency of the APEO degradation products is perhaps 100000 times less than that of estradiol in stimulating or causing vitellogenesis i e vitellogenin production and reduced testicular size Concentrations of 203 ygL NP and 48 ygL OP were the lowest doses tested that caused vitellogenin production in rainbow trout ie these concentrations were the LOEL The corresponding NOAELs were 50 and 16ygL respectively for NP and OP In these experiments fish were exposed to the indicated concentrations for 3 weeks thus given the actual river water concentrations the LOELs indicate a good probability for a real environmental effect Jobling and Sumpter 1998 used cultured hepatocytes liver cells to test the effects of various alkyl phenols with various alkyl chain lengths Aquatic toxicology 273617372 a They hypothesized that production of vitellogenin was under control of estrogen As in the later studies with fish inrvivo this inevitro study also observed increases in vitellogenin production The estrogenic control of vitellogenin production was confirmed by incubating the antiestrogenic chemical tamoxifen with the hepatocytes in the presence of the alkylphenols Vitellogenin production was suppressed as a result They also noted that the longer the alkyl chain then the less potent was the alkyl phenol in inducing vitellogenin synthesis Figure 7A see OP bar which is octylphenol g P p Page9ofll ESRP 531 Fundamentals of Environmental Toxicology Fall 2005 E a GSI vitellogeninngml ma39 quotquot31 m 0quot quotP NEE mo tnlttal mm 2 op w NPtEE NPon rols Figure 397 Effect of estrogenic alkylphenolic compounds 30 ygL and EE2 2 ngL on the synthesis of vitellogenin and testicular growth in male rainbow trout exposed for 3 weeks NPlEC is nonylphenoxycarboxylic acid NP2EO is quot FF quot 1 i very potent synthetic estrogen The latter two compounds are intermediate breakdown products on the pathway leading to nonylphenol OP is octylphenol The vitellogenin was measured in the blood plasma and testicular growth was measured as gonadosomatic index GSI Jobling et al 1996 Environ Toxicol amp Chem 15 1947202 2 Inevitro studies have shown both NP and OP to have estrogenic activity a Soto et al 1991 pinonylphenol an estrogenic xenobiotic released from modified polystyrene Environ Health Perspectives 92 1677173 b White R et al 1994 Environmentally persistent alkylphenolic compounds are estrogenic Endocrinology 135 1757182 Surnpter who was res onsible for pointing out the estrogenic activity found in sewage ef uents several years ago hypothesized that the alkyl phenols may not be the main culprit in inducing vitellogenin production in fis a In papers published in fall of 1998 his group showed that natural estrogens may be the main culprit that cause endocrine effects in fish near sewage outfalls Desbrow C Routledge E J Brighty G C Sumpter J P and Waldock M Identification of estrogenic chemicals in STW effluent 1 Giemical fractionation and in vitro biological screening 1998 Environ Sci amp Technol 321549 1558 Routledge E Jet al Identification of estrogenic chemicals in STW effluent 2 In vivo responses in trout and roach 1998 ESampT 32 155971565 4 On the other hand research in Spain did not rule out the possible contribution nonylphenol to vitellogenin induction in male carp Sole et al 2000 Environ Sci Technol 34507675083 a Note that male carp collected in the vicinity of STPs showed elevated levels of vitellogenin Compare Table 1 data with data in Figure 8 ESRP531 Lecture 9 CaseStudy doc Page 10 of 11 ESRP 531 Fundamentals of Envrronrnental Toxicology Fall 2005 Table l 60 wmm Mwbm 0000 rdener Anola a lgualada Flgure 8 vltellogenln content is expressed as the percent relauve to a posluve control rnale sh lnjected wth synthetlc estrogen Control sh were not exposed to alkylphenols 5 Regardless of the cause of fernrnrzatron m ml39p collected near sTPs Euro e England France Germany and the Scandmzvlzn countnes have use 1 household cleamng products and l avo unta bano AP tnctrons on rndustnal cleanng appllmuons are set to follow n 2000 lt 111997 ESampT31316A D ls there relevance for human 1 57 l henols be ublqultouslnfood uentheretal 2002 676 6 Found NPs ln all food ltems concentratrons from o l ugkg e 19 pigkg b lntake esurnated at 7 5 yday lnfznts fed breast mllk and formula at o 2 pigday and l 4 yday respectwely Putatwe sour s formulzuon resldues from pesucrde appllcztlons msnonylphenolphosphlte anuoxrdant used ln plasuc packzglng surfactants ln dlslnfectants ESRPSBI Lecture 9 CaseStudy doc Page ll of ll