Acute nephrotoxicities and hepatotoxicities of 1,2-dibromo-3-chloropropane and 1,2-dibromoethane in male and female F344 rats

Four consecutive intraperitoneal (i.p.) injections with 40 mg/kg of 1,2-dibromo-3-chloropropane (DBCP) reduced the in vitro accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA) by slices of renal cortex and increased blood urea nitrogen (BUN) concentration in both male and female rats, but elevated serum glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) activities in females only. Four consecutive treatments with 1,2-dibromoethane (EDB) reduced the accumulation of PAH in male rats, but failed to alter TEA accumulation, BUN concentration or GPT and GOT activities in rats of either sex. Single i.p. injections of EDB or DBCP (40 mg/kg, approximately one-half of the acute, i.p. LD50 values) were without effect on serum GPT and GOT activities, BUN concentration or the accumulations of PAH and TEA in male rats when measured 24, 48 or 96 h after treatment, except that PAH accumulation was reduced at 96 h.These results indicate that BUN and the accumulations in vitro of PAH and TEA by renal cortical slices are appropriate endpoints for studying DBCP nephrotoxicity. Measurements of serum GOT and GPT activities detected DBCP hepatotoxicity in female rats only. The nephrotoxicity of EDB was indicated by measurement of TEA accumulation only.     

Respiratory pathology in rats and mice after inhalation of 1,2-dibromo-3-chloropropane or 1,2 dibromoethane for 13 weeks

Seventy F344 rats and 144 B6C3F1 mice were subdivided into seven groups. Three groups were each exposed via inhalation to 1, 5, or 25 ppm of 1,2-dibromo-3-chloropropane (DBCP) for 6 h per day, 5 days per week for 13 weeks. Three additional groups were each similarly exposed to 3, 15, or 75 ppm of 1,2-Dibromoethane (EDB). The remaining group was exposed to room air under the same conditions. At 13 weeks, rats and mice showed severe necrosis and atrophy of the olfactory epithelium in the nasal cavity after inhalation of 5 or 25 ppm DBCP and 75 ppm EDB. Lower concentrations induced squamous cell metaplasia, hyperplasia and cytomegaly of the epithelium of the respiratory nasal turbinals. Squamous metaplasia, hyperplasia and cytomegaly of the epithelium was also seen in larynx, trachea, bronchi and bronchioles. Other compound related toxic lesions in rats were seen in the liver, kidney and testes.     

The integrity of liver protein synthesis in male rats treated with 1,2-dibromo-3-chloropropane

Male rats treated with a single dose of 1,2-dibromo-3-chloropropane (DBCP) were tested for their ability to carry out the synthesis of liver proteins. In animals treated for 12 h, we found no changes in the uptake of [14C]orotic acid into liver RNA or the uptake of [3H]leucine into liver or serum protein. Uptake of [3H]leucine into the soluble fraction of the enlarged liver increased in proportion to liver size, while the uptake of [14C]orotic acid was unchanged. Examination of the ultrastructure of liver cells from rats treated for 12, 24, or 48 h revealed that the structure of the rough and smooth endoplasmic reticulum (RER; SER) were modified. An absence of ordered stacks of the RER and the presence of tangled nets of SER were noted.     

Glutathione conjugation of chlorobenzylidene malononitriles in vitro and the biotransformation to mercapturic acids in rats

The glutathione conjugation of 2-chloro-, 3-chloro-, 4-chloro- and 2,6-dichlorobenzylidene malononitrile (chloroBMNs) was investigated in vitro. In incubation mixtures containing rat liver cytosol (9000 g), the decrease in the initial amount of glutathione due to the various chloroBMNs ranged from 40 to 60% and occurred both enzymatically and spontaneously at physiological conditions (37°C, pH7.4). 2,6-DichloroBMN, however, depleted glutathione largely spontaneously (38±3%). The steric hindrance of the two chlorosubstituents probably plays an important role during the glutathione-S-transferase catalyzed reaction.The hydrolysis of the chloroBMNs to the corresponding chlorobenzaldehydes and malononitrile was studied in a mixture of buffer pH 7.4 and ethanol. The rate of hydrolysis of 2,6-dichloroBMN was slower than those of the related chloroBMNs. This means that 2,6-dichloroBMN will be the most stable compound in the presence of water.Only IP administration of 2-chloroBMN (CS) to adult male Wistar rats gave enhancement of urinary thioether excretion. A thioether could be isolated and was identified as the N-acetyl-S-[2-chlorobenzyl]-L-cysteine. The quantity of this benzylmercapturic acid in the urine of rats amounted to 4.4% dose (0.07 mmol/kg, n=12).After IP administration of 2-chloro- and 3-chlorobenzaldehyde to rats benzylmercapturic acid excretion in the urine was found to be 7.6 and 1.1% of the dose, respectively. Administration of the related 4-chloro- and 2,6-dichlorobenzaldehyde, however, resulted in _no urinary mercapturic acid excretion.It is very likely that in rats the initial biotransformation of chloroBMNs is mainly hydrolysis to corresponding chlorobenzaldehydes, leading in the case of 3-chloro-, 4-chloro- and 2,6-dichloroBMN to no mercapturic acid excretion in the urine.Nevertheless, 2,6-dichloroBMN will be the most reactive compound with proteins and therefore the best haptene in comparison with the related chloroBMNs.This work was financially supported by a grant from the Dutch Foundation for Medical Research FUNGO, grant no. 13-28-57     

Reproductive tract defects induced in adult male rats by postnatal 1,2-dibromo-3-chloropropane exposure

The reproductive tract of the male rat may be particularly susceptible to chemical injury during the early postnatal period since significant developmental changes occur in the tract at that time. The subcutaneous administration of relatively low doses of 1,2-dibromo-3-chloropropane (DBCP, 5-20 mg/kg) on alternate days, from 2 to 20 days of life, resulted in a marked dose-related reduction in the testis, epididymis, and seminal vesicle weights. Histological evaluation revealed degenerative cellular changes in the testes of the 5 mg/kg treated group and obliteration of the seminiferous tubules in the 10 mg/kg treated animals. Biochemical studies showed that the in vitro androgen production capacity per unit weight of testicular tissue was elevated, as a function of the DBCP dose, correlating with the apparent increases in the Leydig cell concentrations observed histologically in the treated animals. Due to the marked reduction in the testes weight of DBCP-treated animals, the in vitro testicular androgen production rate, when expressed on the basis of testes pair weight, was reduced; it was consistent with the observed DBCP-induced decrease in serum androgen levels. Early DBCP exposure also obliterated the androgen responsiveness of the seminal vesicle and epididymis in the adult rat, which may also contribute to the diminution in the weights of these androgen-dependent organs. The present study also indicated that immature rats were more susceptible than sexually matured male rats to DBCP toxicity. Moreover, the results of the critical period study indicated that the first 10 days of life are of particular importance in the reproductive tract toxicity of DBCP.     

Glutathione conjugation and mercapturic acid formation in the developing rat, in vivo and in vitro.

1. The levels of GSH-S-epoxidetransferase (GSH-S-transferase E, EC 2.5.1.18), gamma-glutamyl transpeptidase (EC 2.3.2.2) and S-substituted cysteine N-acetyltransferase have been measured in the liver and kidney of neonatal to adult rats. 2. GSH-S-epoxidetransferase and S-substituted cysteine N-acetyltransferase activities were less than 10% of the adult values in neonatal rats, rising gradually to reach adult values at about 40 days of age. Renal gamma-glutamyl transpeptidase activity was 27% of the adult value 2 days after birth and increased after 15 days reaching adult levels by 40 days. 3. The percentages of the doses of 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) and of 1,2-epoxybutane, administered at the same dose level to rats aged 4 days to adult, excreted as the corresponding mercapturic acids in 24 h, were not significantly different. 4. Adult and 10 day old rats doses at the same dose level with ENPP excreted N-acetyl-S-[2-hydroxy-3-(p-nitrophenoxy)propyl]-L-cysteine (ENPP-MA) at the same rate. 5. In addition to ENPP-MA, dosed rats under 13 days of age excreted the corresponding substituted cysteine. 6. The correlation between results in vitro and in vivo is discussed.     

1,2-dibromo-3-chloropropane (DBCP)-induced infertility in male rats mediated by a post-testicular effect

The potential for the chemosterilant 1,2-dibromo-3-chloropropane (DBCP) to reduce male fertility by acting at a site in the genital tract beyond the testis was evaluated in male, Fischer 344 rats. A single sc treatment with 100 mg/kg DBCP reduced fertility in male rats 2 to 7 days postexposure without affecting mating frequency. Doses of 10, 20, or 40 mg/kg DBCP given sc once daily for 7 days caused a dose-dependent reduction in the metabolism of glucose to CO2 by epididymal sperm, as measured in vitro. Conversion of glucose to lactate was not reduced, indicating inhibition of energy metabolism at a step post-glycolysis. No clinical signs of toxicity were observed in these studies. Direct addition of DBCP to epididymal sperm being incubated in vitro also inhibited the metabolism of glucose to CO2. Inhibition was concentration related, and the minimal inhibitory concentration was 0.316 mM. These data indicate that DBCP may cause a nearly immediate infertility via a direct effect on post-testicular sperm. A possible mechanism of this infertility is inhibition by DBCP of glucose metabolism in the ejaculated sperm.     

Glutathione conjugation of nitro compounds by monkey glutathione S-transferases

The distribution in Japanese monkey tissues of glutathione S-transferase activity toward some aromatic nitro compounds was examined by measuring the release of the nitro group as nitrite ion. The activity was especially high in liver, kidney and small intestine when compounds such as 4-nitroquinoline N-oxide, 5-nitrofurfural diacetal and o-dinitrobenzene were used as substrates. The nitrite-releasing activity of the major enzyme purified from rhesus monkey liver was also tested on fifty-two nitro compounds including nineteen nitrofuran derivatives. Among the thirty-three nitro compounds other than the nitrofuran derivatives tested as substrates, the purified enzyme showed activity only toward o-dinitrobenzene, 4-nitroquinoline N-oxide, 3,4-dinitrobenzoic acid, p-dinitrobenzene, 2,5-dinitrobenzoic acid, 2,5-dinitrophenol, tetra-chloronitrobenzene and 2,4-dinitrobenzoic acid. The crude supernatant fraction of rhesus monkey liver showed activity in substrate specificity roughly similar to that of the purified enzyme. On the other hand, among at least ten carcinogenic 2-substituted 5-nitrofran derivatives tested, 4,6-diamino-2-(5-nitro-2-furyl)-s-triazine, 5-nitro-2-furaldehyde semicarbazone, N-[[3-(5-nitro-2-furyl)-1,2,4-oxadiazol-5-yl]methyl] acetamide, and N-[5-(5-nitro-2-furyl)-1-3,4-thiadiazol-2-yl)acetamide were shown to be enzymatically conjugated with reduced glutathione. Among the other nine 2-substituted 5-nitrofuran derivatives tested, six compounds could be the substrates of the enzyme, and 5-nitrofurfural and 5-nitrofurfural diacetal were especially good substrates. There was, however, little apparent correlation between their carcinogenicity and susceptibility to glutathione S-transferase. The bulky substituents at position 2 appeared to decrease the susceptibility of these nitrofuran derivatives to the enzyme. Both Vmax and Km values of the purified enzyme varied greatly among the substrates, and the optimum pH fell between 7.5 and 9.0 in most cases.     

Glutathione conjugation of 4-hydroxy-trans-2,3-nonenal in the rat in vivo, the isolated perfused liver and erythrocytes.

The formation of glutathione (GSH) conjugates of racemic 4-hydroxy-trans-2,3-nonenal (4-HNE) in the rat in vivo in the perfused rat liver and rat erythrocytes has been studied. An HPLC system was developed for the assay of 4-HNE-glutathione conjugates (HNE-SG). The very sensitive electrochemical detection method (detection limit 5 pmol) can also be used to study endogenously formed HNE-SG. Three diastereomeric HNE-SG conjugates could be separated by this system. Rat liver cytosol catalyzed the formation of 2 of the 3 conjugates. When 17 micromol/kg [(3)H] 4-HNE was injected intravenously in the rat, 21% of the radioactivity was excreted within 90 min in bile and 37% in urine. Most of the 4-HNE in bile was present as 2 of the HNE-SG conjugates (molecular mass 463). In addition, 25% was excreted as a third GSH conjugate (molecular mass of 461), which was identified as the lactone of the 4-hydroxynonenoic acid glutathione conjugate. Erythrocytes in vitro eliminated 4-HNE very rapidly, in part by GSH conjugation, suggesting that they may also play an important role in vivo. To study the role of the liver selectively, we used the recirculating perfused rat liver without erythrocytes in the perfusion medium; the same conjugates were found, but the third conjugate was a minor component. These results present direct evidence for the in vivo formation of 4-HNE glutathione conjugates in which the liver may play an important role.     

Effects of 1,2-dibromo-3-chloropropane on hepatic heme synthesis

Previous studies showed that 1,2-dibromo-3-chloropropane (DBCP) caused a decrease in hepatic microsomal cytochrome P-450 [D.E. Moody, B. Head, and E.A. Smuckler (1979) J. Environ. Pathol. Toxicol. 3, 177-190; D.E. Moody, G.A. Clawson, C.H. Woo, and E.A. Smuckler (1982) Toxicol. Appl. Pharmacol. 66, 278-279], suggesting that hepatic heme metabolism may be affected by DBCP treatment. This study tested this hypothesis. Various parameters of hepatic heme synthesis were measured at intervals ranging from 0 to 72 hr in male Sprague-Dawley rats given a single oral dose (200 mg/kg) of DBCP. Incorporation of the radiolabeled heme precursor [delta-14C]aminolevulinic acid (14C-ALA) into liver, protein, extracted heme, and subcellular fractions of liver homogenates was significantly decreased to 75, 58, and 81% of controls, respectively, at 24 hr. At 48 and 72 hr after DBCP treatment, the accumulation of 14C-ALA label after 4 hr in liver homogenates and subcellular fractions was significantly increased in comparison to controls. These changes in 14C-ALA uptake were accompanied by decreases in total liver and microsomal heme, but not mitochondrial heme. Decreases were found in the spectral content of two heme proteins, cytochromes P-450 and b5, and the activity of another heme protein, catalase. Heme oxygenase activity increased to 130, 151, 209, and 186% of control values at 12, 24, 48, and 72 hr after DBCP, respectively. A slight, but significant, increase in ALA-synthetase to 112% of controls occurred at 24 hr, and slight, but significant, decreases in ALA-dehydratase to 90 and 80% of control occurred at 12 and 24 hr, respectively. No significant changes in uroporphyrinogen-1-synthetase or ferrochelatase at the time points tested was noted. The porphyrin content of liver was increased to 130% of control, while the serum and urine porphyrin levels were decreased to 30% of the control values at 24 hr. Liver ALA content was not significantly altered through the time period studied, but serum and urine levels were increased at 24 hr to 176 and 130% of the control values, respectively. In conclusion, the decreases in liver heme proteins following a single oral dose of DBCP are accompanied by alterations in heme turnover, particularly a prolonged increase in heme oxygenase activity.     

Toxicokinetics of 1,2-dibromo-3-chloropropane (DBCP) in the rat

The objective of this study was to determine the kinetics of absorption, distribution, and elimination of DBCP after intravenous (iv) administration in plasma, and after oral administration in water or corn oil, to conscious, fed, male Fischer 344 rats. Rats were prepared with an external jugular vein cannula and were dosed with 0.1, 1, or 10 mg/kg DBCP into the penile sinus or orally as a solution in water or in corn oil (1 mg/kg only). Blood was sampled at various times up to 12 hr, concentrations of DBCP were determined by gas chromatography, and data were evaluated by classical pharmacokinetic techniques. After oral administration in water, absorption of DBCP was rapid, and the distribution and elimination phase was biexponential. There did not appear to be any saturation of DBCP absorption, distribution, or elimination at the high oral or iv dose. After oral administration of DBCP in a corn oil vehicle, absorption was prolonged, suggesting retention of DBCP in the stomach; this could contribute to the toxic effects of DBCP on the forestomach when chronically administered in corn oil. The areas under the blood concentration/time curve were similar regardless of vehicle, suggesting that systemic toxicity might be independent of the vehicle.     

Glutathione conjugation of busulfan produces a hydroxyl radical-trapping dehydroalanine metabolite

1. The Phase 2 drug metabolism of busulfan yields a glutathione conjugate that undergoes a β-elimination reaction. The elimination product is an electrophilic metabolite that is a dehydroalanine-containing tripeptide, γ-glutamyldehydroalanylglycine (EdAG). In the process, glutathione lacks thiol-related redox properties and gains a radical scavenging dehydroalanine group.

2. EdAG scavenged hydroxyl radical generated in the Fenton reaction in a concentration-dependent manner was monitored by electron paramagnetic resonance (EPR) spectroscopy. The apparent rate of hydroxyl radical scavenging was in the same range as published values for known antioxidants, including N-acyl dehydroalanines.

3. A captodatively stabilized carbon-centered radical intermediate was spin trapped in the reaction of EdAG with hydroxyl radical. The proposed structure of a stable product in the Fenton reaction with EdAG was consistent with that of a γ-glutamylserylglycyl dimer.

4. Observation of the hydroxyl trapping properties of EdAG suggests that the busulfan metabolite EdAG may contribute to or mitigate redox-related cytotoxicity associated with the therapeutic use of busulfan, and reaffirms indicators that support a role in free radical biology for dehydroalanine-containing peptides and proteins.

     

Glutathione conjugation and induction of a 32,000 dalton stress protein

Chinese hamster ovary cells were exposed to various concentrations of diethylmaleate in order to produce various levels of intracellular glutathione (GSH) depletion. Exposure to a 20 microM concentration or more of diethylmaleate depleted the intracellular glutathione concentration by 80% or more and resulted in enhanced synthesis of two 32 kDa proteins which exhibited a pI of about 6.5. Exposure of cells to 50 microM buthionine sulfoximine for 24 hr reduced GSH levels by 95% but did not enhance the synthesis of this protein. Addition of diethylmaleate to buthionine sulfoximine-treated cells resulted in enhanced synthesis of the 32 kDa protein however. Exposure to 0.4 mM diamide triggered the synthesis of several heat shock proteins but did not induce the synthesis of the 32 kDa protein. These results indicated that enhanced synthesis of the 32 kDa protein occurred only after glutathione depletion exceeded 80% and required formation of a glutathione conjugate.     

Toxicologic and reproductive effects of inhaled 1,2-dibromo-3-chloropropane in male rabbits

Groups of 10 male New Zealand white rabbits were exposed by inhalation to 0, 0.1, 1.0 or 10 ppm of 1,2-dibromo-3-chloropropane (DBCP) vapor for 6 hours/day, 5 days/week for 14 weeks, except that the 10 ppm group was exposed for only 8 weeks due to mortality. The semen of rabbits was evaluated on a weekly basis during the exposure period and at periodic intervals during a recovery period (32 weeks for all groups except the 10 ppm groups which was for 38 weeks). In order to assess the fertility of the exposed rabbits, each male was allowed to mate with an unexposed female at the 14th and 41st week of the study. Exposure of rabbits to 1 and 10 ppm of DBCP by inhalation produced adverse reproductive effects as well as decreases in sperm count, motility and viability. Rabbits treated at 1 and 10 ppm had decreased sperm counts between the 8th and 14th weeks of the study. All of the 10 ppm rabbits were infertile when mated during the 14th week. The effects of DBCP on spermatogenesis were shown to be essentially reversible in rabbits exposed to 1 ppm; however, at 10 ppm, recovery was not complete under the conditions of the test. Rabbits exposed to 10 ppm had severe testicular alterations as early as 4 weeks into the study and these progressed to severe testicular atrophy by 8 weeks. Those exposed to 1 ppm for 14 weeks developed moderate testicular atrophy (approximately 50% reduction in size). Following the recovery period, the rabbits in the 10 ppm group had evidence of partial reversibility of the testicular atrophy. Electron microscopic evaluation of testicular tissue confirmed findings by light microscopy effects and also indicated increased numbers of abnormal sperm within the seminiferous tubules of rabbits at both the 10 and 1 ppm exposure levels. Those exposed to 0.1 ppm had an equivocal increase in abnormal sperm after the 14-week exposure period but not after the recovery period. Based on these results 0.1 ppm level of DBCP is considered as a no effect level for reproductive parameters.     

Glutathione conjugation of aflatoxin B1 exo- and endo-epoxides by rat and human glutathione S-transferases.

Much evidence supports the view that the rate of conjugation of glutathione (GSH) with aflatoxin B1 (AFB1) exo-epoxide is an important factor in determining the species variation in risk to aflatoxins and that induction of GSH S-transferases can yield a significant protective effect. An assay has been developed in which the enzymatic formation of the conjugates of GSH and AFB1 exo-epoxide and the recently described AFB1 endo-epoxide is measured directly. 1H NMR spectra are reported for both the AFB1 exo- and endo-epoxide-GSH conjugates. Structural assignments were made by comparison with AFB1 exo- and endo-epoxide-ethanethiol conjugates, for which nuclear Overhauser effects were measured to establish relative configurations. The endo-epoxide was found to be a good substrate for GSH conjugate formation in rat liver cytosol while mouse liver cytosol conjugated the exo-epoxide almost exclusively. Human liver cytosol conjugated both epoxide isomers to much lower extents than did cytosols prepared from rats or mice. Purified rat GSH S-transferases catalyzed the formation of the AFB1 exo-epoxide-GSH conjugate in the order 1-1 approximately 4-4 approximately 3-3 greater than 2-2 greater than 4-6 (7-7 and 8-8 did not form the exo-epoxide-GSH conjugate at levels above the nonenzymatic rate). The only rat GSH S-transferases that conjugated the endo-epoxide were 4-4 and 4-6, with 4-4 being the more active.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of P-450 activity and glutathione levels in 1,2-dibromo-3-chloropropane tissue distribution, renal necrosis and in vivo DNA damage

Treatments known to alter P-450 activity and glutathione levels were used to elucidate the involvement of P-450 and glutathione S-transferase metabolism in 1,2-dibromo-3-chloropropane (DBCP) organ toxicity in the rat. Phenobarbital pretreatment abolished DBCP-induced renal necrosis, whereas it had only a small effect on initial renal DNA damage. The DBCP levels in plasma and tissues were markedly reduced by phenobarbital pretreatment. Perdeuterated DBCP had much higher plasma and tissue levels than protio-DBCP in phenobarbital-pretreated animals, but perdeuteration was without effect in uninduced animals. This indicates that P-450 metabolism of DBCP is of major importance only in phenobarbital-pretreated animals. In order to study the effects of decreased glutathione levels on renal distribution and toxicity, rats were pretreated with either diethyl maleate or buthionine sulfoximine. The DBCP levels in plasma and tissues showed transitory elevations after diethyl maleate and buthionine sulfoximine pretreatment compared to the control situation. Despite the fact that diethyl maleate and buthionine sulfoximine pretreatments are known to block DBCP-induced DNA damage in vitro, these pretreatments did not significantly alter DBCP-induced renal necrosis nor DNA damage. Thus, a role for glutathione conjugation in DBCP-induced in vivo renal toxicity could not be established in the present study.     

Glutathione conjugation of methazolamide and subsequent reactions in the ciliary body in vitro

Conjugation reaction of methazolamide with glutathione and its subsequent reactions were studied in vitro. Glutathione, cysteinylglycine, and cysteine conjugates of methazolamide were chemically synthesized. All of the three compounds showed absorbance below 330 nm, with maximal absorbance at approximately 300 nm. At the wavelengths below 220 nm, absorbance was proportional to the number of the amino acids each compound had. Amino acid analysis of the glutathione conjugate showed that the conjugation reaction involved the cysteine residue of glutathione. In order to identify the chemical structure of the reaction product, cysteine conjugate was subjected to infrared, proton nuclear magnetic resonance, and mass spectral analyses. These studies indicated that the cysteine conjugate was S-(5-acetylimino-4-methyl-delta 2-1,3,4-thiadiazolinyl)cysteine. The reaction with glutathione was not catalyzed by glutathione S-transferases, but proceeded in the absence of the enzyme. The glutathione conjugate was degraded by bovine ciliary body homogenate to the cysteinylglycine conjugate and then to the cysteine conjugate.     

The role of glutathione conjugation in the mutagenicity of 1,2-dibromoethane

Two mechanisms for the toxic actions of 1,2-dibromoethane have been postulated, both of which involve biotransformation. The first is oxidation to 2-bromoacetaldehyde, a highly reactive substance, the second a possible direct conjugation to glutathione, giving rise to a reactive half-mustard. It was the purpose of this investigation to determine to what extent these two reactive species are responsible for the mutagenicity of 1,2-dibromoethane. To assess quantitatively the importance of the conjugation to glutathione in vivo, rats were administered single doses of 1,2-dibromoethane; 30–55 per cent of the dose was excreted as mercapturic acid. The conjugation of 1,2-dibromoethane to glutathione was also studied in vitro. Specific activities of the metabolizing systems used in the mutagenicity experiments were determined. The mutagenicity of 1,2-dibromoethane towards Salmonella typhimurium TA100 was considerably enhanced by the addition of 100,000 g supernatant fraction, whereas the addition of microsomes had no effect, indicating that the primary glutathione adduct is responsible for the mutagenic effect. As a model for the mutagenic intermediate, S-2-bromoethyl-N-acetyl-cysteine methyl ester was synthesized. This proved to be a very reactive and highly mutagenic compound, which can be further metabolized and thereby detoxified by glutathione conjugation. A similar phenomenon is likely to occur in the mutagenicity test with 1,2-dibromoethane, where after an initial rise in the number of mutants with increasing amounts of glutathione, the number of mutations decreases again. These results clearly indicate that glutathione conjugation plays an important role in the mutagenicity of 1,2-dibromoethane.