Impact of endocrine-active compounds on adrenal androgen production in pigs during neonatal period.

This study investigated the impact of neonatal exposure to endocrine-active compounds (EACs): flutamide (antiandrogen), 4-tert-octylphenol (an estrogenic compound), and methoxychlor (an organochlorine insecticide exhibiting estrogenic, antiestrogenic and antiandrogenic activities) on androgen production within porcine adrenal glands. The expression of genes related to androgen synthesis and the level of androgen production were analyzed (i) in the adrenal glands of piglets exposed to EACs during the first 10 days of life (in vivo study), and (ii) in adrenal explants from sow-fed or formula-fed 10-day-old piglets incubated with EACs (ex vivo study). EACs affected the expression of genes linked to adrenal androgen biosynthesis. The prominent effect of methoxychlor on downregulation of StAR, CYP11A1 and HSD3B and upregulation of CYP17A1 and SULT2A1 were demonstrated. Furthermore, our study revealed divergent response to EACs between sow-fed and formula-fed piglets, suggesting that natural feeding may provide protection against adverse EACs effects, particularly those interfering with estrogens action.

Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors.

In this paper, we investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities on ovarian follicles of adult pigs. Piglets were injected with MXC (20 µg/kg body weight) or corn oil (controls) from postnatal Day 1 to Day 10 (n = 5 per group). Then, mRNA expression, protein abundance and immunolocalization of growth and differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), anti-Müllerian hormone (AMH) and cognate receptors (ACVR1, BMPR1A, BMPR1B, TGFBR1, BMPR2, and AMHR2), as well as FSH receptor (FSHR) were examined in preantral and small antral ovarian follicles of sexually mature gilts. The plasma AMH and FSH levels were also assessed. In preantral follicles, neonatal exposure to MXC increased GDF9, BMPR1B, TGFBR1, and BMPR2 mRNAs, while the levels of AMH and BMP15 mRNAs decreased. In addition, MXC also decreased BMP15 and BMPR1B protein abundance. Regarding small antral follicles, neonatal exposure to MXC upregulated mRNAs for BMPR1B, BMPR2, and AMHR2 and downregulated mRNAs for AMH, BMPR1A, and FSHR. MXC decreased the protein abundance of AMH, and all examined receptors in small antral follicles. GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles of control and treated ovaries. All analyzed receptors were detected in the oocytes and granulosa cells of preantral follicles, and in the granulosa and theca cells of small antral follicles. The exception, however, was FSHR, which was detected only in the granulosa cells of small antral follicles. In addition, MXC decreased the plasma AMH and FSH concentrations. In conclusion, the present study may indicate long-term effects of neonatal MXC exposure on GDF9, BMP15, AMH, and FSH signaling in ovaries of adult pigs. However, the MXC effects varied at different stages of follicular development. It seems that neonatal MXC exposure may result in accelerated initial recruitment of ovarian follicles and impaired cyclic recruitment of antral follicles.

Effects of neonatal methoxychlor exposure on the ovarian transcriptome in piglets.

Methoxychlor (MXC) is a man-made organochlorine insecticide capable of disrupting endocrine functions due to its mixed steroidal properties (estrogenic, anti-estrogenic and/or anti-androgenic). Retarded follicle development was recently reported in neonatal pigs treated with MXC. The goal of the current study was to better understand the mechanism of MXC action in the ovary of newborn piglets. By employing RNA-Seq we studied the expression of protein coding (mRNA) and long non-coding RNA (lncRNA) transcripts in the ovary of the MXC-treated piglets. Piglets were injected (sc) daily with MXC (100 mg/kg body weight) or corn oil (controls) between postnatal Days 1 and 10 (n = 3 piglets/group). The ovaries excised from 11-day-old piglets were processed for total RNA isolation and subsequent RNA sequencing. Four hundred sixty differentially expressed genes (DEGs) and 143 differentially expressed lncRNAs (DELs) were identified in the ovaries of MXC-treated piglets (P-adjusted < 0.05; abs(log2FC) > 1). Functional enrichment analysis showed that MXC altered the expression of genes associated with intracellular and membrane transport, intra-ovarian signaling as well as cell-cell junction and communication. Moreover, positive and negative correlations determined between the identified DEGs and DELs suggest that some lncRNAs may mediate the MXC action in the ovary. The results support the hypothesis that MXC-induced changes in the expression of genes involved in neonatal ovarian folliculogenesis increase the risk of fertility problems in adults.

Bioaccumulation and toxicity of methoxychlor on Chinese mitten crab (Eriocheir sinensis).

Chinese mitten crab, a featured macrobenthos, has been one of the most important economical aquatic species in China. This study assessed the accumulation of an organochlorine pesticide methoxychlor (MXC) in Chinese mitten crab during exposure to 1 mg/L of MXC. The results showed the residual concentration of MXC in the ovary and hepatopancreas reached 55.07 ±â€¯2.64 ng/g and 34.51 ±â€¯2.35 ng/g, respectively. After exposure, tubular vacuolization of epithelial tissues, condensed egg cells and obvious intervals between egg cell wall and stroma were observed in the hepatopancreas and ovary, respectively. Significant changes of three key metabolic enzymes in hepatopancreas were observed upon exposure to MXC. Compared to the control, acetylcholinesterase level was significantly higher at day 7 (0.15 ±â€¯0.01 vs. 0.06 ±â€¯0.00 U/mgprot); glutathione S-transferase level was elevated at both day 4 (12.01 ±â€¯0.48 vs. 3.20 ±â€¯0.44 U/mgprot) and day 7 (12.84 ±â€¯1.01 vs. 8.22 ±â€¯0.81 U/mgprot); superoxide dismutase was sharply increased at day 4 (21.20 ±â€¯0.24 vs. 3.66 ±â€¯0.60 U/mgprot) but decreased at day 7 (3.74 ±â€¯0.12 vs. 9.44 ±â€¯0.85 U/mgprot). Overall, dissolved MXC accumulated in lipid-rich tissues could cause damages on epithelial cells and egg cells and change metabolic activities of enzymes involved in antioxidative stress and detoxification processes.

Transcriptional networks associated with the immune system are disrupted by organochlorine pesticides in largemouth bass (Micropterus salmoides) ovary.

Largemouth bass (Micropterus salmoides) inhabiting Lake Apopka, Florida are exposed to high levels of persistent organochlorine pesticides (OCPs) and dietary uptake is a significant route of exposure for these apex predators. The objectives of this study were to determine the dietary effects of two organochlorine pesticides (p, p'-dichlorodiphenyldichloroethylene; p, p' DDE and methoxychlor; MXC) on the reproductive axis of largemouth bass. Reproductive bass (late vitellogenesis) were fed one of the following diets: control pellets, 125ppm p, p'-DDE, or 10ppm MXC (mg/kg) for 84days. Due to the fact that both p,p' DDE and MXC have anti-androgenic properties, the anti-androgenic pharmaceutical flutamide was fed to a fourth group of largemouth bass (750ppm). Following a 3 month exposure, fish incorporated p,p' DDE and MXC into both muscle and ovary tissue, with the ovary incorporating 3 times more organochlorine pesticides compared to muscle. Endpoints assessed were those related to reproduction due to previous studies demonstrating that these pesticides impact the reproductive axis and we hypothesized that a dietary exposure would result in impaired reproduction. However, oocyte distribution, gonadosomatic index, plasma vitellogenin, and plasma sex steroids (17ß-estradiol, E2 and testosterone, T) were not different between control animals and contaminant-fed largemouth bass. Moreover, neither p, p' DDE nor MXC affected E2 or T production in ex vivo oocyte cultures from chemical-fed largemouth bass. However, both pesticides did interfere with the normal upregulation of androgen receptor that is observed in response to human chorionic gonadotropin in ex vivo cultures, an observation that may be related to their anti-androgenic properties. Transcriptomics profiling in the ovary revealed that gene networks related to cell processes such as leukocyte cell adhesion, ossification, platelet function and inhibition, xenobiotic metabolism, fibrinolysis, and thermoregulation were altered by p, p' DDE, MXC, and flutamide. Interestingly, immune-related gene networks were suppressed by all three chemicals. The data suggest that p, p' DDE and flutamide affected more genes in common with each other than either chemical with MXC, consistent with studies suggesting that p, p' DDE is a more potent anti-androgen than MXC. These data demonstrate that reproductive health was not affected by these specific dietary treatments, but rather the immune system, which may be a significant target of organochlorine pesticides. The interaction between the reproductive and immune systems should be considered in future studies on these legacy and persistent pesticides.

Hepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: Comparison between in vivo and in vitro and across species.

In vitro tools using isolated primary fish hepatocytes have been proposed as a useful model to study the hepatic metabolism of xenobiotics in fish. In order to evaluate the potential of in vitro fish hepatocyte assays to provide information on in vivo metabolite patterns of pesticides in farmed fish, the present study addressed the following questions: Are in vitro and in vivo metabolite patterns comparable? Are species specific differences of metabolite patterns in vivo reflected in vitro? Are metabolite patterns obtained from cryopreserved hepatocytes comparable to those from freshly isolated cells? Rainbow trout and common carp were dosed orally with feed containing the pesticide methoxychlor (MXC) for 14days. In parallel, in vitro incubations using suspensions of freshly isolated or cryopreserved primary hepatocytes obtained from both species were performed. In vivo and in vitro samples were analyzed by thin-layer chromatography with authentic standards supported by HPLC-MS. Comparable metabolite patterns from a qualitative perspective were observed in liver in vivo and in hepatocyte suspensions in vitro. Species specific differences of MXC metabolite patterns observed between rainbow trout and common carp in vivo were well reflected by experiments with hepatocytes in vitro. Finally, cryopreserved hepatocytes produced comparable metabolite patterns to freshly isolated cells. The results of this study indicate that the in vitro hepatocyte assay could be used to identify metabolite patterns of pesticides in farmed fish and could thus serve as a valuable tool to support in vivo studies as required for pesticides approval according to the EU regulation 1107.

Degradation of 2,4 dichlorobiphenyl via meta-cleavage pathway by Pseudomonas spp. consortium.

Two bacterial isolates (Pseudomonas sp. GSa and Pseudomonas sp. GSb) were in close association able to assimilate 2,4 dichlorobiphenyl (2,4 CB), a PCB congener. GC-MS analysis of spent culture medium of the consortium with 2,4 CB as substrate showed 90 % degradation (according to Electron capture detection values) with catechol as one of the important intermediate compounds through meta-cleavage pathway. Further, ability of the consortium to utilise PCB congeners, Methoxychlor, Aroclor 1016, Chlorobenzoic acids and Monoaromatic compounds indicated that the consortium of GSa and GSb would be an ideal candidate for in situ bioremediation of PCB.

Interactions of endosulfan and methoxychlor involving CYP3A4 and CYP2B6 in human HepaRG cells.

Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation.

Effects of methoxychlor and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane on human and rat 17α-hydroxylase/17,20-lyase activity.

Exposure to methoxychlor, an agricultural pesticide, has been associated with reduced testicular androgen secretion. However, methoxychlor is converted to 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) in the liver, which then acts as its biologically active metabolite. Both methoxychlor and HPTE have been credited with estrogenic properties and have a weak anti-androgenic activity. However, the exact mechanisms of steroidogenic enzyme inhibition remain to be clarified. In the present study, human and rat testis microsomes were employed to investigate the inhibitory activities of methoxychlor and HPTE on 17α-hydroxylase/17,20-lyase (CYP17A1). The CYP17A1 enzyme is critical for androgen biosynthesis and catalyzes conversion of progesterone into androstenedione. The results demonstrated that HPTE directly inhibited human and rat CYP17A1 activities, while methoxychlor had no effects on this enzyme activity even at a concentration of 100 µM. The IC50 values of HPTE were 1.13±0.10 (human) and 6.87±0.13 µM (rat), respectively. When HPTE was incubated with rat immature Leydig cells, it also inhibited CYP17A1 activity with an IC50 value of 6.29±0.1 µM. Results of enzyme inhibition were supported by the observation that HPTE inhibited luteinizing hormone-stimulated 5α-androstane-3α,17ß-diol and testosterone secretion by immature Leydig cells with IC50 values of 6.61±0.03 and 3.78±0.003 µM, respectively. The mode of action of HPTE on CYP17A1 activity was determined to be uncompetitive with the substrate progesterone. In conclusion, HPTE, the metabolite of MXC, directly inhibited human and rat testis CYP17A1 activities.

A role of Bradyrhizobium elkanii and closely related strains in the degradation of methoxychlor in soil and surface water environments.

We have reported that a leguminous bacterial strain, Bradyrhizobium sp. strain 17-4, isolated from river sediment, phylogenetically very close to Bradyrhizobium elkanii, degraded methoxychlor through O-demethylation and oxidative dechlorination. In the present investigation, we found that B. elkanii (USDA94), a standard species deposited in the Culture Collection, degraded methoxychlor. Furthermore, Bradyrhizobium sp. strain 4-1, also very close to B. elkanii, isolated from Japanese paddy field soil, degraded methoxychlor. These B. elkanii and closely related strains degraded methoxychlor through almost identical metabolic pathways, and cleaved the phenyl ring and mineralized. In contrast, another representative Bradyrhizobium species, B. japonicum (USDA110), did not degrade methoxychlor at all. Based on these findings, B. elkanii and closely related strains are likely to play an important role not only in providing the readily biodegradable substrates but also in completely degrading (mineralizing) methoxychlor by themselves in the soil and surface water environment.

O-Demethylation and successive oxidative dechlorination of methoxychlor by Bradyrhizobium sp. strain 17-4, isolated from river sediment.

O-Demethylation of insecticide methoxychlor is well known as a phase I metabolic reaction in various eukaryotic organisms. Regarding prokaryotic organisms, however, no individual species involved in such reaction have been specified and characterized so far. Here we successfully isolated a bacterium that mediates oxidative transformation of methoxychlor, including O-demethylation and dechlorination, from river sediment. The isolate was found to be closely related to Bradyrhizobium elkanii at the 16S rRNA gene sequence level (100% identical). However, based on some differences in the physiological properties of this bacterium, we determined that it was actually a different species, Bradyrhizobium sp. strain 17-4. The isolate mediated O-demethylation of methoxychlor to yield a monophenolic derivative [Mono-OH; 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane] as the primary degradation product. The chiral high-performance liquid chromatography (HPLC) analysis revealed that the isolate possesses high enantioselectivity favoring the formation of (S)-Mono-OH (nearly 100%). Accompanied by the sequential O-demethylation to form the bis-phenolic derivative Bis-OH [1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane], oxidative dechlorination of the side chain proceeded, and monophenolic carboxylic acid accumulated, followed by the formation of multiple unidentified polar degradation products. The breakdown proceeded more rapidly when reductively dechlorinated (dichloro-form) methoxychlor was applied as the initial substrate. The resultant carboxylic acids and polar degradation products are likely further biodegraded by ubiquitous bacteria. The isolate possibly plays an important role for complete degradation (mineralization) of methoxychlor by providing the readily biodegradable substrates.

Reductive dechlorination of methoxychlor by bacterial species of environmental origin: evidence for primary biodegradation of methoxychlor in submerged environments.

Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] is an organochlorine insecticide that undergoes dechlorination in natural submerged environments. We investigated the ability to dechlorinate this compound in seven environmental bacterial species ( Aeromonas hydrophila , Enterobacter amnigenus , Klebsiella terrigena , Bacillus subtilis , Achromobacter xylosoxidans , Acinetobacter calcoaceticus , and Mycobacterium obuense ) and the enteric bacterium Escherichia coli as a positive control. In R2A broth at 25 °C under aerobic, static culture, all species except Ach. xylosoxidans were observed to convert methoxychlor to dechlorinated methoxychlor [1,1-dichloro-2,2-bis(4-methoxyphenyl)ethane]. The medium was aerobic at first, but bacterial growth resulted in the consumption of oxygen and generated microaerobic and weakly reductive conditions. Replacement of the headspace of the culture tubes with nitrogen gas was found to decrease the dechlorination rate. Our findings suggest that extensive bacterial species ubiquitously inhabiting the subsurface water environment play an important role in the primary dechlorination of methoxychlor.

An environmental fate study of methoxychlor using water-sediment model system.

Agricultural waste water containing pesticides can reach the sea via rivers and estuaries, including brackish lakes. We studied the metabolic fate of methoxychlor [MXC; 1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] in a model system consisting of sediment and associated water collected from two sampling sites: a brackish lake and a freshwater river. MXC degraded rapidly and was finally mineralized in both sediment systems. The first step of degradation was dechlorination to yield 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethane [de-Cl-MXC] or CN-replacement to yield 2,2-bis(4-methoxyphenyl)acetonitrile [MXC-CN], followed by O-demethylation. Although the metabolites were common to the two sediments, the dynamics of the metabolites over time were clearly distinct. In the brackish lake sediment, de-Cl-MXC accumulated transiently, whereas in the river sediment, it was rapidly converted to its demethylated metabolite. We also found that dechlorination and CN-replacement proceeded in autoclave-sterilized river sediment. In the river sediment, the abiotic reaction mediated by abundant humic acid and low oxygen level also appeared to contribute to the overall MXC metabolism.

Persistent organic pollutants in ringed seals from the Russian Arctic.

Organochlorine compounds total DDT (ΣDDT), total HCH isomers (ΣHCH), toxaphenes (sum of Parlar 26, 50, 62), mirex, endrin, methoxychlor, total chlorinated benzenes (ΣCBz), total chlordane compounds (ΣCHL), polychlorinated biphenyls (total of 56 congeners; ΣPCBs), polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), and polybrominated diphenyl ethers (sum of 7 tri- to hepta congeners; ΣPBDEs) were analysed in the blubber of adult ringed seals from the four areas of the Russian Arctic (White Sea, Barents Sea, Kara Sea and Chukchi Sea) collected in 2001-2005. Ringed seals from the south-western part of the Kara Sea (Dikson Island - Yenisei estuary) were the most contaminated with ΣDDTs, ΣPCBs, ΣCHL, and mirex as compared with those found in the other three areas of Russian Arctic, while the highest mean concentrations of ΣHCHs and PCDD/Fs were found in the blubber of ringed seals from the Chukchi Sea and the White Sea, respectively. Among all organochlorine compounds measured in ringed seals from the European part of the Russian Arctic, concentrations of ΣDDT and ΣPCBs only were higher as compared with the other Arctic regions. Levels of all other organochlorine compounds were similar or lower than in seals from Svalbard, Alaska, the Canadian Arctic and Greenland. ΣPBDEs were found in all ringed seal samples analysed. There were no significant differences between ΣPBDE concentrations found in the blubber of ringed seals from the three studied areas of the European part of the Russian Arctic, while PBDE contamination level in ringed seals from the Chukchi Sea was 30-50 times lower. ΣPBDE levels in the blubber of seals from the European part of the Russian Arctic are slightly higher than in ringed seals from the Canadian Arctic, Alaska, and western Greenland but lower compared to ringed seals from Svalbard and eastern Greenland.

Bioconcentration and biotransformation of [¹4C]methoxychlor in the brackish water bivalve Corbicula japonica.

To obtain basic information on the metabolic fate of xenobiotics in the brackish water, bivalve Corbicula japonica, bioconcentration and biotransformation experiments were performed using methoxychlor (MXC) as a model compound. Bivalves were exposed to [ring-U-¹4C]MXC (10 µg L⁻¹) for 28 days under semi-static conditions followed by a 14-day depuration phase. The ¹4C concentration in the bivalves rapidly increased and reached a steady state after exposure for 7 days (BCFss = 2010); however, it rapidly decreased with a half-life of 2.2 days in the depuration phase. Mono- and bis-demethylated MXC, and their corresponding sulphate conjugates, were identified as minor metabolites. No glycoside conjugates (including glucuronide and glucoside) were detected. Despite this biotransformation system, bivalves were found to excrete retained MXC mostly unchanged although its relatively hydrophobic nature.

Increased sensitivity of estrogen receptor alpha overexpressing antral follicles to methoxychlor and its metabolites.

Methoxychlor (MXC), an organochlorine pesticide, and its metabolites, mono-hydroxy MXC (MOH) and bis-hydroxy MXC (HPTE) are known ovarian toxicants and can cause inhibition of antral follicle growth. Since these chemicals bind to estrogen receptor alpha (ESR1), we hypothesized that ovaries overexpressing ESR1 (ESR1 OE) would be more susceptible to toxicity induced by MXC and its metabolites because the chemicals can bind to more ESR1 in the antral follicles. We cultured antral follicles from controls and ESR1 OE mouse ovaries with either the vehicle dimethylsulfoxide (DMSO), MXC, MOH, or HPTE. The data show that at 96 h, the cultured antral follicles from ESR1 OE antral follicles are more susceptible to toxicity induced by MXC, MOH, and HPTE because low doses of these chemicals cause follicle growth inhibition in ESR1 OE mice but not in control mice. On comparing gene expression levels of nuclear receptors in the cultured antral follicles of ESR1 OE and control follicles, we found differential messenger RNA (mRNA) expression of Esr1, estrogen receptor beta (Esr2), androgen receptor (Ar), progesterone receptor (Pr), and aryl hydrocarbon receptor (Ahr) between the genotypes. We also analyzed mRNA levels of Cyp3a41a, the enzyme metabolizing MOH and HPTE, in the cultured follicles and found that Cyp3a41a was significantly lower in DMSO-treated ESR1 OE follicles compared with controls. In ESR1 OE livers, we found that Cyp3a41a levels were significantly lower compared with control livers. Collectively, these data suggest that MXC and its metabolites cause differential gene expression in ESR1 OE mice compared with controls. The results also suggest that the increased sensitivity of ESR1 OE mouse ovaries to toxicity induced by MXC and its metabolites is due to low clearance of the metabolites by the liver and ovary.

Methoxychlor induces apoptosis via mitochondria- and FasL-mediated pathways in adult rat testis.

In the past few years, there has been much concern about the adverse health effects of environmental contaminants in general and organochlorine in particular. Studies have shown the repro-toxic effects of long-term exposure to methoxychlor, a member of the organochlorine family. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. In the present study we sought to elucidate the mechanism(s) underpinning the gonadal effects within hours of exposure to methoxychlor. Experimental rats were divided into six groups of four each. Animals were orally administered with a single dose of methoxychlor (50mg/kg body weight) and killed at 0, 3, 6, 12, 24, and 72h post-treatment. The levels and time-course of induction of apoptosis-related proteins like cytochorome C, caspase 3 and procaspase 9, Fas-FasL and NF-kappaB were determined to assess sequential induction of apoptosis in the rat testis. DNA damage was assessed by TUNEL assay and flowcytometry. Administration of methoxychlor resulted in a significant increase in the levels of cytosolic cytochrome c and procaspase 9 as early as 6h following exposure. Time-dependent elevations in the levels of Fas, FasL, pro- and cleaved caspase 3 were observed. The DNA damage was measured and showed time-dependent increase in the TUNEL positive cells, and also by flowcytometry of testicular cells. The study demonstrates induction of testicular apoptosis in adult rats following exposure to a single dose of methoxychlor.

Metabolism of methoxychlor by the P450-monooxygenase CYP6G1 involved in insecticide resistance of Drosophila melanogaster after expression in cell cultures of Nicotiana tabacum.

Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane, 1) against the background of endogenous enzymes of the corresponding non-transgenic culture. The Cyp6g1-transgenic cell culture metabolized 96% of applied methoxychlor (45.8 microg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 microg), whereas inhibition in the non-transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid (6; 20 microg, 24 h) in the Cyp6g1-transgenic culture by 82% in the presence of piperonyl butoxide (200 microg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor (1). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.

Anaerobic biodegradation of organochlorine pesticides in contaminated soil - significance of temperature and availability.

Anaerobic biodegradation of the pesticides: gamma-hexachlorocyclohexane, methoxychlor, o,p'- and p,p'-DDT in field polluted soil was tested at 12, 22 and 30 degrees C, using methanogenic granular sludge as inoculum. The contaminants were removed quite effectively at all temperatures and their removal rates increased 1.2-1.7 times with the increase in temperature. In most cases pesticide concentrations after an initial substantial decline remained almost constant until the end of experiment. These residual concentrations were also temperature dependent and they were 1.4-8.2 times higher at 12 degrees C than at 30 degrees C. DDT was degraded via DDD and accumulation of this metabolite was lower (19-64%) than the corresponding amount of removed DDT, especially at higher temperatures. Further transformation of DDD was confirmed by formation of p,p'-dichlorobenzophenone. Additional experiment demonstrated that removal was limited to readily desorbing fractions of pesticides, while their desorption-resistant fractions persisted in the soil. However, DDD metabolite was only partially removed despite its good desorbability.