Effect of single and repeated in vitro exposure of ovarian follicles to o,p'-DDT and p,p'-DDT and their metabolites

The aim of the presented study was to compare the effect of o,p'-DDT [1,1-dichloro-2,2-bis-(p,p'-chlorophenyl)-ethylene] and p,p'-DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane] and their metabolites DDE and DDD on estradiol secretion by ovarian follicles, the target organs of environmental estrogens. Theca interna (Tc) and granulosa cells (Gc) were collected from medium size porcine follicles and cultured as a monolayer. The cells were initially cultured for 24 h to allow attachment to the plates and then media were changed for the new ones and o,p'-DDT and p,p'-DDT and their metabolites: o,p'-DDE, p,p'-DDE and o,p'-DDD were added at doses of 4, 40, 400 ng and 4 microg/ml medium to investigate dose-dependent effects. Media were collected after 24 h and frozen for estradiol content determination. When the effect of single and repeated exposure was investigated, the lowest dose of 4 ng/ml and the highest one of 4 microg/ml were chosen on the basis of the results of Experiment 1. o,p'-DDT exerted antiestrogenic action at all doses used while its metabolites and p,p'-DDT and its metabolites decreased estradiol secretion only when present in the medium at a dose of 4 ng/ml. The highest doses caused the increase in estradiol secretion. Parent o,p'-DDT and its metabolites showed antiestrogenic action after single exposure to 4 ng/ml while parent p,p'-DDT and its metabolites caused estrogenic action. All investigated compounds, except o,p'-DDT, increased estradiol secretion after single exposure to the dose of 4 microg/ml. Repeated exposure resulted in a massive antiestrogenic action of all investigated chemicals. In conclusion, our study points to time-dependent effect of DDT and its metabolites on ovarian follicles with the strongest estrogenic properties observed after single exposure and antiestrogenic action caused by repeated exposure. Given the duration of folliculogenesis, one can imagine many different potential mechanisms by which DDT could influence steroidogenesis.     

HCH and DDT residues in human placentas in Murcia (Spain)

Organochlorinated insecticides are ubiquitous toxicants that are transplacentally transferred from mother to fetus and are reported to produce adverse health effects in pregnant woman and neonates. To investigate hexachlorocyclohexane (HCH) and 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) exposure in pregnant woman, a total of 102 placentas were analyzed by a gas chromatograph equipped with electron capture detector. Organochlorine residues were found in 69.6% of the samples. Lindane was detected in 24.5%, alpha-HCH in 2.9%, beta-HCH in 6.9%, p,p'-DDE in 44.1%, p,p'-DDT in 14.7% and p,p'-DDD in 10.8% of the samples. The pattern of dispersal by these substances in Murcia is similar to that described by different authors in other countries. Therefore, the past use of DDT and the present employment of lindane seem to be reflected in body tissues of the residents of this area.     

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.     

DDT and its metabolite DDE alter steroid hormone secretion in human term placental explants by regulation of aromatase activity

Placental explants were used to compare the effects of two isomers of DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane), p,p'-DDT and o,p'-DDT and their metabolites p,p'-DDE and o,p'-DDE (1,1,-dichloro-2,2-bis(p-chlorophenyl)ethylene) on steroid hormone secretion (estradiol (E2) and progesterone (P4)). Explants were treated with 1, 10, 100ng/ml or 1microg/ml of each compound for 24h. We found that all investigated compounds at all doses caused reductions of estradiol secretion. Moreover, it was shown that the inhibition of estradiol secretion was due to direct action on aromatase activity. Twenty-four-hour exposure to p,p'-DDE, o,p'-DDT or o,p'-DDE at doses of 100ng/ml or 1microg/ml increased P4 secretion, suggesting that these compounds act on P450scc. The fluorometric assay confirmed that all investigated compounds inhibited aromatase activity at a concentration of 100ng/ml. Our findings suggest that by decreasing estradiol secretion with concomitant stimulation of progesterone secretion, DDT could be a factor that influences the outcome of pregnancy.     

The short- and long-term effects of two isomers of DDT and their metabolite DDE on hormone secretion and survival of human choriocarcinoma JEG-3 cells

JEG-3 cells were used to compare the effects of two isomers of DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane), p,p'-DDT and o,p'-DDT and their metabolite DDE (1,1,-dichloro-2,2-bis(p-chlorophenyl)ethylene) on progesterone (P4) and human chorionic gonadotropin (hCG) secretion and cell apoptosis. Cells were treated with 1, 10, 100 ng/ml or 1 mug/ml of each compound for 24 or 72 h. Twenty four hours of exposure at 1 mug/ml of p,p'-DDT and o,p'-DDT decreased, whereas both DDEs, at all investigated concentrations, increased P4 secretion. Seventy two-hour exposure to all concentrations of both isomers of DDT and their metabolite DDE stimulated progesterone secretion. Statistically significant decrease in hCG secretion after 24 h and increase in hCG secretion after 72 h exposure for all investigated compounds was noted. Decrease in caspase-3 activity was observed in cells exposed to both isomers of DDT and its metabolites. These findings indicate that both isomers of DDT and their metabolite DDE are able to alter main placental hormone production and survival of JEG-3 cells in the concentration- and time-dependent manner.     

The effect of p,p′-DDT (Dicophane) and its metabolites on ATPase activity in rat liver mitochondria

The effect of DDT and its main metabolites on ATPase activity in rat liver mitochondria was studied. These compounds stimulated ATPase activity in intact mitochondria of rat liver and had no effect on ATPase activity in damaged mitochondria. This suggests that the uncoupling of the oxidative phosphorylation in mitochondria may be responsible for the toxic effect of DDT in mammals.

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Abbreviations Used DDT or p,p-DDT 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane - DDE 1,1-dichloro-2,2-bis (p-chlorophenyl)ethylene - DDOH 2,2-bis (p-chlorophenyl)ethanol - DDA bis (p-chlorophenyl)acetic acid - DNP 2,4-dinitrophenol - ATPase ATP phosphohydrolase (EC 3.6.1.3.)     

COMPARATIVE PHARMACODYNAMICS OF CYP2B INDUCTION BY DDT,DDE, AND DDD IN MALE RAT LIVER AND CULTURED RAT HEPATOCYTES

In this study the pharmacodynamics were characterized of rat hepatic cytochrome P450 2B (CYP2B) induction by the pesticide DDT \[1,1,1-trichloro-2,2-bis(p -chlorophenyl) ethane] and its metabolites DDE \[1,1-dichloro-2,2-bis(p -chlorophenyl)ethylene], which is bioretained, and DDD \[1,1-dichloro-2,2-bis(p -chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction ( benzyloxyresorufin O -dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and 620 ppm in diet (14 d of exposure). The efficacies ( E values) for induction of benzyloxyresorufin O -dealkylation by DDT, max DDE, and DDD were 24-, 22-, and 21-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and 0.51 mu M, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and 5.9 mumol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and 2.7 mu M , respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.     

Thiol stimulation of the cytochrome p-450-dependent reduction of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) to 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD)

The enzymatic reduction of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) to 1,1 dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) was studied with rat hepatic microsomal fractions. The reaction required NADPH and was inhibited by dioxygen and carbon monoxide, which shows that the reaction is catalyzed by cytochromes P-450. Moreover, when the reaction was studied in the presence of deuterium oxide, no deuterium was incorporated into the DDD, which suggests that a one-electron reduction of DDT to the 1,1-dichloro-2,2-bis(p-chlorophenyl)ethyl radical followed by hydrogen atom abstraction accounts for the formation of DDD. The microsomal reduction of DDT to DDD was stimulated markedly by several thiols, including glutathione. The stimulatory effect of thiols was concentration dependent and was not due to conservation of cytochrome P-450, because nonthiol antioxidants failed to stimulate the reaction. The mechanism of the stimulation is not understood, but thiols do not promote the formation of DDD by preventing the alkylation of microsomal lipids and, thereby, stimulating the formation of a reduced alkane. Finally, these results show that soluble, lowmolecular weight compounds may enhance the activity of membrane-bound enzymes.     

Depression of the Ca(2+)-ATPase activity of the rat liver endoplasmic reticulum by the liver tumour promoters, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane and phenobarbital.

The effects of a short-term in vivo administration of two liver tumour promoters (phenobarbital and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane on rat liver endoplasmic reticulum Ca(2+)-ATPase were investigated. The specific activity values of this membrane-bound enzyme significantly decreased (P less than 0.01) by 51% for phenobarbital-treated rats and by 48% for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane-treated rats compared with control animals. The depression of liver endoplasmic reticulum Ca(2+)-ATPase appears to be a manifestation of the toxicological effect of tumour promoters.     

Metabolism of methoxychlor by hepatic P-450 monooxygenases in rat and human. 1. Characterization of a novel catechol metabolite

Previous investigations demonstrated that the incubation of the chlorinated hydrocarbon pesticide methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] with rat liver microsomes generates phenolic estrogenic metabolites. The current study shows that the incubation of liver microsomes from untreated and phenobarbital-treated rats and human donors, in the presence of NADPH, yields three phenolic metabolites. Identification of the metabolites was achieved by TLC, HPLC, GC/MS, and LC/MS and by hydrodynamic voltammetric analysis. These metabolites were identified as the mon- and didemethylated phenolic derivatives (mono-OH-M and bis-OH-M, respectively) and as a novel trihydroxy derivative (tris-OH-M). The tris-OH-M was demonstrated to be a catechol [1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3,4-dihydroxyphenyl)ethane]. Furthermore, the tris-OH-M becomes radiolabeled by [methyl-3H3]-S- adenosylmethionine (SAM) in a reaction catalyzed by catechol O-methyltransferase (COMT), indicating that tris-OH-M behaves like a catechol. Incubation of the monohydroxy metabolite with liver microsomes from phenobarbital-treated rats (PB microsomes) yields the dihydroxy and the trihydroxy metabolites. Furthermore, the time course of methoxychlor metabolism by PB microsomes demonstrated a rapid appearance and disappearance of the monohydroxy metabolite with the subsequent formation of the dihydroxy and trihydroxy metabolites. On the basis of these findings, it is proposed that the metabolic route of methoxychlor by mono-oxygenases involves sequential demethylations to the dihydroxy derivative and a subsequent ring hydroxylation.     

Correlation of chlorinated pesticides concentration in semen with seminal vesicle and prostatic markers

Semen samples of fertile and infertile men were analysed by gas liquid chromatography (GLC) for the presence of dichlorodiphenyltrichloroethane (DDT) and its metabolites (1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene) (pp'-DDE); (1,1,1-trichloro-2,2-bis(p-chlorophenyl ethane) (pp'-DDT); (1,1-dichloro-2,2 bis(p-chlorophenyl) ethane) (pp'-DDD); (1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane) (op'-DDT), hexachlorocyclohexane (HCH) and its isomers alpha (alpha), beta (beta), gamma (gamma), delta (delta) and aldrin. The biochemical analysis of seminal vesicle and prostatic marker was done by spectrophotometer. The concentrations of alpha-, beta-, gamma-HCH, pp'-DDE and pp'-DDD were higher in the semen of infertile than in that of fertile men. An elevation in the fructose level along with decrease in the gamma-glutamyl transpeptidase and acid phosphatase activity was noticed in infertile men as compared to fertile subjects. The data showed correlation between alpha-, beta-, gamma-HCH, pp'-DDE, pp'-DDD and seminal vesicle marker fructose and prostatic marker gamma-gluatamyl transpeptidase and acid phosphatase in infertile men. The study suggests that the chlorinated pesticides may influence the semen quality by affecting the seminal and prostatic functions in infertile men.     

Role of hepatic monooxygenases in generating estrogenic metabolites from methoxychlor and from its identified contaminants

Previous investigations demonstrated that methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] contains estrogenic contaminants and that methoxychlor per se is not an estrogen but is a proestrogen being metabolized in vivo into estrogenic products. The present study examined structurally identified methoxychlor contaminants as to their estrogenic or proestrogenic properties. Also, the estrogenic activity of demethylated metabolites of methoxychlor and of one contaminant was determined. To examine these properties, we utilized an assay developed by us that monitors whether a given compound, incubated with isolated rat uteri, can diminish the uterine cytosolic estrogen receptor and elevate the nuclear estrogen receptor and whether metabolic intervention by hepatic microsomal monooxygenase(s) is required by the respective compound for this cellular redistribution of the receptor. Of the 15 compounds examined which constitute with methoxychlor 99.5% of total technical grade methoxychlor, two compounds, 1,1-dichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethene (mono-OH-MDDE) and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-methoxychlor), were active per se and two compounds, 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethene (MDDE) and methoxychlor, required metabolic transformation for estrogenic activity to be manifested. Subsequently, it was shown that the mono- and bis-OH metabolites of MDDE and of methoxychlor were active estrogens and that the order of activity, either by the above procedure or in terms of relative binding affinity to rat uterine cytosolic receptor, was as follows: bis-OH-MDDE much greater than bis-OH-methoxychlor greater than mono-OH-MDDE greater than mono-OH-methoxychlor. Following the in vitro observations, the activity of MDDE and bis-OH-MDDE was determined in vivo in immature rats. It appears that both compounds are estrogenic, yielding marked elevation in ornithine decarboxylase (EC 4.1.1.17) levels and moderate increase in uterine weight. A comparison with methoxychlor and bis-OH-methoxychlor [1,1,1-trichloro-2,2-bis(p-hydroxyphenyl)ethane] demonstrates that the order of potencies is similar to that observed in the in vitro determinations. These studies demonstrate the usefulness of the in vitro assay for determining the estrogenic and proestrogenic properties of compounds of which limited quantities are available, often insufficient for in vivo determination. Also, whereas the in vitro assay is simple and rapid, a lengthy investigation might be required to determine in vivo whether a given compound is an estrogen or a proestrogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Organochlorine compounds in adipose tissue of Greenlanders and southern Danes.

Abdominal fat tissue samples from the general population of Greenland and from southern Denmark were analyzed for o,p'-DDE and p,p'-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], p,p,-DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane], o,p'-DDT and p,p'-DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], lindane (1,2,3,4,5,6-hexachlorocyclohexane), aldrin (1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-endo-exo-1,4:5,8-dimethanonaphthalene), dieldrin (1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-endo-exo-1,4:5,8-dimethanonaphthalene), heptachlor (1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene), heptachlor epoxide, and polychlorinated biphenyls (PCBs). Fat tissue from Greenlanders contained significantly higher amounts of p,p'-DDE (p less than or equal to 0.05), p,p'-DDT (p less than or equal to 0.01), and total DDT (SIGMA DDT, the sum of DDT and its metabolites) (p less than or equal to 0.01) than southern Danes. Lindane, aldrin-like residue, dieldrin heptachlor-like residue, heptachlor epoxide, and PCBs were present in adipose tissue of both groups and there were no significant differences between the groups. The p,p'-DDE level in Greenland was lower than that in the United States, eastern Europe, and India. Among Greenlanders of different ages the highest sigma DDT was found in the age group of 22-45 yr; the content of PCBs increased with age. Among southern Danes the highest sigma DDT was found in a higher age group than in Greenlanders. Among Greenlanders the content of aldrin-like residue decreased with age and that of dieldrin increased with age. A significant correlation was found (on the basis of wet weight) between p,p'-DDE and PCB content in southern Danes (p less than or equal to 0.02). The correlation between sigma DDT and PCB content was also significant in this population (p less than or equal to 0.01). These two relationships were not significantly correlated for the Greenlanders. The DDE/PCB and sigma DDT/PCB ratios were higher in Greenlanders than in southern Danes. These ratios are age-dependent and are highest in the age group 22-45 yr among the Greenlanders. A low DDE/PCB ratio and a low sigma DDT/PCB ratio have been proposed as markers for industrialization. In Greenlanders p,p'-DDE represented about 70% of sigma DDT. For southern Danes this level was about 90%. The data obtained are presented and discussed on the basis of both lipid and wet weight levels.     

Efficient removal of the organochlorine pesticide and heavy-metal residues in<Emphasis Type="Italic">Epimedium brevicornum Maxim</Emphasis> by supercritical fluid extraction

A method involving depuration of 12 organochlorine pesticides (OCPs) and 7 heavy metals from Epimedium brevicomum Maxim was developed using supercritical fluid extraction (SPE). The pesticides in the study consisted of alpha, beta-, gamma-, and delta-Benzene hexachloride, Pentachloronitrobenzene (PCNB), Pentachloroaniline (PCA), Heptachlor (HEPT), Methyl -pentachlorophenyl sulfide (MPCPS), pp'-DDE[1,1-dichloro-2, 2-bis (p-chlorophenyl) ethylene], op'-DDT [1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane], pp'-DDD [1,1-dichloro-2-2-bis(p-chlorophenyl)]ethane, pp'-DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane]. A series of experiments was conducted to optimize the final extraction conditions as following: pure CO2, extraction pressure of 15 Mpa, extration temperature of 60 degrees C, extraction time of 10 min, flow rate at 55 kg/h. A GC method with electron capture detection was employed to determination of the OCPs, and an atomic absorption spectrometry (AAS) was designed for the determination of 7 heavy metals including of Pb, Cd, Cu, Fe, Zn, As, Hg in Epimedium brevicomum Maxim. A HPLC method was developed for the quantitative determination of active constituents. The SFE was used to remove the organochlorine pesticide and heavy metals from Epimedium brevicornum Maxim, receiving high decontamination rate of pesticide residue and low loss of active constituents.     

Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6beta-hydroxycortisol/cortisol (6beta-OHC/FC) ratio. POP exposures were assessed by measuring their concentrations in serum lipid. The results showed a unimodal distribution of the 6beta-OHC/FC ratio with values ranging from 0.58 to 27.38. Women had a slightly higher 6beta-OHC/FC ratio than men (p=0.07). Confounder-adjusted multiple regression analysis showed significant associations between 6beta-OHC/FC ratios and summation PCB, PCB-TEQ and p,p'-DDE, o,p'-DDT and HCB, respectively, but the associations were statistically significant for men only.     

In vitro analysis of 2,2-bis(p-chlorophenyl) acetic acid (DDA) handling by rat kidney and liver

On exposure of renal cortical slices or liver slices to [¹⁴C]DDA (2,2-bis[p-chlorophenyl] acetic acid), both tissues accumulate DDA in a concentration- and time-dependent manner. At 10 μm DDA maximum tissue to medium ratios were approximately 20 in both tissues. Incubation under N₂ or addition of metabolic inhibitors reduced uptake by 50%. Other organic acids depressed energy-dependent DDA uptake, but did not influence uptake in the presence of metabolic inhibitors. Neither the parent pesticide, DDT (1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane), nor organic bases significantly inhibited DDA accumulation. Accumulation of a second organic acid, p-aminohippurate (PAH), was depressed by DDA but not by DDT. Thus, DDA is apparently accumulated by an energy-dependent carrier-mediated process in both liver and kidney. The specificity of this process indicates that it is the organic acid transport system.     

Reassessment of ecotoxicities of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane in Japanese quail under the OECD draft new avian one-generation reproduction test guideline

Ecotoxicological hazards of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDT) were investigated by a one-generation reproduction study using Japanese quail (Coturnix coturnix japonica) under an Organization for Economic Co-operation and Development (OECD) draft new test guideline 206 following acute and subchronic toxicity studies. In the subchronic feeding toxicity study, tremors, convulsions, and deaths were observed with a clear sex difference, males being more susceptible than females. The estimated total number of sperm tended to decrease in a dose-dependent manner at the end of 6-week treatment. In the one-generation reproduction study conducted at dose levels of 0, 6, 30, and 150 ppm, the estimated total number of sperm tended to decrease in a dose-dependent manner with a significant difference at 150 ppm. Tremors were observed in the majority of hatchlings in the 150 ppm group and at lower incidences in the 30 ppm group. Significantly high mortality rate in chicks persisted from treatment week 3-6 in the 150 ppm group and at treatment weeks 4 and 5 in the 30 ppm group. Despite of these severe adverse effects of p,p'-DDT on hatchlings and chicks, fertilization, egg laying, eggshell thickness or embryonic development was hardly impaired by p,p'-DDT or its metabolites. From these results, it appears that the OECD draft new avian one-generation reproduction test guideline is effective for ecological hazard assessment of chemicals.     

Noradrenergic influence on the prepulse inhibition of acoustic startle

Oral administration of p,p'-1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), a chemical believed to increase neuronal membrane excitability increased the acoustic startle responsiveness of rats. Inhibition of the acoustic startle response with a brief, prepulse white-noise stimulus was evident at 12.5 to 25 mg/kg of p,p'-DDT, but not at 50 mg/kg. Pretreatment of rats with phenoxybenzamine, an adrenergic receptor antagonist, attenuated the effects of 12.5 mg/kg of p,p'-DDT on the acoustic startle reflex, and decreased the maximum magnitude reduction produced by the prepulse stimulus in DDT-exposed rats. These data extend previous work showing that p,p'-DDT augments startle reactivity in rats and is in accord with the existence of an excitatory norepinephrine(NE)-containing pathway modulating motor outflow in the acoustic startle reflex arc.     

Chlorinated pesticide concentration in semen of fertile and infertile men and correlation with sperm quality

In recent years controversy has evolved regarding the role of environmental pollutants especially chlorinated pesticides and heavy metals on male infertility. Previous data generated on the correlation of chlorinated pesticides with human semen are scarce and controversial. The objective is to explore the possibility of correlation if any between the chlorinated pesticides and sperm count and motility. Semen analysis were performed in 50 samples collected each from fertile and infertile men and pesticides estimation for dichlorodiphenyltrichloroethane (DDT) and its metabolites dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE); 1,1,1-trichloro-2,2-bis(p-chlorophenyl ethane) (pp'DDT); 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (pp'DDD), 1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane (op'DDT), aldrin, hexachlorocyclohexane (HCH) isomers alpha (α), beta (β), gamma (γ), delta (δ) were done by gas liquid chromatography. The higher concentrations of pesticide, viz. α-, β-, γ-, δ-HCH, DDT and its metabolites (pp'DDE and pp'DDD) were detected in semen samples of infertile males. The data showed significant association between β-HCH, γ-HCH, pp'DDE, pp'DDD with semen quality parameters.     

Glucuronidation of the oxidative cytochrome P450-mediated phenolic metabolites of the endocrine disruptor pesticide: methoxychlor by human hepatic UDP-glucuronosyl transferases.

Methoxychlor, a currently used pesticide, is a proestrogen exhibiting estrogenic activity in mammals in vivo. Methoxychlor undergoes oxidative metabolism by cytochromes P450, yielding 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M) and 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane (bis-OH-M) as main metabolites. Since humans may be exposed to these estrogenic metabolites, which are potential substrates of UDP-glucuronosyltransferases (UGTs), their glucuronide conjugation was investigated with human liver preparations and individual UGTs. Incubation of both mono-OH-M and bis-OH-M with human liver microsomes formed monoglucuronides. The structures of the glucuronides were identified by liquid chromatography/tandem mass spectometry. Examination of cDNA-expressed recombinant human hepatic UGTs revealed that several catalyze glucuronidation of both compounds. Among the cDNA-expressed UGT1A enzymes, UGT1A9 seemed to be the main catalyst of formation of mono-OH-M-glucuronide, whereas UGT1A3 seemed to be the most active in bis-OH-M-glucuronide formation. Furthermore, the chiral selectivity of mono-OH-M glucuronidation was examined. The results of the incubation of single enantiomers generally agreed with the chiral analyses of mono-OH-M derived from the glucuronidase digestion of the glucuronides of the racemic mono-OH-M. There was a relatively slight but consistent enantioselective preference of individual UGT1A1, UGT1A3, UGT1A9, and UGT2B15 enzymes for glucuronidation of the S- over the R-mono-OH-M, whereas in human liver microsomes differences were observed among donors in generating the respective R/S-mono-OH-M ratio. Since it was previously shown that human liver microsomes demethylate methoxychlor mainly into S-mono-OH-M, the observation that UGT1A isoforms preferentially glucuronidate the S-mono-OH-M suggests a suitable mechanism for eliminating this major enantiomer. This enantiomeric preference, however, is not extended to all samples of human liver microsomes that we tested.