Secretory function of ovarian cells and myometrial contractions in cow are affected by chlorinated insecticides (chlordane, heptachlor, mirex) in vitro.

The aim of the study was to investigate the effect of chlordane, heptachlor and mirex, on hormonal regulation of the force of myometrial contractions. Myometrial, endometrial, granulosa and luteal cells as well as strips of myometrium from non-pregnant cows were incubated with three insecticides at environmentally relevant doses (0.1, 1 or 10ng/ml). None of the insecticides affected the viability of studied cells. Chlordane stimulated, while heptachlor and mirex inhibited, secretion of testosterone and estradiol from granulosa cells as well as secretion of progesterone from luteal cells, respectively. Secretion of oxytocin (OT) from granulosa cells was increased after incubation with all studied insecticides. Only mirex stimulated OT secretion from luteal cells, while heptachlor inhibited this effect. None of them affected synthesis of OT in luteal cells and prostaglandins (PGF2 and PGE2) secretion from uterine cells, except PGE2 secretion from endometrial cells was decreased when the cells were incubated with 0.1ng/ml of chlordane. Basal and OT-stimulated myometrial contractions were increased by mirex and decreased by heptachlor. The data show that the insecticides altered secretory function of ovarian cells. Heptachlor and mirex affected also myometrial contractions in vitro, but uterine secretion of prostaglandins were not involved in the mechanism of that adverse effect of insecticides. The data indicate on potential of these insecticides to disturb fertilisation, blastocyst implantation or even the length of gestation.

Characteristic molecular signature for early detection and prediction of persistent organic pollutants in rat liver.

Persistent organic pollutants (POPs) are degradation-resistant anthropogenic chemicals that accumulate in the food chain and in adipose tissue, and are among the most hazardous compounds ever synthesized. However, their toxic mechanisms are still undefined. To investigate whether characteristic molecular signatures can discriminate individual POP and provide prediction markers for the early detection of POPs exposure in an animal model, we performed transcriptomic analysis of rat liver tissues after exposure to POPs. The six different POPs (toxaphene, hexachlorobenzene, chlordane, mirex, dieldrin, and heptachlor) were administered to 11-week-old male Sprague-Dawley rats, and after 48 h of exposure, RNAs were extracted from liver tissues and subjected to rat whole genome expression microarrays. Early during exposure, conventional toxicological analysis including changes in the body and organ weight, histopathological examination, and blood biochemical analysis did not reflect any toxicant stresses. However, unsupervised gene expression analysis of rat liver tissues revealed in a characteristic molecular signature for each toxicant, and supervised analysis identified 2708 outlier genes that discerned the POPs exposure group from the vehicle-treated control. Combination analysis of two different multiclassifications suggested 384 genes as early detection markers for predicting each POP exposure with 100% accuracy. The data from large-scale gene expression analysis of a different POP exposure in rat model suggest that characteristic expression profiles exist in liver hepatic cells and multiclassification of POP-specific molecular signatures can discriminate each toxicant at an early exposure time. The use of these molecular markers may be more widely implemented in combination with more traditional techniques for assessment and prediction of toxicity exposure to POPs from an environmental aspect.

Exposure to persistent organic pollutants (POPs) and DNA damage as an indicator of environmental stress in fish of different feeding habits of Coatzacoalcos, Veracruz, Mexico.

The region of Coatzacoalcos, Veracruz hosts one of the largest and most important industrial areas of Mexico and Latin America. Industrial development and rapid population growth, have triggered a severe impact on aquatic ecosystems of the region. The aim of this study was to determine the levels of POPs in sediment and in muscle tissue of five fish species from different trophic levels in downstream residents of the Coatzacoalcos River, and their integration with DNA damage in the fish, evaluated with the comet assay in whole blood as a biological indicator of stress, in order to obtain a baseline of the ecological condition of the region. The compounds detected in sediment and in muscle tissue were hexachlorobenzene (HCB), α-, ß-, γ-hexachlorocyclohexane (HCH), dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), mirex and polychlorinated biphenyls (PCBs). Sediment concentrations of these pollutants (except for mirex) exceeded the values of protection provided by international guidelines, suggesting a potential risk to aquatic life in the region. DNA damage recorded in the fish species is evidence of exposure to a mix of genotoxic pollutants, which combined with exposure to POPs, reflects the degree of environmental stress of aquatic organisms in the region. The results of this study show the importance of determining the presence of contaminants in the environment, the bioaccumulation in tissues and their effects on exposed organisms, providing an integrated approach in assessing the health of aquatic ecosystems.

Demographic, endocrine and behavioral responses to mirex in the South polar skua.

Population consequences of chronic exposure to multiple pollutants at low environmental doses remain speculative, because of the lack of appropriate long-term monitoring surveys. This study integrates proximate and ultimate aspects of persistent organic pollutants (POP) burden in free-living vertebrates, by coupling hormonal and behavioral endpoints, life-history traits, and population dynamics. Blood samples (N=70) were collected in South polar skuas during two breeding periods, in 2003 and 2005, and individuals were annually monitored until 2011. Multi-state mark recapture models were used to test the effects of POP levels on demographic traits. Survival rate and long-term breeding probability were not related to individual POP levels, whereas long-term breeding success significantly decreased with increasing blood levels of mirex, an organochlorine insecticide. At the proximate level, corticosterone (stress hormone) and prolactin (parental care hormone) levels were not linked to individual POP burden. Nest defense in 2005 was significantly less intensive in chick-rearing skuas bearing higher mirex levels, suggesting reproductive behavioral impairment. Matrix population models were then built to project the rate of population decline according to increasing mirex burden. Although mirex levels were 2.8 times higher in 2003 than in 2005, the population-level effect of mirex was only detected in 2005, the year of higher corticosterone levels. The combination of endocrine traits with demographic analysis thereby enables to provide new support of synergistic interactions between pollutants and stress levels on long-term breeding outputs and population dynamics.

A carcinogenicity assay of Mirex in Charles River CD rats.

The long-term administration of 50 and 100 ppm of Mirex in the diets of male and female Charles River CD rats was associated with a spectrum of liver lesions, from foci or areas of cellular alteration and neoplastic nodules to hepatocellular carcinoma. Statistically significant numbers of neoplastic nodules were observed in the livers of male rats receiving the high dose. Neoplastic nodules and hepatocellular carcinomas were not observed in control rats.

The chlorinated pesticide mirex is a novel nonphorbol ester-type tumor promoter in mouse skin.

The hepatocarcinogenic organochlorine pesticide, mirex, was examined as a tumor promoter in the mouse skin initiation-promotion model. Female CD-1 mice were initiated with 200 nmol 7,12-dimethylbenz[a] anthracene and topically promoted three times weekly for 20 weeks with doses of 25, 50, 100, or 200 nmol mirex. Mirex promoted tumors at all dose levels in a dose-dependent manner. At 20 weeks, mice promoted with 25, 50, 100, and 200 nmol mirex developed an average of 0.2, 4, 10, and 16 tumors per mouse with a 10, 60, 93, and 96% incidence of tumor-bearing mice, respectively. With continued treatment to 34 weeks, mice promoted with 25, 50, and 100 nmol mirex developed an average of 0.7, 7, and 12 tumors per mouse with a 27, 85, and 100% incidence of tumor-bearing mice, respectively. These results demonstrate that mirex is a very effective tumor promoter in mouse skin. The effect of mirex on several biochemical and morphological events associated with tumor promotion was then investigated. Mirex did not stimulate epidermal protein kinase C activity in vitro. Unlike the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, a single topical application of mirex (200 nmol) did not increase [3H]thymidine incorporation into epidermal DNA up to 108 h after application. Furthermore, multiple applications of 200 nmol mirex (3 times weekly for 4 weeks) resulted in only a very weak proliferative response; mirex increased the number of nucleated epidermal cell layers from 1 to 2 in acetone-treated controls to 2 to 3 while 2 nmol 12-O-tetradecanoylphorbol-13-acetate produced 6 to 7 nucleated cell layers. Mirex (200 nmol) did not induce ornithine decarboxylase activity up to 56 h after a single topical application. Collectively, these data indicate that mirex is a novel nonphorbol ester-type tumor promoter in mouse skin.

Hepatic microsomal p-nitroanisole O-demethylase. Effects of chlordecone or mirex induction in male and female rats.

The effects of chlordecone (CD) or mirex treatment on the hepatic microsomal monooxygenase system of male and female rats were investigated, and kinetic parameters (apparent Vmax and apparent Km) for p-nitroanisole O-demethylase were studied in detail. Both pesticides elevated the levels of cytochrome P-450 in a time- and dose-dependent manner. The maximum rate of p-nitroanisole metabolism in males was increased about 100 and 50% and the apparent Km was elevated about 40- and 18-fold by CD and mirex respectively. p-Nitroanisole metabolism in females was reduced slightly by treatment with either agent, and the apparent Km was increased about 14-fold by CD but was relatively unaffected by mirex treatment.

Altered hepatic energy status in chlordecone (Kepone)-potentiated CCl4 hepatotoxicity.

Previous studies have demonstrated that increased intracellular calcium, depletion of glycogen, and suppressed hepatocellular division resulting in progression of hepatic lesion without recovery are associated with chlordecone (CD)-potentiated CCl4 hepatotoxicity. Since these phenomena are indicative of compromised hepatic energy status, the present studies were designed to investigate this possibility. Neither hepatic ATP content nor mitochondrial Mg2(+)-ATPase was altered significantly in rats maintained on diets contaminated with either CD (10 ppm), or phenobarbital (PB; 225 ppm) alone for 15 days. Similarly, CCl4 (100 microL/kg) administration alone did not alter hepatic ATP levels or mitochondrial Mg2(+)-ATPase activity in rats maintained on a normal diet. However, CCl4 administration to CD pretreated rats resulted in significantly decreased hepatic ATP content as early as 1 hr (36%), and this decrease was irreversibly progressive with time (81% at 6 hr). Oligomycin-sensitive Mg2(+)-ATPase was decreased significantly only starting at 6 hr (21%) after CCl4 administration, indicating that depletion of ATP at early time points was most likely due to rapid utilization consequent to toxic events. CCl4 administration to mirex or PB pretreated rats resulted in a smaller decrease in ATP levels (18-24%) only at 24 hr, returning to normal levels by 36-48 hr, in accord with rapid recovery from limited liver injury. These findings indicate that CCl4 administration to CD but not to PB or mirex pretreated rats results in a severely compromised energy status of the liver. The progressive and early depletion of liver ATP and the inhibition of Mg2(+)-ATPase in CD + CCl4 treated rats indicate the association of compromised energy status with altered Ca2+ homeostasis, depletion of glycogen, and suppressed cell division in CD-potentiated CCl4 toxicity.

Evaluation of mirex, photomirex and chlordecone in the terrestrial aquatic laboratory model ecosystem.

The insecticides mirex and chlordecone and the mirex photodegradation product, photomirex, were evaluated in a terrestrial aquatic laboratory model ecosystem. Although chlordecone was to some extent degraded during the 33 days, neither mirex nor photomirex produced identifiable levels of decomposition products in the water or in any of the organisms of the model ecosystem. All three compounds accumulated significantly in the organisms of the model ecosystem, with chlordecone the least bioaccumulative and photomirex the most bioaccumulative.

Minimal role of enhanced cell proliferation in skin tumor promotion by mirex: a nonphorbol ester-type promoter.

Mirex, a chlorinated hydrocarbon previously used as a systemic insecticide and flame retardant, is a nongenotoxic hepatocarcinogen in both rats and mice. In liver, mirex induced biochemical responses and hyperplasia characteristic of increased cell proliferation, which is consistent with its role as a liver tumor promoter. We have recently shown that mirex is a potent nonphorbol ester-type skin tumor promoter in 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated mice. However, unlike its effect in liver, a single topical application of mirex to skin does not induce the acute biochemical responses, such as increased epidermal DNA synthesis and ornithine decarboxylase activity, indicative of increased cell proliferation. Multiple topical applications of mirex over a 1 month period induced only a minimal increase in the number of epidermal nucleated cell layers, which contrasts with definitive hyperplasia induced by a comparable tumor-promoting dose of 12-O-tetradecanoylphorbol-13-acetate (TPA). Collectively, these data indicated that mirex is promoting through a novel mechanism. Further evidence that mirex promotes tumors through a mechanism distinct from that of the prototypical skin tumor promoter, TPA, was obtained by examining the effect of their simultaneous co-treatment. The co-application of mirex and TPA yielded a tumor multiplicity greater than the sum of the responses of each promoter individually. In summary, our results demonstrate that mirex, a carcinogenic and hyperplastic agent in liver, is also a very effective tumor promoter in mouse skin, but suggest that mirex operates via a novel mechanism in skin that may involve only a minimal role for enhanced cell proliferation.

Quantitative determination of endocrine- disrupting polychlorinated biphenyls and organochlorinated pesticides in human serum using gas chromatography with electron-capture detection and tandem mass spectrometry.

A sensitive, selective and reliable procedure was developed and validated to determine organochlorinated compounds, which have endocrine-disrupting effects, in human serum. Target compounds were selected between polychlorinated biphenyls and organochlorinated pesticides. Sample workup consisted of (1) extraction of serum with organic solvents, (2) clean-up of the organic extract using acid treatment with H(2)SO(4), (3) elution of the cleaned-up extract through a liquid column chromatographic system and (4) analysis of the fraction eluted by gas chromatography with electron capture detection (ECD) and tandem mass spectrometry (MS/MS) detection. Performance characteristics, such as linearity, sensitivity, precision, accuracy and recovery, of both chromatographic methods were studied. The proposed analytical methodology was applied to determine the target compounds in serum samples from women living in agricultural areas of Almería (Spain). The results show the advantage of MS/MS over ECD in the analysis of real human serum samples where matrix interferences can be confused with target pesticides.

17beta-estradiol is a hormonal regulator of mirex tumor promotion sensitivity in mice.

Mirex, an organochlorine pesticide, is a potent non-phorbol ester tumor promoter in mouse skin. Previous studies have shown that female mice are 3 times more sensitive to mirex tumor promotion than male mice and that ovariectomized (OVX) female mice are resistant to mirex promotion, suggesting a role for ovarian hormones in mirex promotion. To determine whether the ovarian hormone 17-beta estradiol (E2) is responsible for the sensitivity of female mice to mirex promotion, female mice were initiated with DMBA; 2 weeks later groups of mice were OVX and implants, with or without E2, were surgically implanted subcutaneously. These mice were treated topically twice weekly with mirex for 26 weeks. E2 implanted OVX mice demonstrated high normal physiologic levels of serum E2 throughout the tumor promotion experiment. E2 implants restored by 80% the intact mirex-sensitive phenotype to the OVX mice. Consistent with a role for E2 and ERalpha and ERbeta, treatment of DMBA-initiated female mice with topical ICI 182,780, an estrogen-receptor antagonist, reduced mirex tumor multiplicity by 30%. However, in cells co-transfected with ERalpha or ERbeta and estrogen-responsive promoter reporter, mirex did not stimulate promoter reporter activity, suggesting that the promotion effect of mirex is downstream of ERalpha/beta. Finally, a tumor promotion study was conducted to determine whether E2 implants could increase the sensitivity of male mice to mirex promotion. E2 implants in male mice did increase sensitivity to mirex promotion; however, the implants did not produce the full female sensitivity to mirex tumor promotion. Collectively, these studies indicate that E2 is a major ovarian hormone responsible for mirex tumor promotion sensitivity in female mice.

Ninety-day toxicity of photomirex in the male rat.

Photomirex (8-monohydromirex) is a demonstrated environmental contaminant and was observed in previous short-term studies to produce lesions in the liver, thyroid and testes of male rats. The present study was undertaken to confirm those observations and to determine the effects after a longer period of exposure. Male rats were fed photomirex for 13 weeks at levels of 0.20, 1.0, 5.0, 25 and 125 ppm in the diet. Deaths were observed in animals receiving the highest dose. Decreased body weight gain and food intake were also observed in that group. Liver weights were increased at 5.0 ppm photomirex and higher. Photomirex caused changes in several biochemical parameters including serum sorbitol dehydrogenase and hepatic aniline hydroxylase activities. Dose-related histological abnormalities were observed in the thyroid and liver starting at the lowest dose level. These results confirm earlier findings and show that photomirex is a potent hepato- and thyrotoxin.

Reversibility of the toxicological changes induced by photomirex and mirex.

Photomirex (8-monohydromirex) is a known environmental contaminant. In a previous 28-day and 90-day study, photomirex was shown to cause a number of biochemical and histological alterations in male rats. The present study was undertaken to determine the reversibility of those changes and to compare both effects and reversibility with the present compound mirex. Male rats were fed diets containing 0, 0.05, 0.5, 5.0 and 50 ppm photomirex and 5 ppm or 50 ppm mirex for 28 days. Ten animals/group were killed at this time and the remainder placed on clean feed. Ten animals/group were killed after another 12, 24, and 48 weeks. The lower body weight gain in animals fed 50 ppm photomirex and 50 ppm mirex observed after 28 days on the test was not evidence after 12 weeks on the clean diet or thereafter. Reduced food consumption produced by these groups persisted for 12 weeks but not 24 weeks. Liver hypertrophy was observed in the 5.0-ppm and 50-ppm photomirex groups after 28 days and persisted in the highest dose group for 24 weeks on clean diet. Elevated serum sorbitol dehydrogenase activity was observed in the 50-ppm photomirex group and persisted for 12 weeks on clean diet. Histological changes in liver and thyroid were present up to 48 weeks on clean diet, whereas treatment-related testicular changes observed after 28 days were not evident after 12 weeks. Significant residues of photomirex were still evident in all tissues examined up to 48 weeks postexposure in the 50-ppm photomirex group. The data indicate that photomirex and mirex are extremely persistent compounds and can result in a variety of biochemical and histological changes long after exposure has ceased.

Effects of oral administration of chlordecone and mirex on brain biogenic amines in mice.

Levels of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and the 5-HT metabolite, 5-hydroxyindole acetic acid (5-HIAA) were determined in brains of mice after daily oral administration of 10, 25 or 50 mg/kg of chlordecone or mirex until mortality occurred. Significant decreases in whole brain and striatal DA levels were observed in chlordecone-treated mice exhibiting tremors. Mirex had no effect at the 3 doses tested. In mice treated with chlordecone or mirex, the 5-HT levels were elevated only in animals exhibiting severe tremors or diarrhea, respectively. NE levels were not altered by chlordecone or mirex. These results suggest that chlordecone-induced neurotoxicity may be due in part to a decrease in the inhibitory state of dopamine neurons.

Chlordecone interaction with catecholamine binding and uptake in rat brain synaptosomes.

The in vitro and in vivo effects of chlordecone on rat brain synaptosomal binding and uptake of 3H-dopamine and 3H-norepinephrine were determined. The data show that chlordecone decreases both binding and uptake of the 3H-catecholamines in vitro and in vivo. The uptake of 3H-dopamine was more sensitive to chlordecone than binding. 3H-norepinephrine binding and uptake was decreased to a lesser degree as compared to dopamine. In general, chlordecone at about 25-50 microM concentration produced a 50% decrease of the ligands binding and uptake. Mirex, a non-neurotoxic compound and a structural analog of chlordecone altered neither binding nor uptake of the ligands tested. These studies suggest that chlordecone may be modulating the pre and post synaptic function.

Effects of chlordecone and mirex on amino acids incorporation into brain and liver proteins in the mouse.

Effects of chlordecone and mirex on 3H-leucine or 3H-amino acids incorporation into mouse brain and liver proteins were investigated. In mice treated with chlordecone by oral intubation, 25 mg/kg/day for 3 days, a significant decrease in 3H-leucine incorporation into brain trichloroacetic acid (TCA)-precipitable proteins was observed. On the other hand, mirex significantly increased 3H-leucine incorporation into TCA-precipitable proteins in the liver but not in that of the brain. These phenomena also were confirmed by the data obtained from 3H-amino acids mixture incorporation into both brain and liver proteins in mice which were orally administered with chlordecone or mirex, 25 mg/kg/day for 4 days. These results are also substantiated by a single dose of chlordecone (125 or 200 mg/kg, p.o.) or mirex (250 or 350 mg/kg, p.o.). These results suggest that although structurally similar, these two analogs differ significantly in their effects on amino acids incorporation into brain and liver proteins.

Ultrastructural alterations in the liver of rats fed photomirex (8-monohydromirex).

Alterations in the liver of rats fed photomirex or mirex for 28 d have been studied by electron microscopy. A dose of 0.05 or 0.5 ppm photomirex produced selective ultrastructural damage to the mitochondria of hepatocytes. The parenchymal cells had a proliferation of smooth-surfaced endoplasmic reticulum (SER) with a concomitant depletion of glycogen, a reduction of rough-surfaced endoplasmic reticulum (RER) profiles, an abnormal accumulation of lipid droplets, and pleomorphic nuclei at the 5 ppm photomirex level. In the animals receiving 50 ppm photomirex there were degenerating foci in the organ. There was an increase of electron-dense bodies in the hepatocytes from the 5 ppm mirex group but these cells, in the animals receiving 50 ppm mirex, had SER proliferation and glycogen depletion. It is concluded that photomirex causes more severe ultrastructural changes in the livers of rats than mirex.

Histopathologic lens changes in mirex-exposed rats.

The early histologic lesions and sequential changes in the development of mirex-induced cataracts were studied in the offspring of lactating female Sherman rats given oral doses of mirex for 5 consecutive days post partum. The earliest histologic change, seen at neonatal day 7, was slight swelling of the individual cortical lens fibers. At day 9, many swollen but intact fibers were observed, and at days 11 and 13, extensive degeneration and necrosis appeared throughout the cortex of the affected lenses.

The effects of mirex on the neonatal rat lens in vitro, with a comparison to kepone.

Lenses were removed from 12-day-old rats and incubated in medium containing 20 microM mirex. Lenses were removed from the medium at 2, 4, 6, and 8 h and assayed for K+ and Na+ concentrations and hydration. Mirex had no in vitro effect on these lens parameters compared to controls, and produced no changes in lens clarity. In contrast, incubation for 8 h in medium containing 20 microM kepone resulted in elevated lens Na+ and lower lens K+. Thus, cataracts induced by mirex in vivo are probably not due to a direct toxic effect on the lens.