A genome-wide screen identifies yeast genes required for tolerance to technical toxaphene, an organochlorinated pesticide mixture.

Exposure to toxaphene, an environmentally persistent mixture of chlorinated terpenes previously utilized as an insecticide, has been associated with various cancers and diseases such as amyotrophic lateral sclerosis. Nevertheless, the cellular and molecular mechanisms responsible for these toxic effects have not been established. In this study, we used a functional approach in the model eukaryote Saccharomyces cerevisiae to demonstrate that toxaphene affects yeast mutants defective in (1) processes associated with transcription elongation and (2) nutrient utilization. Synergistic growth defects are observed upon exposure to both toxaphene and the known transcription elongation inhibitor mycophenolic acid (MPA). However, unlike MPA, toxaphene does not deplete nucleotides and additionally has no detectable effect on transcription elongation. Many of the yeast genes identified in this study have human homologs, warranting further investigations into the potentially conserved mechanisms of toxaphene toxicity.

Serum gastrin and gastric enterochromaffin-like cells during estrous cycle, pregnancy and lactation, and in response to estrogen-like agents in rats.

Histamine-containing enterochromaffin-like (ECL) cells are numerous in the gastric mucosa. They operate under the control of gastrin. ECL-cell tumors (gastric carcinoids) may arise as a consequence of sustained hypergastrinemia. For reasons unknown, such tumors have a female preponderance both in laboratory animals and humans. The present study consisted of four experiments exploring the possibility that gender-related factors might affect rat ECL cells. 1) A gender difference in terms of serum gastrin concentration and oxyntic mucosal histidine decarboxylase (HDC) activity appeared in Sprague-Dawley but not Wistar rats. Ultrastructural appearance of the ECL cells did not differ between genders. 2) During the different phases of the estrous cycle, the serum gastrin concentration, HDC activity and histamine concentration did not change. 3) During pregnancy, the serum gastrin concentration was suppressed, while it was increased during lactation. The HDC activity and the histamine concentration of the oxyntic mucosa were correlated with the levels of circulating gastrin. 4) Twelve-month treatment with estrogen-like agents, dieldrin and/or toxaphene (alone or in combination) was without any effect on the ECL cells neither in male nor in female rats. In conclusion, the ECL cells are under the control of gastrin, but probably not hormones that involve in the estrous cycle and pregnancy and lactation in rats. Possible gender-related factors behind the female preponderance of ECL-cell tumors remain unknown.

Determination of toxaphene specific congeners in fish liver oil and feedingstuff using gas chromatography coupled to high resolution mass spectrometry.

A new method for the determination of nine toxaphene specific congeners in fish liver oil and feedingstuff has been developed. The samples were extracted using pressurized liquid extraction followed by a purification on silica and florisil columns. Identification and quantification were conducted using GC-(EI)-HRMS, and comparison with MS/MS detection was performed, using electron ionization and negative chemical ionization. Limits of detection were ranged from 0.01 to 0.22 microg kg(-1) (12% moisture) as required for feed samples. The calibration curves showed a good linearity for all congeners (R(2)>0.99). Repeatability was below 9% for all the congeners and recoveries were in-between 73 and 86%. This analytical method was applied to the quantification of thirteen real samples collected within national monitoring plans for further risk assessment.

In vitro modulation of prolactin mRNA by toxaphene and 3,3',4,4'-tetrachlorobiphenyl.

It is now well recognized that many environmental contaminants are capable of disrupting endocrine processes in a variety of species, including birds, mammals, reptiles, and fish. Among these contaminants are toxaphene and polychlorinated biphenyls (PCBs), two of the most prevalent contaminants present in aquatic food chains of the Great Lakes and the Canadian Arctic region. We set out to investigate the possible endocrine-modulating activities of toxaphene, the PCB congener 3,3',4,4'-tetrachlorobiphenyl (TeCB), an equimolar mixture of both compounds (toxaphene/TeCB), and estradiol (E(2)) (E(2)/toxaphene, E(2)/TeCB) on prolactin (PRL) mRNA expression. Concentrations ranging from 10(-7) to 10(-11)M for both toxaphene and TeCB were assayed but only toxaphene modulated PRL mRNA levels, as determined by relative quantitative reverse transcriptase-polymerase chain reaction. Maximal induction by toxaphene was seen at 10(-7)M, resulting in a fourfold increase in PRL mRNA levels. No interactions were observed for combinations of the test substances. Our study demonstrates that toxaphene may exhibit estrogen-like activity by modulating PRL mRNA levels in GH(3) cells.

Toxaphene, but not beryllium, induces human neutrophil chemotaxis and apoptosis via reactive oxygen species (ROS): involvement of caspases and ROS in the degradation of cytoskeletal proteins.

Chemicals of environmental concern are known to alter the immune system. Recent data indicate that some contaminants possess proinflammatory properties by activating neutrophils, an area of research that is still poorly investigated. We have previously documented that toxaphene activates human neutrophils to produce reactive oxygen species (ROS) and accelerates apoptosis by a yet unknown mechanism. In this study, we found that toxaphene induces another neutrophil function, chemotaxis. Furthermore, we found that toxaphene induces both chemotaxis and apoptosis via a ROS-dependent mechanism, since these responses were blocked by the addition of catalase to the culture. In addition, toxaphene was found to induce the degradation of the cytoskeletal proteins gelsolin, paxillin, and vimentin during apoptosis, and this was reversed by the addition of z-VAD-FMK (caspase inhibitor) or catalase, demonstrating the importance of caspases and ROS in this process. In contrast to toxaphene, we found that beryllium does not induce superoxide production, and, this correlates with its inability to induce chemotaxis and apoptosis. We conclude that toxaphene induces chemotaxis and apoptosis via ROS and that caspases and ROS are involved in the degradation of cytoskeletal proteins.

Molecular basis for the constitutive activity of estrogen-related receptor alpha-1.

Some orphan nuclear receptors, including estrogen-related receptor alpha-1 (ERRalpha-1), can activate gene transcription in a constitutive manner. Little is known about the molecular basis of the constitutive activity of these receptors. Our results from site-directed mutagenesis experiments have revealed that Phe-329 (analogous to Ala-350 in estrogen receptor alpha (ERalpha)) is responsible for the constitutive activity of ERRalpha-1. The ERRalpha-1 mutant F329A lost the transactivation activity and acted as a dominant negative mutant. The mammalian cell transfection experiments revealed that the ERRalpha-1 mutant F329A, like wild-type ERalpha, recognized toxaphene (an organochlorine pesticide) as an agonist. This compound was previously shown to be an antagonist of wild-type ERRalpha-1. On the other hand, like wild-type ERRalpha-1, the ERalpha mutant A350F was found to be constitutively active (as demonstrated by mammalian cell transfection and yeast two-hybrid assays). These results indicate that Phe-329 in ERRalpha-1 and Ala-350 in ERalpha play important roles in both ligand binding and transactivation function.

Activation of human neutrophils by technical toxaphene.

Toxaphene is a persistent organic pollutant (POP) known to be composed of numerous congeners. Toxaphene technical mixture applied as a pesticide consists of over 800 congeners. Among these, T(2) and T(12) are the two environmentally prevalent forms found in humans. Although toxaphene is known to exert some toxic effects, including potential proinflammatory properties, little is known concerning its action on cells of the human immune system, especially neutrophils. In the present study, we found that toxaphene was not necrotic for human neutrophils incubated for up to 24 h with concentrations ranging from 0.1 to 50 microg/ml. Toxaphene was found to induce neutrophil superoxide production (O(-)(2)) in a concentration-dependent manner. The potency and the kinetics of toxaphene-induced O(-)(2) by neutrophils were found to be similar to that of the classical neutrophil agonists phorbol 12-myristate 13-acetate (PMA). Furthermore, the use of various transduction signal inhibitors (genistein, pertussis toxin, staurosporine, H-7, and HA-1077), suggests that, as for PMA, toxaphene mediates its effect primarily via PKCs and, to a lesser extend, via tyrosine kinases. In this respect, staurosporine, H-7, and genistein were found to inhibit toxaphene- and PMA-induced O(-)(2) production by 52, 72, and 31% and by 63, 62, and 23%, respectively. Toxaphene was also found to significantly enhance neutrophil phagocytosis of opsonized sheep red blood cells and to induce neutrophil apoptosis. The induction of neutrophil apoptosis was paralleled with a decrease in CD16 expression. T(2) and T(12), the two prevalent congeners found in humans, were also found to significantly increase the O(-)(2) production in neutrophils at a concentration of 5 microg/ml. We conclude that neutrophils are important targets for toxaphene, as this POP can activate O(-)(2) production by a PKC- and tyrosine kinase-dependent mechanism, induce phagocytosis, and accelerate the apoptotic rate. This is the first study that focuses on toxaphene/human neutrophil interactions.

Toxaphene is antiestrogenic in a human breast-cancer cell assay.

Toxaphene is a complex mixture of chlorinated bornanes, bornenes, and bornadienes and was a heavily used insecticide in the United States until its use was restricted in 1982. There are conflicting reports regarding the potential for toxaphene to induce estrogenic responses in human and nonhuman animals. Due to the public concern over environmental estrogens, the estrogenicity of toxaphene was examined in a human breast-cancer cell assay, the MCF-7 focus assay, which is based on in vitro postconfluent cell proliferation and tissue restructuring. In this assay, 0.1-1 nM 17beta-estradiol (E2) produces maximum postconfluent proliferation and formation of multicellular nodules or foci. Toxaphene was also tested for its ability (1) to bind the estrogen receptor (ER) in a competitive binding assay using recombinant human ERalpha (rhER) and in a whole-cell competitive ER binding assay, and (2) to alter the catabolism of E2 in MCF-7 cell cultures. Results from the MCF-7 focus assay showed: (1) Toxaphene alone was not estrogenic between the concentrations of 0.5 nM and 10 microM, (2) toxaphene in binary combinations with chlordane, dieldrin, or endosulfan (alpha or beta) was not estrogenic, and (3) toxaphene was weakly antiestrogenic (it reduced the number of foci induced by 0.1 nM and 0.01 nM E2). Results from the competitive binding assays showed that (1) toxaphene alone did not bind rhER or ER in MCF-7 cells, and (2) toxaphene in binary combinations with other pesticides did not bind rhER. Results from the growth assay and radiometric analysis of E2 catabolism showed that (1) toxaphene did not alter the growth rate of MCF-7 cell cultures over 13 d, and (2) toxaphene did not alter the catabolism of E2. In conclusion, results from the MCF-7 focus assay demonstrate that toxaphene is weakly antiestrogenic rather than estrogenic.

Two organochlorine pesticides, toxaphene and chlordane, are antagonists for estrogen-related receptor alpha-1 orphan receptor.

Estrogen-related receptor (ERR) alpha-1 shares a high amino acid sequence homology with estrogen receptor alpha. Although estrogens are not ligands of ERR alpha-1, our recent results suggest that toxaphene and chlordane, two organochlorine pesticides with estrogen-like activity, behave as antagonists for this orphan nuclear receptor. The two compounds increased ERR alpha-1-mediated expression of the reporter enzyme beta-galactosidase in a yeast-based assay. The screen was developed by expressing the hERR alpha-1-yeast Gal 4 activation domain fusion protein in yeast cells carrying the beta-galactosidase reporter plasmid, which contains an ERR alpha-1-binding element. In transfection experiments using mammalian cell lines, such as the SK-BR-3 breast cancer cell line, the compounds were found to have an antagonist activity against ERR alpha-1-mediated expression of the reporter chloramphenicol acetyltransferase. In contrast to the findings with ERR alpha-1, the two compounds were found to slightly induce the estrogen receptor a-mediated expression of chloramphenicol acetyltransferase in SK-BR-3 cells. In a ligand-independent manner, the ERR alpha-1 activity in SK-BR-3 cells was induced 3-fold by cotransfection with the GRIP1 coactivator expression plasmid. Toxaphene was found to be capable of suppressing the GRIP1 coactivator-induced ERR alpha-1 activity in SK-BR-3 cells. In addition, a stable ERR alpha-1 expressing HepG2 hepatoma cell line was generated, and the aromatase activity in the transfected cell line was found to be twice that in the untransfected cell line. The enzyme aromatase converts androgens to estrogens, and aromatase expression in HepG2 cells is regulated in part by an ERR alpha-1-modulating promoter. A 24-h incubation of an ERR alpha-1-transfected HepG2 cell line with 10 microM toxaphene reduced its aromatase activity to the level in the untransfected cell line. Because toxaphene is not an inhibitor of aromatase, it is thought that the decrease of the aromatase activity in ERR alpha-1 transfected HepG2 cells following toxaphene treatment resulted from a suppression of the aromatase expression by toxaphene acting as the antagonist of ERR alpha-1. Toxaphene and chlordane are among the 12 persistent organic pollutants identified by the United Nations Environment Programme as requiring urgent attention. Their antagonistic effects on ERR alpha-1 should not be overlooked.

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.

Investigation of hepatic cytochrome P-450 enzyme induction and DNA adduct formation in male CD/1 mice following oral administration of toxaphene.

Exposure of experimental animals to toxaphene induces hepatic cytochrome P-450 (CYP). Although chronic administration of toxaphene to mice was found to cause an increased incidence of liver tumors, a mechanism for its carcinogenicity has yet to be elucidated. We investigated two potential mechanisms of toxaphene-induced carcinogenicity: peroxisomal proliferation and DNA binding. Peroxisomal proliferation was evaluated by measuring the level of immunodetectable CYP 4A1, an isozyme of CYP that is specifically induced by peroxisomal proliferators, in hepatic microsomes from CD1 mice that were treated by oral gavage for seven consecutive days with corn oil vehicle or 10, 25, 50 or 100 mg kg(-1) toxaphene. In comparison to control mice, toxaphene-treated mice had increased liver weight, increased liver/body weight ratios and increased levels of total hepatic CYP and cytochrome b5. No increase in the level of immunodetectable levels of CYP 4A1 was found in hepatic microsomes from toxaphene-treated mice when compared to controls. In contrast, increases in immunodetectable CYP 4A1 were detected in hepatic microsomes from mice treated with the peroxisomal proliferator clofibrate. These findings suggest that toxaphene-induced induction of CYP may not involve CYP 4A1 and that peroxisomal proliferation may not be involved in toxicity. Significant increases in immunodetectable levels of CYP 2B were, however, detected in toxaphene-treated mice, and are consistent with earlier reports demonstrating that toxaphene, like many other pesticides, induces the phenobarbital-inducible subfamily of CYP. Analysis of DNA adduct levels in the livers of toxaphene-treated mice by DNA 32P-post-labeling showed no evidence of DNA adduct formation.

Effects of some persistent halogenated environmental contaminants on aromatase (CYP19) activity in the human choriocarcinoma cell line JEG-3.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB126), a technical PCB mixture (Aroclor 1016), and a technical toxaphene mixture (Camphechlor) on aromatase (CYP19) activity were investigated in human choriocarcinoma JEG-3 cells. After 18 h incubation with TCDD, PCB126, Aroclor 1016 or toxaphene, ethoxyresorufin-O-deethylase (EROD), and aromatase activity were determined. To exclude serum effects, incubations were carried out with or without fetal calf serum in the medium. EROD activity was induced by both TCDD and PCB126 in the presence or absence of serum, which indicates that JEG-3 cells are responsive toward dioxin-like chemicals. Neither Aroclor 1016 nor toxaphene affected EROD activity in these cells. Calculated EC50 values for induction of EROD activity were 0.71 and 0.40 nM for TCDD, and 48 and 20 nM for PCB126 in presence or absence of serum, respectively. Incubation with TCDD or PCB126 with or without serum caused a concentration-dependent decrease in the aromatase activity of up to 4.9-fold. Calculated EC50 values for this effect were 52 pM and 13 nM for TCDD, and 75 and 48 nM for PCB126 in the presence and absence of serum, respectively. Aroclor 1016 and toxaphene had no effect on aromatase activity at concentrations up to 1.0 microM for Aroclor 1016 or 3.0 microM for toxaphene. These results show that aromatase activity can be decreased in a concentration dependent way within the same range where EROD activity is increased. In view of these results, possible effects of dioxin-like compounds on estrogen producing and androgen target cells should be studied in more detail.

Effect of toxaphene on estrogen receptor functions in human breast cancer cells.

Toxaphene (polychlorinated camphenes) is an insecticidal mixture of >670 chemicals, which was widely used until the mid 1980s. Due to their lipophilic and volatile nature, these chemicals accumulate in animal and human tissues and continue to be a major contaminant in marine and freshwater biota. Cytotoxic and genotoxic effects in mammalian test systems suggest that toxaphene is a carcinogen and reports support the hypothesis that toxaphene could have tumor-promoting potential in human breast tissue. In order to examine the potential of toxaphene as an environmental endocrine disrupter, we investigated its effect on the estrogen receptor (ER) function in human breast cancer MCF-7 cells. Using transient gene expression experiments, we observed approximately 60% and 80% inhibition of the constitutive and 17beta-estradiol induced ER-dependent transactivation, respectively. The involvement of the ER in the ability of toxaphene to block the estrogen action was verified by cotransfection studies in ER-negative MDA-MB-231 cells. The interference of toxaphene with the ER mediated responses was supported by a significant suppression of endogenously expressed pS2 RNA and decreased levels of secreted pS2 protein. These reproducible results indicate that toxaphene can disturb hormonal signals mediated by the ER and suggest that these environmental chemicals have potential endocrine disrupting activities which may affect the reproductive health and increase the risk of carcinogenesis.

Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism.

The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.

Synergistic activation of estrogen receptor with combinations of environmental chemicals.

Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.

SCE induction and cell-cycle delay by toxaphene.

Toxaphene is genotoxic in mammalian cell systems and also inhibits cell replication. It was therefore used to investigate possible masking of SCE induction due to cell-cycle delay. In this study, toxaphene-treated Chinese hamster lung (Don) cells exhibited a dose-dependent decrease in cell-cycle progression compared with untreated cells. At high, nontoxic toxaphene levels (15 micrograms/ml), cell cycling also slowed as the toxaphene treatment time was increased. Toxaphene induced significantly higher numbers of SCEs in treated cells, demonstrating a dose- and treatment time-relationship. Slopes of dose-response curves were 0.29, 0.43 and 0.77 SCE/micrograms toxaphene for 20.5 h, 24.5 h and 28.5 h incubation, respectively. There were no changes in SCE values in control cultures even when slower dividing cells were sampled e.g. at longer incubation times. Thus, higher SCE values in Chinese hamster cells were not associated per se with slower or more delayed cells. The results demonstrate that longer toxaphene treatment times were not necessary for obtaining sufficient harlequin-stained cells for SCE analysis, but that higher numbers of SCEs occurred in slower dividing cells, following prolonged incubation of cultures treated with toxaphene.

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.