Organochlorine compounds in human adipose tissue from north Texas.

This paper reports a preliminary study that was conducted to determine the concentrations of organochlorine compounds in the adipose tissue of residents of North Texas. Thirty-five human adipose tissue samples were obtained during autopsy between 1987 and 1988 from persons who had no known occupational exposure to organochlorine pesticides. These samples were analyzed by electron-capture gas chromatographic methods for the presence of beta-benzene hexachloride, o,p'-DDE, p,p'-DDE, o,p'-DDT, p,p'-DDT, dieldrin, oxychlordane, and heptachlor epoxide. The findings indicate greater than 97% occurrence for each compound with the exception of o,p'-DDE and o,p'-DDT, each of which occurred in 54% of the population sampled. Statistical analyses of the data showed strong positive correlations between adipose tissue concentrations and age for oxychlordane, heptachlor epoxide, p,p'-DDT, p,p'-DDE, and total DDT (the normalized sum of DDT and its analogues) (p less than or equal to .05). Statistically significant differences (p less than or equal to .05) in geometric mean concentrations between all age groups were found for total DDT and p,p'-DDE. However, the group means comparison test was only significantly different between the 41-60 yr and 61 and over age groups for p,p'-DDT, oxychlordane, and heptachlor epoxide. No statistically significant difference was found between sexes in this study. The geometric means of pesticide concentrations in adipose tissue were compared with those reported in previous U.S. Environmental Protection Agency surveys for the general population. This comparison clearly indicates a declining temporal trend in environmental exposure for banned DDT analogues; however, a consistent temporal trend exists for oxychlordane and heptachlor epoxide--pesticides whose uses are currently restricted but not proscribed.     

Organochlorine pesticide residues in chicken eggs: a survey

One hundred and five chicken egg samples were taken from seven geographical locations in Kenya and analyzed for organochlorine pesticide residues using gas-liquid chromatography. Nine organochlorine compounds were detected: alpha- and gamma-HCH/BHC (hexachlorocyclohexane/benzene hexachloride), aldrin, dieldrin, p,p'-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], p,p'-DDD (TDE) [2,2-bis(p-chlorophenyl)-1,1-dichloroethane] (tetrachlorodiphenylethane), o,p'-DDD [2-(o-chlorophenyl)-2-(p-chlorophenyl)-1,1-dichloroethane], o,p'-DDT [2-(o-chlorophenyl)-2-(p-chlorophenyl)-1,1,1-trichloroethane], and p,p'-DDT. Eighteen of the samples contained DDT levels exceeding the practical residue limit (0.5 ppm). The mean DDT residue content was 0.68 ppm, a result influenced greatly by the high contamination in the 18 samples. Both individual and mean residue levels for other organochlorine pesticides detected were well within the respective practical residue limits.     

The use of primary hepatocytes from brown trout (Salmo trutta lacustris) and the fish cell lines RTH-149 and ZF-L for in vitro screening of (anti)estrogenic activity of wood extractives.

Wood extractives are constituents of wood present in pulp and paper mill effluents, which may cause reproductive disturbances in fish. In the present study, we examined three cellular in vitro bioassays in order to assess (anti)estrogenic potencies of the wood extractives dehydroabietic acid (DHAA), isopimaric acid (IPA), betulinol (BET), hydroxymatairesinol (HMR), a phytosterol preparation (ULT), an oxidized phytosterol preparation (OX) and the model estrogen 17beta-estradiol (E2). The test systems used were primary hepatocyte cultures from brown trout and two piscine liver cell lines, RTH-149 and ZF-L. Estrogenicity was measured as vitellogenin (Vtg) secretion in cell culture medium. The primary hepatocytes cultures responded to E2 in a dose-dependent way. Vtg induction was inhibited with a simultaneous exposure to 4-hydroxytamoxifen (4-HT) indicating an estrogen receptor mediated response. DHAA and ULT induced a weak statistically non-significant Vtg production, and weak additive effects were found in some combination treatments of wood extractives and E2. Additionally, a pulp mill effluent tested on primary hepatocytes induced Vtg production when exposed at a 1% dilution. The cell lines secreted negligible amounts of Vtg upon E2 stimulation, which was neither dose-dependent nor inhibited by 4-HT. In conclusion, trout primary hepatocytes could be useful for assessing (anti)estrogenic potencies of compounds, and the wood extractives and a pulp mill effluent showed only weak or no estrogenic activity in this model system.     

The effects of twenty-two organochlorine pesticides as inducers of the hepatic drug-metabolizing enzymes

The effects of 22 organohalogen pesticides as inducers of hepatic drug-metabolizing enzymes in the immature male Wistar rat have been determined, this group includes four isomeric hexachlorocyclohexanes, technical chlordane, alpha-chlordane, gamma-chlordane, oxychlordane, trans-nonachlor, heptachlor, heptachlor epoxide, aldrin, dieldrin, kepone, toxaphene, mirex, hexachlorobenzene (HCB) and several DDT analogs. With the exception of HCB, all of the pesticides induced microsomal dimethylaminoantipyrine, N-demethylase and aldrin epoxidase activities and the cytochrome P-450 content of microsomes from animals pretreated with most of the compounds was also increased compared to control rats. These pesticides all resembled phenobarbitone in their mode of induction. The effects of HCB as a microsomal enzyme inducer resembled those observed after coadministration of phenobarbitone plus 3-methylcholanthrene.     

Fecundity, 17beta-estradiol concentrations and expression of vitellogenin and estrogen receptor genes throughout the ovarian cycle in female Eastern mosquitofish from three lakes in Florida

Previous studies of Eastern mosquitofish in contaminated Lake Apopka, Florida, have documented reduced sperm count and sexual behaviour in males but increased fecundity and liver weight in females, compared to nearby reference lakes. Liver weight can be an indicator of vitellogenin (Vtg) synthesis in fish, such as the mosquitofish. It was therefore hypothesized that estrogenic organochlorine pesticides, present at elevated concentrations in animals from Lake Apopka, could cause the reproductive disorders in males, as well as increase female fecundity. We initiated a test of this hypothesis by examining the relationship between 17beta-estradiol (E2) tissue concentrations, hepatic estrogen receptor alpha (ERalpha) and Vtg A, B and C gene expression and fecundity in sexually mature female Eastern mosquitofish from Lake Apopka and two reference lakes, Lake Woodruff and Lake Orange. We observed that female Eastern mosquitofish from one site in contaminated Lake Apopka produced fewer but bigger embryos than females from the other Lake Apopka site and two reference sites. However, female E2 concentrations and hepatic ERalpha and Vtg A, B and C gene expression showed no overall differences among the four sites, and it is therefore unlikely that the differences in fecundity were caused by estrogenic EDCs. In addition, we observed no induction of any of the three Vtg genes in male Eastern mosquitofish at the two Lake Apopka sites. Based on the well-documented high sensitivity of Vtg induction as a biomarker of xenoestrogen exposure, the evidence from the present study does not support the hypothesis that estrogenic EDCs are affecting reproduction in Eastern mosquitofish living in Lake Apopka. Our experimental design tested specifically for effects mediated via the ER, and e.g. antiandrogenic DDT metabolites might still be of importance for mosquitofish reproduction in Lake Apopka.     

Evaluation of estrogenic activities of aquatic herbicides and surfactants using an rainbow trout vitellogenin assay.

Estrogenic potencies of four herbicides (triclopyr, 2,4-dichlorophenoxyacetic acid (2,4-D), diquat dibromide, glyphosate), two alkylphenol ethoxylate-containing surfactants (R-11 and Target Prospreader Activator (TPA)), and the binary mixture of surfactants with the herbicides were evaluated using an in vivo rainbow trout vitellogenin assay. Juvenile rainbow trout exposed to 2,4-D (1.64 mg/l) for 7 days had a 93-fold increase in plasma vitellogenin (Vtg) levels compared with untreated fish, while rainbow trout exposed to other pesticides alone did not show elevated vitellogenin levels compared to the control fish. When combined with surfactants, trends indicated enhanced estrogenicity for all combinations, but only 2,4-D and triclopyr caused significant induction of Vtg. Concentration-response studies demonstrated that the lowest observed effect concentrations (LOECs) for 2,4-D and triclopyr were 0.164 mg/l and 1 mg/l, respectively. In terms of measured 4-nonylphenol (4-NP), the LOECs of R-11 and TPA were 20 micro/l and 9.5 microg/l, respectively. Binary mixtures of TPA and 2,4-D showed a greater than additive estrogenic response at the lowest concentrations tested, but a less than additive response at the highest combined concentrations. Binary mixtures of TPA with triclopyr also caused greater than additive Vtg responses in two middle concentrations when compared to TPA or triclopyr alone. When trout were exposed to water collected from a site where triclopyr was used in combination with TPA, a concentration-dependent increase in Vtg expression was observed. Measured values of 4-NP were 3.7 microg/l, and triclopyr concentrations were below detection (<5 ng/l). Estradiol equivalents (EEQs) of the lake water were calculated from an estradiol concentration-response curve and were similar (8.5 +/- 7.7 ng/l) to the mean values for the combined triclopyr + TPA treatments (9.9-12.2 ng/l) in the laboratory, suggesting the estrogenicity of the water may have been due to the treatment. These results demonstrated the binary mixture of alkylphenol ethoxylate-containing surfactants with two aquatic pesticides possessed greater than additive estrogenic responses in fish under laboratory conditions and in a field setting.     

Demographic and seasonal influences on human serum pesticide residue levels

This study was intended to characterize more fully the distribution of serum concentrations of 16 pesticide residues with regard to key demographic and seasonal variables in a subsample of persons from the Second National Health and Nutrition Examination Survey between the ages of 12 and 74 yr old. Blood sera in 2-ml aliquots were analyzed, and the results were confirmed for 5994 persons. Almost all participants (99.5%) had p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) concentrations greater than or equal to 1 ppb, ranging as high as 378.6 ppb. For the other pesticide residues, only beta-benzene hexachloride (beta-BHC) (quantified in 17.2% of the sera), dieldrin (10.6%), and another DDT-related residue, p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT) (35.7%) were found at quantifiable levels in more than 10% of the serum specimens. Of the remaining analytes, hexachlorobenzene (HCB), trans-nonachlor (TNC), and heptachlor epoxide (HE) were found at quantifiable concentrations in 1-10% of the specimens, whereas o,p'-DDT, o,p'-DDE, p,p'-DDD, mirex, alpha-BHC, gamma-BHC, heptachlor, delta-BHC, and aldrin were found in less than 1% of the serum specimens. Results showed that increasing age, residing on a farm, or being a male conferred increased risks of exposure to most of the pesticide residues, independent of all other demographic and seasonal factors. In a pattern less consistent across the different pesticide residues and for fewer of the pesticides, persons who lived below the national poverty level, were nonwhite, resided in the South or West, or were examined in the spring or winter also seemed to have an increased likelihood of having quantifiable serum levels.     

Serum free BG-1 cell proliferation assay: a sensitive method for determining organochlorine pesticide estrogen receptor activation at the nanomolar range.

Most xenobiotic estrogeniety assay methods rely on direct agonist action on the estrogen receptor (ER) to approximate activation potential. Such methods do have drawbacks since some ER activating pesticides are weak or non-agonistic in ligand-binding assays. This study discusses a method that detects pesticide estrogenic actions regardless of ER ligand binding ability. Using a serum-free BG-1 ovarian cell culture model, we investigated the ability of several organochlorine (OC) pesticides to stimulate known estrogenic actions. We observed concentration dependent ER mediated cell proliferation in BG-1 cells using heptachlor epoxide (HE), beta-hexachlorohexane (beta-HCH), and endosulfan (Endo). In addition, we observed upregulation of the ERE-dependent proteins progesterone receptor and PS2. Gel-shift/EMSA studies for ERE binding further supported these OC's ERE activating abilities. All of these effects were abolished using ICI 164,384(ICI). Using the same culture conditions, we tested the blocking action of growth factor antibodies for erbB2(9G6) and insulin-like growth factor (IGF-Ab) receptors and discovered they inhibited BG-1 proliferation (9G6: HE and beta-HCH/ IGF-Ab: Endo.) This experiment confirms the existence of a possible cross-talk between ER and growth factor receptors in OC ligand-dependent activation and also validates this sensitive method for determining both ligand-dependent and independent estrogenic activity of selected pesticides.     

Investigation of the role of estrogenic action and prostaglandin E2 in DDT-stimulated rat uterine contractions ex vivo.

Previous work in our laboratory showed that o,p'-DDT increases the frequency of rat uterine contractions in vitro. The present study investigated whether this response was related to prostaglandin E2 (PGE2) release from the uterine strips or to the estrogenicity of o,p'-DDT. Contraction frequency was evaluated by recording isometric spontaneous contractions in longitudinal uterine strips from pregnant rats. Assessment of PGE2 levels in the muscle bath showed no significant differences between control and DDT-treated strips, although significant amounts of PGE2 were detected in both groups and increased contraction frequency was observed in o,p'-DDT-treated strips. Furthermore, a role for direct estrogenic action in the medication of o,p'-DDT-stimulated uterine contraction was not supported by the contractility data, because: (i) unlike o,p'-DDT, 17-beta-estradiol had no stimulatory effect, but instead exerted a significant inhibitory effect on uterine contraction; (ii) the estrogen antagonist, tamoxifen, failed to block the stimulatory effect of o,p'-DDT; and (iii) p,p'-DDD, a non-estrogenic DDT analogue, significantly stimulated contraction frequency, similar to o,p'-DDT. These results suggest that the stimulatory effect of o,p'-DDT on contraction frequency is not dependent on PGE2 release or direct estrogen receptor-related action.     

Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout (Oncorhynchus mykiss)

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZ's mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout (Oncorhynchus mykiss) a dose of either 2 or 200 μg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 (CYP) 1A and pi-class glutathione S-transferase (GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked.     

Evaluation of the genotoxicity of environmental contaminants in sediments to rainbow trout hepatocytes

Rainbow trout hepatocytes were used as an in vitro bioassay to assess the genotoxic potential of single chemicals and marine sediment extracts. Freshly prepared trout hepatocytes were exposed to either benzo[a]pyrene, N-methyl-N′-nitro-N-nitrosoguanidine, β-naphtoflavone, or organic extracts of marine sediments for 24 h at 15°C. Genotoxicity was assayed using the nick translation assay, which makes use of a nonradioactive nucleotide (biotin-dUTP), and the DNA alkaline precipitation assay followed by fluorometric detection of DNA strands. Exposure to benzo[a]pyrene or methyl-N′-nitro-N-nitrosoguanidine, known indirect-and direct-acting genotoxins respectively, produced genotoxicity to rainbow trout hepatocytes with both assays. β-Naphtoflavone displayed genotoxic activity in trout hepatocytes. Sediment extracts and reference sediment extracts displayed high toxicity and genotoxicity to trout hepatocytes. Chemical analyses showed that these sediments contained significant amounts of organochlorine pesticides, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons. Cell toxicity was correlated with total levels of organochlorine pesticides and polychlorinated biphenyls but not total levels of polycyclic aromatic hydrocarbons. No positive correlation was found with the nick translation assay between total levels of chemicals and genotoxicity in marine sediments. Genotoxicity obtained with the alkaline precipitation assay was correlated with levels of the organochlorine pesticide DDT. However, more tests would be required to further substantiate possible links with other specific chemicals. © by John Wiley & Sons, Inc.     

Relation between the content of organochlorine compounds in Finnish human milk and characteristics of the mothers

Neutral organochlorine pesticide and PCB residues were analyzed by GC-MS technique in 183 human milk samples obtained in 1984-1985 from 165 women living in different parts of Finland. The effect of the donors' age, body mass, place of residence, number of children, dietary habits, smoking habits, occupational history, and weight loss on the organochlorine content of human milk were studied. Of all the milk samples analyzed, p,p'-DDE concentrations were above the detection limit in 99.5%, p,p'-DDD + p,p'-DDT in 57.9%, isomers of HCH in 30.0%, cis-chlordane in 4.9%, oxychlordane in 3.3%, trans-nonachlor in 6.0%, heptachlor in 12.0%, and heptachlor epoxide in 6.6%. Mirex was not found in any of the milk samples, whereas the signals of chlorinated terpenes (toxaphenes) were detected but could not be quantitatively determined. The mean fat adjusted residue levels above the detection limit in Finnish human milk samples of primipara mothers were 0.66 ppm for total DDT compounds, 0.08 ppm for HCB, 0.93 ppm for PCBs, 0.41 ppm for chlordane compounds, 0.20 ppm for isomers of HCH, and 0.10 ppm for heptachlor epoxide. The geometric means were 0.46, 0.06, 0.57, 0.02, 0.02, and 0.01 ppm, respectively. The age of the mothers positively correlated with the DDE concentrations in human milk. The residues of OC compounds in human milk did not differ in women living in plywood industry regions, those actually working in the industry, and other mothers. Small differences were detected in the levels of organochlorine compounds in different parts of Finland. No relation was found between the OC content and the fish consumption, smoking habits, weight loss, or social group of the donors.     

Estrogenic potencies of several environmental pollutants, as determined by vitellogenin induction in a carp hepatocyte assay.

Estrogenic potencies of several xenoestrogens were determined in vitro, using cultured hepatocytes from a genetically uniform male carp strain (Cyprinus carpio). Estrogenicity was measured as induction of the yolk protein precursor vitellogenin (Vtg), and compared to Vtg induction by 17beta-estradiol (E2). The order of estrogenic potency was: methoxychlor (MXCL) > o,p-DDT > chlordecone approximately/= bisphenol-A approximately/= 4-t-pentylphenol. Estrogenic potencies of these compounds varied from 1 x 10(-3) to 1 x 10(-4) relative to E2. The synthetic estrogen DES had a relative estrogenic potency of 0.5, whereas dieldrin, beta-endosulfan, o,p-DDE, and toxaphene (technical mixture) did not induce vitellogenesis at concentrations up to 100 microM. Experiments in which cells were simultaneously exposed to E2 and these xenoestrogens showed that the Vtg-inducing activities of E2 and 4-t-pentylphenol or bisphenol-A were (partially) additive, whereas E2 antagonized the estrogenic effects of MXCL and o,p-DDT. The effect of cytochrome P4501A (CYP1A)-induction on the estrogenicity of MXCL was studied by co-exposing cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD (10 pM) caused a greater than 50-fold induction of CYP1A, measured as ethoxyresorufin O-deethylase (EROD) activity, but Vtg induction by MXCL was not significantly affected. This indicates that CYP1A is not involved in the bioactivation of MXCL to more potent estrogenic metabolites in carp. The CARP-HEP (hepatocyte) assay can detect xenoestrogens with a potency > or = 2 x 10(-5) relative to E2. It allows simultaneous testing of more than 10 compounds for both estrogenic and antiestrogenic effects, which makes it a promising tool for the screening of suspected xenoestrogens.     

Increased ErbB-2 tyrosine kinase activity, MAPK phosphorylation, and cell proliferation in the prostate cancer cell line LNCaP following treatment by select pesticides.

The oncogene erbB-2 codes for a receptor tyrosine kinase that functions as a key mitotic signal in a variety of cell types. Amplification or overexpression of erbB-2 occurs in many forms of cancer, such as of the breast, colon, and prostate, and is an indicator of poor prognosis in those diseases. In the human prostate cancer cell lines LNCaP and PC-3, erbB-2 kinase was activated by pesticides of different chemical classes: (1) the organochlorine insecticides beta-hexa-chlorocyclohexane (beta-HCH), o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT), and heptachlor epoxide; (2) the pyrethroid insecticide trans-permethrin, and (3) the fungicide chlorothalonil. o,p'-DDT also causes phosphorylation of mitogen-activated protein kinase (MAPK) and cellular proliferation of the androgen-dependent LNCaP line. However, no proliferative effect was observed in the androgen-independent PC-3 line. The proliferative effect of o,p'-DDT in LNCaP could not be blocked by the androgen receptor antagonist p,p'-dichlorodiphenyldichloroethene (p,p'-DDE), indicating that this effect of o,p'-DDT does not occur through direct interaction with the androgen receptor. Together these data demonstrate a putative mechanism for the action of certain pesticides in hormonal carcinogenesis.     

No-effect levels of organochlorine pesticides based on induction of microsomal liver enzymes in short-term toxicity experiments

A number of organochlorine pesticides were given in the diet of rats during 2 weeks. The induction of microsomal liver enzymes by different dose levels of these compounds was studied. In this way a no-effect level was established for each pesticide. This was then compared with no-effect levels from the literature, based on histopathological abnormalities in the liver in long-term experiments.In general, good agreement was found, except for some compounds, such as chlorfenson, kelthane and chlordane, for which a much lower no-effect level was found in this study. The capacity for inducing enzymes decreased in the order: heptachlor, DDT, chlorfenson, dieldrin, dicofol, chlordane, tetrasul, V110, hexachlorobenzene (HCB), lindane, endosulfan. Polychlorinated biphenyl (PCB) was also studied. This substance had a very high enzyme-inducing capacity for aniline hydroxylase (AH), but only a medium one for aminopyrine demethylase (APDM) and hexobarbital oxidase (HexOx). Methoxychlor and dichlobenil did not show any enzyme induction at the dose levels tested.     

Effects of sub-lethal levels of dichlorodiphenyltrichloroethane and dichlorodiphenyldichloroethylene on in vitro steroid biosynthesis by ovarian follicles or steroid metabolism by embryos of rainbow trout (Oncorhynchus mykiss)

This study examined the possibility that DDT and DDE, at sub-lethal exposure levels, exert direct effects on the biotransformation of gonadal steroids by rainbow trout (Oncorhynchus mykiss) ovarian follicles and embryos. Ovarian follicles were co-incubated with DDT or DDE at 0.01 or 1 mg l-1 to examine effects of the pesticides on basal or cAMP-activated steroidogenesis. Ovarian preparations were incubated with radiolabelled [3H]pregnenolone ([3H]P5), and the tritiated metabolites of [3H]P5 metabolism were separated using high-performance liquid chromatography (HPLC). Testosterone (T) and 17beta-estradiol (E2) production were also measured using radioimmunoassay (RIA). Embryos were either exposed to the pesticides in ovo, or co-incubated in vitro with the pesticides. The effect of the pesticides on embryo steroid biotransformation was examined using a range of radioactively labelled substrates, including [3H]P5, [3H]progesterone ([3H]P4), [3H]T and [3H]E2. At the concentrations used, the pesticides had no significant effect on the relative amounts of unconjugated radiolabelled steroids formed by the biotransformation of [3H]P5 under conditions of basal or cAMP-stimulated ovarian steroidogenesis. However, DDT and DDE appeared to reduce the basal accumulation of androgen as a product of P5 biotransformation by ovarian follicles. Basal or cAMP-stimulated total estrogen production was not affected. In addition, DDT at 1 mg l-1 and DDE at 0.01 mg l-1 significantly increased and decreased cAMP-stimulated T accumulation, respectively. Also DDT at 0.01 mg l-1 and DDE at 1 mg l-1 significantly increased and decreased basal E2 accumulation, respectively. The steroid metabolites synthesized from the different substrates by embryos were essentially similar in both controls and pesticide-exposed groups, and the survival of embryos to hatch was not significantly affected by pesticide exposure, in ovo, with an approximately 90% hatchability in all treatment groups. This study suggests that although DDT and DDE may affect ovarian androgen synthesis under some conditions, under the conditions of the present study, they do not impact on overall rates of gonadal estrogen synthesis. Similarly, the pesticides do not appear to directly affect steroid biotransformation by embryos.     

Assessing combined toxicity of estrogen receptor agonists in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes

The presence of highly complex mixtures of chemicals in the environment challenges our ability to assess single chemical effects and the interaction that occurs with cellular receptor targets and regulation of endocrine processes. In this study concentration addition (CA) and independent action (IA) prediction models were used to assess the combined toxicity of mixtures of environmental relevant estrogen receptor (ER) agonists (hormones and anthropogenic pollutants) in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes using the ER-mediated production of vitellogenin (Vtg) as a biological marker (biomarker) for estrogenicity. Nine of the eleven tested chemicals induced the production of Vtg and the parameters from the fitted concentration-response curves were used to model four mixtures containing four (17β-estradiol, estrone, estriol and diethylstilbestrol), five (musk ketone, 4-tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene), seven (17β-estradiol, estrone, estriol, diethylstilbestrol, 4-tert-octylphenol, bisphenol A and o,p′-DDT) and nine compounds (17β-estradiol, estrone, estriol, diethylstilbestrol, musk ketone, 4-tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene). The CA and IA prediction model proved to be a good estimation for the combined effect of mixtures of ER agonists at low relative mixture concentration (e.g. relative to the maximum mixture concentrations used), but a deviation from the prediction models was observed when exposing hepatocytes to high relative mixture concentrations. The CA and IA prediction models’ ability to predict the combined estrogenic effect of complex mixtures, especially in the low concentration-response range, is of ecological relevance since organisms in the environment generally encounter low concentrations of chemicals from a wide array of chemical groups that may not elicit estrogenic effects on their own.     

The presence of environmental pollutants in the follicular fluid of farm animals (cattle,sheep, goats,and pigs)

Organochlorine pesticides and polychlorinated biphenyls are widely used in agriculture and industry, respectively. The present study assessed the burden of environmental pollutants in the follicular fluid of farm animals (cattle, sheep, goats, and pigs). An analytical method combining a solid-phase extraction with (C(18)) for clean-up and GC-electron capture detection using a capillary column was implemented for isolation and determination of organochlorine pesticides and polychlorinated biphenyls (PCBs). Of the organochlorine pesticides, hexachlorobenzene (HCB), hexachlorocyclohexane (HCH, alpha-, beta-, and gamma-isomers), dieldrin, heptachlor epoxide, and the DDT-related chemicals (o,p'-DDE, p,p'-DDE, p,p'-DDD, p,p'-DDT) were detected and of the PCBs, the congeners PCB-52, -101, -138, -153, and -180 were detected. In all species of farm animals, the most frequently detected pollutant was gamma-HCH (90-100% of samples) followed by HCB (80-100%), and p,p'-DDE (75-90.91%). Species differences in the concentrations of HCB, beta-HCH, heptachlor epoxide, and DDT-related chemicals in follicular fluid were noted as well as differences in the concentrations of some pollutants within the same species.     

Estrogenic activity of procymidone in primary cultured rainbow trout hepatocytes (Oncorhynchus mykiss).

It has been shown that procymidone, a dicarboximide fungicide, alters sexual differentiation in vivo and in vitro. The aim of this study was to evaluate the estrogenic activity of this fungicide using the synthesis of vitellogenin (Vtg) in rainbow trout hepatocyte as a biological marker. The cells were treated for 24 h with procymidone 150 microM, using 17beta-estradiol 20 microM as a positive control. The doses were chosen on the basis of cell viability (Neutral Red and MTT tests) and solubility. The results show that procymidone leads to a qualitative and quantitative increase in Vtg synthesis. In Western immonoblots, the 170 and 30 kDa bands, which respectively correspond to the monomeric form of Vtg and posvitine, were brighter in cells treated with procymidone and 17beta-estradiol than those corresponding to the negative controls (cells treated for 24 h with DMSO 0.1% alone); ELISA showed that the cells treated with the fungicide and 17beta-estradiol had a 48 and 76%, respectively, higher Vtg concentration than the negative controls (P<0.01). Western blotting also revealed the induction of HSP27 (27 KDa), which further confirms the estrogenic acitivity of procymidone as it is known that the 3' region of HSP27/28 containing the gene mRNAs is induced by estrogen treatment. Procymidone increased also the production of both HSP70 protein (70 KDa) and free oxygen radicals. This last finding is in agreement with the toxic mechanism of dicarboximide fungicides. It can therefore be presumed that the estrogenic activity of procymidone in primary cultured trout hepatocytes is related to oxidative damage which, as many other studies have shown, can increase the levels of estrogens such as 17beta-estradiol, and thus increase Vtg synthesis     

Mechanisms of interaction of endocrine-disrupting chemicals with glutamate-evoked secretion of gonadotropin-releasing hormone.

In previous studies, we detected a dichlorodiphenyltrichloroethane (DDT) derivative in the serum of children with sexual precocity after migration from developing countries. Recently, we reported that DDT stimulated pulsatile gonadotropin-releasing hormone (GnRH) secretion and sexual maturation in the female rat. The aim of this study was to delineate the mechanisms of interaction of endocrine-disrupting chemicals including DDT with GnRH secretion evoked by glutamate in vitro. Using hypothalamic explants obtained from 15-day-old female rats, estradiol (E2) and DDT caused a concentration-related increase in glutamate-evoked GnRH release while p,p'-dichlorodiphenyldichloroethene and methoxychlor had no effect. The effective DDT concentrations in vitro were consistent with the serum concentrations measured in vivo 5 days after exposure of immature rats to 10 mg/kg/day of o,p'-DDT. Bisphenol A induced some stimulatory effect, whereas no change was observed with 4-nonylphenol. The o,p'-DDT effects in vitro were prevented partially by a selective antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptors. A complete prevention of o,p'-DDT effects was caused by an estrogen receptor (ER) antagonist as well as an aryl hydrocarbon receptor (AHR) antagonist and inhibitors of protein kinases A and C and mitogen-activated kinases. While an intermittent incubation with E2 caused no change in amplification of the glutamate-evoked GnRH release for 4 h, continuous incubation with E2 or o,p'-DDT caused an increase of this amplification after 3.5 h of incubation. In summary, DDT amplifies the glutamate-evoked GnRH secretion in vitro through rapid and slow effects involving ER, AHR, and AMPA receptor mediation.