In Utero Chlordecone Exposure and Thyroid, Metabolic, and Sex-Steroid Hormones at the Age of Seven Years: A Study From the TIMOUN Mother-Child Cohort in Guadeloupe.

Background: Chlordecone is an endocrine-disrupting chemical with well recognized estrogenic and progestagenic properties. This organochlorine insecticide was extensively used in the French West Indies from 1973 to 1993 to control the banana root borer. Due to its poor degradation in the environment, permanently polluted soil is responsible for the current contamination of the food chain and human beings. We aimed to examine the relationship of in utero exposure to chlordecone and thyroid (thyroid stimulating hormone [TSH], free tri-iodothyronine [FT3], free thyroxine [FT4]), metabolic (insulin growth-factor 1, leptin, adiponectin), and sex-steroid (dehydroepiandrosterone [DHEA], total testosterone [TT], dihydrotestosterone [DHT], estradiol [E2]) hormone levels in children at the age of seven years who participated in TIMOUN, an ongoing birth cohort in Guadeloupe. Methods: Chlordecone concentrations were measured in cord-blood at delivery. Thyroid, metabolic, and sex-steroid hormone levels were determined in the blood of children at seven years of age. Associations between in utero chlordecone exposure and hormone levels at seven years of age were assessed by multiple linear or logistic regression, controlling for confounding factors. Results: Among the study population (210 boys and 228 girls), chlordecone and hormone measurements were available for 124 boys and 161 girls. We found the third quartile of in utero chlordecone exposure relative to the lowest quartile to be associated with elevated TSH levels in girls and elevated DHEA, TT, and DHT levels in both sexes. Complementary non-linear analysis (spline regression) confirmed a significant non-linear trend for TSH in girls and DHEA and DHT in boys. Conclusion: In utero chlordecone exposure was associated with elevated levels of selected thyroid (TSH) and sex-steroid (DHEA, TT, and DHT) hormones at seven years in a non-monotonic dose response (inverted U) relationship. The implications for future health and reproductive function in puberty and adulthood should be determined.

Expression of biotransformation and oxidative stress genes in the giant freshwater prawn Macrobrachium rosenbergii exposed to chlordecone.

Chlordecone is a persistent organochlorine pesticide widely used between 1972 and 1993 in the French West Indies to control the root borer in banana fields. Chlordecone use resulted in long-term pollution of soils, contamination of waters, of aquatic organisms, and of fields. Chlordecone is known to be neurotoxic, to increase prostate cancer, and to have negative effects on cognitive and motor development during infancy. In Guadeloupe, most of the freshwater species living in contaminated rivers exceed the French legal limit of 20 µg·kg(-1) wet weight. In the present study, we chose a transcriptomic approach to study the cellular effects of chlordecone in the giant freshwater prawn Macrobrachium rosenbergii, an important economical species in Guadeloupe. Quantitative PCR revealed an induction of genes involved in defense mechanism against oxidative stress (catalase and selenium-dependent glutathione peroxidase) in prawns exposed to low environmental concentrations of chlordecone after 12 and 24 h of exposure. In prawns reared in a contaminated farm, transcription of genes involved in the biotransformation process (cytochrome P450 and glutathione-S-transferase (GST)) were induced after 8 days of exposure. Our results provide information on the mechanims of defense induced by chlordecone in aquatic crustacean species. This gene expression study of selected genes should be further strengthened by proteomic analyses and enzymatic activity assays to confirm the response of these biomarkers of stress in crustaceans and to give new insights into the mechanism of toxicity by chlordecone.

Pharmacodynamic model of the rat estrus cycle in relation to endocrine disruptors.

Several strains of laboratory rats have a high background incidence of mammary tumors and develop a persistent, anovulatory estrus condition at about 12 mo of age. The increased tumor incidence is believed to be associated with elevated estradiol (E2) and prolactin during the period of persistent estrus. A pharmacodynamic estrus cycle (PD-EC) model for the Sprague-Dawley rats has been developed in an attempt to analyze the physiological basis of early-onset persistent estrus and to examine the potential sites of interactions in the hypothalamic-pituitary-ovarian axis for endocrine-modulating xenobiotics that accelerate the onset of persistent estrus. This initial estrus cycle model focused solely on cyclical changes in E2 and luteinizing hormone (LH). An LH surge was scheduled when a hypothetical estrus cycle-related protein (EC-RP) under transcriptional control by the E2 receptor reached a critical concentration. In the model, aging-related cumulative hypothalamic E2 exposure impaired the LH surge by reducing the rate of production of the EC-RP. The progressively decreasing intercycle resynthesis rate leads first to longer, variable-length cycles and finally to persistent estrus at about 12 mo of age. This model construct is consistent with early-onset persistent estrus related to neonatal E2 exposures, with acyclicity associated with high-dose E2 exposure in the adult, and with persistent estrus conditions associated with exposures to xenobiotic endocrine modulators that are either weak E2 antagonists or weak E2 agonists. With further development these pharmacodynamic estrus cycle models should be useful in aiding risk assessments for compounds causing mammary-tissue tumors associated with persistent estrus states.

Amplified interactive toxicity of chemicals at nontoxic levels: mechanistic considerations and implications to public health.

It is widely recognized that exposure to combinations or mixtures of chemicals may result in highly exaggerated toxicity even though the individual chemicals might not be toxic. Assessment of risk from exposure to combinations of chemicals requires the knowledge of the underlying mechanism(s). Dietary exposure to a nontoxic dose of chlordecone (CD; 10 ppm, 15 days) results in a 67-fold increase in lethality of an ordinarily inconsequential dose of CCl4 (100 microliters/kg, ip). Toxicity of closely related CHCl3 and BrCCl3 is also enhanced. Phenobarbital (PB, 225 ppm, 15 days) and mirex (10 ppm, 15 days) do not share the propensity of CD in this regard. Exposure to PB + CCl4 results in enhanced liver injury similar to that observed with CD, but the animals recover and survive in contrast to the greatly amplified lethality of CD + CCl4. Investigations have revealed that neither enhanced bioactivation of CCl4 nor increased lipid peroxidation offers a satisfactory explanation of these findings. Additional studies indicate that exposure to a low dose of CCl4 (100 microliters/kg, ip) results in limited injury, which is accompanied by a biphasic response of hepatocellular regeneration (6 and 36 hr) and tissue repair, which enables the animals to recover from injury. Exposure to CD + CCl4 results in suppressed tissue repair owing to an energy deficit in hepatocytes as a consequence of excessive intracellular influx of Ca2+ leading initially to a precipitous decline in glycogen and ultimately to hypoglycemia. Supplementation of cellular energy results in restoration of the tissue repair and complete recovery from the toxicity of CD + CCl4 combination. In contrast, only the early-phase hepatic tissue repair (6 hr) is affected in PB + CCl4 treatment, but this is adequately compensated for by a greater stimulation of tissue repair at 24 and 48 hr resulting in recovery from liver injury and animal survival. A wide variety of additional experimental evidence confirms the central role of stimulated tissue repair as a decisive determinant of the final outcome of liver injury inflicted by CCl4. For instance, a 35-fold greater CCl4 sensitivity of gerbils compared to rats is correlated with the very sluggish tissue repair in gerbils. These findings are consistent with a two-stage model of toxicity, where tissue injury is inflicted by the well described "mechanisms of toxicity," but the outcome of this injury is determined by whether or not sustainable tissue repair response accompanies this injury.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatic polyamines and related enzymes following chlordecone-potentiated carbon tetrachloride toxicity in rats.

Chlordecone potentiation of the hepatotoxic and lethal effects of CCL4 has been well established. Recent studies have shown that the suppression of hepatocellular regeneration results in an accelerated progression of liver injury leading to complete hepatic failure. Since polyamines are involved in cell division, these studies were designed to investigate the polyamine levels and associated enzymes in the livers of rats treated with a low-dose combination of CD and CCl4. For comparison, a large toxic dose of CCl4 was also employed. The extent of liver toxicity in rats fed 10 parts per million chlordecone (CD) for 15 days and subsequently injected with a single dose of CCl4 (100 microL/kg body weight) or a high dose of CCL4 alone (2.5 mL/kg body weight) was similar 6 and 24 hr later as assessed by plasma transaminase levels. There was significant elevation in liver ornithine decarboxylase, S-adenosylmethionine decarboxylase, and putrescine at 24 hr and spermidine N1-acetyltransferase, N1-acetylputrescine, putreanine, putrescine, and N1-acetylspermidine at 6 hr in rats treated with the high dose of CCl4 alone compared to the combination treatment. Spermidine levels decreased up to 6 hr and then increased up to 24 hr for both treatments. Spermine continuously decreased up to 24 hr for the CD and CCl4 low-dose combination treatment compared to rats treated with a high dose of CCl4 alone. Spermidine levels were lower than in controls and rose towards control value between 6 and 24 hr after the combination treatment and the high dose of CCl4. Results indicate that the CD and CCl4 low-dose combination treatment increased liver toxicity, resulting in compromised polyamine metabolism that is coincidental with suppressed hepatocellular regeneration, which leads to an accelerated progressive phase of liver injury and culminates in complete hepatic failure.

Modulation of adrenal ornithine decarboxylase by chlordecone, p,p'DDT and permethrin.

Adrenal ornithine decarboxylase (ODC) is greatly elevated in rats dosed once with one of several insecticides; chlordecone, p,p'DDT or permethrin. An abrupt rise in the dose-response curve was apparent between 25 and 50 mg chlordecone/kg body wt, corresponding to the range where major behavioral changes occur. The latter dose of chlordecone resulted in a persistent elevation of adrenal ODC for at least 4 days, longer than that caused by the other insecticides examined. No equivalent changes were found in levels of hypothalamic or hippocampal ODC after chlordecone treatment. Hypophysectomy did not reduce the response of adrenal ODC to chlordecone, after the diminished size of the adrenal gland in operated animals was taken into account. Isolated adrenal cortical cells of a mouse tumor line (Y-1), when incubated in media containing 10(-5) M chlordecone, responded with increased ODC activity. These data suggest that the mechanism of adrenal activation by chlordecone may possess a direct component, in addition to the well-characterized stimulation of pituitary ACTH secretion caused by chlordecone acting on the hypothalamo-hypophyseal system.

Pharmacological modification of tremor and enhanced acoustic startle by chlordecone and p,p'-DDT.

Pretreatment of rats with phenoxybenzamine (5 mg/kg; SC), an alpha adrenergic antagonist, decreased the peak tremor power and startle magnitude of rats subsequently given DDT (75 mg/kg; PO) or chlordecone (60 mg/kg; IP), without having a significant effect on control animals. Pretreatment with an intracerebroventricular injection of calcium (3.75 microM in 5 microliters NaCl) decreased the peak tremor power due to subsequently administered DDT, while increasing the tremor response in rats later dosed with chlordecone. The effects of phenoxybenzamine are postulated to be due to a blockade of an excitatory influence of the adrenergic system. Calcium may decrease DDT-induced tremor by acting as a neuronal stabilizer. Potentiation of the tremorigenic effect of chlordecone by calcium may be due to increased levels of intracellular calcium, resulting in augmented release of neurotransmitters in chlordecone-exposed animals.

Effects of chlordecone on neuroendocrine function of female rats.

The effects of chlordecone on reproductive function was examined in adult females. Chlordecone was shown to mimic estrogen by producing persistent vaginal estrus in ovariectomized and intact females. In ovariectomized females, chlordecone reduced serum levels of luteinizing hormone (LH) and increased prolactin (PRL). In intact females, chlordecone reduced the preovulatory surges of both LH and PRL but had no effect on serum hormones of diestrous females. Chlordecone failed to mimic estrogen in priming the ovariectomized female for behavioral receptivity, and instead appeared to antagonize the priming action of estrogen. Examination of neurotransmitter changes suggested that chlordecone disrupted the usual sequence of events occurring throughout the female's estrous cycle and that this disturbance of neurotransmitter equilibrium contributed to the reproductive dysfunction. The hypothesis that chlordecone disrupts reproductive function by acting as a weak estrogen received considerable support. However, the results of several studies indicated that the neural effects of chlordecone and estradiol were not identical. We have proposed that chlordecone mimics many of the estrogen-receptor mediated neural events. However, because of the persistence of chlordecone in the organism, chlordecone fails to initiate the sequential changes which characterize estrous cyclicity.

Effect of chlordecone (Kepone) on the rat brain concentration of 3-methoxy-4-hydroxyphenylglycol: evidence for a possible involvement of the norepinephrine system in chlordecone-induced tremor.

3-Methoxy-4-hydroxyphenylglycol (MHPG) is the major metabolite of norepinephrine (NE) in the rat brain. A single injection of tremorigenic doses of chlordecone to adult male Fischer-344 rats resulted in significant increases in MHPG concentrations in hypothalamus, brain stem, cerebellum, and caudate nucleus. The increase in MHPG was accompanied by a decrease in NE in the hypothalamus, suggesting that chlordecone treatment caused an increase in the turnover of NE in the brain. There was a dose- and time-related correlation between the increases in the concentrations of MHPG in hypothalamus, brain stem, and cerebellum and tremor in rats. The increase in MHPG in hypothalamus and brain stem occurred as early as 1 hr postdosing; this preceded the earliest measurable sign of tremor and initial hypothermia. Whether the alterations in the brain NE system are involved in the expression of the tremor and the initial hypothermia induced by chlordecone or whether they are merely associated with these changes is not clear.

Effects of chlordecone exposure on brain neurotransmitters: possible involvement of the serotonin system in chlordecone-elicited tremor.

The purpose of this study was to correlate the chlordecone-elicited tremor activity with alterations of brain neurotransmitters. A single injection of chlordecone (80 mg/kg, ip) significantly increased the brain levels of 5-hydroxyindoleacetic acid (5-HIAA) but did not affect the concentrations of dopamine, dihydroxphenylacetic acid, aspartate, taurine, glutamate, glycine, and gamma-aminobutyric acid (GABA). There was a dose- and time-related correlation between the increases in striatal 5-HIAA levels and tremor after chlordecone treatment. A subsequent study with pargyline indicated that the increase in striatal 5-HIAA level represented an increase in the turnover of serotonin. This study plus the previous finding that pizotifen (BC-105), a serotonin receptor blocker, attenuated chlordecone-elicited tremor strongly suggests a possible involvement of the serotonin system in mediating the tremor elicited by this insecticide.

Evaluation of neonatal chlordecone neurotoxicity during early development: initial characterization.

The effects of neonatal exposure of rats to chlordecone were assessed in the preweaning period of development. On day 4 postpartum, pups received a SC injection (20 microliter) of either dimethylsulfoxide (DMSO) or 1 mg/pup of chlordecone dissolved in DMSO. Body weights on days 14 and 21 were slightly (9-14%), but significantly, depressed in both sexes by the neonatal chlordecone exposure. Whole body movements, monitored via a spectral analyzer, indicated a significant high frequency tremor in the chlordecone-exposed pups on postnatal days 10, 14, and 18. The auditory startle response to a 4 kHz, 110 dB (SPL) tone was examined on days 12, 16, and 20. A significant enhancement of startle responsiveness was noted in chlordecone-exposed pups relative to vehicle-injected littermates; this effect was localized primarily at 16 days of age and in the female pups. Evaluation of undifferentiated motor activity, as assessed by testing individual pups in the presence of homecage shavings, indicated significant chlordecone-induced hypoactivity in both sexes on postnatal day 15. Hypoactivity persisted through 21 days of age in the female, but not male, chlordecone-exposed pups. This neurotoxic profile of tremor, depressed body weight, and altered responsiveness to novel/stressful environments is detectable in early life and is similar to that observed when adult rats are exposed to chlordecone. The profile also bears similarity to the primary signs and symptoms of chlordecone exposure in adult humans. Collectively, the present observations offer a tentative working model for the further investigation of the developmental neurotoxicity of chlordecone.

Subchronic dietary exposure of rats to chlordecone (Kepone) modifies levels of hypothalamic beta-endorphin.

Rats were exposed for extended times to low doses of chlordecone (1 and 6 ppm in the diet). After 105 days of exposure of female rats to this diet, levels of circulating prolactin and growth hormone were not significantly altered, but at the higher dose of chlordecone, hypothalamic beta-endorphin levels were selectively reduced. Since no changes were seen in pituitary levels of beta-endorphin of treated rats, chlordecone had some regional specificity. Levels of dopamine, serotonin, and their metabolites were unchanged in the caudate nucleus of female rats exposed to chlordecone. Thus, low levels of chlordecone appear to selectively affect hypothalamic beta-endorphin suggesting the hypothalamus may be especially sensitive to this neurotoxicant. The gestationally and lactationally-exposed female offspring of these rats showed a major increase in the levels of growth hormone at 100 days of age. However, other parameters assayed remained unaltered. These consisted of biogenic amines within the caudate nucleus, pituitary and hypothalamic met-enkephalin and circulating prolactin, growth hormone, and estrogen.