Endrin potentiates early-stage adipogenesis in 3T3-L1 cells by activating the mammalian target of rapamycin.

Obesogens are a type of endocrine-disrupting chemicals (EDCs) that disrupt the human endocrine system, resulting in obesity and metabolic disease. Several obesogens, including bisphenol A, tolylfluanid, and some pesticides, have been identified and studied previously; however, the underlying molecular mechanisms by which obesogens interfere with adipogenesis and induce insulin resistance in adipocyte remain unknown. This study aims to determine which type of chemical is the most potent obesogen and to investigate its effect on adipogenesis-related gene expressions. 3T3-L1, a pre adipocyte cell line, was differentiated into mature adipocytes with either vehicle or various obesogens, including bisphenol A, tolylfluanid, and endrin, as well as corticosterone, at the same dose. Subsequently, intracellular and secreted triglyceride levels were measured, and the expression of genes and proteins involved in adipogenesis and lipogenesis was investigated. We found that endrin was the most potent regulator of adipogenic differentiation, as compared to tolylfluanid, bisphenol A, and corticosterone. Endrin increased intracellular and secreted triglyceride levels and enhanced the expression of adipogenic transcription factors as well as the terminal differentiation marker in a dose-dependent manner. During the early stages of differentiation, endrin enhanced mammalian target of rapamycin (mTOR) activity, which was suppressed by the pharmacological blockade of the protein kinase B-mTOR pathway, with repressed adipogenic differentiation. However, endrin did not change the expression levels of the downstream members of the mTOR signaling pathway or proteins related to lipolysis in response to insulin. Thus, we suggest that endrin potentiates early-stage adipogenic differentiation by activating the mTOR pathway.

Environmental endocrine disruptors promote adipogenesis in the 3T3-L1 cell line through glucocorticoid receptor activation.

The burgeoning obesity and diabetes epidemics threaten health worldwide, yet the molecular mechanisms underlying these phenomena are incompletely understood. Recently, attention has focused on the potential contributions of environmental pollutants that act as endocrine disrupting chemicals (EDCs) in the pathogenesis of metabolic diseases. Because glucocorticoid signaling is central to adipocyte differentiation, the ability of EDCs to stimulate the glucocorticoid receptor (GR) and drive adipogenesis was assessed in the 3T3-L1 cell line. Various EDCs were screened for glucocorticoid-like activity using a luciferase reporter construct, and four (bisphenol A (BPA), dicyclohexyl phthalate (DCHP), endrin, and tolylfluanid (TF)) were shown to significantly stimulate GR without significant activation of the peroxisome proliferator-activated receptor-gamma. 3T3-L1 preadipocytes were then treated with EDCs and a weak differentiation cocktail containing dehydrocorticosterone (DHC) in place of the synthetic dexamethasone. The capacity of these compounds to promote adipogenesis was assessed by quantitative oil red O staining and immunoblotting for adipocyte-specific proteins. The four EDCs increased lipid accumulation in the differentiating adipocytes and also upregulated the expression of adipocytic proteins. Interestingly, proadipogenic effects were observed at picomolar concentrations for several of the EDCs. Because there was no detectable adipogenesis when the preadipocytes were treated with compounds alone, the EDCs are likely promoting adipocyte differentiation by synergizing with agents present in the differentiation cocktail. Thus, EDCs are able to promote adipogenesis through the activation of the GR, further implicating these compounds in the rising rates of obesity and diabetes.

Six high-priority organochlorine pesticides, either singly or in combination, are nonestrogenic in transfected HeLa cells.

There have been increasing concerns that environmental chemicals may adversely affect the health of humans and wildlife by acting as endocrine modulators. These concerns have been augmented by the realization that human exposure occurs not just to single chemical agents, but typically to mixtures of chemicals that could exhibit estrogenic activity qualitatively and/or quantitatively different from that of individual components. To address these concerns, we have evaluated the ability of six organochlorine pesticides (4, 4'-DDT, 4,4'-DDD, 4,4'-DDE, aldrin, dieldrin, or endrin, all classified high priority by ATSDR) to modulate transcriptional activation of an estrogen-responsive reporter gene in transfected HeLa cells. In these assays, HeLa cells cotransfected with an expression vector encoding estrogen receptor and an estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid were exposed to these pesticides individually and in defined combinations. While estradiol consistently elicited 10- to 23-fold dose-dependent inductions in these assays, the six organochlorine pesticides showed no detectable dose-related response when tested individually. When tested in binary combinations, the pesticide mixtures showed no additional estrogenicity. Thus, the pesticides tested, singly or as mixtures, showed virtually no evidence of estrogenicity.

Endrin inhibits adipocyte differentiation by selectively altering expression pattern of CCAAT/enhancer binding protein-alpha in 3T3-L1 cells.

The effects of selected chlorinated cyclodiene pesticides on the adipocyte differentiation process were examined using the 3T3-L1 adipocyte model in vitro. Endrin was found to cause a dose-dependent inhibition of adipocyte differentiation in 3T3-L1 cells. Aldrin and dieldrin were less potent than endrin in interfering with the adipogenic process. Endrin's inhibitory effect was effective only when the pesticide was present in the medium during the first 48 h after exposure of 3T3-L1 cells to adipogenic inducers. Immunoblots analysis revealed that endrin caused a dose-dependent, selective inhibition of the intracellular levels of CCAAT enhancer binding protein (C/EBP)alpha without altering the expression patterns of C/EBPbeta or C/EBPdelta along the differentiation. Supershift analysis showed that DNA-binding capacity of C/EBPalpha was affected most by endrin treatment. Endrin also caused a decrease in the elevation of the adipogenic factor peroxisome proliferator-activated receptor (PPAR)gamma elicited by the adipogenic inducers. However, the cotreatment with troglitazone, a thiazolidinedione known to activate PPARgamma, did not suppress the antiadipogenic action of endrin, indicating that its direct action site is not PPARgamma receptor. Endrin also altered the pattern of activation of nuclear factor-kappaB, a factor activated by 12-O-tetradecanoylphorbol-13-acetate and tumor necrosis factor-alpha, which are known to interfere with adipocyte differentiation. Thus, endrin inhibited the normal decrease in nuclear factor-kappaB-DNA binding observed as cells are acquiring the adipocyte phenotype at a late stage of differentiation. Our results suggest that endrin inhibits adipocyte differentiation through the specific suppression of C/EBPalpha.

TCDD endrin and lindane induced increases in lipid metabolites in maternal sera and amniotic fluids of pregnant C57BL/6J and DBA/2J mice.

TCDD, endrin and lindane induce an oxidative stress and enhance lipid peroxidation in fetal and placental tissues of mice. The levels of the products resulting from altered lipid metabolism, including malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT) and acetone (ACON) have been determined in maternal sera and amniotic fluids of pregnant C57BL/6J and DBA/2J mice after oral administration of single fetotoxic doses of TCDD, endrin and lindane on day 12 of gestation, using high pressure liquid chromatography (HPLC). Under these conditions, TCDD given at a dose of 30 micrograms/kg body weight to C57BL/6J mice produced 2.5-3.9 and 1.7-4.0 fold increases in the levels of the four metabolites in maternal sera and the amniotic fluids, respectively. TCDD given to DBA/2J mice at a dose of 60 micrograms/kg produced 1.5-1.7 and 1.7-2.2 fold increases in the levels of these metabolites in maternal sera and the amniotic fluids, respectively. Endrin, when given at a dose of 4.5 mg/kg body weight to either mouse strain, produced increases of 1.9-3.1 and 1.7-3.2-fold in the levels of the four metabolites in maternal sera and the amniotic fluids, respectively, in the C57BL/6J mice and increases of 1.4-1.6 and 1.2-1.5 fold in the levels of these metabolites in maternal sera and the amniotic fluids, respectively, in the DBA/2J mice. Lindane given at a dose of 30 mg/kg body weight to C57BL/6J mice produced 1.5-2.0 and 1.3-1.7-fold increases in the four metabolites in maternal serum and amniotic fluids, respectively, while administration of this same dose to DBa/2J mice produced 1.2-1.5 and 1.1-1.5 fold increases, respectively. Increases in the levels of lipid metabolites occur in maternal serum and amniotic fluid as a result of enhanced lipid peroxidation in response to TCDD, endrin and lindane. Lipid peroxidation may participate in the fetotoxic effects of these xenobiotics and these effects are observed regardless of the Ah-responsiveness of the mice, although higher levels of the metabolites are produced by TCDD in Ah-responsive mice.

Protective effects of lazaroid U74389F (16-desmethyl tirilazad) on endrin-induced lipid peroxidation and DNA damage in brain and liver and regional distribution of catalase activity in rat brain.

Endrin, a poly-halogenated cyclic hydrocarbon, induces hepatic lipid peroxidation, modulates calcium homeostasis, decreases membrane fluidity, and increases nuclear DNA damage. Little information is available on the neurotoxicity of endrin. The effects of endrin on lipid peroxidation, DNA damage, and regional distribution of catalase activity were assessed in rat brain and liver 24 h following an acute oral dose of 4.5 mg endrin/kg. Lipid peroxidation associated with whole brain mitochondria increased 2.4-fold, whereas microsomal lipid peroxidation increased 2.8-fold following endrin administration. Lipid peroxidation also increased 2.0-fold both in hepatic mitochondria and microsomes. Catalase activity decreased 24% in the hypothalamus, 23% in the cortex, 38% in the cerebellum, and 11% in the brain stem in response to endrin. A 4.3-fold increase in brain nuclear DNA-single strand breaks (SSB) was observed in endrin-treated rats. Pretreatment of rats intraperitoneally with the lazaroid U74389F (16-desmethyl tirilazad) (10 mg/kg in two doses) attenuated the biochemical consequences of endrin-induced oxidative stress. The administration of U74389F in citrate buffer (pH 3.8) provided better protection than administering the lazaroid in corn oil, decreasing endrin-induced lipid peroxidation by 50-80% and DNA-SSB by approximately 72% in liver and 85% in brain, while ameliorating the suppressed catalase activity. The data suggest an involvement of an oxidative stress in the neurotoxicity and hepatotoxicity induced by endrin, which can be attenuated by the lazaroid U74389F.

Protective effects of antioxidants against endrin-induced hepatic lipid peroxidation, DNA damage, and excretion of urinary lipid metabolites.

Oxidative stress is believed to play a pivotal role in endrin-induced hepatic and neurologic toxicity. Therefore, the effects of the antioxidants vitamin E succinate and ellagic acid have been examined on hepatic lipid peroxidation, DNA single-strand breaks (SSB), and the urinary excretion of lipid metabolites following an acute oral dose of 4.5 mg endrin/kg. Groups of rats were pretreated with 100 mg/kg vitamin E succinate for 3 d followed by 40 mg/kg on day 4, or 6.0 mg ellagic acid/kg for 3 d p.o. followed by 3.0 mg/kg on day 4 or the vehicle. Endrin was administered p.o. on day 4 2 hr after treatment with the antioxidant. All animals were killed 24 h after endrin administration. Vitamin E succinate pretreatment decreased the endrin-induced increase in hepatic mitochondrial and microsomal lipid peroxidation by approximately 60% and 40%, respectively. Ellagic acid pretreatment reduced the endrin-induced increased in mitochondrial and microsomal lipid peroxidation by approximately 76 and 79%, respectively. Both vitamin E succinate and ellagic acid alone produced small but nonsignificant decreases in hepatic mitochondrial and microsomal lipid peroxidation. A 3.3-fold increase in the incidence of hepatic nuclear DNA single-strand breaks was observed 24 h after endrin administration. Pretreatment of rats with vitamin E succinate, vitamin E, and ellagic acid decreased endrin-induced DNA-SSB by approximately 47%, 22%, and 21%, respectively. Pretreatment of rats with vitamin E succinate decreased the endrin-induced increase in the urinary excretion of malondialdehyde, acetaldehyde, formaldehyde, and acetone by approximately 68, 65, 70, and 55%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes from endrin-treated rats.

Recent studies have shown that the administration of endrin to rodents induces lipid peroxidation in various tissues and decreases glutathione content. These results suggest that endrin produces reactive oxygen species and/or free radicals. We have therefore examined the effect of endrin (4.5 mg/kg) on the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes in rats. The effects of endrin on hepatic mitochondrial and microsomal lipid peroxidation and membrane fluidity as well as the incidence of hepatic nuclear DNA damage were also examined. Twenty-four hours after endrin administration, significant increases in the production of chemiluminescence by the three tissue fractions were observed. Furthermore, peritoneal macrophages from endrin-treated animals resulted in 3.0- and 2.8-fold increases in cytochrome c and iodonitrotetrazolium (INT) reduction, indicating enhanced production of superoxide anion. Endrin administration also resulted in significant increases in lipid peroxidation of mitochondrial and microsomal membranes as well as decreases in the fluidity of these two membranous fractions. A significant increase in hepatic nuclear DNA single-strand breaks also occurred in response to endrin administration. The results indicate that macrophage, mitochondria, and microsomes produce reactive oxygen species following endrin administration, and these reactive oxygen species may contribute to the toxic manifestations of endrin.

Endrin-induced production of nitric oxide by rat peritoneal macrophages.

The effect of oral endrin administration to rats on the production of nitric oxide (NO) by peritoneal macrophages was investigated. Nitric oxide formation was measured as nitrite. Endrin (4.5 mg/kg) enhanced the secretion of NO by approx. 300%. The effect of endrin on NO formation was both dose- and time-dependent. Ellagic acid, which has been shown to be a potent antioxidant, inhibited the elevation of NO production induced by endrin. These results suggest that the toxicity of endrin may at least in part be due to the production of an oxidative stress.

Unusual effects of benzodiazepines and cyclodiene insecticides on an expressed invertebrate GABAA receptor.

We have previously reported [(1991) EMBO J. 10, 3239-3245] the sequence of an invertebrate gamma-aminobutyric acid (GABA) type A (GABAA) receptor polypeptide which forms homo-oligomeric GABA-gated, bicuculline-sensitive, chloride-ion channels upon heterologous expression. We now demonstrate that the benzodiazepines Ro5-4864 (4'-chlorodiazepam) and diazepam, that are active at mammalian peripheral benzodiazepine sites, and not those benzodiazepines specific for central sites, directly active the homo-oligomeric receptor and evoke larger maximal responses than those elicited by GABA. In addition, members of the cyclodiene class of insecticides block the channel of the receptor in a manner indistinguishable from that of picrotoxin.

Prolonged exposure to GABA activates GABA-gated chloride channels in the presence of channel-blocking convulsants.

1. In assays of 36Cl- uptake into mouse brain vesicles, 100 microM GABA markedly increased both the initial rate of 36Cl- uptake and the total amount of chloride taken up over a 120-sec incubation period. Specific GABA-dependent 36Cl- uptake (the difference between total and background uptake) was essentially complete within 15 sec of incubation. 2. Incubation with GABA following preincubation with 10 microM endrin, a polychlorocycloalkane insecticide and established blocker of GABA-gated chloride channels, showed a stimulation of uptake over background levels that was much slower in onset than that observed with GABA alone but nevertheless achieved virtually the same level of stimulation above background levels after 90 sec of incubation with GABA. 3. In electrophysiological assays of GABA receptors expressed in Xenopus oocytes following injection with rat brain mRNA, endrin (20 microM) effectively blocked the transient currents elicited by brief exposure of oocytes to GABA (200 microM). However, prolonged exposure to GABA in the absence of perfusion produced a large, slowly-developing inward current. 4. The actions of several known GABA antagonists were also compared as inhibitors of GABA-dependent 36Cl- uptake into mouse brain vesicles at short (4 sec) and long (120 sec) incubation times using concentrations of inhibitors known to produce approximately 70-90% inhibition of GABA-dependent chloride uptake in 4-sec incubations. Picrotoxinin and TBPS, like endrin, were completely ineffective as inhibitors in 120-sec incubations. In contrast, bicuculline was almost as effective at 120 sec as at 4 sec, and avermectin Bla produced approximately 50% inhibition of the GABA response after 120 sec.(ABSTRACT TRUNCATED AT 250 WORDS)

Endrin-induced depletion of glutathione and inhibition of glutathione peroxidase activity in rats.

1. Recent studies have shown that endrin induces lipid peroxidation and may produce toxicity through an oxidative stress. We have therefore examined the effect of endrin administration to rats on glutathione content and the activities of glutathione metabolizing enzymes. 2. The oral administration of endrin resulted in dose- and time-dependent decreases in hepatic and renal glutathione content with maximum depletion (90%) occurring in liver at approximately 24 hr post-treatment. 3. Decreases in glutathione content were also observed in lung, brain, spleen and heart. 4. Endrin (4 mg/kg) decreased selenium dependent glutathione peroxidase activity in liver and kidney by 64 and 50%, respectively, while small increases were observed in the activities of glutathione reductase and glutathione S-transferase. 5. The toxicity of endrin may be at least in part related to oxidative tissue damage associated with depletion of glutathione and inhibition of glutathione peroxidase activity.

Effects of lindane on the glucose metabolism in rat brain cortex cells.

The influence of 0.5 mM gamma-hexachlorocyclohexane (gamma-HCH, lindane) on glucose transport has been investigated using the analog 3-O-methyl-D-(U-14C)glucose. The glucose uptake was lineal for at least 10 sec. Preincubation of dissociated brain cortex cells with lindane decreased the transport of glucose with respect to the controls. The treatment of brain cortex cells with other organochlorine compounds indicated that the alpha-, delta-HCH isomers and dieldrin reproduced the same inhibitory pattern, while beta-HCH and endrin were inactive. The total radioactivity incorporated into CO2 from (U-14C) glucose in the cerebral cortex is also inhibited by lindane in a time dependent manner.

Block of an insect central nervous system GABA receptor by cyclodiene and cyclohexane insecticides.

The effects of a cyclodiene (endrin) and a cyclohexane (lindane) insecticide have been tested on gamma-aminobutyric acid (GABA) receptors in the central nervous system of the cockroach (Periplaneta americana), by using electrophysiological methods and an in vitro functional receptor assay. In electrophysiological experiments on an identified motor neuron (Df), endrin blocked the GABA response with a 50% inhibition concentration of 5.0 x 10(-7) M in a non-competitive manner. The actions of endrin were irreversible under the experimental conditions adopted. Increasing the intracellular chloride concentration reduced the effectiveness of endrin, whereas a change in the potassium concentration failed to influence the block by endrin of GABA responses. Lindane exhibited similar actions to endrin on insect GABA receptors, but was approximately an order of magnitude less effective. In a microsac preparation from cockroach nerve cords, endrin, at a concentration of 1.0 x 10(-5) M, completely blocked GABA-stimulated 36Cl- uptake, whereas the same concentration of lindane was less potent, only blocking about 40% of uptake under similar conditions. Neither insecticide had any effect on L-glutamate-activated chloride channels. The results demonstrate that endrin and lindane block functional insect neuronal GABA receptors.

Polychlorinated convulsant insecticides potentiate the protective effect of NaCl against heat inactivation of [3H]flunitrazepam binding sites.

Six polychlorinated convulsant insecticides that potently inhibit t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes also potentiate the protective effect of NaCl (200 mM) against heat inactivation of [3H]flunitrazepam binding sites on the same membranes. Similar effects were obtained with all "cage" convulsants tested. The rank order of potencies as heat protection potentiators was similar to the rank order of potencies as inhibitors of [35S]TBPS binding (alpha-endosulfan greater than endrin greater than dieldrin greater than toxaphene greater than lindane). alpha-Endosulfan and endrin are more potent in both respects than any previously reported picrotoxin-like (cage) convulsant, but are much less toxic to mammals. The greatly reduced toxicities of alpha-endosulfan and endrin in mammals may reflect partial gamma-aminobutyric acid (GABA)-neutral properties of these compounds. Time courses of heat inactivation of [3H]flunitrazepam binding sites in the presence of 200 mM NaCl plus saturating concentrations of endrin or picrotoxin revealed monophasic components constituting about 88% of the binding sites, suggesting that virtually all [3H]flunitrazepam binding sites are coupled to picrotoxin binding sites in the GABA/benzodiazepine/picrotoxin receptor complex. Protection against heat inactivation constitutes a useful tool for characterizing the various allosterically linked binding sites in neurotransmitter receptor complexes.

Environmental stress and parasitic infections: II. Effects of gramoxone and hexadrin (pesticides) on the survival characteristics of Fasciola gigantica miracidia.

Miracidia of Fasciola gigantica have a maximum longevity of 9 h. Miracidial survival was found to be age dependent and the instantaneous mortality rate increased exponentially with miracidial age and pesticide treatment. Mortality of the pesticide-treated miracidia differs significantly from that of the control. The LC50 value for Hexadrin was calculated at 1.93 ppm and for Gramoxone at 0.75 ppm. Dosage-mortality lines when tested statistically show no significant difference between the two pesticides at 95% level of confidence. The toxic effect of the pesticides was enhanced by synergism between the pesticides.

Environmental stress and parasitic infections: I. The effects of Gramoxone and Hexadrin on embryo and hatching of Fasciola gigantica miracidia.

The effect of two chemical compounds commonly used in agriculture, Gramoxone (a herbicide) and Hexadrin (an insecticide) on embryonic development and hatching of Fasciola gigantica miracidia were experimentally assessed. These two pesticides were introduced in varying quantities into petri dishes containing unembryonated eggs of the trematode for a period of 30 days. LC50 (lethal concentration for 50% hatching) values were determined for them. For gramoxone it was 2 ppm and for Hexadrin it was 2.5 ppm. Results also show that at 4 ppm and 5.5 ppm the pesticides achieve 99.0% mortality of eggs. At sublethal concentrations they cause prolongation of embryonic processes and inhibition of hatching of miracidia.

Ontogenesis and properties of the convulsant recognition site(s) of the gamma-aminobutyric acid (GABA) receptor complex in chicken embryo.

Ontogenesis of the convulsant (picrotoxinin or t-butylbicyclophosphorothionate (TBPS] site(s) was defined in chicken embryonic brain with the radioligands [35S]TBPS and [3H]t-butylbicycloorthobenzoate [( 3H]TBOB). Binding of the radioligands is detectable from day 6 of incubation. The increase in binding after day 14 of incubation is due to an increase in the number of binding sites. The pharmacological properties of the embryonic recognition site(s) do not undergo significant changes during hatching based on the affinity of embryonic and chick brain membranes for [3H]TBOB; the rate of association and dissociation for TBOB; non-competitive inhibition of [3H]TBOB binding and modulation of 1R, alpha S-cis-cypermethrin interaction with the recognition site(s) by GABA; and inhibition of radioligand binding by endrin, picrotoxinin and TBPS. Early development and in vitro susceptibility of the recognition site to convulsive toxicants makes the embryo a possible target for a variety of drugs and environmental toxicants acting at this site.

Monogenic inheritance of cyclodiene insecticide resistance in mosquitofish, Gambusia affinis.

Certain populations of the mosquitofish, Gambusia affinis, developed high levels of resistance to endrin and other cyclodiene insecticides as a result of inadvertent exposure to agricultural sprays. Genetic crossing studies show that endrin resistance is inherited as a single, autosomal, intermediate gene.