Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III.

Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson's disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.

Estrogen-responsive transient expression assay using a brain aromatase-based reporter gene in zebrafish (Danio rerio).

Whereas endogenous estrogens play an important role in the development, maintenance, and function of female and male reproductive organs, xenoestrogens present in the environment disrupt normal endocrine function in humans and wildlife. Various in vivo and in vitro assays have been developed to screen these xenoestrogens. However, traditional in vivo assays are laborious and unsuitable for large-scale screening, and in vitro assays do not necessarily replicate in vivo functioning. To overcome these limitations, we developed a transient expression assay in zebrafish, into which a brain aromatase (cyp19a1b)-based estrogen-responsive reporter gene was introduced. In response to 17beta-estradiol (10(-6) M) and heptachlor (10(-6) M), zebrafish embryos carrying the reporter construct expressed enhanced green fluorescent protein in the olfactory bulb, telencephalon, preoptic area, and mediobasal hypothalamus. This system will serve to model the in vivo conversion and breakdown of estrogenic compounds and thus provide a rapid preliminary screening method to estimate their estrogenicity.

Impacts of molt-inhibiting organochlorine compounds on epidermal ecdysteroid signaling in the fiddler crab, Uca pugilator, in vitro.

Because of their chemical stability and lipophilicity, many organochlorine compounds (OCs) can readily accumulate in fatty tissues of crustaceans. Several OCs have been reported to inhibit crustacean molting. To determine whether the disruption of crustacean molting by these OCs involves interference with ecdysteroid signaling in the epidermis, the impacts of five molt-inhibiting OCs on the level of N-acetyl-beta-glucosaminidase (NAG, EC 3.2.1.30) mRNA in cultured epidermal tissues from the fiddler crab, Uca pugilator, were investigated using quantitative real-time PCR. The NAG mRNA was found to be inducible by 20-hydroxyecdysone (20-HE) in cultured epidermal tissues. The inducibility of NAG mRNA in cultured epidermal tissues by 20-HE is not only further direct evidence that epidermal expression of NAG gene in U. pugilator is controlled by the molting hormone but also validates the use of the NAG mRNA as a biomarker for epidermal ecdysteroid signaling. When Aroclor 1242, 2,4,5-trichlorobiphenyl (PCB29), endosulfan or kepone was administered alone, the expression of NAG gene in cultured epidermal tissues was upregulated, while heptachlor had no effects. Under binary exposure to both 20-HE and an OC, a condition similar to the natural hormonal milieu of epidermal tissues of animals impacted by OCs, both Aroclor 1242 and endosulfan were found to be capable of antagonizing ecdysteroid signaling in cultured epidermal tissues. This antagonizing effect on epidermal ecdysteroid signaling can at least partly explain the inhibitory effects of these two agents on crustacean molting. PCB29, when given together with 20-HE, produced an additive effect on epidermal ecdysteroid signaling but such an additive effect was not observed when kepone was combined with 20-HE.

Heptachlor and o-p'DDT effects on protein kinase activities associated with human placenta particulate fractions.

Organochlorine pesticides have been detected in placenta. The ability of heptachlor (HC) and 1,1,1-tricholoro-2-(2-chlorophenyl)-2-4-chlorophenyl)ethane (o-p'DDT) to interfere with protein phosphorylation was evaluated. In vitro incubations of cell-free placental villi homogenates with a concentration range 1-100 microM were performed. In particulate fractions, total serine/threonine kinase activity was increased by 10 microM HC and o-p' DDT (59% and 82%, respectively). Maximum eightfold increase was observed with 10 microM o-p' DDT on protein kinase A activity. By contrast, protein kinase C activity was reduced by 10 microM HC and o-p' DDT (40% and 52%, respectively). Endogenous substrate phosphorylation studies demonstrated that slight but significant increase in 24-kDa band labeling was produced in nuclear samples with 1, 10, and 100 microM HC and 100 microM o-p' DDT. Exposition to 100 microM HC increased 85-kDa band labeling. In mitochondrial fractions, 10 microM HC and o-p' DDT increased 24- and 65-kDa bands' labeling. These data indicate that both pesticides affect protein kinase activities in particulate fraction. Nuclear compartmentalization of these compounds, insertion in membranes, and chemical stress production may be associated to the observed effects, thus suggesting deleterious consequences in signaling pathways.

Effects of o,p'-DDE, heptachlor, and 17beta-estradiol on vitellogenin gene expression and the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus.

Effects of two endocrine disruptors, o,p'-DDE and heptachlor, and 17beta-estradiol (E(2)) on vitellogenin (Vg) and the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis were examined in male tilapia. In the first experiment, fish were given 5 weekly injections of either E(2), o,p'-DDE or heptachlor (5 microg/g). E(2) treatment increased plasma Vg and hepatic expression of three Vg genes (Vgs A, B, and C) and estrogen receptor alpha (ERalpha), while reducing plasma levels of IGF-I and suppressing the expression of IGF-I, the GH receptor (GHR2) and the putative somatolactin receptor (GHR1). Neither pesticide greatly affected the other parameters examined, except for a significant reduction in expression of GHR2 and increased plasma IGF-I. In the second experiment, fish were given a single injection of o,p'-DDE or heptachlor (100 microg/g), or E(2) (5 microg/g) and sacrificed 5 days post-injection. Treatment with E(2) stimulated expression of all three Vg genes. Both o,p'-DDE and heptachlor increased expression of VgB, whereas only o,p'-DDE increased VgA expression. There was no effect of o,p'-DDE or heptachlor on VgC expression or plasma Vg levels. Treatment with o,p'-DDE and heptachlor as well as E(2) increased ERalpha and ERbeta transcript levels. Similarly, both pesticides increased GHR1 and IGF-I expression, whereas no significant effect of E(2) was observed on GHR1, GHR2 or IGF-I expression. These results indicate that o,p'-DDE and heptachlor have varying temporal and dose effects on modulation of Vg and the GH/IGF-I axis that are distinct from E(2).

Gender differences in brain peripheral benzodiazepine receptor (PBR) expression and seizures produced by heptachlor during development.

Heptachlor has been widely used as an insecticide. It is a GABA-A antagonist and causes seizures. It also increases peripheral benzodiazepine receptors (PBRs) in brain. PBRs are found on the outer mitochondrial membrane in glia, rather than in neurons, and are necessary for steroidogenesis in brain. We compared the effects of acute oral administration of heptachlor (60 mg/ml oil/kg body wt) at 10 ages from postnatal day (PND) 0 to 60 on brain PBR expression and seizure severity in both male and female rats at 1 and 2 hr after administration. From PND 10 through 60, brain PBR expression was increased about 175-225% of controls at both 1 and 2 hr after heptachlor in females. In males however, PBRs were only increased at 30-60 days at 1 hr but not at any age at 2 hr. At 2 hr after heptachlor at 30-60 days in males, PBRs were significantly lower than at 1 hr and even tended to be lower than control levels. By contrast, seizure intensity was greater in males than in females from 10 through 20 days of age at 1 hr and was even greater at 2 hr from 16 through 30 days of age, reflecting the lower PBR levels at 2 hr than at 1 hr in males. Thus, the gender difference in PBR expression was the opposite of the gender difference in seizure intensity. PBRs in brain synthesize several neurosteroids, including allopregnanolone, which is a potent anticonvulsant agent. We hypothesize that the gender differences in seizure intensity after heptachlor were due to the action of heptachlor in greatly increasing PBR expression in females but not in males. Thus the greater expression of PBRs in females would result in more synthesis of allopregnanolone than in males. Therefore, because of allopregnanolone's anticonvulsant effects, seizure intensity was less in females than in males. By comparison, maximal electroshock (MES) caused seizures and increased PBRs in brain in both male and female developing rats with no gender differences at 10-20 days of age.

Alteration of thrombine-signaling mechanism by heptachlor in human platelets.

Heptachlor is a persistent organochlorine insecticide that has been detected in human tissues and fluids. The ability of heptachlor to interfere with platelet phosphoinositides metabolism and related signaling events stimulated by thrombin was evaluated. In vitro incubations with a concentration range of 1-100 microM heptachlor, prior to platelets activation, were performed. Experiments showed that 10 microM increased protein Kinase C (PKC) activity and phosphatidylinositolbiphosphate and phosphatidic acid phosphorylation. Simultaneously phosphatidylcholine and phosphatidylethanolamine breakdown were prevented. Similar effects were observed with HC 1 microM. However, heptachlor 100 microM increased phosphatidylinositolbiphosphate phosphorylation but reduced serine/threonine kinases activity. We propose that signal transduction steps downstream phospholipase C (PLC) are unphysiologically activated by heptachlor and facilitated by the increase in phosphatidylinositolbiphosphate, the substrate for PLC activity, thus producing an accumulation of phosphatidic acid. The elevated level of this compound itself or the transient increase in diacylglycerol produced may cause calcium mobilization and the activation of PKC. In contrast with the alterations observed in phospholipids and protein phosphorylation, no changes in aggregation properties were observed.

Modulation of CPP32 activity and induction of apoptosis in Human CEM X 174 lymphocytes by heptachlor, a chlorinated hydrocarbon insecticide.

Heptachlor is an organochlorine insecticide used worldwide for the control of pests both agriculturally and domestically. Its lipophilic structure allows it to bioaccumulate and pass through the food chain, exposing those who come in contact with it to its tumor promoting and possible carcinogenic effects. As a mechanism of tumor promotion, we explored the possibility of heptachlor suppressing the apoptotic process in human CEM x 174 lymphocytes. In this article, we describe the effect of heptachlor on the activity of the apoptosis protease CPP32. We show that heptachlor by itself was able to stimulate CPP32 activity at relatively high concentrations. When combined with the chemotherapeutic agent doxorubicin, a known CPP32 activator, a dual effect was observed. Low concentrations of heptachlor (5 microM-10 microM) suppressed doxorubicin-induced CPP32 activity, and high concentrations of heptachlor (80 microM-120 microM) augmented it. We also showed that heptachlor alone at relatively high concentrations induced apoptosis-associated changes in CEM x 174 cells including high molecular weight (HMW) DNA cleavage and chromatin condensation. From these results, it appears that heptachlor has tumor promoting-like effects at lower concentrations, and at higher concentrations induces apoptosis as a mechanism of cytotoxicity.

Differential regulation of the major cyclin-dependent kinases, cdk2 and cdc2, during cell cycle progression in human lymphocytes exposed to heptachlor.

Heptachlor, a chlorinated hydrocarbon insecticide, has been considered an environmental contaminant with potential adverse health effects. Exposure to heptachlor may impair immune functions including the inhibition of leukocyte chemotaxis, which causes defects in host defense mechanisms. This study addresses the effects of heptachlor on the cell cycle progression of human lymphocytes. It has been found that addition of heptachlor to cultured lymphocytic cells prevents the cells from progression into the S phase of the cell cycle, with a concomitant accumulation of cells in G1 phase. An accompanying decrease (deactivation) in cyclin-dependent kinase cdk2 and dephosphorylation (activation) of cdc2 was observed. The altered cell cycle progression may trigger the cell's apoptotic potential, as indicated by the reduced amount of Bcl-2 synthesized inside heptachlor-treated cells. The interference of cell cycle progression by heptachlor was also seen with chlordane and toxaphene, two other chlorinated hydrocarbon insecticides.

Suppression of chemokine-induced chemotaxis of monkey neutrophils and monocytes by chlorinated hydrocarbon insecticides.

Chemokines, characterized as pro-inflammatory chemicals made by the immune system, consist of a family of low molecular weight proteins with potent in vitro chemotactic activity causing leukocyte accumulation in vivo. This study determines the effects of organochlorine pesticide exposure on the chemotactic functions of monkey neutrophils and monocytes, using a 48-well chemotaxis chamber. Chemokines IL-8 (interleukin-8) and RANTES were used as the chemoattractants to induce chemotaxis among these monkey leukocytes. Monkey neutrophils or monocytes were first treated with heptachlor, chlordane or toxaphene for 1 hour at 37 degrees C, and the number of cells migrating toward 200 ng/ml IL-8 (for neutrophils) or 100 ng/ml RANTES (for monocytes) were scored. Inhibition of chemotaxis was seen with all samples after treatment with heptachlor, chlordane and toxaphene at concentrations from as low as 10(-14) M to 10(-5) M. Among the three compounds studied, toxaphene was the least effective in preventing monocytes from migrating toward RANTES. The ability of these pesticides to inhibit chemotaxis did not correlate directly with their potential apoptotic effects on the monkey leukocytes. These studies suggest that exposure to organochlorine pesticides may alter leukocyte-related immune functions.

Effect of lindane and heptachlor on delta-aminolaevulinate synthase and its regulation.

The effect of lindane and heptachlor on haem metabolism was studied with the aim to elucidate the mechanism of their porphyrinogenic action. The effects of these compounds on delta-aminolaevulinate synthase (ALA-S) and ferrochelatase were evaluated and the mechanism of increase of ALA-S activity was especially studied. The results indicated the following: (1) Lindane and heptachlor produced increases in ALA-S activity; this effect was dependent on the drug dose, the time of treatment, and the development of the animal, the maximum response being obtained prior to hatching. Lindane was observed to have a greater effect on ALA-S than heptachlor. In fact, when effects of lindane and heptachlor were compared we observed that lindane produced: (a) greater increases in ALA-S activity (six fold vs four fold), both with respect to dimethyl sulphoxide (DMSO) controls (3.8+/-0.3 nmol ALA/g liver per h); (b) earlier ALA-S response (1.5 h vs 4 h); (c) responses at lower doses (0.3 mg/egg vs 1 mg/egg). (2) The increase in ALA-S activity produced by lindane or heptachlor is an induction and not an activation process since it depends on protein synthesis and the drugs per se have no effect. Thus, our results obtained from studies in ovo with actinomycin D and cycloheximide suggest that lindane is acting at the translational level while heptachlor interferes at the level of transcription. (3) The study of ALA-S subcellular distribution indicated no accumulation in the cytosol of DMSO controls and in the lindane or heptachlor treated embryos, neither of the chlorinated pesticides alter the normal subcellular distribution of this regulatory enzyme in the liver. (4) Exogenous haem was able to prevent or decrease the induction of ALA-S elicited by both pesticides, thus showing that lindane or heptachlor-induced ALA-S respond to haem regulation. (5) Lindane had no effect on ferrochelatase activity at the doses and times assayed, but heptachlor decreased this enzyme activity. The porphyrinogenic mechanism of lindane and heptachlor is discussed on the basis of the present results.

The Drosophila cytochrome P450 gene Cyp6a2: structure, localization, heterologous expression, and induction by phenobarbital.

The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is located on the right arm of chromosome 2 at position 43A1-2 and comprises two exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increased production of the CYP6A2 protein. DNA from the Cyp6a2 promoter region was functional when linked to a luciferase reporter gene and transfected into D. melanogaster Schneider cells. Moreover, a dose-dependent induction of luciferase activity by phenobarbital indicated that elements necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 protein in lepidopteran cells infected with a Cyp6a2-recombinant baculovirus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 protein produced in this system metabolized aldrin and heptachlor to their epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6-hydroxypyrimidine. Metabolism in lysates of cells infected with recombinant baculovirus was greatly enhanced by the addition of purified housefly NADPH cytochrome P450 reductase and cytochrome b5. These results show that CYP6A2 catalyzes the metabolism of organophosphorus insecticides and they implicate Cyp6a2 overexpression in metabolic resistance. The Cyp6a2 gene appears to be a suitable model for a genetic analysis of the phenobarbital induction process.

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells.

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 microg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 microg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 microg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 microg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 microg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing 'initiated' cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.

Modulation by the insecticides heptachlor and chlordane of the cell-mediated immune proliferative responses of rhesus monkeys.

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood samples from twelve young, healthy male rhesus monkeys. Triplicate cultures were made in RPMI 1640 with 10% heat-inactivated homologous plasma with or without the test mitogens (phytohemagglutinin, concanavalin A and pokeweed mitogen). Under a defined assay condition, although a different monkey responded to a different degree (3 to 4-fold difference in stimulation index) to a specific mitogen, the proliferative responses of all monkeys were affected significantly by the addition of the chlorinated hydrocarbon heptachlor or chlordane to the test cultures. The insecticides, at 10-40 microM, may also act as mitogens for the unstimulated monkey PBMC or stimulate the release of IL-2 from a mitogen-stimulated culture. At 80 microM, both heptachlor and chlordane completely suppressed the proliferation and IL-2 release of the monkey lymphocytes. These studies suggest immunomodulatory effects of the insecticides heptachlor and chlordane.

Induction of differentiation in human myeloblastic leukemia ML-1 cells by heptachlor, a chlorinated hydrocarbon insecticide.

Effects of heptachlor, an organochlorine pesticide, on human myeloblastic leukemia ML-1 cells were determined. Similar to 12-O-tetradecanoylphorbol-13-acetate (TPA), a known tumor promoter, heptachlor induced cell adherence and formation of extended cytoplasmic pseudopodia in ML-1 cells. The growth of ML-1 was slightly stimulated by low concentrations (less than 30 nM) of heptachlor. A dose-responsive cell death was also observed when ML-1 cells were treated with heptachlor at concentrations greater than 80 microM. Examination by light microscopy of the cells treated with 80 microM heptachlor revealed a gradual appearance of differentiation characteristics in the culture. On Day 3 of the treatment, 41% of the cells remained unchanged as ML-1, 39% of the cells showed changes and apparent cell differentiation, and 20% of the cells were induced to differentiate to monocyte- or macrophage-like cell type. Electron microscopy also revealed cellular differentiation and the presence of monocyte- and macrophage-like cell types (22%) was confirmed by positive esterase staining.

The effect of the insecticide heptachlor on ras proto-oncogene expression in human myeloblastic leukemia (ML-1) cells.

Expression of the ras proto-oncogene mRNA in human myeloblastic leukemia (ML-1) cells was analyzed as a function of cDNA amplification by polymerase chain reaction (PCR). By using a pair of oligonucleotides that flank exon-2 from opposite strands (5' and 3') of H-ras cDNA for PCR amplification, ML-1 cells were found to express a 112 bp segment of the ras transcript. A rapid decline in the expression of this transcript was seen in cells treated with heptachlor, a chlorinated hydrocarbon insecticide. Expression of the same ras segment was not affected by treatment of ML-1 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, addition of serum to quiescent, heptachlor-treated cultures of ML-1 cells inhibited the effect of heptachlor and restored the expression of the ras protooncogene mRNA.

Blocking actions of heptachlor at an insect central nervous system GABA receptor.

The actions of the polychlorocycloalkane insecticide heptachlor, and its epoxide metabolite, were examined on GABA receptors in insects and vertebrates. Electrophysiological experiments on the cell body of the cockroach (Periplaneta americana) fast coxal depressor motor neuron (Df), and GABA-activated 36Cl- uptake experiments on microsacs prepared from cockroach ventral nerve cords showed that both heptachlor and heptachlor epoxide blocked functional GABA receptors. The block appeared to be non-competitive and was voltage-independent over the membrane potential range -75 mV to -110 mV. There was no significant difference between the potencies of heptachlor and heptachlor epoxide in the functional assays for insect GABA receptors. Both compounds inhibited [35S]-t-butylbicyclophosphorothionate [( 35S]TBPS) binding in insects and vertebrates. The findings provide further evidence for block of an insect GABA receptor/Cl- channel by the cyclodiene class of polychlorocycloalkanes, and reveal differences in the insecticide-[35S]TBPS binding site interactions of insects and vertebrates.

Effects of halogenated hydrocarbons on rumen microorganisms.

Halogenated hydrocarbons such as polychlorinated biphenyls (PCB's), heptachlor (HEP), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane (DDT), and pentachlorophenol (PCP) are environmental contaminants and, at times, can bioaccumulate in the food chain. Cattle have been contaminated in a variety of ways, but generally it is believed that they are only affected by high concentrations of the chemicals. Rumen microorganisms, however, may be affected at lower doses, thus possibly affecting the cow's growth and milk production. Polychlorinated biphenyls, HEP, DDT, and PCP were tested by a 1-stage in vitro fermentation procedure. Substrate utilization was determined by measuring percent dry matter disappearance. Four concentrations (0, 10, 50, and 100 ppm) were studied, and in vitro incubations were conducted for 24 and 48 hr. Samples were removed from 48-hr incubations to determine if the chlorinated hydrocarbons were metabolized during fermentation. Dry matter disappearance proved to be a reliable method to determine microbial activity in the presence of chemicals. Substrate dry matter disappearance for controls and all concentrations of PCB's, HEP, and DDT was approximately 50 and 80% at 24 and 48 hr, respectively. The PCP significantly (P less than 0.05) depressed the percent dry matter disappearance in 50- and 100-ppm cultures to 45 and 30% at 24 hr and 70 and 50% at 48 hr, respectively. Metabolic changes in the test chemicals were not detected by gas chromatographic analysis.