Metabolism Of Bromobenzene Research Articles

Toxicogenomics of bromobenzene hepatotoxicity: a combined transcriptomics and proteomics approach

Toxicogenomics is a novel approach integrating the expression analysis of thousands of genes (transcriptomics) or proteins (proteomics) with classical methods in toxicology. Effects at the molecular level are related to pathophysiological changes of the organisms, enabling detailed comparison of mechanisms and early detection and prediction of toxicity. This report addresses the value of the combined use of transcriptomics and proteomics technologies in toxicology. Acute hepatotoxicity was induced in rats by bromobenzene administration resulting in depleted glutathione levels and reduced average body weights, 24 hr after dosage. These physiological symptoms coincided with many changes of hepatic mRNA and protein content. Gene induction confirmed involvement of glutathione- S-transferase isozymes and epoxide hydrolase in bromobenzene metabolism and identified many genes possibly relevant in bromobenzene toxicity. Observed glutathione depletion coincided with induction of the key enzyme in glutathione biosynthesis, γ-glutamylcysteine synthetase. Oxidative stress was apparent from strong upregulation of heme oxygenase, peroxiredoxin 1 and other genes. Bromobenzene-induced protein degradation was suggested from two-dimensional gel electrophoresis, upregulated mRNA levels for proteasome subunits and lysosomal cathepsin L, whereas also genes were upregulated with a role in protein synthesis. Both protein and gene expression profiles from treated rats were clearly distinct from controls as shown by principal component analysis, and several proteins found to significantly change upon bromobenzene treatment were identified by mass spectrometry. A modest overlap in results from proteomics and transcriptomics was found. This work indicates that transcriptomics and proteomics technologies are complementary to each other and provide new possibilities in molecular toxicology.

P3B67 - Nephrotoxicity of bromobenzene in rats: The effect of pretreatment by 3-methyl-cholanthrene on bromobenzene metabolism

P3B67 - Nephrotoxicity of bromobenzene in rats: The effect of pretreatment by 3-methyl-cholanthrene on bromobenzene metabolism

Effects of vitamins E, C and catalase on bromobenzene- and hydrogen peroxide-induced intracellular oxidation and DNA single-strand breakage in Hep G 2 cells

Background/Aims: Water-soluble vitamin E (Trolox C), ascorbic acid and catalase were shown in our previous study to protect isolated rat hepatocytes against bromobenzene-induced toxicity. Methods: In order to study the mechanisms of this protection and the pathogenesis of bromobenzene-induced hepatocellular injury, a fluorometric assay for the investigation of intracellular oxidation, indicated by conversion of dichlorofluorescein diacetate to dichlorofluorescein, was used. Single-strand DNA breakage was also evaluated in Hep G 2 cells by a radio-labelling method. Results: Bromobenzene (2.4 and 4.8 mM) induced a significant increase in dichlorofluorescein fluorescence intensity compared to the controls. Trolox C, ascorbic acid or catalase significantly inhibited bromobenzene-induced enhancement of fluorescence intensity ( p<0.05-0.001), as well as reduced auto-intracellular oxidation in untreated Hep G 2 cells. Hydrogen peroxide (H 2O 2) evoked a dose-dependent increase in dichlorofluorescein fluorescence intensity in Hep G 2 cells, and the effect was completely blocked by Trolox C (2.0 mM) and catalase (4800 unit/ml). Bromobenzene caused significant single-strand DNA breakage in Hep G 2 cells during 2 h suspension incubation and 24 h primary incubation. H 2O 2 (400 μM) led to marked single-strand DNA breakage in 20 min, and the effect was attenuated by Trolox C. Conclusions: Metabolism of bromobenzene in Hep G2 cells induces production of H 2O 2, indicated by enhancement of dichlorofluorescein fluorescence intensity, or other free radicals, which leads to single-strand DNA breakage in the cells. Vitamins E and C and catalase display strong intracellular antioxidative effects. Vitamin E could partially inhibit H 2O 2-induced single-strand DNA breakage in the cells.

Protective effects of trolox C, vitamin C, and catalase on bromobenzene-induced damage to rat hepatocytes.

The protective effects of trolox C (water-soluble vitamin E), vitamin C, and catalase on bromobenzene (BB)-induced toxicity to isolated rat hepatocytes were evaluated. The glutathione (GSH) content of the hepatocytes exposed to BB was measured. BB caused acute damage to the cells during 2 h of incubation (short) when BB was added directly to the culture wells, whereas a late-occurring and time-dependent increase in lactate dehydrogenase (LDH) leakage rate was observed during 24 h of incubation (long) when BB was dissolved in a different way. Incubation of the cells with trolox C (0.5-2.0 mM) prevented the hepatocellular damage induced by BB at 2.4 mM during the long-term incubation. Vitamin C (0.1-1.0 mM) had a protective effect on BB-induced toxicity during both the short- (BB, 1.6 mM) and the long- (BB, 2.4 mM) term incubations. Catalase (3200 U/ml) also showed a beneficial effect on the cells during the short-term BB exposure. Trolox C (2.0 mM) and vitamin C (0.5 mM) restored BB-induced GSH depletion in the cells. BB induced two patterns of LDH leakage from isolated hepatocytes on the basis of different ways of BB exposure and incubation periods. Trolox C, vitamin C, and catalase exerted protective effects on BB-induced toxicity during short- or/and long-term incubations. The effects were concentration-dependent. Restoration of GSH content in BB-exposed hepatocytes suggests that trolox C and vitamin C could reduce GSH consumption during BB metabolism and exert an antioxidant effect.

Potential tolerance against bromobenzene-induced acute hepatotoxicity due to prior subchronic exposure

Groups of adult male Sprague-Dawley rats were treated i.p. with 0, 0.3 and 0.5 mmole bromobenzene (BB)/kg per day in corn oil, 5 days a week for 4 weeks. Thereafter, one half of each of these groups was treated i.p. with a single acute toxic dose of 2.5 mmole BB/kg. Urines were then collected for 24 h and the animals were then sacrificed. The hepatotoxicity induced by an acute dose of BB was significantly reduced due to prior subchronic exposure to BB at 0.5 mmol/kg, but not so at 0.3 mmol/kg. These data indicate a potential tolerance against acute hepatotoxicity of BB due to prior subchronic exposure. A significant increase in the urinary excretion of thioethers or mercapturic acids of BB combined with a significant increase in the urinary level of p-bromocatechol due to prior subchronic treatment with 0.5 mmol BB/kg relative to those due to acute treatment alone was observed. Thus enhanced bromobenzene metabolism could partly explain such potential tolerance against its acute hepatotoxicity. Such protection may also be related to certain cellular events which might occur subsequent to metabolic activation of BB.

Enzymatic and chemical syntheses of cis-dihydrodiol derivatives of monocyclic arenes

Metabolism of bromobenzene and iodobenzene by growing cultures of Pseudomonas putida UV4 gave the corresponding cis-dihydrodiol products 1 and 2 in high yields; subsequent direct chemical substitution of the halogen atoms in these metabolites provided a new range of enantiomerically pure cis-dihydrodiols of known absolute configuration.

Antagonism of bromobenzene-induced hepatotoxicity by phentolamine: Evidence for a metabolism-independent intervention

A previous study has revealed that phentolamine markedly antagonizes the bromobenzene-induced hepatotoxicity and lethality in B6C3F1 mice. One potential mechanism by which phentolamine may diminish the bromobenzene-induced hepatotoxicity is by a direct or indirect interference with the metabolism of bromobenzene to toxic metabolites. In the present study, phentolamine cotreatment failed to alter the elimination of bromobenzene from serum or the distribution of bromobenzene to liver. This suggests that phentolamine cotreatment does not indirectly interfere with bromobenzene bioactivation secondary to changes in bromobenzene absorption, distribution, or elimination. Further, a phentolamine concentration 10- to 20-fold greater than those measured in vivo failed to alter the in vitro metabolism of bromobenzene to its ortho- and para-phenolic metabolites. It is believed that para-bromophenol represents the rearrangement product of the hepatotoxic 3,4-epoxide and that ortho-bromophenol is a product of the nonhepatotoxic 2,3-epoxide pathway. Thus, it appears that phentolamine does not antagonize bromobenzene-induced hepatotoxicity by inhibiting the formation of hepatotoxic intermediates, nor by enhancing metabolism via the nonhepatotoxic pathway. On the basis of these studies, we conclude that phentolamine antagonism of bromobenzene-induced hepatotoxicity occurs through a mechanism independent of bromobenzene bioactivation.

Effect of the antioxidant N,N1-diphenyl-p-phenylenediamine (DPPD) on bromobenzene metabolism and toxicity in isolated hepatocytes.

Bromobenzene induced cytotoxicity in isolated hepatocytes was partly prevented by the antioxidant DPPD (N,N'-diphenyl-p-phenylenediamine). This protection did not appear to be a result of an inhibition of lipid peroxidation but rather to an inhibition of the metabolic activation of bromobenzene. The extent of DPPD dependent inhibition of cytochrome P-450 catalyzed reactions appears to depend on the substrate being metabolized. Thus, whereas cytochrome P-450 catalyzed metabolism of bromobenzene and harmine was inhibited by DPPD, the N-dealkylation of aminopyrine was not. The results presented in this paper indicate the importance of evaluating the effect of antioxidants such as DPPD as inhibitors of bioactivation prior to drawing definitive conclusions regarding involvement of lipid peroxidation in drug induced cytotoxicity.

The contribution of bromobenzene to our current understanding of chemically-induced toxicities

The metabolism and toxicity of bromobenzene has been investigated for well over one hundred years. The urinary excretion of mercapturic acids was first reported in 1879, in animals treated with bromobenzene. Bromobenzene has since proven to be a valuable tool in efforts to unravel the complexities involved in chemical- induced toxicities. For example, the importance of metabolic activation via the cytochrome(s) P-450; the role of glutathione in the detoxification of reactive metabolites; and the toxicological significance of covalent binding, enzyme inactivation and lipid peroxidation have all been illustrated in studies with bromobenzene. Thus, many of the principles involved in chemical-induced toxicity have been exemplified in studies with bromobenzene. These studies have provided substantial insight into the role of chemically reactive metabolites in the genesis of xenobiotic-mediated cytotoxicity.

Effects of chemical and enzymic probes on microsomal covalent binding of bromobenzene and derivatives. Evidence for quinones as reactive metabolites.

1. The chemical reactivity of bromobenzene metabolite(s) responsible for its protein covalent binding was investigated by determining the effects of many chemical and enzymic probes on the metabolism and covalent binding of [3,5-3H]bromobenzene with rat liver microsomes in vitro. 2. Classical cytochrome P-450 enzyme inhibitors decreased both metabolism and binding in parallel, whereas scavenging agents for reactive oxygen species and free radicals exhibited little or no effect. Sulphur nucleophiles were extremely efficient in decreasing binding with little or no effect on metabolism. Reducing agents such as ascorbate and diaphorase decreased binding slightly more than metabolism. 3. UDP-Glucuronic acid inhibited neither metabolism nor binding, but all three mono-bromophenols decreased binding more than metabolism. Trichloropropene oxide was unique in decreasing metabolism more than binding. 4. The effects of ascorbate, glutathione, bisulphite and butylated hydroxytoluene (BHT) on metabolism and binding of five ortho-substituted bromobenzene derivatives (o-BrC6H4X; X = OCH3, CH3, Br, CF3, and CN) were similar to their effects on the metabolism and binding of bromobenzene. 5. Collectively these results support a major role for quinones as the reactive metabolites responsible for the majority of the protein covalent binding of bromobenzene and its ortho-substituted derivatives in microsomal systems in vitro.

Metabolism of bromobenzene. Analytical chemical and structural problems associated with studies of the metabolism of a model aromatic compound.

Our studies of bromobenzene metabolism have shown that the 3,4-oxide is metabolized to 3- and 4-bromophenol through an extended glutathione pathway. The mechanism of sulfur elimination from a dihydrobromobenzene metabolite is not known, although it is known that the aromatization reaction will occur in a 9000-g supernatant fraction of rat liver. The hepatotoxic and nephrotoxic metabolites of bromobenzene are most likely bromthiocatechols and a bromothiopyrogallol, respectively.

Effect of a cysteine prodrug (L‐2‐oxothiazolidine‐4‐carboxylic acid) on the metabolism and toxicity of bromobenzene: A repeated exposure study

The relationship between dose, toxicity, and metabolism of bromobenzene and the use of urinary metabolite excretion as an index of internal exposure to the reactive electrophilic intermediate bromobenzene 3,4-epoxide after repeated treatments with bromobenzene in presence or absence of L-2-oxothiazolidine-4-carboxylic acid (OTCA) were evaluated in mice. Repeated treatments with bromobenzene doses of 0.5, 0.75, and 1.0 mmol/kg ip twice a day for 18 d produced a marked reduction in 24-h urinary excretion of bromophenylmercapturic acid and p-bromophenol; this was accompanied by increases in plasma transaminases. Treatment with OTCA (1.0-3.0 mmol/kg) prevented toxicity and enhanced the 24-h urinary excretion of various bromobenzene metabolites by approximately 30-75, 104-145, and 164-269% for bromobenzene doses of 0.5, 0.75, and 1.0 mmol/kg, respectively. The effect of OTCA was further characterized by investigating the metabolism of bromobenzene given as a challenge dose of 4.0 mmol/kg to mice pretreated with bromobenzene and OTCA for 18 d. Pretreatment with bromobenzene reduced the 0- to 6-h urinary excretion of all metabolites after the challenge dose; this effect was virtually reversed by OTCA. It is concluded that repeated bromobenzene administration reduces its own detoxification to mercapturic acid and phenolic metabolites and elicits toxicity. This phenomenon is reversed after OTCA administration. This study further provides evidence that internal exposure to the reactive electrophilic intermediate bromobenzene 3,4-epoxide could be assessed more accurately by summing the urinary excretion of bromophenylmercapturic acid and p-bromophenol after OTCA treatment.

Effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid, on the metabolism and toxicity of bromobenzene: an acute study.

The effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid (OTCA), on certain aspects of the metabolism and toxicity of bromobenzene administered acutely to mice was investigated by (i) characterizing the influence of OTCA on the metabolic profile of low and high bromobenzene dose at 0-6, 6-12, and 12-24 h, (ii) determining the effective doses range and administration time for OTCA, as well as the optimum period for urine sampling; and (iii) measuring the efficacy of OTCA for protection against bromobenzene induced toxicity. Coadministration of OTCA and bromobenzene enhanced the urinary excretion of mercapturic acid and phenolic metabolites, during 6-12 h, by approximately 152 and 193%, respectively. Maximum efficacy was observed when OTCA (16.0 mmol/kg) was administered concomitantly with bromobenzene (4.0 mmol/kg). Finally, OTCA administration was found to afford substantial protection against elevation of plasma transaminases used as indices of bromobenzene-induced hepatotoxicity. N-acetylcysteine, another cysteine prodrug, had essentially similar effects on the metabolism and toxicity of bromobenzene. Thus, administration of cysteine prodrugs enhances the urinary excretion of several metabolites of bromobenzene and affords protection against bromobenzene-induced hepatotoxicity.

Metabolism of bromobenzene : Analytical chemical and structural problems associated with studies of the metabolism of a model aromatic compound

Our studies of bromobenzene metabolism have shown that the 3,4-oxide is metabolized to 3- and 4-bromophenol through an extended glutathione pathway. The mechanism of sulfur elimination from a dihydrobromobenzene metabolite is not known, although it is known that the aromatization reaction will occur in a 9000- g supernatant fraction of rat liver. The hepatotoxic and nephrotoxic metabolites of bromobenzene are most likely bromothiocatechols and a bromothiopyrogallol, respectively.

Effect of sodium selenite upon bromobenzene toxicity in rats: II. Metabolism

The effects of sodium selenite (12.5 or 30 μmol/kg, ip) upon bromobenzene metabolism were examined in male rats treated with selenite at 72 hr prior to bromobenzene exposure (7.5 mmol/kg, ip). The inhibitory nature of selenium treatment upon xenobiotic metabolism and increased production of hepatic thiol suggested that selenite might affect metabolic activation and detoxification of bromobenzene. Selenite treatment lowered in vivo covalent binding of [ 14C]bromobenzene while the in vitro covalent binding of [ 14C]bromobenzene in microsomes isolated from selenite-treated rats was unaffected compared to control. When the rate of [ 14C]bromobenzene decline in whole blood was evaluated in selenite-treated rats over 48 hr after bromobenzene administration, no significant differences were observed when compared to control. Furthermore, values of glutathione conjugates and hydroxylated metabolites of bromobenzene were similar in urine samples collected over 48 hr from selenite and control rats. Mechanistically, reduction of bromobenzene hepatotoxicity by selenite is not mediated through an altered metabolism of bromobenzene, but by alteration of cellular events occurring after metabolic activation.

Influence of mercuric chloride on the metabolism and hepatotoxicity of bromobenzene in rats

When male Sprague-Dawley rats were treated with 1 mg mercuric chloride (HgCl2)/kg, sc 6 hr prior to or simultaneously with a single 2.5-mmole/kg ip dose of bromobenzene and sacrificed 48 hr after the bromobenzene dose, the activities of serum transaminases (SGOT and SGPT) were found to be significantly reduced when compared with those obtained in bromobenzene-alone-treated rats. Similar phenomena were observed when rats were treated simultaneously with 1 or 2 mg HgCl2/kg and 1 mmole bromobenzene/kg, but not when bromobenzene was given 6 hr prior to HgCl2 injection. When 5 mmole bromobenzene/kg and 1 mg HgCl2/kg were given simultaneously to the animals, such an apparent reduction in bromobenzene toxicity was again observed. In each case, HgCl2 alone had no effect on the transaminase activities. HgCl2 (1 mg/kg, sc) treatment reduced the hepatic microsomal cytochrome P-450 content. Treatment with 1 mg HgCl2/kg 6 hr prior to bromobenzene injection (2.5 mmole/kg) significantly reduced the urinary excretion of para- and metabromophenols, and parabromocatechol during 0 to 24-hr period without affecting the urinary thioethers. These data suggest a possible reduction in the rate of formation of bromobenzene epoxide intermediate due to mercury pretreatment, resulting in a lowering of the steady state level of this epoxide so that an inhibition of hepatotoxicity due to bromobenzene could occur. However, simultaneous treatments of HgCl2 and bromobenzene failed to modify the urinary metabolic excretion pattern of bromobenzene. When rats were given 10, 50, and 100 ppm of HgCl2 in drinking water daily for 4 weeks prior to an ip injection of 2.5 mmole bromobenzene/kg and were sacrificed 48 hr after the dose, no changes in SGOT and SGPT activities were observed. These results indicate that changes in the metabolism and hepatotoxicity of bromobenzene due to mercury depend on (a) the dose and time of mercury administration, and (b) the mode of administration of mercury, acute or chronic.

Influence of selenium on the metabolism of bromobenzene and a possible relationship to its hepatotoxicity

When male Sprague-Dawley rats were treated with sodium selenite (1 mg/kg, sc) 24 hr prior to or simultaneously with bromobenzene (2.5 mmol/kg, ip) and sacrificed 48 hr after the bromobenzene dose, increased levels of the activities of serum transaminases (serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT)) induced in the bromobenzene- treated rats were significantly reduced in the presence of selenium. However, no such reduction in the transaminases activities were observed when rats were either pretreated with selenite for 48 hr or pretreated with 0.1, 0.2, or 0.5 mg/kg of selenite. Although selenium alone had no effect on the hepatic microsomal drug metabolism, simultaneous treatment of selenite (1 mg/kg) with bromobenzene resulted only an increase in the activity of aniline hydroxylase after 48 hr as compared to that in the bromobenzene-treated group. When rats were given 2.5, 10, and 20 ppm of selenite in drinking water daily for 4 weeks prior to an ip injection of 2.5 mmol/kg of bromobenzene and were sacrificed 48 hr after bromobenzene administration, a reduction in the SGOT activities in all the pretreated groups and a reduction of SGPT activity in 20 ppm selenite-treated group were observed when compared with those in the bromobenzene-treated groups. A dose-dependent increase in hepatic GSH concentrations were observed due to such chronic selenium treatment. Treatment with selenite (1 mg/kg) 24 hr prior to bromobenzene injection (2.5 mmol/kg) increased initially both o and p-bromophenols in the rat urine at 0–7.5 hr without affecting urinary thioethers. On the contrary, the ratio of thioethers to p-bromophenol was significantly higher in both 2.5 and 10 ppm selenite-pretreated (4 weeks) rats as well as a significant increase in the ratio of thioethers to total phenolic metabolites in 10 ppm and an increase close to significant in 2.5 ppm selenite-treated rats were observed initially at 0–7.5 hr urine samples. These results indicate that acute selenium pretreatment under certain conditions, favors increased hydroxylation of the intermediate bromobenzene epoxides, whereas higher detoxification of the epoxides involving hepatic glutathione (GSH)GSH transferases pathway is more favored due to increased biosynthesis of GSH in certain chronic selenium treated rats.

Influence of zinc chloride on the metabolism and hepatotoxicity of bromobenzene in rats

In studying the possible interactive effects of various heavy metals on bromobenzene hepatotoxicity and metabolism, zinc chloride (ZnCl2) (0.5, 2.0, and 10.0 mg/kg) was given ip 24 hr prior to ip administration of bromobenzene (2.5 mmol/kg body wt). Animals were sacrificed 48 hr after bromobenzene. A significant increase in the activities of serum transaminases (serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT)) was observed at 0.5 mg/kg ZnCl2 and such an effect was not observed at the two higher doses of ZnCl2. However, no such increase in the transaminases activities was observed when rats were treated with identical doses of ZnCl2 48 hr before the administration of bromobenzene. When rats were treated with 2 mg/kg ZnCl2 6 hr prior to the bromobenzene dose, a potentiation of the hepatotoxicity due to bromobenzene was again observed, whereas simultaneous treatment of bromobenzene and ZnCl2 produced no such effect. Treatment with ZnCl2 (0.5 mg/kg) 24 hr prior to bromobenzene injection failed to modify the pattern of the urinary metabolites of bromobenzene. When rats were given 50, 250, or 500 ppm of ZnCl2 in drinking water daily for 4 weeks prior to an ip injection of 2.5 mmol/kg bromobenezene, a reduction in the activities of serum transaminases was observed in 250 ppm ZnCl2-treated rats only. Such a reduction in the hepatotoxicity of bromobenzene is accompanied by a simultaneous reduction of the in vivo metabolism of bromobenzene by zinc, as substantiated by reduction of its urinary thioethers as well as of its total urinary metabolites. The present study has shown that changes in the metabolism and hepatotoxicity of bromobenzene depend on the dose of zinc administered, as well as on the temporal relationship between the zinc and bromobenzene administrations. The definitive mechanism responsible for such interactions remains to be elucidated.

The role of 4-bromophenol and 4-bromocatechol in bromobenzene covalent binding and toxicity in isolated rat hepatocytes

4-Bromophenol and 4-bromocatechol are formed as metabolites of bromobenzene in vivo and in isolated rat hepatocytes. Both of these metabolites may potentially contribute to the hepatotoxicity of bromobenzene. Bromobenzene metabolism in hepatocytes isolated from phenobarbital-treated rats forms 0.12 to 0.17 m m 4-bromophenol and 4-bromocatechol in 2 hr, with 1 to 3 m m bromobenzene. The role of activated metabolites derived from 4-bromophenol and 4-bromocatechol in bromobenzene covalent binding and toxicity was investigated with isolated hepatocytes in suspension. The covalent binding of the phenol and the catechol was increased four- to eightfold by the addition of unlabeled bromobenzene. Two-hour incubations of 0.25 m m 14C-labeled 4-bromophenol or 4-bromocatechol with hepatocytes isolated from phenobarbital-treated rats resulted, under these conditions, in no significant toxicity, and approximately 4 and 25%, respectively, of the covalent binding associated with bromobenzene itself. Two- and six-hour incubations with higher 4-bromophenol and 4-bromocatechol concentrations demonstrated that 1 to 3 m m substrate concentrations were required for cytotoxicity. These results show that metabolically produced 4-bromophenol and 4-bromocatechol do not play significant roles in the production of bromobenzene cytotoxicity in isolated hepatocytes, and that they contribute only modestly to bromobenzene covalent binding.

Metabolism of bromobenzene to glutathione adducts in lung slices from mice treated with pneumotoxicants

Recent studies showing that the bronchiolar Clara cell and alveolar Type II cell are major loci of cytochrome P-450 monooxygenases in the lung suggested that measurement of xenobiotic metabolizing enzyme activity might provide a useful and sensitive index of injury to these cell types. Accordingly, an assay has been developed for quantitating the rate of formation of reactive bromobenzene metabolites in lung slices which is based upon measuring the rate of formation of bromobenzene glutathione adducts. To demonstrate that monitoring adduct formation would yield quantitatively similar data to the traditional covalent binding assay for measuring the formation of reactive bromobenzene intermediates, covalent binding and conjugate formation were assayed in incubations of phenobarbital-induced hepatic microsomes conducted in the presence of various cytochrome P-450 monooxygenase inhibitors. Incubation conditions which decreased the rate of covalent binding (incubations done in the absence of glutathione) resulted in similar decreases in conjugate formation (incubations done in the presence of glutathione). In lung slices, the metabolism of bromobenzene to glutathione conjugates was linear for 20 min and continued to increase with time over the entire 160 min of the study. The formation of bromobenzene glutathione adducts in lung slices from piperonyl butoxide-treated animals occurred at a significantly lower rate than control. Likewise, lung slices from animals treated with butylated hydroxytoluene or carbon tetrachloride, agents known to injure alveolar epithelial cells, metabolized bromobenzene to glutathione conjugates at significantly slower rates than control. In contrast, treatment with naphthalene or dichloroethylene, agents which damage the bronchiolar epithelial cells, had little or no effect on conjugate formation. Similarly, there were no significant differences in the rate of bromobenzene glutathione conjugate formation between lungs of air- and ozone-exposed (1.0 ppm × 4 hr) mice killed 2,24,48,72, or 120 hr after exposure. These studies suggest that monitoring the rate of bromobenzene glutathione conjugate formation in lung slices may be a useful and sensitive biochemical index of injury to certain cells of the lung but that severe damage to the nonciliated bronchiolar epithelial cells has little effect on the rate of metabolic activation of this aromatic hydrocarbon.