Glutathione S-transferase-mediated chlorothalonil metabolism in liver and gill subcellular fractions of channel catfish

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is a broad spectrum fungicide that is a potent acute toxicant to fish. Therefore, the metabolism of chlorothalonil was investigated in liver and gill cytosolic and microsomal fractions from channel catfish (Ictalurus punctatus) using HPLC. All fractions catalyzed the metabolism of chlorothalonil to polar metabolites. Chlorothalonil metabolism by cytosolic fractions was reduced markedly when glutathione (GSH) was omitted from the reaction mixtures. The lack of microsomal metabolism in the presence of either NADPH or an NADPH-regenerating system indicated direct glutathione S-transferase (GST)-catalyzed conjugation with GSH without prior oxidation by cytochrome P450. Cytosolic and microsomal GSTs from both tissues were also active toward 1-chloro-2,4-dinitrobenzene (CDNB), a commonly employed reference substrate. In summary, channel catfish detoxified chlorothalonil in vitro by GST-catalyzed GSH conjugation in the liver and gill. The present report is the first to confirm microsomal GST activity toward CDNB in gill and toward chlorothalonil in liver, and also of gill cytosolic GST activity towards chlorothalonil, in an aquatic species.     

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.     

Deficient induction of sulfobromophthalein conjugating activity by phenobarbital in hamster liver

Administration of phenobarbital, a known inducer of glutathione S-transferase activity in rat liver, failed to stimulate sulfobromophthalein (BSP) conjugation by liver cytosol in hamsters. The latter displayed poor ability to conjugate this substrate, despite very high glutathione-conjugating activity with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB). Of the six substrates tested, in this species, 1,2-epoxy-3-(4-nitrophenoxy)propane (ENPP) was the only one whose conjugation was greatly enhanced by phenobarbital (+172%). Nevertheless, hamsters proved as responsive to phenobarbital induction as rats, since it increased their relative liver weight and microsomal enzyme activity. The deficient induction of liver BSP-conjugating activity observed with phenobarbital is consistent with the finding that it did not affect the hepatic transport of this substrate in hamsters.     

Metabolism of hexachloro-1,3-butadiene in mice: in vivo and in vitro evidence for activation by glutathione conjugation

1. The metabolism of 14C-hexachloro-1,3-butadiene (HCBD) was studied in mice and in subcellular fractions from mouse liver and kidney. 2. In the presence of glutathione (GSH), liver microsomes and cytosol transformed HCBD to S-(pentachlorobutadienyl)glutathione (PCBG). PCBG formation in subcellular fractions from mouse kidney was very limited. Oxidative metabolism of HCBD by cytochrome P-450 could not be demonstrated. 3. Cysteine conjugate beta-lyase was present in mitochondria and cytosol from mouse liver and kidney. 4. After an oral dose of 30 mg/kg 14C-HCBD, mice eliminated 67.5-76.7% of dose in faeces; urinary elimination accounted for 6.6-7.6%. 5. Metabolites of HCBD identified are: S-(pentachlorobutadienyl)glutathione in faeces; S-(pentachlorobutadienyl)-L-cysteine, N-acetyl-S-(pentachlorobutadienyl)-L-cysteine and 1,1,2,3-tetrachlorobutenoic acid in urine. 6. The results suggest that conjugation of HCBD with GSH in liver, followed by renal processing of the glutathione S-conjugates and beta-lyase-catalysed formation of reactive intermediates, accounts for the organ specific toxicity of HCBD in mice.     

Cytosolic factors that alter the metabolism of N,N-dimethyl-4-aminoazobenzene by rat liver microsomes

N,N-Dimethyl-4-aminoazobenzene (DAB), an azo dye carcinogen, is N-demethylated and 4'-hydroxylated by rat liver microsomes. Addition of hepatic cytosol to the microsomal system stimulated both pathways. This occurred in the presence of added NADPH or an NADPH-generating system. Cytosol was effective only when present prior to addition of substrate; no stimulation was seen when added after the reaction had begun. This suggested a direct effect on the microsomes rather than a chemical interaction with one or more metabolic intermediates of DAB. The degree of stimulation was somewhat different when using microsomes from phenobarbital- or beta-naphthoflavone-treated animals, implying a selectivity of the cytosolic effect for various isozymes of cytochrome P-450. Some loss of stimulatory activity occurred with dialysis. Activity was restored by adding back glutathione (GSH) which can stimulate DAB metabolism even in the absence of cytosol. DAB metabolism is also stimulated by EDTA. Although both EDTA and cytosol inhibit lipid peroxidation, cytosol stimulated DAB metabolism even in the presence of EDTA. Therefore, suppression of lipid peroxidation does not explain satisfactorily the cytosolic effect. Separation of cytosolic proteins by gel filtration revealed a factor which inhibits N-demethylation but not 4'-hydroxylation of DAB. Heating at 100 degrees partially inactivated the stimulatory activity. However, inhibitory activity was less susceptible to heat inactivation than was stimulatory activity. These results indicate that, in the whole cell, microsomal metabolism of xenobiotics is regulated to an appreciable extent by macromolecular cytosolic substances.     

Evidence for the metabolism of mitozantrone by microsomal glutathione transferases and 3-methylcholanthrene-inducible glucuronosyl transferases

The metabolism of mitozantrone, a chemotherapeutic agent used in the treatment of breast cancer, has been studied in vitro using rat liver subcellular fractions. This compound would appear to be metabolized by two interesting pathways. One involves conjugation with glucuronic acid, catalyzed most effectively by a 3-methylcholanthrene-inducible glucuronosyl transferase. The other pathway appears to be a glutathione conjugation reaction which requires prior metabolism by cytochrome P-450. The reaction with glutathione appears to be enzymatic as 1-chloro-2,4-dinitrobenzene was a potent inhibitor of this reaction. Liver cytosol did not enhance the microsomal rate of glutathione-conjugate formation, suggesting an important role for the microsomal glutathione transferases in the disposition of this compound. The relationship between these reactions and the mode of action of mitozantrone is discussed.     

Stereoselective glutathione conjugation of the separate alpha-bromoisovalerylurea and alpha-bromoisovaleric acid enantiomers in the rat in vivo and in rat liver subcellular fractions

Glutathione (GSH) conjugation of the separate alpha-bromoisovalerylurea (BIU) enantiomers was studied in the rat. Administration of (R)-BIU resulted in excretion of a single glutathione conjugate in bile (IU-S-G/I) and a single mercapturate in urine (IU-S-MA/B). The other enantiomer, (S)-BIU, was exclusively metabolized to the other diastereomeric conjugates, IU-S-G/II and IU-S-MA/A. Thus, the conjugation of BIU with glutathione was completely stereospecific. Both the GSH conjugate and mercapturate derived from (R)-BIU were excreted two to three times more rapidly than their diastereomeric (S)-BIU counterparts. The enantiomers did not influence each others metabolism as reflected by identical metabolite excretion rates when the BIU enantiomers were administered either separately or as the racemic mixture. A similar rate difference for GSH conjugation of the separate BIU enantiomers was observed in incubations with rat liver cytosol as source of GSH transferases, suggesting that the stereoselectivity in vivo was due to glutathione conjugation properly. Similar results were obtained with a rat liver microsomal fraction, indicating that microsomal GSH transferases are active towards BIU and have a similar stereoselectivity as the cytosolic enzymes. Comparison of the GSH conjugation of BIU with that of its analogue alpha-bromoisovaleric acid (BI, which lacks the amide-linked urea group) revealed an opposite stereoselectivity: while (R)-BIU was conjugated faster than (S)-BIU, the (R) enantiomer of the acid was conjugated more slowly than (S)-BI. The alpha-bromocarbonyl compounds BI and BIU present a new type of substrate for the GSH transferases and allow studies of these enzymes in intact organisms as well as investigations on the stereoselectivity of GSH conjugation.     

Enzymatic conjugation of hexachloro-1,3-butadiene with glutathione. Formation of 1-(glutathion-S-yl)-1,2,3,4,4-pentachlorobuta-1,3-diene and 1,4-bis(glutathion-S-yl)-1,2,3,4-tetrachlorobuta-1,3-diene

The glutathione-dependent metabolism of the nephrotoxin and nephrocarcinogen hexachloro-1,3-butadiene (HCBD) was investigated in subcellular fractions from rat liver and kidney. HCBD was metabolized by hepatic glutathione S-transferases to (E)- and (Z)-1-(glutathion-S-yl)-pentachlorobuta-1,3-diene (GPCB) in a ratio of 20:1, which were identified by secondary ion MS and by GC-MS after acid hydrolysis. The formation of GPCB was dependent on time and on protein and glutathione concentrations. Microsomal glutathione S-transferases from rat liver catalyzed GPCB formation more efficiently than did cytosolic glutathione S-transferases; very low rates of GPCB formation were observed in kidney subcellular fractions. GPCB is also a substrate for glutathione S-transferases and is metabolized to a diglutathione conjugate, which was identified by secondary ion MS and 13C NMR spectrometry as 1,4-bis(glutathion-S-yl)-1,2,3,4-tetrachlorobuta-1,3-diene (BTCB). BTCB formation from GPCB was dependent on time and on protein, glutathione, and GPCB concentrations. Hepatic cytosol catalyzed BTCB formation more efficiently than did hepatic microsomes; significant amounts of BTCB were also formed in kidney cytosol. Hepatic formation of glutathione S-conjugates, translocation of the S-conjugates to the kidney, and renal processing to form reactive intermediates may be the cause of HCBD-induced nephrotoxicity and, perhaps, nephrocarcinogenicity.     

Metabolism of hexachloro-1,3-butadiene in mice: in vivo and in vitro evidence for activation by glutathione conjugation

1. The metabolism of ¹⁴C-hexachloro-1,3-butadiene (HCBD) was studied in mice and in subcellular fractions from mouse liver and kidney.

2. In the presence of glutathione (GSH), liver microsomes and cytosol transformed HCBD to S-(pentachlorobutadienyl)glutathione (PCBG). PCBG formation in sub-cellular fractions from mouse kidney was very limited. Oxidative metabolism of HCBD by cytochrome P-450 could not be demonstrated.

3. Cysteine conjugate β-lyase was present in mitochondria and cytosol from mouse liver and kidney.

4. After an oral dose of 30 mg/kg ¹⁴C-HCBD, mice eliminated 67˙5-76˙7% of dose in faeces; urinary elimination accounted for 6˙6-7˙6%.

5. Metabolites of HCBD identified are: S-(pentachlorobutadienyl)glutathione in faeces; S-(pentachlorobutadienyl)-L-cysteine, N-acetyl-S-(pentachlorobutadienyl)-L-cysteine and 1,1,2,3-tetrachlorobutenoic acid in urine.

6. The results suggest that conjugation of HCBD with GSH in liver, followed by renal processing of the glutathione S-conjugates and β-lyase-catalysed formation of reactive intermediates, accounts for the organ specific toxicity of HCBD in mice.     

Detoxification of molinate sulfoxide: comparison of spontaneous and enzmatic glutathione conjugation using human and rat liver cytosol

Previous lab studies implicated the sulfoxidation pathway of molinate metabolism to induce testicular toxicity. Once molinate is metabolized to molinate sulfoxide, it undergoes further phase II metabolism either spontaneously, enzyme catalyzed, or both to form glutathione-conjugated molinate. This study compared the metabolic capability of rat and human liver cytosol to form a glutathione (GSH)-conjugated metabolite of molinate. The GSH conjugation of molinate sulfoxide in rat cytosol was described by the constants Km of 305 microM and Vmax of 4.21 nmol/min/mg cytosol whereas the human values were 91 microM and 0.32 nmol/min/mg protein for Km and Vmax, respectively. At the same 1 mM GSH concentration, the in vitro bimolecular nonenzymatic rate constant of 3.02 x 10(-6) microM(-1) min(-1) was calculated for GSH conjugation of molinate sulfoxide. Specific activity for rat and human glutathione transferase was calculated to equal 1.202 +/- 0.25 and 0.809 +/- 0.45 micromol/min/mg protein, respectively by 1-chloro-2,4-dinitrobenzene (CDNB) assay. Compared to a conventional GSH depletion model (BSO + DEM combination), molinate alone was nearly as effective in reducing GSH levels by approximately 90 and 25% in liver and testes, respectively. The impact of molinate sulfoxide's ability to adduct glutathione transferase and inhibit the production of the glutathione conjugated metabolite was examined and found to be negligible.     

Glutathione S-transferases and chloroform toxicity in streptozotocin-induced diabetic rats

The glutathione S-transferase activity in liver and kidney cytosol was significantly decreased in short term diabetes induced with streptozotocin, whereas no decrease in the transferase was observed in phenobarbital-treated diabetic rats. Toxicity of chloroform was potentiated in streptozotocin- or phenobarbital-treated rats. The decrease in liver cytosolic and microsomal glutathione S-transferase activity was observed in long term diabetic rats, and only microsomal transferase activity was restored by insulin treatment. There was no release of glutathione S-transferases into the serum in the diabetic rats, and the transferases were not inhibited by streptozotocin in vitro. These results showed that glutathione S-transferase activity decreased during diabetes, and this decrease may contribute to altering drug metabolism and toxicity in diabetes.     

Subcellular distribution of glutathione S-transferase in Chinese fetal liver

Subcellular fractions were isolated from Chinese fetal liver at 4-8 months of age for the determination of glutathione S-transferase (GST). Using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, GST activity was found to be 66 +/- 34 nmol/(min.mg protein), mainly in the cytosol. The GST activities were detected principally in microsomes and their values were 66 +/- 31 and 144 +/- 83 nmol/(min.mg protein), respectively, when assayed with p-nitrobenzyl chloride (PNB) and ethacrynic acid (EA) as substrates. There were no age and sex-related differences in GST activities for any of the substrates studied during fetal development. The Km values of GST for CDNB, PNB and EA were 1112, 1039 and 205 mumol/L, respectively. The conjugation of GST may play an important role in fetal hepatic metabolism of toxic electrophiles.     

Effect of dietary clofibrate on epoxide hydrolase activity in tissues of mice

The effects of dietary clofibrate on the epoxide-metabolizing enzymes of mouse liver, kidney, lung and testis were evaluated using trans-stilbene oxide as a selective substrate for the cytosolic epoxide hydrolase, cis-stilbene oxide and benzo[a]pyrene 4,5-oxide as substrates for the microsomal form, and cis-stilbene oxide as a substrate for glutathione S-transferase activity. The hydration of trans-stilbene oxide was greatest in liver followed by kidney greater than lung greater than testis. Its hydrolysis was increased significantly in the cytosolic fraction of liver and kidney of clofibrate-treated mice and in the microsomes from the liver. Isoelectric focusing indicates that the same enzyme is responsible for hydrolysis of trans-stilbene oxide in normal and induced liver and kidney. Clofibrate induced glutathione S-transferase activity on cis-stilbene oxide only in the liver. Hydrolysis of both cis-stilbene oxide and benzo[a]pyrene 4,5-oxide was highest in testis followed by liver greater than lung greater than kidney. Hydration of cis-stilbene oxide was induced significantly in both liver and kidney by clofibrate but that of benzo[a]pyrene 4,5-oxide was induced only in the liver. These and other data based on ratios of hydration of benzo[a]pyrene 4,5-oxide to cis-stilbene oxide in tissues of normal and induced animals indicate that there are one or more novel epoxide hydrolase activities which cannot be accounted for by either the classical cytosolic or microsomal hydrolases. These effects are notable in the microsomes of kidney and especially in the cytosol of testis.     

Conjugation reactions in hepatocytes isolated from streptozotocin-induced diabetic rats

The activities of three drug conjugation reactions, glutathione, glucuronic acid and sulphate conjugation and the synthesis of glutathione, have been measured in hepatocytes isolated from streptozotocin-induced male diabetic rats. The intracellular content of reduced glutathione (GSH) was decreased in diabetic rat hepatocytes compared with controls. Following depletion of the intracellular GSH stores with diethylmaleate, the resynthesis of GSH in the presence of 0.5 mM L-methionine, occurred faster in diabetic rat hepatocytes than in those from control rats indicating that the cystathione pathway may be more efficient in the diabetic animals. In contrast, there was no significant difference in the resynthesis of GSH between control and diabetic rat hepatocytes in the presence of L-cysteine. The GSH conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB) was deficient in diabetic rat hepatocytes, although only the effect on the former reaction was statistically significant (P less than 0.05). The Vmax for CDNB conjugation was significantly lower (P less than 0.05) in cytosolic fractions prepared from diabetic rat liver than in control rat liver fractions. This was accompanied by an increase in the affinity of the enzyme for CDNB. In contrast, the Vmax and Km for the conjugation of DCNB in cytosolic fractions were unaffected by the induced-diabetes. Glucuronic acid conjugation of both 1-naphthol and phenolphthalein was markedly deficient in diabetic rat hepatocytes. The intracellular concentrations of the cofactor for glucuronidation, UDP-glucuronic acid, were decreased in diabetic rat liver and this was thought to contribute to the defect in glucuronidation. The sulphation of 1-naphthol was not significantly altered by the induced diabetes. Deficiencies in glutathione and glucuronic acid conjugation in streptozotocin-induced diabetic rats may result in an increased susceptibility to xenobiotic induced cytotoxicity.     

Effect of protein-calorie malnutrition on cytochromes P450 and glutathione S-transferase.

Protein-calorie malnutrition (PCM) can develop both from inadequate food intake and as a consequence of diseases such as cancer and AIDS. Several studies have shown that PCM can alter drug clearance but little information is available on the effect of PCM on individual cytochrome P450 isoforms and phase II conjugation enzymes. The aim of the present study was to begin a systematic evaluation of the effect of PCM on the activity of individual drug metabolizing enzymes in a rat model of PCM. Control and PCM rats received isocaloric diets which contained either 21% or 5% (deficient) protein. After 3 weeks, the animals were sacrificed and microsomal and cytosolic fractions prepared. Ethoxyresorufin O-deethylation (EROD), chlorzoxazone 6-hydroxylation, dextromethorphan N- and O-demethylation and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation were used as measures of CYP1A, CYP2E1, CYP3A2, CYP2D1 and glutathione S-transferase (GST) activity, respectively. Additionally, NADPH-cytochrome P450 reductase activity was measured in the liver microsomes. PCM significantly reduced the maximum velocity (Vmax) of all model reactions studied. However, differential effects were observed with respect to K(m) values of the reactions. The K(m) values for EROD and dextromethorphan N-demethylation were significantly increased in PCM animals, whereas the K(m) values for chlorzoxazone 6-hydroxylation and dextromethorphan O-demethylation were decreased. In contrast, the K(m) value for CDNB conjugation was unchanged. When NADPH-cytochrome P450 reductase activity was compared, a 29% reduction in reductase activity was noted in PCM animals as compared to controls. Thus, it appears that PCM decreases the overall activity of certain phase I and phase II metabolism enzymes in rat liver while exhibiting differential effects on K(m). Furthermore, this reduction in activity may be due in part to diminished activity of cytochrome P450 reductase.     

Bioactivation of tetrachloroethylene. Role of glutathione S-transferase-catalyzed conjugation versus cytochrome P-450-dependent phospholipid alkylation

The metabolism of [14C]tetrachloroethylene (Tetra) and its metabolite S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) was investigated with in vitro systems to substantiate metabolic pathways of Tetra deduced from in vivo experiments. In the presence of NADPH, rat hepatic microsomal fractions metabolized Tetra to soluble metabolites, which were identified as trichloroacetic acid and oxalic acid by gas chromatography/mass spectroscopy and a metabolite largely bound to microsomal macromolecules. The majority of the alkylated macromolecules were identified as N-trichloroacetylated phospholipids by high performance liquid chromatography and GC/MS. When Tetra was incubated with hepatic microsomes and cytosol in the presence of 10 mM glutathione, but in the absence of NADPH, the formation of a polar metabolite other than trichloroacetic acid and oxalic acid was observed. This metabolite was identified, after hydrolysis to the corresponding cysteine conjugate, as S-(1,2,2-trichlorovinyl)-glutathione (TCVG). Microsomal GSH S-transferases catalyzed TCVG formation more efficiently than cytosolic GSH S-transferases; the competitive substrate 1-chloro-2,4-dinitrobenzene inhibited TCVG formation. In the presence of both NADPH and GSH, TCVG formation in microsomes was decreased, indicating that oxidative metabolism and GSH conjugation of Tetra are competitive reactions. The Tetra metabolite TCVC was cleaved by bacterial cysteine conjugate b-lyase to dichloroacetic acid and pyruvate. The obtained results substantiate the postulated pathways of Tetra biotransformation and demonstrate that both oxidative and conjugative reactions occur in hepatic Tetra metabolism. Phospholipid alkylation, which occurs during oxidative metabolism, may be a deactivation reaction, whereas TCVG formation, renal metabolism to TCVC, and cleavage of TCVC by b-lyase under formation of mutagenic intermediates may contribute to the nephrocarcinogenic effect of Tetra.     

In vitro metabolism of the epoxide substructure of cryptophycins by cytosolic glutathione S-transferase: species differences and stereoselectivity

The enzyme kinetics of the glutathione (GSH) conjugation of cryptophycin 52 (C52, R-stereoisomer) and cryptophycin 53 (C53, S-stereoisomer) by cytosolic glutathione S-transferases (cGSTs) from human, rat, mouse, dog and monkey liver were studied. Vmax, Km, and CLint values for glutathione conjugation of C52 (R-stereoisomer) were 0.10 +/- 0.01 nmol min-1 mg-1, 3.24 +/- 0.23 microM, and (3.15 +/- 0.09) x 10(-2) ml min-1 mg-1, respectively, in human cytosol. Due to limited solubility relative to the Km, only CLint values were determined in rat ((7.76 +/- 0.10) x 10-2 ml min-1 mg-1) and mouse ((7.61 +/- 0.50) x 10(-2) ml min-1 mg-1) cytosol. Enzyme kinetic parameters could not be determined for C53 (S-stereoisomer). Microsomal GSH conjugation in human, rat, and mouse was attributed to cytosolic contamination. No GSH conjugation was seen in any biological matrix from dog or monkey. There was little GSH conjugation of C53 by cytosol or microsomes from any species. The metabolism of C52 and C53 by epoxide hydrolase was also investigated. No diol product was observed in any biological matrix from any species. Thus, cGSTs are primarily responsible for C52 metabolism.     

Cytosolic glutathione S-transferases in drosophila melanogaster

Post-microsomal supernatants from Drosophila melanogaster and rat liver homogenates were investigated with respect to their glutathione S-transferase (GST) activity. It appeared that the Drosophila transferase did not conjugate the epoxides styrene-7,8-oxide and 1,2 epoxy(p-nitrophenoxy)-propane.Attempts to isolate the Drosophila GST isozymes by means of the well-documented method for the purification of the rat liver transferases failed, because the Drosophila transferases did not bind to CM-cellulose. Purification by subsequent ion exchange on DEAE-cellulose, molecular sieving on Sephadex G-100 and hydroxylapatite chromatography, revealed three active fractions from Drosophila cytosol and five active fractions from rat liver cytosol, using 1-chloro-2,4-dinitrobenzene as the electrophilic substrate. None of the Drosophila active fractions catalyzed the conjugation of glutathione with the epoxides mentioned.It is concluded that there are important differences between the GST systems of both species, resulting in differences in the metabolic fate of chemicals that are substrates for glutathione conjugation. This has to be taken into account in the evaluation of genotoxicity testing in Drosophila of potentially harmful compounds.     

The effect of tridiphane (2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane) on hepatic epoxide-metabolizing enzymes: indications of peroxisome proliferation

Administration of tridiphane (Tandem, DOWCO 356, 2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane) to male Swiss-Webster mice for 3 days at 100, 250, and 500 mg/kg (ip) resulted in increases in liver weight accompanied by an increase in mitotic index and increases in large particle and microsomal protein. Epoxide hydrolase (EH) activity towards cis-stilbene oxide (CSO, microsomal EH) was elevated in microsomes and cytosol, a decrease in microsomal cholesterol EH was found, and hydrolysis of trans-stilbene oxide (TSO, cytosolic EH) was elevated in the cytosol but not in the microsomes. Glutathione S-transferase (GST) activity was elevated in cytosol for CSO, TSO, and 1,2-dichloro-4-nitrobenzene (DCNB), with inconsistent responses found with 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP). Microsomal GST was not consistently effected by tridiphane. Clofibrate (500 mg/kg, 3 daily ip injections) treatment resulted in similar responses in liver size, microsomal protein, and the EHs. The increase in cytosolic EH activity previously has been noted only in animals treated with peroxisome proliferators. Examination of livers from mice treated with 250 mg/kg tridiphane revealed that an increase in hepatic peroxisomes was apparent after 3 days of treatment. This was accompanied by decreases in serum cholesterol and triglyceride levels and increases in liver carnitine acetyl transferase and cyanide-insensitive oxidation of palmitoyl-CoA. This study demonstrates that tridiphane does have in vivo effects on mammalian epoxide-metabolizing enzymes and extends the association of increased cytosolic epoxide hydrolase activity with peroxisome proliferation.     

Metabolism of formaldehyde during in vitro drug demethylation: Effectiveness of protection by semicarbazide

Formaldehyde formation is usually determined as a measure of the metabolism of drug substrates in vitro. We have studied the fate of formaldehyde added to incubates of rat liver microsomes or 9000 g supernatant fraction as well as the effectiveness of semicarbazide in protecting formaldehyde from metabolic degradation. Formaldehyde is known to be oxidized by a cytosolic NAD- and GSH-dependent (form)aldehyde dehydrogenase. We found that, in addition, some other NAD-independent reactions take place in the cytosol. We observed, moreover, that formaldehyde is also metabolized by the 9000 g supernatant fraction fortified with cofactors for hepatic monooxygenase in the absence of NAD. This finding could be attributed to a Hitherto unknown, cytosolic NADP-dependent, GSH-requiring dehydrogenase. The microsomal fraction metabolized formaldehyde only to a small extent. Therefore, in order to use formaldehyde formation as a parameter of drug metabolism, semicarbazide is necessary to protect formaldehyde from further metabolism in the 9000 g supernatant fraction and microsomes. By determining amounts of both formaldehyde and p-chlor-aniline duringp-clor-N-methylanilinedemethylation, it was shown that semicarbazide (4 mM) only partially protected for-maldehyde from further metabolism in the 9000 g supernatant, although semicarbazone formation from the added formaldehyde and semicarbazone progressed more rapidly than formaldehyde metabolism. As higher semicarbazide concentrations inhibit microsomal demethylations, it is concluded that determination of formaldehyde is not a suitable method for determining drug demethylation by the 9000 g liver supernatant. In microsomal incubates, only a low semicarbazide concentration (1.0 mM) was necessary to protect formaldehyde from further metabolism.