Rat 17 beta-hydroxysteroid dehydrogenase type IV is a novel peroxisome proliferator-inducible gene.

To better understand the molecular mechanisms of the pleiotropic responses induced by exposure to peroxisome proliferator chemicals (PPCs), we conducted a systematic search for genes whose mRNA levels are modulated by the PPC WY-14,643 (WY) in rat liver. The sequence of one up-regulated cDNA (2480 bp) was predicted to encode a protein of 735 aa with 82% identity to the porcine 17 beta-hydroxysteroid dehydrogenase type IV (HSD IV). Like the porcine enzyme, the rat HSD IV contains' a region homologous to yeast hydratase-dehydrogenase-epimerases and to sterol carrier proteins, indicating that the rat HSD IV has broad substrate specificity and contributes to cholesterol metabolism. The rat HSD IV was regulated by diverse PPCs via two distinct mechanisms. Induction of HSD IV and acyl-CoA oxidase (ACO) proteins in rat liver at different treatment times and concentrations of gemfibrozil and di-n-butyl phthalate were almost identical, indicating that HSD IV mRNA induction involves the peroxisome proliferator-activated receptor alpha, a regulator of ACO. In contrast, HSD IV protein levels were only weakly induced by WY, a strong inducer of ACO protein, even though the levels of HSD IV and ACO mRNA were strongly stimulated by WY and gemfibrozil. Thus, HSD IV protein levels were uniquely regulated pretranslationally by WY via a novel mechanism. Increased conversion of estradiol to the less-active estrone by HSD IV induction may explain how phthalate exposure leads to decreases in serum estradiol levels and suppression of ovulation.

Characterization of the methionine S-oxidase activity of rat liver and kidney microsomes: immunochemical and kinetic evidence for FMO3 being the major catalyst.

Methionine is oxidized to methionine sulfoxide by rat liver and kidney microsomes in an O2- and NADPH-dependent manner. In all microsomal assays, no methionine sulfone was detected. Use of a monoclonal antibody to rat liver cytochrome P-450 reductase, various cytochrome P-450 and peroxidase inhibitors, antioxidants, and competitive flavin-containing monooxygenase (FMO) substrates suggested that methionine sulfoxidation was exclusively mediated by FMOs. At 5 mM methionine, the d-isomer of methionine sulfoxide was preferentially detected over the l-isomer in both liver (ratio, 5:1) and kidney microsomes (ratio, 12:1); however, at 30 to 40 mM methionine concentrations, the diastereomeric ratio was reduced to approximately 3:1 in both tissues. The Vmax/K(m) ratios determined for the liver and kidney microsomes were similar. Because cDNA-expressed rabbit FMO3 and FMO1 were previously shown to preferentially catalyze methionine and S-benzyl-L-cysteine (SBC) sulfoxidations, respectively, these substrates were used to isolate two distinct S-oxidase activities from the same rat liver microsomal preparation. The purified activities have apparent molecular weights of approximately 55 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings that the methionine S-oxidase reacted intensely with antibodies raised against rabbit FMO3 and the SBC S-oxidase reacted intensely with antibodies raised against rabbit FMO1 provide evidence for these activities being associated with FMO3 and FMO1, respectively. The apparent methionine K(m) determined with the purified methionine S-oxidase was 3.4 mM, whereas the apparent methionine K(m) determined with the purified SBC S-oxidase was 48 mM. The methionine sulfoxide d:l diastereomeric ratio obtained with methionine S-oxidase was 15:1, whereas the diastereomeric ratio obtained with SBC S-oxidase was only 2:1. These results provide strong evidence for the expression of both FMO1 and FMO3 in rat liver microsomes and suggest that FMO3 is the major catalyst of methionine sulfoxidation in rat liver and kidney microsomes.

Paradoxical increase in peroxisomal cyanide-insensitive respiration following dietary exposure to WY-14,643 in the perfused liver.

WY-14,643, a lipid-lowering drug, increases basal rates of oxygen uptake in perfused livers. Because peroxisomes consume oxygen for H2O2 production and are induced by WY-14,643 treatment, it is possible that peroxisomal beta-oxidation can account for some of this increase in cellular respiration. Therefore, cyanide, an inhibitor of mitochondrial cytochrome oxidase, was infused into livers of WY-14,643-fed rats (0.1% WY-14,643 in laboratory rat chow for 1, 21, and 105 days) to assess peroxisomal cyanide-insensitive respiration. As expected, the addition of cyanide abolished oxygen uptake nearly completely; however, after approximately 20 min oxygen consumption unexpectedly returned to basal levels in 105-day WY-14,643-treated animals but not in untreated controls. Urea synthesis, a process dependent upon ATP, was decreased and remained low during cyanide infusion in livers from both groups, indicating that mitochondria were not responsible for this unusual increase in oxygen uptake in the presence of cyanide. Methanol metabolism, which requires oxygen to form H2O2, was decreased from 37 +/- 5 to 6 +/- 1 micromol/g/hr in all groups treated with cyanide; however, it was increased significantly about 20 min later to 25 micromol/g/hr in livers from WY-14,643-treated rats, indicating that oxygen for peroxisomal H2O2 production is involved in cellular respiration in the presence of cyanide. Fasting abolished the recovery of both oxygen uptake and methanol metabolism in WY-14,643-fed rats, suggesting that ATP for acyl CoA synthetase, an enzyme which metabolizes fatty acids to acyl CoA compounds, is provided by glycolysis. Indeed, oleate significantly increased methanol metabolism in fed control rats from 8 +/- 4 to 26 +/- 3 micromol/g/hr in the presence of cyanide, indicating that fatty acid supply is necessary for peroxisomal respiration. Taken together, these experiments demonstrate that when mitochondrial respiration is inhibited, livers from rats fed WY-14,643 chronically have the unique ability of metabolizing fatty acids through the peroxisome using glycolytic ATP.

Gemfibrozil-induced peroxisome proliferation and hepatomegaly in male F344 rats.

Gemfibrozil is a widely used hypolipidemic drug in humans that causes peroxisome proliferation and hepatocarcinogenesis in rodents. The induction of hepatomegaly and hepatic peroxisome proliferation (measured as peroxisomal acyl CoA oxidase activity), was determined and compared to another peroxisome proliferator, WY-14,643 (0.1% in the diet) in male F344 rats. In a 21-day study, dietary no-observable-effect and lowest-observable-effect levels of gemfibrozil for both hepatomegaly and peroxisome proliferation were 0.002% and 0.005%, respectively. In a 42-day study, dietary concentrations of 0.9-2.0% gemfibrozil induced a similar magnitude of hepatomegaly to WY-14,643 (2.3-fold) but a higher level of peroxisome proliferation (16-18-fold) than the maximum induction for WY-14,643 (13-fold). The plateau in magnitude of gemfibrozil-induced peroxisome proliferation across the 0.9-2.0% dietary concentrations was associated with a plateau in serum concentration of gemfibrozil (approximately 20 micrograms/ml), similar to concentrations reported in human subjects receiving oral gemfibrozil. These results indicate that maximal induction of peroxisome proliferation by gemfibrozil can exceed that of a more potent compound such as WY-14,643, and further suggest that maximal induction of peroxisome proliferation can be limited by steady-state serum concentrations. Moreover, the reported lack of hepatic responses to gemfibrozil in humans is unlikely to be the result of inefficacy or unavailability of this drug, compared to other peroxisome proliferators, in rodents.

Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: relationship to mitochondrial alterations.

Recent studies have indicated a lack of correlation between hepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and the carcinogenicity of peroxisome proliferators (PP) and suggested that DNA in intact hepatic nuclei may be insensitive to increases in 8-OHdG resulting from PP exposure. The possibility that PP-induced elevations in acyl CoA oxidase (ACO) activity might result in oxidative damage to mitochondrial DNA (mtDNA) was therefore investigated by feeding male F344 rats the hepatocarcinogenic PP Wy-14,643 (Wy, 0.1% in the diet) for 3, 6, 11, or 22 weeks, or clofibric acid (CA, 0.5% in the diet) for 22 weeks. Following the respective PP exposures, hepatic peroxisomal acyl CoA oxidase activity was determined and DNA isolated from either mitochondria or unfractionated liver homogenates and analysed for the presence of 8-OHdG. PP treatment caused an increase in ACO activity (10- to 15-fold) at all time points examined and an increase of 8-OHdG (1.5- to 2-fold) in DNA isolated from unfractionated liver homogenates following PP treatment for 11 or 22 weeks. No increase of 8-OHdG in mtDNA was detected. However, quantitation of a PCR amplified region from the D-loop of mtDNA demonstrated a 2- to 3-fold increase in the relative amount of mtDNA in DNA isolated from unfractionated liver homogenates following 3, 11, and 22 weeks exposure to Wy or CA (22 weeks only). In addition, a slight increase in the mitochondrial volume density (1.4-fold) was observed in electron micrographs of liver samples from rats exposed to Wy for 22 weeks. These results (i) demonstrate that PP treatment, at levels which cause an increase in ACO activity, does not cause oxidative damage to mtDNA, and (ii) suggest that one reason for the observed increase of 8-OHdG in DNA from unfractionated liver homogenates may be an increase in the amount of mtDNA present in these samples. Furthermore, these studies provide additional evidence against a role of oxidative DNA damage, measured as 8-OHdG, in PP-induced rodent hepatocarcinogenesis and suggest that alterations in mitochondria or other effects may be more pertinent to PP-related carcinogenesis.

Methimazole protection of rats against gentamicin-induced nephrotoxicity.

Methimazole was previously shown to protect rats, mice, and (or) dogs against cisplatin-, cephaloridine-, 2-bromohydro-quinone-, and S-(1,2-dichlorovinyl)-L-cysteine-induced nephrotoxicity. In this study, methimazole effects on gentamicin (GM) induced nephrotoxicity were examined. Rats given GM (40 mg/kg) twice daily for 10 days exhibited higher blood urea nitrogen (BUN) concentrations and severe necrosis of virtually all proximal tubules compared with saline-treated controls. Rats cotreated with methimazole (20 mg/kg) exhibited minimal proximal tubular necrosis and were protected against GM-induced increase in BUN concentrations, despite having higher kidney GM concentrations. Rats given GM alone for 3 days exhibited no proximal tubular necrosis and no elevation of BUN values. However, these rats exhibited an increase in nonprotein disulfide concentrations and a decrease in renal protein thiol and protein disulfide concentrations, as opposed to rats given GM and methimazole. Together the results show that methimazole was an effective antagonist of GM-induced nephrotoxicity. Methimazole did not inhibit GM renal uptake but may protect against GM-induced nephrotoxicity by acting as an antioxidant within the kidneys.

Roles of cysteine conjugate beta-lyase and S-oxidase in nephrotoxicity: studies with S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-cysteine sulfoxide.

Effects of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and its putative metabolite DCVC sulfoxide (DCVCO) on renal function in vivo and in vitro were investigated to assess the role of sulfoxidation in the mechanism of toxicity of cysteine S-conjugates. Both conjugates were potent nephrotoxicants in rats in vivo, but at equimolar doses, DCVCO produced greater renal injury (i.e., increases in blood urea nitrogen levels and anuria and more severe and widespread proximal tubular necrosis) than DCVC. Pretreatment of rats with aminooxyacetic acid (AOAA), a selective cysteine conjugate beta-lyase (beta-lyase) inhibitor, did not protect against DCVCO nephrotoxicity, whereas rats given DCVC and AOAA exhibited partial protection. These results suggest that in addition to cleavage by the beta-lyase, sulfoxidation by the cysteine conjugate S-oxidase (S-oxidase) may play a role in DCVC nephrotoxicity. In isolated rat kidney proximal tubular (PT) and distal tubular (DT) cells, both DCVC and DCVCO produced time- and concentration-dependent increases in the release of lactate dehydrogenase. Because DCVC was generally more toxic in PT cells and DCVCO was more toxic in DT cells, an attempt was made to correlate in vitro cytotoxicity with the cellular distribution of the beta-lyase and S-oxidase. The finding that beta-lyase activity exhibited a 2-fold higher Vmax/Km ratio in PT cells than in DT cells, the greater inhibition of both beta-lyase activity and DCVC toxicity by AOAA in PT cells than in DT cells and the lower (40%) S-oxidase activity in PT cells than in DT cells provide evidence for the importance of the beta-lyase in DCVC toxicity in PT cells. The finding that DCVCO was more toxic in DT cells than in PT cells and the inability of AOAA to protect DT cells from DCVC-induced cytotoxicity, however, provide further evidence for DCVC bioactivation by S-oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization and purification of the flavin-dependent S-benzyl-L-cysteine S-oxidase activities of rat liver and kidney microsomes.

Previously, we provided evidence that cysteine conjugate S-oxidase (S-oxidase) activities of rat liver and kidney microsomes may be associated with flavin-containing monooxygenases (FMOs). In this study, the biochemical properties of these activities were further investigated. When NADPH was replaced by NADH, the S-oxidase activities were reduced significantly. Removal of the flavin moiety from microsomes significantly reduced the S-oxidase activities; however, addition of exogenous FAD or FMN restored the activities of the flavin-depleted microsomes. Solubilization of hepatic or renal microsomes with Emulgen 911, Nonidet P-40, Triton X-100, or 3-[(3-cholamidopropyl)dimethyl-ammonio]-1- propane sulfate or inclusion of the sulfhydryl-reactive agents Hg2+, N-ethylmaleimide, or iodoacetamide did not affect the S-oxidase activities, whereas solubilization of either hepatic or renal microsomes by cholate or heating of renal microsomes in the absence of NADPH significantly reduced the S-oxidase activities. In addition to male rat hepatic and renal microsomes, the S-oxidase activities were detected in lung microsomes of male rats and hepatic and renal microsomes of male mice and female rats and rabbits. The male rat kidney maintained the highest S-oxidase activity of all species and tissues examined. Whereas the aforementioned results provided further evidence for the S-oxidase activities being associated with FMOs, unambiguous evidence for this hypothesis was provided by the purification of the activities from rat liver (580-fold) and kidney (700-fold) microsomes and by the use of the isolated proteins in polyacrylamide gel electrophoresis, flavin content determinations, amino-terminal amino acid sequence analysis, amino acid composition analysis, and substrate kinetic studies. The findings that the S-oxidases were immunoreactive with antibodies raised against the pig liver 1A1 isozyme but not with antibodies raised against the rabbit lung 1B1 isozyme and that the liver S-oxidase amino-terminal amino acid sequence was more comparable to the amino-terminal amino acid sequences of pig and rabbit liver 1A1 isozymes than to those of rabbit lung 1B1 and liver 1D1 isozymes provide evidence that the S-oxidases are related to the known FMO 1A1 isozymes.

Methimazole protection of rats against chemically induced kidney damage in vivo.

Because methimazole has antioxidant properties, the effects of methimazole treatment on cephaloridine, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), 2-bromohydroquinone (2-BHQ) and cis-diaminedichloroplatinum (II) (cisplatin)-induced nephrotoxicity were investigated. Rats given cephaloridine (1 g/kg), cisplatin (5 mg/kg), DCVC (100 mg/kg) or 2-BHQ (157 mg/kg) i.p. exhibited significant elevations in blood urea nitrogen concentrations, which correlated with appearance of distinct renal histopathological changes. Cephaloridine, DCVC or 2-BHQ-induced nephrotoxicity was reduced only when methimazole (20-40 mg/kg) was given 30 min before the nephrotoxicant, whereas cisplatin-induced nephrotoxicity was reduced when methimazole was given 30 min before and up to 4 hr after cisplatin. Because the renal organic acid transport system plays an important role in the nephrotoxicity of cephaloridine, cisplatin and DCVC, the role of the organic acid transport system in the renal uptake of methimazole was investigated. With rat kidney cortical slices, methimazole uptake was time- and concentration-dependent; however, the organic acid transport substrates, probenecid (1 mM) and p-aminohippuric acid (7.5 mM), were ineffective in blocking methimazole uptake. Furthermore, cephaloridine (1 mM) uptake by kidney cortical slices was not affected by methimazole (5 mM). Rats given methimazole (40 mg/kg) 30 min before cephaloridine (2 g/kg) had serum and kidney cephaloridine concentrations similar to rats given cephaloridine only, but the methimazole-pretreated rats were significantly protected against cephaloridine-induced oxidation of renal nonprotein thiols. These results show that methimazole does not inhibit the transport of cephaloridine into the kidneys, but may protect against cephaloridine-induced renal damage by acting as an antioxidant within the kidneys.

Reactivity of cysteine S-conjugate sulfoxides: formation of S-[1-chloro-2-(S-glutathionyl)vinyl]-L-cysteine sulfoxide by the reaction of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide with glutathione.

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) sulfoxide, a putative metabolite of the toxic cysteine S-conjugate DCVC, was synthesized by the reaction of DCVC with H2O2 and characterized by fast atom bombardment mass spectrometry (FAB-MS) and proton nuclear magnetic resonance spectroscopy. DCVC sulfoxide was stable when kept at room temperature overnight in phosphate buffer (pH 6.8-7.8) or when heated in phosphate buffer (pH 7.2 or 7.6) or H2O (pH 3.5 or 10.5) for 20 min at 37 degrees C. However, in the presence of glutathione (GSH), DCVC sulfoxide was readily converted to S-[1-chloro-2-(S-glutathionyl)vinyl]-L-cysteine sulfoxide (I), a product formed by the Michael addition of GSH to DCVC sulfoxide followed by the loss of HCl. Evidence for the mechanism of this reaction was obtained by the finding that DCVC, which cannot act as a Michael acceptor, did not react with GSH under conditions similar to those used with DCVC sulfoxide. When the reaction of DCVC sulfoxide with GSH was carried out at room temperature and pH 7.4, formation of I was complete at 5 min, but when the reaction was carried out for 2 h at pH 6.0 or 4.4 at 37 degrees C, product formation was nearly 37 or 3% of that formed at pH 7.4, respectively; product formation did not increase when the reaction was carried out at pH 8.5. When DCVC sulfoxide (100 mg/kg) was administered to rats, hepatic and renal reduced nonprotein thiol concentrations were decreased at 1 h to 74 and 27% of that in control rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cysteine conjugate S-oxidase. Characterization of a novel enzymatic activity in rat hepatic and renal microsomes.

Cysteine conjugate S-oxidase activity, with S-benzyl-L-cysteine as substrate, was found mostly in the microsomal fractions of rat liver and kidney. In the presence of oxygen and NADPH, S-benzyl-L-cysteine is converted to S-benzyl-L-cysteine sulfoxide; no S-benzyl-L-cysteine sulfone was detected. The Vmax for S-benzyl-L-cysteine sulfoxide formation by kidney microsomes was nearly 3-fold greater than the rate measured with liver microsomes. Inclusion of catalase, superoxide dismutase, glutathione, butylated hydroxyanisole, the peroxidase inhibitor, potassium cyanide, the cytochrome P-450 inhibitors, 1-benzylimidazole and metyrapone, or a monoclonal antibody to cytochrome P-450 reductase did not inhibit the metabolic reaction. Flavin-containing monooxygenase alternate substrates, N,N-dimethylaniline, n-octylamine, and methimazole inhibited the S-oxidase activities. Analogues of S-benzyl-L-cysteine, S-methyl-L-cysteine, and S-(1,2-dichlorovinyl)-L-cysteine inhibited the S-benzyl-L-cysteine S-oxidase activities, whereas S-carboxymethyl-L-cysteine and S-benzyl-L-cysteine methyl ester had no effect. These results provide clear evidence against the involvement of reactive oxygen intermediates or cytochrome P-450 in the sulfoxidation of S-benzyl-L-cysteine and indicate that the S-oxidase activities may be associated with flavin-containing monooxygenases which exhibit selectivity in the interaction with cysteine S-conjugates.