Microsomal Cytochrome P-450 Content Research Articles

The effects of cadmium, mercury and lead in vitro on hepatic microsomal mixed function oxidase and lipid peroxidation.

In this study, the authors investigated the effect of cadmium, mercury and lead in vitro on hepatic microsomal mixed function oxidase and lipid peroxidation. The results showed that these metals could inhibit the activity of hepatic microsomal aniline hydroxylase, decrease the concentration of hepatic microsomal cytochrome P-450 and increase the concentration of the inactive form of hemoprotein, P-420. Besides, they could enhance hepatic microsomal lipid peroxidation. There were marked concentration-dependent responses in these microsomal reactions. Thus, it may be suggested that cadmium, mercury and lead are capable of impairing hepatic microsomal mixed function oxidase in vitro by stimulating membrane lipid peroxidation.

Effect of interferon inducers on superoxide anion generation from rat liver microsomes detected by lucigenin chemiluminescence

Microsomal superoxide anion ( O − 2 ) production was detected using the chemiluminigenic probe, bis-N-Methylacridinium nitrate (lucigenin). Superoxide dismutase (SOD) inhibited 55% of the light emission but in the presence of a detergent (Triton X100) SOD inhibited the light emission by 94%. Lucigenin chemiluminescence from rat liver microsomes supplemented with NADPH was found to be selective and sensitive in detecting the O − 2 production. Treatment of rats with poly IC and LPS resulted in a decrease of the hepatic microsomal cytochrome P450 content by 44% and 37% respectively. The decrease in the cytochrome P450 contents was accompanied by a decrease in LgC1 from the hepatic microsomal fractions by 61% for the poly IC and by 51% for the LPS treated rats. This is the first report to demonstrate that decreased P450 in the presence of normal amounts of cytochrome P450(c) reductase produce correspondingly less O − 2 from the microsomes.

No effect of chronic ethanol administration on the activity of alcohol dehydrogenase and aldehyde dehydrogenases in the near-term pregnant guinea pig

The objective of this study was to determine the effect of chronic maternal administration of moderate-dose ethanol on alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the guinea pig at near-term pregnancy. The activity of each enzyme in the maternal liver, fetal liver, and placenta of the guinea pig at 59 days of gestation (term, 66 days) was determined spectrophotometrically following chronic daily oral administration of two doses of 1 g ethanol/kg maternal body weight or isocaloric sucrose solution. There was no experimental evidence of ethanol-induced malnutrition in the mother or growth retardation in the fetus. There was a statistically significant increase (65%) in the microsomal cytochrome P-450 content of the maternal liver for the ethanol treatment compared with the sucrose treatment. The alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the maternal liver, fetal liver, and placenta were not statistically different for the ethanol-treated compared with the sucrose-treated animals. This also was the case for the maternal blood and fetal blood ethanol and acetaldehyde concentrations, determined at 2h after maternal administration of 1 g ethanol/kg maternal body weight. These data demonstrate that the ethanol- and acetaldehyde-oxidizing enzyme activities in the maternal-placental-fetal unit of the guinea pig at near-term pregnancy were not changed by chronic administration of moderate-dose ethanol.

Differences between induction effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene and phenobarbitone

The inductive effects of phenobarbitone (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) were compared in C57BL/6J mice. Induction parameters included six substrates: ethylmorphine (EM), benzphetamine (Bph), biphenyl, ethoxycoumarin (EtoC), pentoxyresorufin and dichloro- p-nitroanisole (DPNA). In order to validate this descriptive approach the comparison was extended to diazepam, rifampicin, warfarin, and pregnenolone-16α-carbonitrile (PCN). All inducers were clearly distinguishable from each other. Warfarin was similar to PB, rifampicin was similar to PCN. TCPOBOP differed significantly from PB in relative liver weight, cytochrome P-450 content of liver microsomes, EM-, Bph- and DPNA-demethylations, biphenyl-hydroxylations, EtoC de-ethylation and absorption maximum of reduced CO-cytochrome P-450. TCPOBOP, as an inducer, was less “specific” than PB: total metabolic rates were excessively increased due to microsomal protein (1.5 times) and cytochrome P-450 (4 times) augmentation, whereas cytochrome P-450-related metabolic rates were less increased than those after PB. Thus TCPOBOP does not seem to be as similar to PB as was suggested in the first description of its inducing potency.

In vivo toxicity of cisplatin and carboplatin on the leydig cell function and effect of the human choriogonadotropin

In order to characterize the respective toxicity of cisplatin (CDDP) and carboplatin (CBDCA) on the male reproductive system, we have investigated their in vivo effects on the steroidogenesis function of rat leydig cells. Animals were treated at the respective ld 50 of platin compounds, and we analyzed plasmatic testosterone level, microsomal cytochrome P-450 and platinum concentrations in the testis. CDDP induces a dramatic change in both the testosterone level and the microsomal cytochrome P-450 concentration. In contrast, CBDCA was found to be less toxic than CDDP, probably due to its different accumulation at the testis level. We also investigated the potential action of human chorionic gonadotropin which allows a full restoration of the steroidogenesis function.

Effect of 1,3-butanediol on rat liver microsomal NDMA demethylation and other monooxygenase activities

The administration of 1,3-butanediol (BD) previously has been shown to elevate blood concentrations of ketone bodies, to potentiate carbon tetrachloride hepatotoxicity, and to increase the hepatic microsomal content of cytochrome P450 and the activity of aniline hydroxylase. In the present study, oral treatment (10 g/kg) with racemic BD and each of its enantiomers ( RBD and S-BD) induced NDMA demethylase activity by approx. 1.5-fold in rat hepatic microsomes obtained 12 h later, suggesting an induction of P450IIE1, the acetone/ethanol-inducible form of P450. The results agreed with an immunochemieally determined increase in the levels of this isozyme. No change in P450 content, NADPH-cytochrome- c reductase, or in pentoxyresorufm dealkylase activity were detected. Blood levels of acetone were determined during a 10-h period after BD administration and showed a higher initial rate of increase by RBD, possibly due to stereoselective metabolic oxidative metabolism. However, no difference in the induction of NDMA demethylase activity by the enantiomers could be detected. Induction of P450IIE1 probably contributes to the previously described potentiation of haloalkane-induced hepatotoxicity by BD administration.

Mechanism of Inhibition of Aflatoxin B1- Dna Binding in the Liver by Butylated Hydroxyanisole Pretreatment of Rats

Previous studies have demonstrated that pretreatment of rats with butylated hydroxyanisole (BHA) inhibited aflatoxin B1 (AFB1)-DNA binding and AFB1 hepatocarcinogenesis. Our detailed studies on the effect of BHA pretreatment of rats on both AFB1-DNA binding and AFB1-glutathione (AFB1-SG) conjugation with hepatosubcellular fractions, isolated hepatocytes and in intact animals are reviewed. For these studies, young male rats were fed a semisynthetic diet with or without 0.75% BHA for 2 wks. Even though there were no significant differences either in microsomal cytochrome P-450 content or microsome-mediated AFB1 binding to exogenous DNA with cytochrome P-450 from control or treated rats, there were large differences in GSH S-transferase activity with treated cytosols showing 100% higher activity than the controls. Kinetics of cytosolic inhibition of microsome-mediated AFB1-DNA binding and formation of AFB1-SG conjugate indicated that the inhibition of AFB1-DNA binding was greater with cytosols from treated compared to the controls with the concomitant formation of AFB1-SG conjugate. Reconstitution studies with intact nuclei, microsomes and cytosol indicated more AFB1-DNA binding with the control than with BHA-treated animals. In isolated hepatocyte system, at 2 μM level of AFB1, AFB1-DNA binding in treated hepatocytes was about 15% of controls whereas thiol conjugation was 5 fold higher in the treated than in control hepatocytes. Addition of 1 mM styrene oxide caused about 100% and 9 fold increase in AFB1-DNA binding in control and treated hepatocytes respectively with corresponding decreases in thiol conjugation. In intact rats, BHA treatment reduced hepatic AFB1-DNA binding to 15% of controls with concomitant increase in biliary excretion of AFB1-SG conjugate. These results indicate that the induced cytosolic GSH S-transferases after BHA treatment of rats play a significant role in inhibiting hepatic AFB1-DNA binding and AFB1 hepatocarcinogenesis by inactivation of the reactive AFB1-epoxide.

Characterization of the microsomal mixed-function oxygenase system of the hepatopancreas and green gland of the red swamp crayfish, Procambarus clarkii

1. The microsomal mixed-function oxygenase (MFO) system from the hepatopancreas and green gland of the red swamp crayfish, Procambarus clarkii has been characterized with respect to the constitution of electron transport proteins and the ability to catalyze the metabolism of xenobiotics. 2. Cytochrome P-450 content of hepatopancreas microsomes was approximately 10-fold higher than that of green gland and comparable to that of rat liver. NADPH-cytochrome c reductase activity in hepatopancreas microsomes was approximately 2% of that found in rat liver microsomes. 3. Green gland microsomes catalyzed higher turnover rates of aminopyrine N-demethylase and benzo[a]pyrene hydroxylase than hepatopancreas microsomes. With hepatopancreas microsomes, organic hydroperoxides supported a greater rate of aminopyrine N-demethylation than did NADPH plus O2. 4. P. clarkii hepatopancreas microsomes generally displayed a lower binding affinity (Ks) for a number of type I and type II ligands than did rat liver microsomes.

Effect of glucose administration on various rat hepatic constituents and on the spectral binding of hexobarbital and methadone to rat hepatic cytochrome P450.

The administration of glucose to rats for 48 h resulted in an increase in the duration of anesthesia produced by intraperitoneal injection of pentobarbital. The effect of this glucose treatment on hepatic glycogen and on microsomal protein, cytochrome P450 (P450), cholesterol, lipid and phospholipid content, as well as on the spectral binding of hexobarbital and methadone to microsomal P450, was examined. The glucose treatment appeared to have no effect on microsomal protein or P450 content. The binding of hexobarbital and methadone by P450 produced a type 1 spectrum in both control and glucose-treated animals. The glucose treatment resulted in a decrease in the spectral dissociation constant (Ks) and in the maximal spectral binding (delta Amax) of hexobarbital to P450 while with methadone there was an increase in Ks and a decrease in delta Amax. Total lipid, phosphatidylcholine and phosphatidylethanolamine were increased in the microsomal fractions from glucose-treated animals. The changes in the parameters for drug binding to microsomal P450 probably relate to the increased lipid content of the microsomes although changes in the proportion of P450 isoenzymes could be involved. The previously observed decrease in the microsomal metabolism of hexobarbital and methadone following glucose treatment may be due to decreased binding of these compounds to microsomal P450.

Effect of a Prudhoe Bay crude oil on hepatic and renal peroxisomal beta-oxidation and mixed-function oxidase activities in rats.

The effect of oral administration of a Prudhoe Bay crude oil (PBCO) to male rats (PBCO, 2.6 g/kg body weight, daily) for 5-12 days on hepatic and renal microsomal monooxygenase activities and peroxisomal beta-oxidation has been investigated. PBCO administration leads to liver enlargement. This is associated with induction of microsomal cytochrome P-450 levels (1.6- to 2.0-fold) and dependent mixed-function oxidase activities (7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-depentylase, representing cytochrome P-450I and cytochrome P-450IIB isoenzymes respectively, 9- to 15-fold; omega-oxidation of lauric acid representing the cytochrome P-450IVA1 isoenzyme, 1.4- to 1.5-fold) along with peroxisomal beta-oxidation (palmitoyl CoA oxidation, 2- to 5-fold). It was observed that rats exposed to PBCO showed an increase in renal microsomal cytochrome P-450 content (1.6- to 2.3-fold), cytochrome P-450I activity (5- to 8-fold) and omega-oxidation activity (1.3- to 1.4-fold). However, renal peroxisomal beta-oxidation was unaltered. Serum total triglycerides were lowered by 41-46% after PBCO exposure. These results suggest that induction of peroxisomal beta-oxidation and possibly mono-oxygenases may be related to the carcinogenic/tumorigenic potential of crude oil.

Effect of 3-Methylcholanthrene Pretreatment on Aflatoxin B1Metabolism in Hamster Liver

AbstractThe effects of 3-methylcholanthrene (MC) pretreatment of hamsters on the hepatic metabolism of aflatoxin B1 (AFB1) have been investigated in studies with subcellular, isolated hepatocytes and in vivo. Pretreatment of hamsters with MC (2.5 mg/100 g body wt.) 24 hr. before sacrifice not only increased microsomal cytochrome P-450 content by 70% but also increased the specific activity of cytochrome P-450 dependent metabolic activation of AFB1 as judged by AFB1-DNA binding by 65%. Even in isolated hepatocytes, AFB1-DNA binding was elevated by 65% in MC-treated hepatocytes. The ratios of AFB1-glutathione (AFB1-SG) to AFB1-DNA were about the same in both hepatocyte system. Hepatic AFB1-DNA binding was also increased by 85% in intact animals after MC treatment. It appears that the increased hepatic AFB1-DNA binding after MC treatment of hamsters is a result of higher specific activity and higher levels of cytochrome P-450 species involved in the activation of AFB1.

Coordinate elevations of liver δ-aminolevulinate synthase and cytochrome P-450 RNA by phenobarbital in chicken embryos: The effects of heme

1. 1. The role of heme in the coordinate elevations of liver δ-aminolevulinate (ALA) synthase activity and microsomal cytochrome P-450 concentration induced by phenobarbital (PB) was investigated in the chicken embryo. 2. 2. Eighteen day old chicken embryos were given PB, and the changes in liver content of PB-inducible cytochrome P-450 RNA and of ALA synthase RNA were determined at different times after exposure to the drug. 3. 3. The concentrations of both types of RNA increased rapidly after PB administration, and by 9 hr the level of ALA synthase RNA was 55-fold higher than control and that of cytochrome P-450 RNA was 7-fold higher than normal. 4. 4. While the rate of increase in ALA synthase activity paralleled closely that of the enzyme's RNA concentration, the rate of increase of spectrally active cytochrome P -450 concentration in microsomes lagged behind that of the apoprotein's RNA by several hours. 5. 5. To test whether heme depletion was responsible for the coordinate inductions of the two enzymes, embryos were loaded with ALA 2 hr before exposure to PB. 6. 6. The protocol led to a drop in the PB-inducible ALA synthase RNA concentration and to an increase in that of cytochrome P-450 RNA, measured 6 hr after drug administration. 7. 7. In primary cultures of hepatocytes, hemin in the culture medium caused a modest drop in ALA synthase RNA concentration but had a variable effect on that of cytochrome P-450 RNA in cells incubated with PB for 9 hr. 8. 8. The increased production of apo-cytochrome P-450 could have caused a drop in free heme concentration and led to a derepression of the gene for ALA synthase. That possibility was tested by treating hepatocytes in culture with PB together with cycloheximide (CHX). 9. 9. Not only did CHX fail to block the elevation in ALA synthase RNA concentration, CHX by itself caused increases in the concentrations of both ALA synthase and cytochrome P-450 RNA in the cells. 10. 10. The results indicated that heme depletion was not a prerequisite for ALA synthase induction by phenobarbital.

Contribution of methylsulfonyl metabolites of m-dichlorobenzene to the heme metabolic enzyme induction by the parent compound in rat liver

In the present study, we investigated the contribution of methylsulfonyl metabolites derived from m-dichlorobenzene ( m-DCB) on the heme metabolic enzyme induction by the parent compound in rats. The time courses of the effects of a single ip administration of m-DCB (200 mg/kg, 1.36 mmol/kg) and 2,4- and 3,5-dichlorophenyl methyl sulfones (2,4- and 3,5-DCPSO 2Mes) (each 50 μmol/kg) on hepatic microsomal cytochrome P450 content were almost in parallel with those on the total heme content in liver microsomes. m-DCB significantly increased the heme oxygenase activity, but 2,4- and 3,5-DCPSO 2Mes did not. On the other hand, m-DCB and both methyl sulfones markedly enhanced the δ-aminolevulinic acid (ALA) synthetase activity. No change was observed in percentage saturation of the tryptophan pyrrolase activity after administration of m-DCB, whereas this ratio at 6 hr after injection of 3,5-DCPSO 2Me was increased. In the liver of the dl-buthionine-( S,R)-sulfoximine (BSO)-treated rats dosed with m-DCB, both of 2,4- and 3,5-DCPSO 2Mes were present at significantly lower concentrations than in non-BSO-treated rats. Additionally, the m-DCB did not elevate the ALA synthetase activity in the BSO-treated rat. On the other hand, the administration of either 2,4- or 3,5-DCPSO 2Mes to BSO-treated rats resulted in induction of ALA synthetase. m-DCB and 2,4- and 3,5-DCPSO 2Mes produced a dose-related increase in liver levels of methyl sulfones. The changes in the ALA synthetase activity after the administration of varying doses of m-DCB were similar to those after the administration of 2,4- or 3,5-DCPSO 2Mes, whereas the sum of the concentration of two methyl sulfones in the liver of rats dosed with m-DCB was almost the same as the concentration of methyl sulfone after the administration of either 2,4- or 3,5-DCPSO 2Mes. The results strongly suggest that the methyl sulfones derived from m-DCB, i.e., 2,4- and 3,5-DCPSO 2Mes, contribute highly to the induction of the ALA synthetase activity by the parent compound.

Mouse hepatic cytochrome p-450 isozyme induction by 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene, pyrazole, and phenobarbital

The effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pyrazole on mouse hepatic cytochrome P-450 isozyme expression were compared to the P-450 induction pattern elicited by phenobarbital. TCPOBOP and PB administration caused a similar induction profile by increasing microsomal protein and cytochrome P-450 content and the catalytic activities of several monooxygenases in DBA/2N and AKR/J mice. There were, however, several quantitative and some qualitative differences in the induction profile caused by phenobarbital and TCPOBOP. A few strain-related differences were also observed. Immunoblot analysis with polyclonal anti-coumarin hydroxylase (P-450 Coh) antibody and epitope-specific monoclonal antibodies 1-7-1 and 2-66-3 showed that both phenobarbital and TCPOBOP increase the amount of P450IIB and P-450 Coh. TCPOBOP caused a more pronounced increase in the amount of P-450IIB than phenobarbital, and TCPOBOP also caused an increase in the amount of P-450IA2. These data suggest that in the mouse, TCPOBOP increases mainly the expression of P-450 isozymes responsive to phenobarbital. The effects of pyrazole differed greatly from those caused by TCPOBOP and phenobarbital. In the DBA/2N mice, pyrazole increased coumarin 7-hydroxylation 9.4-fold, whereas in the AKR/J mice the activity was induced only to a level equivalent to the DBA/2N basal level. In immunoblot experiments with anti-P-4500 Coh antibody, the amount of P-450 Coh was considerably higher in DBA 2N mice treated with phenobarbital, TCPOBOP, or pyrazole in comparison with the AKR/J mice, indicating a strain specificity in the inducibility of coumarin 7-hydroxylase by pyrazole.

The effects of perfluorodecanoic acid on hepatic stearoyl-coenzyme A desaturase and mixed function oxidase activities in rats

Perfluorodecanoic acid (PFDA) causes a dioxin-like toxic syndrome and alters the hepatic oleate/stearate ratio in rats. The acute toxic effects of a single ip dose (50 mg/kg) of PFDA on hepatic stearoyl-CoA desaturase and mixed function oxidases were studied in male Fischer-344 rats, 14 days after dosing. PFDA causes a marked decrease in food intake in rats, resulting in severe body weight loss with delayed lethality (2–3 weeks after dosing). To distinguish the effects of hypophagia from those caused by PFDA, pairfed control rats were used in addition to ad libitum-fed controls. Stearoyl-CoA desaturase activity, responsible for the conversion of stearoyl-CoA to oleoyl-CoA, was absent in both PFDA-dosed rats and their pair-fed controls at Day 14. Electron transfer through the desaturase system was significantly reduced in PFDA-treated rats only, and in these rats there was a significant reduction in microsomal cytochrome b 5, an important component of this electron transfer system. Pentobarbital sleeping times were significantly prolonged in both the PFDA-dosed and pair-fed rats, as compared with the ad libitum-fed controls. This effect was more pronounced in PFDA-dosed rats. Waking plasma pentobarbital concentration was similar in all treatment groups. Hepatic microsomal cytochrome P-450 content was unaffected. Aminopyrine N-demethylase activity was greatly reduced in PFDA-dosed rats. Although pair-fed controls also had reduced demethylase activity, it was not as pronounced as in PFDA-dosed rats, and was probably due to the fasted condition of these animals. Although the mechanism of action of PFDA is not known, it is possible that PFDA affects microsomal enzymes by altering the structure and/or function of the membranes in which they are located, through effects on lipid metabolism.

A simple method for assessment of rat cytochrome P-448 isozymes responsible for the mutagenic activation of carcinogenic chemicals

Cytochrome P-448H/L-enriched and cytochrome P-448L-enriched microsomes were prepared from the livers of Sprague-Dawley rats treated with 3-methylcholanthrene (MC) and with a combination of MC and carbon tetrachloride, respectively, and their activities for mediating mutagenic activation of 9 carcinogenic aromatic amines and benzo[ a]pyrene, which are found to be different from cyt. P-450 isozymes as to mutagenic activation, were compared on the basis of microsomal cytochrome P-450 content using Salmonella typhimurium TA98 as a tester bacterium. With regard to the substrate-specificity of cytochrome P-448 isozymes, the present results reflected the reported results with use of a cytochrome P-450-reconstituted system. These findings indicate that the mutation test with cytochrome P-448H/L-enriched and cytochrome P-448L-enriched microsomes could be used as a simple method for the determination of the cytochrome P-448 isozymes responsible for the mutagenic activation of carcinogens and mutagens without the use of a cytochrome P-450-reconstituted system.

The effects of prochloraz on some microsomal parameters in rat liver.

The antifungal agent prochloraz [l-n-propyl-N(2,4,6-trichlorophenoxy ethylcarbamoyl)-imidazole] induces cytochrome P-450 in the liver of vertebrates and interacts with lipid metabolism (Riviere et al. 1984; Laignelet et al. 1986). A study was undertaken to detect changes in lipid composition and fluidity of microsomal membranes. Sprague-Dawley rats (180-190g) were injected i.p. with either 600 Ixmol/kg prochloraz (95% pure; Schering Co.) in corn oil or with corn oil only, 3 days before sacrifice. In the liver homogenates vitamin A was measured (Olson 1979). The following assays were carried out with the microsomal suspensions: benzo(a)pyrene monooxygenase activity (BaPMO) (Depierre et al. 1975), epoxide hydrolase activity (EH) (Oesch et al. 1971), microsomal protein content (Lowry et al. 1951) and cytochrome P-450 content (Omura and Sato 1964). Microsomal lipids were extracted (Bligh and Dyer 1959) and quantitatively determined (Canal et al. 1973); cholesterol content was also measured (Boehringer Mannheim standard kit). Individual phospholipids were separated by two-dimensional TLC (Wolff et al. 1984) and total lipid phosphorus as well as individual phospholipids were thus determined (Ames 1966). Fatty acids were derivatized to their methyl esters (Morrison and Smith 1964) and analysed by GLC. Microsomal fluidity was studied at 37~ by fluorescence depolarization measurement of diphenyl hexatriene (DPH) according to Schinitzki and Barenholdz (1978). The results are summarized in Table 1 and all the differences indicated below are significant (at p < 0.01 level; t test calculation). Prochloraz significantly increased liver weight (+ 27%) and decreased the vitamin A liver burden (-17%). In treated animals the microsomal protein content of whole liver was significantly increased (nearly 40%). In microsomes of treated animals cytochrome P-450 content and BaPMO activity were significantly increased (+21% and + 121%, respectively), although EH activity was not altered. Prochloraz did not change the total lipid and phospholipid contents in microsomal membranes. However, the cholesterol level in these membranes and thus the phospholipid/cholesterol molar ratio were decreased ( -22% and -20%, respectively). Consequently, the microviscosity, evaluated in terms of fluorescence with the probe DPH, was significantly decreased ( 12%). It was reported that prochloraz inhibited lanosterol 14-demethylation (a cytochrome P-450 dependent reaction) in fungi (Schwinn 1983) and increased lanosterol liver content in Japanese quails (Riviere et al. 1984). More-

Protective effects of selenium on acetaminophen-induced hepatotoxicity in the rat

Experiments were undertaken to examine the ability of selenium to protect against acetaminophen-induced hepatotoxicity and to examine possible mechanisms for this protective effect. Pretreatment of male, Sprague-Dawley rats with sodium selenite (12.5 mumol Se/kg, ip) 24 hr prior to acetaminophen administration produced a significant protection against the hepatotoxic effects of acetaminophen as assessed by a decrease in the plasma appearance of alanine aminotransferase and aspartate aminotransferase activities following acetaminophen. This was accompanied by an increase in the hepatic glutathione levels in selenium-treated animals and an inhibition in the decrease in hepatic glutathione content observed in animals receiving hepatotoxic doses of acetaminophen. Selenium pretreatment decreased the in vivo covalent binding of acetaminophen metabolites to hepatic protein, but did not alter hepatic microsomal cytochrome P-450 content or NADPH cytochrome c reductase activity, suggesting that selenium does not significantly alter the metabolism of acetaminophen to reactive electrophilic metabolites by the cytochrome P-450-dependent mixed-function oxidase enzyme system. Selenium produced an increase in the activity of gamma-glutamylcysteine synthetase which may account for the increased glutathione availability in selenium-treated animals and increased the activities of glutathione S-transferase and glucose-6-phosphate dehydrogenase. Examination of the urinary metabolite profile in selenium-treated animals revealed that the urinary excretion of acetaminophen and its metabolites was significantly increased over a 72-hr period. The increase occurred in the AAP-glucuronide metabolite while parent AAP and AAP-sulfate were actually decreased in selenium-treated rats. No change in recovery was observed in the AAP-glutathione or AAP-mercapturate urinary metabolites. While the glutathione conjugating system is enhanced by selenium treatment, amelioration of acetaminophen toxicity is most likely the result of enhanced glucuronidation which effectively diverts the amount of acetaminophen to be converted by the cytochrome P-450 system to the toxic metabolite.

Detection of two heme oxygenase isoforms in the human testis

This study shows heme oxygenase multiplicity is common to rat and human tissues. The isozymes in man and rat, however, are heterogenous proteins that share certain characteristics. Two forms of heme oxygenase, HO-1 and HO-2, were identified in human testis. HO-2 form was the prevalent form. Human and rat HO-1 differed in chromatographic behavior and molecular weight; human HO-1 was a larger molecule (35,400 vs 30,000). The two forms, however, were similar in that immunochemically human HO-1 exhibited reactivity toward antibody to rat HO-1. Human and rat HO-2 also were dissimilar in chromatographic behavior and showed only a weak immunological cross-reactivity. Human and rat HO-1 were essentially the same size. As in rat organs, the microsomal cytochrome P-450 content in human testis was reciprocal to heme oxygenase activity.

Changes in adrenocortical monooxygenase activities in alloxan-diabetic rabbits

Studies were carried out to determine if diabetes mellitus influenced the activities of adrenal steroidogenic enzymes. Adult male rabbits were made diabetic by an i.v. infusion of alloxan (100 mg/kg) and were killed 1 or 2 months later. Mitochondrial cytochrome P-450 concentrations were not affected by diabetes but steroid 11β-hydroxylase activity was greater in the diabetics than in controls after both 1 and 2 months. The type I spectral change produced by 11-deoxycorticosterone, the substrate for 11β-hydroxylation, was also greater in mitochondria from diabetics. By contrast, mitochondrial cholesterol side-chain cleavage activity was similar in controls and diabetics. Microsomal cytochrome P-450 concentrations were unaffected by diabetes but 21-hydroxylase activity was significantly lower in adrenal microsomes from diabetics than from controls. The results indicate that alloxan-induced diabetes alters adrenocortical steroid metabolism which may contribute to changes in the pattern of steroid secretion noted by other investigators.