Biochemical mechanism of GSH depletion induced by 1,2-dibromoethane in isolated rat liver mitochondria. Evidence of a GSH conjugation process.

HPLC measurements of GSH and GSSG levels in isolated rat liver mitochondria, on addition of 1,2-dibromoethane (DBE), revealed the presence of a glutathione (GSH)-conjugating pathway of DBE. This process required the structural integrity of the mitochondrial matrix and inner membrane complex and was inhibited by the uncouplers of oxidative phosphorylation, particularly 2,4-dinitrophenol. On the other hand it was not affected by the energetic state of the mitochondria, since other mitochondrial inhibitors like KCN and oligomycin did not have any effect on it. This process also did not require the involvement of mitochondrial inner membrane transport systems, based on the measurement of the mitochondrial transmembrane potential. The involvement of mitochondrial GSH-S-transferases, located either in the matrix or in the intermembrane space, is discussed.

Induction of calcium efflux from isolated rat-liver mitochondria by 1,2-dibromoethane.

Addition of 1,2-dibromoethane to rat-liver mitochondria induces a concentration-dependent depletion of mitochondrial glutathione. This event seems to be associated with the induction of Ca2+ release from mitochondria pre-loaded with a low pulse of Ca2+. The enhancement of the energy-dissipating process to reaccumulate the released Ca2+ ('Ca2+ cycling') results in a progressive drop of membrane potential. Addition of EGTA (ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid), when the membrane potential has reached the lowest level, restitutes it to a normal value. All these findings and the observation that Ca2+ release also occurs under non cycling conditions (e.g., in the presence of ruthenium red) suggest that 1,2-dibromoethane induces a Ca2+ efflux by activating a selective pathway which is sensitive to critical sulfhydryl groups.

DNA damage by haloalkanes in human lymphocytes cultured in vitro.

The DNA damaging activity of 7 haloalkanes was studied in a short-term in vitro system which utilized human lymphocytes. The parameters studied were the inhibition of scheduled (duplicative) and unscheduled (reparative) DNA syntheses seen as tritiated thymidine uptake. The results obtained suggested that chloromethyl methyl ether (CMME), 1,2-dibromoethane (DBE), trichloroethylene (TCE) and 1,2-dichloroethane (DCE) gave positive results such as DNA damaging agents, while carbon tetrachloride (CTC), chloroform (TCM) and dichloromethane (DCM) gave low or negative results.

Liver cell proliferation induced by the mitogen ethylene dibromide, unlike compensatory cell proliferation, does not achieve initiation of rat liver carcinogenesis by diethylnitrosamine.

The purpose of this investigation was to determine whether mitogen-induced cell proliferation is as effective as compensatory cell proliferation in achieving initiation of carcinogenesis in rat liver. Male Wistar rats were injected with a single non-necrogenic dose of the hepatocarcinogen diethylnitrosamine (DENA) during the peak of DNA synthesis following the administration of the hepatic mitogen ethylene dibromide (EDB) or a necrogenic dose of CCl4. After subjecting the animals to a promoting procedure, the rats were sacrificed and the initiated hepatocytes were monitored as gamma-glutamyltranspeptidase (gamma-GT) positive foci. The results indicate that while DENA administration during compensatory cell proliferation results in the formation of GT positive foci, no enzyme-altered foci were produced when the carcinogen was given during liver hyperplasia induced by EDB, despite the fact that at the time of carcinogen administration, the extent of cell proliferation, as monitored by thymidine incorporation into DNA, was the same in both the groups.

Stimulation of DNA synthesis by rat plasma following in vivo treatment with three liver mitogens.

The present study was undertaken to determine the effect of two different types of liver cell proliferative stimuli, namely compensatory regeneration and direct hyperplasia on DNA synthesis of normal and preneoplastic isolated hepatocytes. Platelet-poor plasma (PPP) isolated from male Wistar rats treated with three different hepato-mitogens, lead nitrate (LN), cyproterone acetate (CPA) and ethylene dibromide (EDB), or subjected to surgical partial hepatectomy (PH), was tested for its ability to stimulate DNA synthesis in normal and preneoplastic hepatocytes in primary cultures. Induction of DNA synthesis was detected as early as 30 min after CPA, EDB and PH administration and persisted up to 5 days after the LN administration. In addition, hepatocytes isolated from preneoplastic liver nodules were also able to respond in culture to the DNA synthesis stimulus induced by these factors.

Studies on the activation of rat liver microsomal glutathione transferase in isolated hepatocytes.

The mechanism of activation of microsomal glutathione transferase in isolated liver cells by diisapropylidene acetone (phorone) was investigated. Phorone (1 mM) causes a time-dependent increase (up to 2.6-fold) in the glutathione transferase activity of microsomes isolated from treated hepatocytes. Since phorone reacts with sulfhydryl groups, the possibility that this compound activated microsomal glutathione transferase directly was studied. It was found that neither the activity of the purified enzyme nor that in isolated microsomes is affected by phorone. It has been suggested [Masukawa T and Iwata H, Biochem Pharmacol 35: 435-438, 1986] that activation of microsomal glutathione transferase by phorone in vivo is mediated through thiol-disulfide interchange involving oxidized glutathione (GSSG). It is shown here that the glutathione transferase activity of isolated microsomes, which was increased by the addition of 10 mM GSSG, can be decreased to the basal level with 0.1 M dithioerythritol. Dithioerythritol, on the other hand, only marginally decreases the glutathione transferase activity in microsomes isolated from phorone-treated hepatocytes. This finding argues against a role for thiol-disulfide interchange in the activation of the enzyme by phorone. Furthermore, the glutathione depletion caused by phorone does not seem to be responsible for activation per se, since other thiol depletors [e.g. diethylmaleate (DEM)] do not affect the activity of the enzyme. Immunoblot analysis of microsomes isolated from phorone-treated hepatocytes did not reveal any partial proteolysis which might have accounted for the activation. It is suggested that activation of microsomal glutathione transferase by phorone proceeds through a mechanism which might reflect an in vivo regulation of this enzyme. Additional compounds which have been shown to activate the microsomal glutathione transferase in vivo were also tested and significant activation was obtained with 1,2-dibromoethane (1.4-fold) but not with DEM or carbon tetrachloride. Activation was also obtained with 1-chloro-2,4-dinitrobenzene (CDNB) (1.6-fold) and to a small extent with t-butyl hydroperoxide (1.2-fold). The activation by 1,2-dibromoethane and CDNB is probably mediated through covalent binding, considering the known alkylating properties of these compounds. CDNB is the first substrate shown to activate the microsomal glutathione transferase implying that electrophilic compounds which are substrates can increase the rate of their own elimination by reacting with this enzyme. In addition, activation by t-butyl hydroperoxide indicates that oxidative stress can activate microsomal glutathione transferase.

Effects of 1,2-dibromoethane on glutathione metabolism in rat liver and kidney.

There are few reports of the effects of glutathione-depleting agents administered for periods longer than 24 hr on the turnover of glutathione (GSH) in mammalian tissues. Studies of such effects are important in relation to the protection of tissues from damage from, for example, reactive metabolites derived from xenobiotics. In the investigation described here, 1,2-dibromoethane dibromide)-a widely used insecticide, nematocide, fungicide and petrol additive, which is hepato- and nephrotoxic-was administered to rats and the effects on non-protein thiol contents and GSH-related enzyme activities were determined in liver and kidney. The classical GSH-depleter diethylmaleate was used in parallel studies for comparative purposes.

Conjugative metabolism of 1,2-dibromoethane in mitochondria: disruption of oxidative phosphorylation and alkylation of mitochondrial DNA.

1,2-Dibromoethane (DBE) is an environmental contaminant that is metabolized by glutathione S-transferases to a haloethane-glutathione conjugate. Since haloethane-glutathione conjugates are known to alkylate nuclear DNA and cytoplasmic proteins, these effects were investigated in isolated rat liver mitochondria exposed to DBE by measuring guanine adducts and several aspects of oxidative phosphorylation including respiratory control ratios, respiratory enzyme activity, and ATP levels. Mitochondrial large-amplitude swelling and glutathione status were assessed to evaluate mitochondrial membrane integrity and function. When exposed to DBE, mitochondria became uncoupled rapidly, yet no large-amplitude swelling or extramitochondrial glutathione was observed. Mitochondrial GSH was depleted to 2-53% of controls after a 60-min exposure to micromolar quantities of DBE; however, no extramitochondrial GSH or GSSG was detected. The depletion of mitochondrial glutathione corresponded to an increase of an intramitochondrial GSH-conjugate which, based on HPLC elution profiles and retention times, appeared to be S,S'-(1,2-ethanediyl)bis(glutathione). Activities of the NADH oxidase and succinate oxidase respiratory enzyme systems were inhibited 10-74% at micromolar levels of DBE, with succinate oxidase inactivation occurring at lower doses. ATP concentrations in DBE-exposed mitochondria in the presence of succinate were 5-90% lower than in the controls. The DNA adduct S-[2-(N(7)-guanyl)ethyl]glutathione was detected by HPLC in mtDNA isolated from DBE-exposed mitochondria. The results suggest that respiratory enzyme inhibition, glutathione depletion, decreased ATP levels, and DNA alkylation in DBE-exposed mitochondria occur via the formation of an S-(2-bromoethyl)glutathione conjugate, the precursor of the episulfonium ion alkylating species of DBE.

Induction of diphtheria toxin-resistant mutants in human cells by halogenated compounds.

The mutagenic power of 1,2-dichloroethane, 1,2-dibromoethane, 1,2-diiodoethane was tested in the human cell line, EUE. In our mutagenic system, based on selection against diphtheria toxin, the halogenated compounds, 1,2-dichloroethane and 1,2-dibromoethane revealed a strong mutagenic effect, whereas 1,2-diiodoethane was not mutagenic at a concentration allowing survival of 41%.

Cytogenetic effects of vincristine sulfate and ethylene dibromide in human peripheral lymphocytes: micronucleus analysis.

Micronuclei kinetics and persistence in mononucleated and binucleated human peripheral lymphocytes following short-term (4 hr) and continuous (until harvest) in vitro exposure to vincristine sulfate (VS) and ethylene dibromide (EDB) were studied. Lymphocytes were exposed to chemicals for various doses and harvested at different culture times. Micronucleus frequencies were scored in both mononucleated and binucleated cells on the same slide. VS-treated cells showed a significantly higher incidence of micronucleus in both mononucleated and binucleated cells than controls (P less than 0.01). The cells treated continuously with VS produced comparatively higher frequencies of micronucleated cells than those treated for 4 hr. Highest micronuclei frequencies were observed 24 hr after chemical treatment in both mononucleated and binucleated cells and decreased later with time. However, the micronucleus frequencies remained significantly higher than the controls even in the cells harvested at 144 hr. VS induced a large number of micronucleated cells with multiple micronuclei. VS also caused a severe decrease in nuclear division due to cytotoxic effect. Lymphocytes treated with EDB for 4 hr and continuously showed a statistically higher incidence of micronuclei in binucleated cells compared to the controls (P less than 0.05), whereas in mononucleated cells higher micronucleus frequencies were observed only in cultures treated continuously. Continuous presence of EDB induced both dose- and time-dependent increase of micronuclei in both mono- and binucleated cells (P less than 0.05). EDB induced relatively few multiple micronucleated cells in comparison with VS. EDB did not affect nuclear divisions even with continuous treatment. High micronucleus frequencies observed at 144 hr harvest following 4 hr treatment of both EDB and VS suggest the persistence of DNA damage in cells. These studies suggest that micronuclei kinetics in human peripheral lymphocytes depends on the genotoxic potentially and cytotoxicity of a genotoxicant.

Transient decrease of liver cytosolic glutathione S-transferase activities in rats given 1,2-dibromoethane or CCl4.

In vivo treatment of fasted male rats with 1,2-dibromoethane (DBE) (0.4 mmol/kg) or carbon tetrachloride (CCl4) (4 mmol/kg) was found to rapidly alter the activities of liver cytosolic and microsomal glutathione S-transferases. Microsomal activities towards chloro-2,4-dinitrobenzene (CDNB) were increased 2 h after either treatment. Cytosolic activities towards CDNB and 3,4-dichloronitrobenzene (DCNB), but not 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP), were selectively and transiently decreased after either treatment. Time course studies in DBE animals indicated that the decrease in cytosolic activity was not evident until 2 h although liver glutathione (GSH) concentrations were diminished within 15 min. In contrast, in CCl4 animals the decrease in cytosolic activity was evident within 15 min and was not accompanied by diminished GSH concentrations. By 4 h, cytosolic activities had rebounded to control levels in both DBE and CCl4-treated animals. Kinetic studies of the enzyme in liver cytosol from animals 2 h after treatment with DBE or CCl4 indicated that both treatments decreased the apparent Vmax while neither treatment altered the apparent Km. This pattern of change allows exclusion of a simple competitive mechanism of enzyme inhibition, but cannot distinguish between reversible non-competitive inhibition and irreversible inhibition. It is possible that the observed decreases in the activities of the abundant cytosal enzyme are due to 'sacrificial' covalent linkages between the enzyme and reactive metabolites of DBE or CCl4.

Modulation of the mitotic action of ethylene dibromide.

Refeeding rats treated with a single high dose of ethylene dibromide (1,2-dibromoethane, EDB) induced liver DNA synthesis. The peak of DNA synthesis, as measured by [methyl-3H]thymidine incorporation was attained after 24 h in refed rats and at 48 h in fasted ones. Fasting enhances the EDB action leading to liver cell necrosis, as shown by elevation of serum enzymes' activities, glutamic pyruvic transaminase (GPT) and sorbital dehydrogenase (SDH). A low dose of EDB administered during 2 and 3 weeks slightly enhanced the liver DNA synthesis and elevated the activity of serum enzymes. Phenobarbitone (PB) treatment of rats together with low dose of EDB during 2 weeks prevented the enzyme activity elevation and attenuated the DNA synthesis. Diethyldithiocarbamate (DDC) pretreatment potentiated the DNA synthesis in fed rats after both a small dose of EDB for 2 weeks and after a single high-dose treatment. In DDC pretreated rats, the high single dose of EDB caused biochemical perturbations in serum and liver representative of liver cell necrosis; changes in serum enzymes' activities also were noticed as early as 2 h after EDB toxication. The possible function of modulators on the mitogenic or the necrogenic action of EDB is discussed.

In vivo potentiation of 1,2-dibromoethane hepatotoxicity by ethanol through inactivation of glutathione-s-transferase.

In the rat, a single ethanol (EtOH) pretreatment (2.5 g/kg b.w., per os) was able to strongly enhance the cytotoxicity of 1,2-dibromoethane (DBE)(87 mg/kg b.w., per os). The principal metabolic routes of DBE involve both oxidative and conjugative transformations. Microsomal cytochrome P450 content and dimethyl nitrosamine demethylase activity were not changed, while a significant loss of cytosolic total GSH-transferase was observed in rats killed 6 h after EtOH pretreatment. Pretreatment with methylpyrazole, an inhibitor of alcohol-dehydrogenase prevented the effects provoked by ethanol. The major EtOH metabolite, acetaldehyde. seemed thus to play a fundamental role in the mechanism responsible for the potentiation of DBE toxicity mediated by EtOH. To further support this hypothesis, disulfiram (75 mg/kg b.w.), an inhibitor of aldehyde dehydrogenase, was given i.p. to rats. When DBE was administered to disulfiram- and EtOH-pretreated rats, a marked increase of liver cytolysis was shown and cytosolic GSH-transferase activity was further inhibited if compared to that induced by EtOH treatment alone. The results are consistent with the hypothesis that EtOH given to rats increases DBE liver toxicity because its major metabolite, acetaldehyde, reduces the DBE conjugates to GSH transferase, with consequent shift of DBE metabolism to the oxidative route and accumulation of reactive oxidative intermediates no longer effectively conjugated with GSH.

Human erythrocyte glutathione S-transferase: a possible marker of chemical exposure.

We have explored the possibility of using glutathione S-transferase (GST) as a biological marker of chemical exposure. All the model compounds tested in the present study (acrolein, propylene oxide, styrene oxide, ethylene dibromide and ethylene dichloride) showed a dose-dependent inactivation of erythrocyte GST in situ as well as the inhibition of purified erythrocyte GST.

Sensitivity of Drosophila melanogaster to low concentrations of gaseous mutagens. II. Chronic exposures.

Sex-linked recessive lethal mutations were induced in Drosophila melanogaster males by gaseous 1,2-dibromoethane at concentrations ranging from 0.2 to 2 parts per million. Significant numbers of mutations could be induced at all these concentrations. Pronounced germ-cell sensitivity differences were observed. For low exposures, spermatids and spermatocytes were about 10--20 times more sensitive than spermatozoa. The dose-effect relation was linear below 60 ppm . h for the 3 cell types. At higher exposures, sterility prevented mutation detection in spermatocytes and in spermatogonia. The lowest effective exposure for spermatozoa was 18 ppm . h (0.25 ppm for 72 h). In spermatids, the lowest exposure tested, 2.3 ppm . h (0.2 ppm for 11 h) induced 4 times the spontaneous mutation rate. Therefore, using prolonged exposure periods one may be able to detect concentrations in the range of parts per billion. Thus, Drosophila appears suitable as a system for detecting very low concentrations of gaseous mutagens in industrial, agricultural and environmental atmospheres.

A procedure for the quantitative measurement of the mutagenicity of volatile liquids in the Ames Salmonella/microsome assay.

We have designed a closed, inert incubation system for testing the mutagenicity of volatile compounds. The containment properties of this system have been investigated using carbon-14 labelled 1,2-dibromoethane. The recovery of this solvent was about 95% following a 48-h incubation at 37 degrees. Using the Ames Salmonella/microsome mutagenicity assay we have determined the mutagenic potency of 10 common halogenated alkane solvents. Of these 10 compounds, only 1,2-dibromoethane and 1-bromo-2-chloroethane give positive results in the standard test procedure, whereas 7 of the 10 give positive results in the closed system. The specificity observed for reversion of the tester strains and the lack of any significant effect of added rat-liver "S9" fractions suggest that these haloalkanes are direct-acting "base-pair" type mutagens. The mutagenic potencies of the 7 positive compounds range from 0.001 revertants per nanomole for 1,2-dichloroethane to 0.172 revertants per nanomole for 1,2-dibromoethane. A minimum or threshold response level for each material has been calculated.

Mutagenicity and cytotoxicity of haloethanes as studied in the CHO/HGPRT system.

When haloethanes were being tested as direct-acting agents in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system, ethylene dibromide (EtBr2) exhibited more cytotoxic and mutagenic activity than ethylene dichloride (EtCl2), and the mixed halogenated congener ethylene bromochloride (EtBrCl) had an intermediate effect. On a molar basis, the relative mutagenic activity of EtBr2 : EtBrCl : EtCl2 was approximately 100 : 6 : 1. Cell survival was reduced to 50% by approx. 3, 6 and 50 mM of EtBr2, EtBrCl and EtCl2, respectively, and declined precipitously with increasing concentrations of the haloethanes. When these 3 haloethanes were assayed in the presence of S9, there was a 5-25-fold increase in cytotoxicity; however, only EtBrCl and EtCl2 also showed a concomitant increase in mutagenicity of 4-fold. The mutagenicity of EtBr2 remained unchanged when assayed in the presence of S9. When NADP was omitted from the S9 mix, which contains a NADPH-regenerating system, the increase in cytotoxicity and mutagenicity observed with the complete S9 mix was abolished. EtBr2 was shown to possess a molar equivalent mutagenic activity to ethyl methanesulfonate under the conditions of the assay. The cytotoxicity of EtBr2 increased as the time of treatment increased up to 24 h, while mutation induction appeared to peak at around 5 h.

New tester strains of Salmonella typhimurium lacking O6-methylguanine DNA methyltransferases and highly sensitive to mutagenic alkylating agents.

Salmonella typhimurium YG7104 and YG7108 are derivatives of the Ames tester strain TA1535, and have chromosomal deletions of the ogtST gene or both the ogtST and adaST genes, respectively. The ogtST and adaST genes encode O6-methylguanine DNA methyltransferases that are involved in the repair of DNA damage caused by alkylating agents. The sensitivities of these strains to 15 mutagens with different structures were tested and compared with those of the parent strain TA1535. Deletion of ogtST or ogtST plus adaST substantially increased the sensitivity of strain TA1535 to the mutagenicity of alkylating agents, such as N-ethyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate or dimethylnitrosamine (DMN). Preincubation of the chemical with S9 mix and bacteria for 20 min at 37 degrees C before pouring them together on agar plates was not necessary to detect the mutagenicity of DMN when strain YG7104 or YG7108 was used as a tester strain. Introduction of plasmid pKM101 did not enhance but rather decreased the sensitivity of YG7104 and YG7108 to alkylating agents. Since the new strains are highly sensitive only to alkylating agents, they will be useful to detect the mutagenicity with high efficiency and to study the mechanism of mutagenesis induced by environmental alkylating agents.

In vivo cytogenetic studies on mice exposed to ethylene dibromide.

The pesticide, ethylene dibromide (EDB), was evaluated with in vivo cytogenetic assays to determine its genotoxicity. CD1 male mice were exposed to EDB through intraperitoneal injections. Bone marrow cells isolated from femora were analyzed for sister-chromatid exchange (SCE), chromosome aberration and micronucleus formation. The results showed that only certain concentrations of EDB tested caused a slight but significant increase in SCEs and chromosome aberrations. However, these increases were not dose-related. No increase in the polychromatic erythrocytes with micronuclei was observed following EDB exposure. Also, EDB did not cause cell-cycle delay in comparison with controls. Thus, it appears that EDB is not an effective genotoxic agent in vivo in mice.

Effect of metabolic activation on the cytotoxicity and mutagenicity of 1,2-dibromoethane in the CHO/HGPRT system.

When ethylene dibromide (EtBr2) was assayed with the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system coupled with a rat liver metabolic activation system (S9), which contains Ca2+ (Ca, Mg-S9), the cytotoxicity of EtBr2 was greatly increased over that obtained when NADP was omitted from the Ca, Mg-S9 or when EtBr2 was assayed as a direct-acting agent. However, on a molar basis, the mutagenicity of EtBr2 remained unaffected. The omission of Ca2+ from the Ca, Mg-S9 metabolic activation system (Mg-S9), with either the addition or omission of NADP, caused approximately a 2-fold decrease in the mutagenicity of EtBr2 when compared to the results obtained by using the Ca, Mg-S9 system. The cytotoxicity of EtBr2 was further increased when a purified microsomal fraction, prepared from the S9 fraction, was used in the presence of Ca2+. In the absence of this calcium ion, this metabolic activation system was extremely cytotoxic to Chinese hamster ovary cells even without the presence of a mutagen or promutagen. The cytotoxicity of EtBr2 in the following assay systems decreased in this order: Ca, Mg-microsomes greater than Ca, Mg-S9 greater than S9 greater than direct-acting agent greater than or equal to Ca, Mg-S9 without NADP greater than or equal to Mg-S9 without NADP. Cytotoxicity appears to be NADP-dependent on the presence of NADP in the S9 system, the mutant yield (number of mutants that could be induced) was higher in its absence. Addition of reduced glutathione to Mg-S9 without NADP increased the mutagenicity of EtBr2 to values that did not exceed those obtained when EtBr2 was tested as a direct-acting agent. On a molar basis, ethyl methanesulfonate (EMS) is less cytotoxic but equally as mutagenic as EtBr2. However the mutant yield of EMS was higher than that of EtBr. Inclusion of Ca, Mg-S9 in the assay system had no effect on the biological activities of EMS.