Introduction

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Daily administration of isoniazid (INH), a highly effective drug in the chemoprophylaxis and treatment of tuberculosis, is associated with mild elevations of liver enzyme activities in plasma in up to 20% of patients (Mitchell et al., 1975) and severe hepatotoxicity (predominantly hepatocellular damage) in approximately 1 to 2% of patients (Barlow et al., 1974). If this hepatotoxicity is not recognized early, it can be fatal. Over 25 years after the toxicity was detected in INH-treated patients, the mechanism remains unknown and the hepatotoxicity remains neither preventable nor treatable.

Our laboratory has developed a model of INH-induced hepatotoxicity in rabbits that closely resembles the toxicity in humans (Sarich et al., 1995). Histopathological changes in INH-induced hepatotoxicity in humans range in severity from focal and diffuse necrosis to multilobular, bridging, and massive necrosis (Maddrey and Boitnott, 1973). Histopathological evaluation of INH-induced hepatotoxicity in rabbits reveals focal and centrilobular inflammatory infiltration and necrosis (Sarich et al., 1995).

Hydrazine is a known hepatotoxin (Yard and McKennis, 1955; Patrick and Back, 1965). We have previously reported a positive correlation between plasma hydrazine levels and severity of INH-induced hepatocellular damage in rabbits (Sarich et al., 1996). This evidence suggesting that hydrazine plays a role in INH-induced hepatotoxicity is supported by previous reports that suggest a role for hydrazine in INH-induced hepatotoxicity in animals and humans (Noda et al., 1983; Peretti et al., 1987; Woo et al., 1992; Gent et al., 1992). The role of acetylhydrazine has also been studied and it has been proposed to be the INH-derived hepatotoxic metabolite in INH-induced hepatocellular damage (Mitchell et al., 1976).

During metabolism of INH, hydrazine can be produced both directly (from INH) and indirectly (from acetylhydrazine; Fig. 1). The direct pathway involves hydrolysis of the amide bond of INH to produce isonicotinic acid and hydrazine. The indirect pathway involves acetylation of INH to acetyl-INH by N-acetyltransferase, hydrolysis of acetyl-INH to isonicotinic acid and acetylhydrazine, and hydrolysis/deacetylation of acetylhydrazine to hydrazine. Both hydrolysis reactions involve an amidase enzyme.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Major pathways of INH metabolism. Direct and indirect pathways of hydrazine production from INH are shown.

A previous study in rats demonstrated that bis-p-nitrophenyl phosphate (BNPP), an amidohydrolase (amidase) inhibitor, prevented acetyl-INH-induced hepatocellular damage (Mitchell et al., 1976). The investigators suggested this to be due to prevention of hydrolysis of acetyl-INH to acetylhydrazine, the suspected INH-derived hepatotoxin at that time. However, Mitchell and colleagues (1976) did not propose the possibility that inhibition of acetyl-INH-induced hepatocellular damage by BNPP could be due to inhibition of production of hydrazine from acetylhydrazine. In 1984, Sendo et al. reported that BNPP acts as a potent amidase inhibitor in vitro, inhibiting the conversion of INH to hydrazine in hepatic microsomal incubations. Because there is evidence that hydrazine is the INH-derived hepatotoxin in our model of INH-induced hepatotoxicity in rabbits, the inhibition of INH-induced hepatocellular damage in our rabbit model by BNPP would provide evidence that the mechanism for the protection is likely due to inhibition of the formation of hydrazine.

Although it has been suggested that hepatic cytochrome P-450s (CYPs) are involved in the mechanism of INH-induced hepatotoxicity (Mitchell et al., 1976), the activity of P-450 enzymes with a potential role have not been studied in this rabbit model. P-450s with a potential role in this model of INH-induced hepatotoxicity include: 1) CYP1A2, known for its role in acetaminophen-induced hepatotoxicity (Raucy et al., 1989); 2) CYP2E1, known also for its role in acetaminophen-induced hepatotoxicity (Raucy et al., 1989), the inhibition and induction of CYP2E1 by INH in humans (Zand et al., 1993), the inhibition of CYP2E1 by INH in rabbits (Sarich et al., 1998), and the ability of CYP2E1 to convert alkylhydrazines into free radical intermediates (Albano et al., 1995); and 3) CYP2B4/5, because it has been observed that an increase in severity of INH-induced hepatocellular damage occurs after pretreatment of rabbits with phenobarbital, possibly due to increased activity of hepatic CYP2B4/5 (Sarich et al., 1995).

The objective of this study was to determine the involvement of INH-amidase activity in the pathogenesis of INH-induced hepatotoxicity in rabbits. The amidase inhibitor BNPP was coadministered with INH and markers of toxicity and plasma hydrazine concentrations in vivo were measured. In addition, the potency of the inhibition of INH-amidase by BNPP was investigated in vitro. The activity of several CYP isozymes were measured during an active phase of INH-induced hepatotoxicity to evaluate their potential role. Hepatic glutathione levels were also measured to determine whether it plays a role in this model of INH-induced hepatotoxicity in rabbits.

	Experimental Procedures

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Animals. Seventy male New Zealand White rabbits weighing 2 to 3 kg were used in this study. Throughout the experiment, the rabbits were housed in pairs in stainless steel cages with a 12-h light/dark cycle and free access to food and water. This project was reviewed and approved by the University of British Columbia Committee on Animal Care.

Materials. INH, BNPP, -NADPH (for the reductase and CYP2E1 assays) and p-nitrophenol (for the CYP2E1 assay) were obtained from Sigma Chemical Co. (St. Louis, MO). Reagents for the argininosuccinic acid lyase (ASAL) assay were obtained as follows: barium argininosuccinate (which was converted to the sodium salt by admixture with sodium sulfate and centrifugation) and 2,4-dichloro-1-naphthol from Sigma; the other reagents required for the ASAL assay were obtained from local chemical suppliers and were all of reagent grade.

INH Injection Protocol. The dosing schedule for INH was as described previously (Sarich et al., 1996, 1998).

Treatment Groups. Animals were randomized into one of five groups, each involving two treatments. The first treatment involved either a BNPP injection of 25 mg/kg (0.074 mmol/kg) or a BNPP-vehicle (VEH) injection (saline). BNPP was dissolved in saline (0.9% NaCl) at 7.65 mg/ml after heating to approximately 60°C (Heymann and Krisch, 1969) and, after cooling, injected i.p. at a volume of 3.3 ml/kg.

Blood Sampling. Blood samples (1 ml) were taken from the lateral ear vein using a heparinized syringe at 0, 12, 24, 32, and 48 h after the first dose of INH, as described previously (Sarich et al., 1996). The animals were sacrificed at 48 h by cervical dislocation and exsanguination.

Biochemical Analysis. Hepatocellular damage was quantitated by measuring peak plasma ASAL activity and peak plasma alanine aminotransferase (ALT) activity. ASAL activity in the plasma has previously been used as a sensitive marker of liver damage (Campanini et al., 1970; Sims and Rautanen, 1975). The quantitation of plasma ASAL activity (expressed as µmol/100 ml/h; Takahara units) was done according to Campanini et al. (1970) with modifications as described in Sarich et al. (1995). ALT activity (U/l), a commonly used marker of liver toxicity, was determined using a kit from Sigma Diagnostics (kit no. 59-20).

Tissue Handling. Immediately after sacrifice of the rabbits, the livers were removed, weighed, and homogenized (using a glass tube and Teflon pestle) with a homogenizing buffer (1.15% KCL, 10 mM EDTA, 10 mM phosphate, pH 7.4). The crude homogenate was centrifuged at 10,000g for 20 min. The supernatant was then centrifuged at 105,000g for 60 min and the resulting pellet was washed by resuspending in the homogenizing buffer and recentrifuging at 105,000g for 60 min. The final pellet was resuspended in a microsomal storage buffer (20% glycerol, 1.15% KCL, 10 mM EDTA, 10 mM phosphate, pH 7.4) and frozen at 60°C before analysis. Additional samples of liver were placed in vials and quick frozen in liquid nitrogen for estimation of tissue glutathione.

Tissue Glutathione. Trichloroacetic acid (TCA) soluble nonprotein thiols, a measure of tissue glutathione levels, was determined according to methods described previously (Moron et al., 1979). Pure reduced glutathione was used as a standard.

Plasma Hydrazine Determination. Hydrazine concentrations (µM) in 24- and 32-h plasma samples were determined based on previous methods (Sarich et al., 1996) with modifications as follows. Plasma was denatured by combining 100 µl of plasma with 100 µl of acetonitrile (100%). This solution was vortexed and allowed to stand for 6 min. One hundred microliters of 0.6 N perchloric acid and 100 µl of HPLC grade water were then added. This mixture was vortexed and centrifuged at 12,700g for 6 min. [Subsequent analyses prompt the authors to recommend addition of 100 µl of a saturated K₂CO₃ solution to precipitate out the perchlorate before the derivatization step to maximize derivatization of hydrazine]. The supernatant from the denaturation procedure was filtered with a Millipore syringe filter (Millex LCR₄, 0.5 µm, 4 mm, Millipore Corporation, Bedford MA). Three hundred microliters of the filtered supernatant was removed and combined with 75 µl of derivatizing reagent [the derivatizing reagent was made up by adding 125 µl 3-methoxybenzaldehyde and 5 ml 10 mM 9-fluorenone solution (internal standard; made up in propanol) to a 50 ml volumetric flask and making up to 50 ml with propanol]. The sample containing solution was placed in the dark for 2 h followed by centrifugation for 6 min and a second filtration with a Millipore syringe filter. The resulting solution containing derivatized hydrazine (3-methoxybenzaldazine; azine) was injected into an HPLC column (4.6 × 100 mm) (Select ODS-2 5 µm; Chromatographic Specialties, Brockville, Ontario, Canada). An ISCO (model 2350) liquid chromatograph and an ISCO V⁴ UV/visible detector set at 300 nm were used for the analysis. The solvent used consisted of 35% 5 mM sodium acetate adjusted to pH 5.0 with glacial acetic acid and 65% HPLC grade acetonitrile in HPLC grade water. The flow rate was 1 ml/min. All solvents were filtered before use (0.45 µm pore, 45 mm Nylon filters, Micron Separation, Inc., Westborough, MA) and degassed in the solvent reservoir.

Amidase Activity Determination. Hepatic amidase activity was determined by incubation of INH with microsomes (Whitehouse et al., 1983; Sendo et al., 1984) followed by measurement of hydrazine using HPLC as described above. Incubation of INH with microsomes was done as follows: One hundred microliters of INH in 67 mM KH₂PO₄ buffer (pH 7.0; 3 mM initial concentration) was incubated with 150 µl hepatic microsomes (3-10 mg protein/ml) and 50 µl 67 mM KH₂PO₄ buffer (pH 7.0) at 37°C for 30 min (300 µl total volume). The reaction was stopped by the addition of 0.3 ml acetonitrile, followed by mixing, standing for 6 min, addition of 0.3 ml 0.6 N perchloric acid and centrifugation at 12,700g for 6 min. The rest of the procedure was the same as for determination of plasma hydrazine levels. In control microsomes, the time course of the reaction was monitored over 120 min and the reaction was found to be linear up to 30 min. A standard curve was prepared (in triplicate) using microsomes spiked with hydrazine. The standard curve had a correlation coefficient (r) of 0.998, a slope of 0.152 (95% confidence intervals 0.140-0.165) mV × s/µM azine and a y-intercept of 1.19 (95% confidence intervals 2.56 to 1.86) mV × s. Hydrazine production was calculated as nmol hydrazine produced/h/mg protein.

Microsomal CYP Enzymes. Hepatic NADPH CYP reductase activity (nmol/min/mg protein) was determined using procedures outlined in Phillips and Langdon (1962), using an extinction coefficient of 19.6 cm¹ mM¹ and a final reaction volume of 1.575 ml. p-Nitrophenol hydroxylase activity (nmol/min/mg protein; used as a marker of CYP2E1 activity) was assayed using an initial substrate concentration (p-nitrophenol) of 100 µM, a peak absorbance of 510 nm and an extinction coefficient of 9.53 cm¹ mM¹ (Reinke and Moyer, 1985; Koop, 1986; Jenner and Timbrell, 1994).

INH, Acetylhydrazine, and Hydrazine Comparison. In a study comparing the toxicity of s.c. administered INH, acetylhydrazine, or hydrazine, these compounds were administered at molar equivalent doses using our standard dosing protocol (0.37 mmol/kg followed by 3 doses of 0.26 mmol/kg at 3-h intervals, for 2 days; Sarich et al., 1996). INH (n = 5), acetylhydrazine (n = 9), and hydrazine (n = 3) were each administered at this equimolar dose. Hydrazine was also administered (n = 8) using the same dosing protocol but at one-half the molar dose (0.19 mmol/kg followed by three doses of 0.13 mmol/kg at 3-h intervals).

Statistics. Plasma ASAL and ALT activities were logarithmically-transformed to give normally distributed data suitable for statistical analysis. For the purpose of clarity, plasma ASAL and ALT activities are presented in the untransformed form (mean ± S.E.; calculated from raw data) in text, tables, and figure legends, but as the log-transformed form in figures. Student's t tests assuming either equal or unequal variances (as determined by F-tests) were used when comparing two groups (Zar, 1984). ANOVA and the Newman-Keul's multiple comparison test were done for comparison of more than two groups (Zar, 1984). All data are presented as mean ± S.E. unless indicated otherwise.

	Results

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In this study, four of seventy animals died before 48 h, the planned time of sacrifice. Three were from the INH-only (VEH-INH) group and one was from the BNPP-INH group. Plasma samples from all four of these animals before death showed highly elevated plasma liver enzymes, indicating hepatocellular damage, and were included in the toxicity analyses. The liver of one of the VEH-INH animals that died between 12 and 24 h was recovered at the time of death and immediately frozen in liquid nitrogen and included in the microsomal preparation and analyses. The livers from the other three were not available for microsomal analyses.

Peak plasma ASAL and ALT activities, as well as hepatic and plasma triglycerides, were significantly increased above control levels only in the VEH-INH group (Table 2).

Plasma hydrazine concentrations at 24 h were not significantly different between the VEH-INH and BNPP-INH groups (Table 3). However, at 32 h, plasma hydrazine concentrations were significantly different (approximately 4 times higher) in the VEH-INH group as compared to the BNPP-INH group (Table 3).

Hepatic amidase activity, determined by measurement of the amount of hydrazine produced after incubation of INH with microsomes for 30 min, was significantly decreased in the VEH-INH group (by 38%) versus the VEH-VEH control group. However, the greatest decrease was found in the BNPP-VEH and the BNPP-INH groups (both decreased to approximately 10% of control levels; Table 4).

Plasma hydrazine levels at 32 h (r = 0.71, r² = 0.50, n = 27, p < .001) correlates with hepatic amidase activity. The correlation, although significant, is not as high between plasma hydrazine levels at 32 h and peak plasma ASAL activity (r = 0.46, r² = 0.21, n = 27, p < .02). Plasma amidase activity did not correlate with any measure of toxicity.

In vitro incubation of BNPP with INH in control microsomes (from VEH-VEH control rabbits) showed that BNPP is a potent inhibitor of hydrolysis of INH to hydrazine with an IC₅₀ of approximately 2.0 µM (Fig. 2). Microsomes were also incubated with various concentrations of INH (0.3, 1, 3, 6, 10, and 15 mM) in the presence of BNPP at concentrations of 0, 1.5, or 3 µM. A Lineweaver-Burk plot of the results (not shown) indicated that at 0 µM BNPP, V_max was 226 nmol/min/mg protein and K_m was 9.3 mM; at 1.5 µM BNPP, V_max was 167 nmol/min/mg protein and K_m was 8.3 mM; and at 3 µM BNPP, V_max was 99.4 nmol/min/mg protein and K_m was 12.1 mM. An Eadie-Hofstee plot of the results (not shown) indicated that at 0 µM BNPP, V_max was 196 nmol/min/mg protein and K_m was 7.8 mM; at 1.5 µM BNPP, V_max was 116 nmol/min/mg protein and K_m was 5.3 mM; and at 3 µM BNPP, V_max was 85.5 nmol/min/mg protein and K_m was 10.0 mM. In general, these data suggest that increasing concentrations of BNPP decreases the V_max of INH-amidase but does not significantly change its K_m.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Concentration-response curve for BNPP in microsomal incubation with INH. INH (3 mM) was incubated in the presence of BNPP (0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 µM) with control microsomes at 37°C for 30 min as described for hepatic INH-amidase activity determination. By measuring the production of hydrazine in the presence of increasing concentrations of BNPP, the IC50 of BNPP was determined (approximately 2 µM). The results shown were derived from three separate incubations of microsomes with INH and BNPP.

Hepatic glutathione content was significantly decreased in the VEH-INH group versus the other four groups (Table 2). There were no significant correlations between hepatic glutathione and log peak plasma ASAL activity, log peak plasma ALT activity, or hepatic triglyceride content in animals receiving INH (BNPP-INH and VEH-INH groups).

The activities of hepatic reductase, EROD, benzoyloxyresorufin-O-dealkylase, PROD, and p-nitrophenol hydroxylase are presented in Table 5.

A negative correlation of EROD activity in the VEH-INH group with hepatocellular damage (peak plasma ASAL, r = 0.66, r² = 0.44, n = 15, p < .01; peak plasma ALT activity, r = 0.70, r² = 0.49, n = 15, p < .005) was observed. In addition, p-nitrophenol hydroxylase activity correlated (negatively) with hepatic steatosis (liver triglycerides; r = 0.71, r² = 0.50, n = 22, p < .001). None of the other P-450 enzyme activities correlated with measures of toxicity.

In the toxicity comparison of s.c. administered INH, acetylhydrazine, or hydrazine, peak plasma ASAL activities in animals administered INH (292 ± 160 Takahara units; n = 5) or acetylhydrazine (142 ± 48 Takahara units; n = 9) resulted in statistically significant increases from baseline (p < .0001). However, in animals administered hydrazine at an equimolar dose, death resulted in the animals (n = 3) within 48 h. Plasma ASAL activity was only obtained in one animal, and the activity was extremely elevated (976 Takahara units). Reducing the dose of hydrazine by one-half did not result in the death of any animals and did cause a statistically significant increase in plasma ASAL activity versus baseline (101 ± 33 Takahara units; n = 8; p < .0001). There was no statistically significant difference in peak plasma ASAL activities between animals treated with INH and acetylhydrazine at full dose or hydrazine at half dose (p = 0.82; ANOVA).

	Discussion

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Treatment with BNPP before INH prevented INH-induced hepatocellular damage, hepatic steatosis, and hypertriglyceridemia. The VEH-INH group was the only group to experience significant INH-induced hepatotoxicity. This is an important and novel observation because it has not been previously demonstrated in an in vivo animal model that INH-induced hepatotoxicity could be prevented using an inhibitor of INH-amidase.

The present study shows that BNPP inhibits the production of hydrazine from INH in vivo and in vitro. The significantly decreased plasma hydrazine levels at 32 h in the BNPP-INH group versus the VEH-INH group shows that BNPP inhibited in vivo formation of hydrazine from INH. BNPP was found to be a long-acting or irreversible inhibitor of INH-amidase because inhibition by BNPP persisted in vitro in microsomes from BNPP-treated animals. This observation is consistent with the phosphorylating mechanism of action of BNPP (Heymann and Krisch, 1967). The present data also suggest that BNPP inhibits amidase via noncompetitive inhibition. The direct conversion of INH to hydrazine in in vitro incubations of control microsomes can be completely inhibited by the addition of BNPP at low µM concentrations. Based upon these observations, a possible mechanism of action of the inhibitory activity of BNPP involves phosphorylation of a hydroxyl group at an allosteric site on the amidase, which results in long-acting/irreversible and noncompetitive enzyme inhibition.

In addition to the inhibition of production of hydrazine, BNPP likely decreased the production of acetylhydrazine from acetyl-INH. Thus, it is possible that the decreased production of acetylhydrazine played a role in the decreased severity of INH-induced hepatotoxicity. However, previous evidence (Sarich et al., 1996) that hydrazine, and not acetylhydrazine, correlates with INH-induced hepatocellular damage suggests that decreased production of hydrazine most likely explains the prevention of INH induced-hepatocellular damage in this rabbit model.

Additional data from a toxicity comparison of INH, acetylhydrazine, and hydrazine were also described in the present study. Administration of hydrazine to rabbits, at the same molar dose as INH in this animal model, resulted in severe toxicity and early death (after severe convulsions). In addition, plasma ASAL activity was extremely elevated (976 Takahara units) in the one animal in which ASAL could be determined. The occurrence of convulsions in this model of INH-induced hepatotoxicity, and the suspected causative role of hydrazine, has been reported previously (Sarich et al., 1995, 1998). When the dose of hydrazine was lowered to one-half the molar dose, the severity of hepatocellular damage was not different from that caused by a full molar dose of INH or acetylhydrazine. On a molar basis, therefore, hydrazine appears to be more toxic than INH or acetylhydrazine. One might have expected an even greater degree of hepatotoxicity from this dose of hydrazine; however, the proportion of hydrazine, administered s.c., that gets to the liver could be quite small, and may not necessarily be a good model for hepatotoxicity of hydrazine directly formed from INH in the liver.

Of course, additional studies are required to fully understand the roles of hydrazine and acetylhydrazine in the mechanism of INH-induced hepatotoxicity in rabbits. This study does, however, confirm that a hydrolysis product(s) of INH is, or becomes, the hepatotoxic species in this model of INH-induced hepatotoxicity, and that hydrolysis product is most likely hydrazine.

The observed decrease in glutathione levels in the INH-treated animals may be due to increased scavenging of reactive substances that are produced as a result of the necrotic and/or steatotic state of the hepatocytes and/or possibly decreased hepatic production of glutathione. Nevertheless, hepatic glutathione levels are not depleted. The 21% decrease in hepatic glutathione stores observed in the food- and water-deprived group suggests that at least some of the decrease in glutathione levels in the VEH-INH group was due to the decreased intake of food and water. This is consistent with previous findings that fasting of rats decreases hepatic glutathione levels possibly via protein deprivation (Pessayre et al., 1979; reviewed in Mandl et al., 1995).

Hepatic EROD activity, a measure of CYP1A1/2 activity (Aix et al., 1994; Rey-Grobellet et al., 1996), was significantly decreased in the VEH-INH, BNPP-INH, and food- and water-deprived VEH-VEH^a groups as compared with controls. Although BNPP itself had no effect, it appears that INH and food and water deprivation both have effects on CYP1A1/2 activity. About 42% of the decrease in the VEH-INH and BNPP-INH groups is due to decreased food and water intake. Although there are no reports of decreased CYP1A1/2 activity due to decreased food and water intake in the literature, there are reports of decreased CYP1A1/2 activity in acetaminophen-induced hepatotoxicity in mice (Snawder et al., 1994) and aflatoxin B₁-induced hepatotoxicity in rabbits (Guerre et al., 1996).

In addition to the effect of food and water deprivation on CYP1A1/2 activity, the activity in the VEH-INH group was significantly decreased as compared with the BNPP-INH group and this group was significantly decreased compared to the VEH-VEH^a group. A significant negative correlation of CYP1A1/2 activity with INH-induced hepatocellular damage (versus ASAL and ALT activity) in the two groups receiving INH (VEH-INH and BNPP-INH) shows that more enzyme activity is inhibited as the severity of hepatocellular damage increases. It is still possible, however, given the 48-h time course of this study that a regulatory effect, for example, decreased RNA, or protein synthesis, caused the decreased CYP1A1/2 activity.

As previously reported, p-nitrophenol hydroxylase (a measure of CYP2E1) activity is significantly decreased after treatment with INH (VEH-INH group; Sarich et al., 1998). Inhibition of CYP2E1 activity has been previously found to occur after acetaminophen-induced hepatotoxicity in mice (Snawder et al., 1994). Aniline hydroxylase activity (another measure of CYP2E1 activity) was also decreased in aflatoxin B₁-induced hepatotoxicity in rabbits (Guerre et al., 1996). Consistent with the findings in the present study, activation of alkylhydrazines into free radical intermediates by CYP2E1 has also been suggested as a mechanism of toxicity of hydrazines (Albano et al., 1995). In acetaminophen-induced hepatotoxicity, CYP1A1/2 and CYP2E1 are involved in the production of reactive and toxic intermediates (Raucy et al., 1989; Patten et al., 1993; Thummel et al., 1993) and have decreased activity after the development of toxicity in mice (Snawder et al., 1994). Interestingly, CYP2E1 activity was increased in the BNPP-INH group (67% greater than the controls) but not in the BNPP-VEH group. BNPP, therefore, resulted in an increase in CYP2E1 activity in the presence of INH, rather than the expected inhibition of CYP2E1 activity in the presence of INH alone. This is possibly due to the inhibitory actions of BNPP on INH hydrolysis.

An interesting finding in this study is that hepatic amidase, EROD, and p-nitrophenyl hydroxylase activities are all significantly decreased in animals treated with INH. Could a common link between these enzymes be that they all come into contact with hydrazine? INH-amidase does because it cleaves hydrazine from INH and it appears that some amidase becomes inactivated in the process. Thus, like INH-amidase, the decreased activities of EROD and p-nitrophenyl hydroxylase may be due to their affinity for hydrazine. It is possible that these P-450s activate hydrazine to a damaging intermediate(s), which results in damage to the producing enzyme and surrounding intracellular components. As has been proposed for acetaminophen and its more reactive analog N-acetyl-m-aminophenol, if a metabolic intermediate is too reactive, as soon as it is produced it reacts with the producing enzyme as a suicide inactivator and the production of reactive metabolites and toxicity becomes self-limiting (Pumford and Halmes, 1997). In this situation, no toxicity would occur to the cell other than to the producing enzyme because the reactive metabolites would immediately bind to the enzyme before causing damage away from the producing enzyme. This reaction, and its inherent potential for causing toxicity, is thus self-limiting. Therefore, if hydrazine is converted to a reactive intermediate by these isozymes, it appears the reactive intermediate is reactive enough to damage and decrease the activity of these isozymes, but not so reactive that some does not get away from the producing isozyme(s) to cause damage to vital intercellular components, causing cell death.

In conclusion, administration of BNPP 30 min before INH administration in this model of INH-induced hepatocellular damage in rabbits prevents elevations of plasma ASAL and ALT activities and hepatic and plasma triglyceride levels, decreases plasma hydrazine levels, and prevents a decrease in the activity of markers for CYP1A1/2 and CYP2E1. These findings suggest that BNPP inhibits a key step in INH metabolism that prevents production of hepatotoxic intermediates. It was found in this study that BNPP is a potent and irreversible inhibitor of hydrolysis of INH to hydrazine with an IC₅₀ of approximately 2 µM. Based on a key previous study implicating hydrazine in the mechanism of INH-induced hepatocellular damage (Sarich et al., 1996), the mechanism by which BNPP prevents INH-induced hepatocellular damage is most likely through the inhibition of the production of hydrazine from INH.