Ethanol potentiation of carbon tetrachloride hepatotoxicity: possible role for the in vivo inhibition of aldehyde dehydrogenase

A potentiation of CCl4-induced hepatotoxicity was observed in rats pretreated with ethanol 18 hr prior to CCl4 exposure. Hepatic microsomal aldehyde dehydrogenase (ALDH) was significantly inhibited in animals sacrificed 1 hr following the sequential exposure, however, no more so than in those animals receiving CCl4 alone. The animals receiving ethanol alone had ALDH activity similar to vehicle treated controls. Twenty-four hours following a potentiating dose of ethanol and CCl4 an 81 and 57% decline in NAD+-dependent microsomal and mitochondrial ALDH activity was observed, respectively. Similar results were observed for microsomal and mitochondrial NADP+-dependent ALDH activity. The decline in membrane-bound ALDH was greater in potentiated animals than in those receiving CCl4 alone. A relatively smaller decline in cytosolic ALDH activity was observed in CCl4 treated rats with or without ethanol pre-exposure. The data suggest that inhibition of membrane bound ALDH may be one of the major mechanisms of in vivo potentiation of CCl4-induced hepatotoxicity by ethanol.     

Evidence for the metabolism of N-nitrosodimethylamine and carbon tetrachloride by a common isozyme of cytochrome P-450

Carbon tetrachloride administration to rats produced a selective loss of hepatic cytochrome P-450-dependent catalytic activities. Of the cytochrome P-450-dependent catalytic activities tested, the metabolism of carbon tetrachloride to phosgene and the low Km N-nitrosodimethylamine demethylase were the most sensitive to destruction by carbon tetrachloride. A 50% or greater loss in these catalytic activities was observed 3 hr after giving 10 microliters carbon tetrachloride/kg. Related catalytic activities, such as the microsomal metabolism of carbon tetrachloride to chloroform and the high Km N-nitrosodimethylamine demethylase, were diminished less than 20% 3 hr after giving 10 microliters carbon tetrachloride/kg. To investigate further the relationship between the metabolism of N-nitrosodimethylamine and carbon tetrachloride, the effect of pyrazole, a known inducer of the low Km N-nitrosodimethylamine demethylase, on carbon tetrachloride metabolism was studied. Pyrazole treatment produced a 5.6-fold increase in the microsomal metabolism of carbon tetrachloride to phosgene and a 1.9-fold increase in the conversion of carbon tetrachloride to chloroform. The similarities between both the loss and the induction of the low Km N-nitrosodimethylamine demethylase and the metabolism of carbon tetrachloride to phosgene suggest that these catalytic activities represent a common isozyme of cytochrome P-450. Analysis of cytochromes P-450 by HPLC provided evidence for an isozyme of cytochrome P-450 inducible by pyrazole and destroyed by carbon tetrachloride.     

Effects of diispropyl 1, 3-dithiol-2-ylidene malonate (NKK-105) on the drug-metabolizing enzymes and fine structure of rat liver (author's transl)]

The mechanism of liver enlargement and anti-fatty liver effect of NKK-105 in the rat were investigated by the mesurement of drug-metabolizing enzyme activities and morphological changes in liver tissue detected using electron microscopy. A single administration of NKK-105(250, 500, 1000 mg/kg, p.o.) induced an apparent increase in liver weight. The elevation of aminopyrine demethylase activity and slight increase in microsomal cytochrome b5 and cytochrome P-450 content were seen with the administration of NKK-105. NKK-105 inhibited lipid peroxide formation in mitochondrial and microsomal fractions. Total lipid content of liver decreased at 12 hr after the administration of NKK-105. Lipid peroxide formation in mitochondrial and microsomal fractions was markedly inhibited by the addition of NKK-105 (1 X 10(-3)M), in vitro. Disarrangement of rough endoplasmic reticulum and increase in smooth endoplasmic reticulum were observed by the administration of NKK-105. The decrease in drug-metabolizing enzymes caused by CCl4 or ethionine was protected in the combination with NKK-105. NKK-105 markedly inhibited the elevation of lipid peroxide formation caused by CCl4 or ethionine. Similar effects on lipid peroxide formation were also obtained in vitro. These results suggest that the enlargement induced by NKK-105 indicates a functional not a toxic response. The inhibition of lipid peroxide formation in mitochondrial and microsomal fractions may thus play an important role in the mechanism of anti-fatty liver effect of NKK-105 on the CCl4 or ethionine-induced fatty liver.     

Diethylthiocarbamic acid methyl ester. A potent inhibitor of aldehyde dehydrogenase found in rats treated with disulfiram or diethyldithiocarbamic acid methyl ester

Rats were treated with disulfiram (Antabuse, DSF) or its metabolite diethyldithiocarbamic acid methyl ester (Me-DDC) and challenged with ethanol. The blood pressure response to ethanol was followed and blood was analyzed for DSF, Me-DDC and diethyldithiocarbamic acid (DDC). The rat liver aldehyde dehydrogenase (ALDH) isozyme activities were measured 2 hr after the ethanol challenge. Both treatments produced a significant fall in the blood pressure when challenged with ethanol, probably caused by a marked decrease in hepatocyte low Km and high Km activities. The mean plasma concentration ranges of Me-DDC and DDC were found to be 49-1241 nmol/l and 182-841 nmol/l, respectively, whereas DSF was undetectable. In addition, it was found that inactivation of hepatocyte low Km ALDH activity was dependent on preoxidation of Me-DDC by the microsomal cytochrome P-450 mixed function oxidases. Me-DDC was found to be oxidized under aerobic conditions in the presence of NADP to form diethylthiocarbamic acid methyl ester (Me-DTC). The structure was confirmed from its MS/EI fragmentation spectrum. Me-DTC was found to be a potent inhibitor of low Km ALDH when added to rat liver homogenate. The compound was also identified as a metabolite in rat blood collected from the DSF and Me-DDC treated rats, and in blood from human alcoholics on DSF treatment. Me-DTC appears to be more selective for the low Km isozymes whereas the opposite seems to be the case for the hydrolytic product, DTC.     

Effects of 8-(2-dimethylaminoethyl)-3-oxo-4-phenyl-1-thia-4,8-diazaspiro [4,5] decane dihydrochloride monohydrate (Y-8845) on carbon tetrachloride-induced liver injury

The effects of 8-(2-dimethylaminoethyl)-3-oxo-4-phenyl-1-thia-4,8-diazaspiro [4, 5] decane dihydrochloride monohydrate (Y-8845) on carbon tetrachloride (CCl4)-induced liver injury were investigated in rats. CCl4-induced attenuation of the plasma cyclic AMP (cAMP) response to glucagon stimulation was significantly prevented by pretreatment with Y-8845. Y-8845 also effectively suppressed the increases in the activities of serum transaminases as well as the decreases in microsomal glucose-6-phosphatase activity and microsomal cytochrome P-450 concentrations induced by CCl4. In rats at 72 hr after CCl4 administration, the plasma cAMP response to glucagon, microsomal glucose-6-phosphatase activity and P-450 concentration were all below the control level. Y-8845 treatment after CCl4 administration rectified these reductions to nearly normal levels. Furthermore, Y-8845 stimulated DNA synthesis during liver regeneration after CCl4 intoxication. These results demonstrate that Y-8845 has a protective effect against CCl4-induced injury in the liver and a stimulating effect on the recovery of the damaged liver.     

Effects of primaquine on hepatic microsomal haemoproteins and drug oxidation

Administration of the antimalarial agent primaquine to male rats (50 mg/kg i.p. daily for 4 days) resulted in a 30% decrease in hepatic microsomal cytochrome P-450 (P-450) content; levels of other microsomal haemoproteins were unaltered. Kinetic analysis of 2 mixed-function oxidase activities (aminopyrine N-demethylase and aniline p-hydroxylase) in primaquine-pretreated rat liver microsomes revealed a significant and similar decrease in the maximal reaction velocities of these enzymes (Vmax), but the apparent Michaelis constants (Km) were not changed. The activity of mitochondrial delta-aminolaevulinic acid synthetase (the rate-limiting step in haem biosynthesis) was normal in primaquine-pretreated rat liver but haem oxygenase activity (the rate-limiting step in haem degradation) was elevated approximately 2-fold. Haem availability for haemoprotein assembly (determined as the haem-saturation ratio of the cytosolic haemoprotein tryptophan pyrrolase) was also normal although the absolute activity of tryptophan pyrrolase was decreased after primaquine pretreatment. In order to facilitate an analysis of the P-450 isozyme profile in control and primaquine-treated rat liver, total microsomal P-450 was isolated by hydrophobic affinity chromatography on n-octylamino-Sepharose 4B. Densitometry of stained polyacrylamide gels following electrophoresis of these partially-purified P-450 fractions indicated that primaquine exposure did not selectively decrease any of the 3 protein bands in the P-450 molecular weight region (48-56 kD). These observations, when considered together, suggest that primaquine may affect P-450 and mixed-function oxidase activity by inhibition of protein synthesis. The characteristic rapid turnover rates of P-450 isozymes may predispose these haemoproteins to the toxic effects of primaquine whereas those haemoproteins that turn over less rapidly, such as cytochrome b5, appear to be less susceptible. Microsomal haem oxygenase activity may be elevated after primaquine administration since lowered haemoprotein requirements for haem could result in excess haem levels within the hepatocyte.     

Involvement of the macrolide antibiotic inducible cytochrome P-450 LM3c in the metabolism of midazolam by microsomal fractions prepared from rabbit liver

This report characterizes the cytochrome P-450 isozyme involved in midazolam metabolism. This study was undertaken into liver microsomal fractions prepared from untreated rabbits or animals treated with drugs known to specifically induce various cytochrome P-450 isozymes such as form LM2 by phenobarbital, LM4 and LM6 by 3-methylcholanthrene and beta-naphthoflavone, LM3a by ethyl alcohol and acetone, and LM3c by macrolide antibiotics (rifampicin, erythromycin and triacetyloleandomycin). Among this library of characterized microsomal preparations, only those obtained from macrolide antibiotic-treated rabbits exhibited a Type I binding spectrum upon addition of midazolam (Ks = 3.2-5.3 micrograms/ml; 10.6-17.5 microM) and significantly metabolized midazolam to its various hydroxylated metabolites (Km = 2.52 +/- 0.22 micrograms/ml; 8.32 +/- 0.73 microM and Vmax = 20 micrograms metabolites formed/min/mg proteins; 66 nmoles metabolites formed/min/mg proteins). The following observations further confirmed the specific involvement of the cytochrome P-450 LM3c isozyme: (i) only anti-cytochrome P-450 LM3c isozyme antibodies intensively inhibited midazolam metabolism, (ii) incubation of microsomes, prepared from TAO-treated rabbits, with midazolam in the presence of potassium ferricyanide which restored the functional cytochrome P-450 LM3c isozyme, increased midazolam metabolism to a similar extent, and (iii) in the presence of Cyclosporin A, a specific substrate of the rabbit cytochrome P-450 LM3c isozyme, midazolam metabolism was inhibited in a concentration-dependent manner. These data demonstrated that the rabbit cytochrome P-450 LM3c isozyme was predominantly involved in midazolam metabolism.     

Induction of cytochrome P-450 mRNA in rainbow trout: in vitro translation and immunodetection

The time course of induction of the rainbow trout microsomal hepatic monooxygenase (MO) system was examined by determination of levels of mRNA and corresponding levels of catalytic activity. Animals were pretreated with beta-naphthoflavone (beta-NF, ip, 100 mg/kg) and terminated at 0, 2, 6, 18, and 48 hr postinjection. Levels of mRNA were determined by immunoprecipitation of in vitro translation products. Levels of mRNA coding for the cytochrome P-450 LM4b isozyme were maximally increased (13-fold) at 18 hr and had decreased almost to pretreatment levels by 48 hr post-treatment. This was in contrast to the catalytic activity in which ethoxyresorufin-O-deethylase (EROD) and ethoxycoumarin-O-deethylase (ECOD) were significantly elevated at both 18 hr (25- and 5-fold, respectively) and 48 hr (46- and 8-fold, respectively). Pretreatment with beta-NF (ip, 100 mg/kg) or 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB, ip, 150 mg/kg) for 18 hr resulted in significant differences in levels of mRNA in only the beta-NF-treated group. The LM2 P-450 isozyme could not be detected by immunoprecipitation with anti-LM2 IgG in trout treated with these same inducers. The results suggest a difference between the time course of induction of the mRNA for cytochrome P-450 LM4b isozyme and the induction of catalytic activity. Under the detection system utilized, the results suggest that the phenobarbital-like inducer, 6-CB, does not induce cytochrome activity nor does it induce the mRNA for cytochrome P-450 LM4b isozyme.     

Differences in the duration of the enhancement of liver mixed-function oxidase activities in ethanol-fed rats after withdrawal

Liver microsomal mixed-function oxidase activities were determined in female Sprague-Dawley rats after 3 weeks of ethanol feeding and for up to 10 days after withdrawal. Ethanol (36% of total calories) was administered in a high fat liquid diet and was replaced isocalorically by carbohydrates in controls. Chronic ethanol feeding similarly enhanced both microsomal cytochrome P-450 content and benzphetamine N-demethylase activity, per mg of protein, and resulted in a disproportionate increase in both aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities. A 6- to 7-day withdrawal period was apparently necessary for the overall disappearance of these effects of ethanol. Marked differences, however, were seen in the time courses of return of these variables to control levels, as also indicated by changes, during this period and specially during the first 24 hr after withdrawal, in the apparent molar activity of the microsomal fraction with the three substrates tested. The results were interpreted as indicating that the distinct ethanol-inducible cytochrome P-450 isozyme, with a high specific activity toward aniline, undergoes a very rapid turnover in liver microsomes. Induction of another form of cytochrome P-450, differing from the former by its slower turnover rate, would explain the induction by ethanol of 7-ethoxycoumarin O-deethylase activity. The withdrawal of ethanol was followed by a rapid but transient increase in benzphetamine N-demethylase activity above the ethanol-induced level, at a time when other activities were rapidly declining. This could suggest that the microsomal content of other cytochrome P-450 isozyme(s), with high specific activity toward this substrate, would also be temporarily altered during ethanol withdrawal. Important alterations in microsomal cytochrome P-450-dependent mixed-function oxidase activities occurred during the initial 24-hr period of withdrawal, even in the absence of a change in microsomal cytochrome P-450 content, indicating that the effects of chronic ethanol ingestion on hepatic drug-metabolizing enzyme activities may also be highly dependent on the proximity of ethanol intake.     

Inhalation pharmacokinetics of carbon tetrachloride in rats based on arterial blood:inhaled air concentration ratios

To estimate the rate of CCl4 metabolism in vivo by using an inhalation pharmacokinetic approach based on arterial blood:air concentration ratios, the blood CCl4 concentrations (Cart) at the end of 5-hr exposure to varying concentrations of CCl4 in inhaled air (Cinh) were determined in male, naive rats and in rats pretreated with po administration of 100 or 200 microliters CCl4/100 g body weight 24 hr before exposure. Hepatic cytochrome P-450 content during and at the end of exposure was also determined. The biphasic nature of the Cart-Cinh curve for naive rats, with a transition at Cinh of about 100 ppm, indicated that CCl4 metabolism is perfusion-limited below 100 ppm and is saturated above 100 ppm. In 100 microliters CCl4-pretreated rats, Cinh at the transition point decreased from 100 to 50 ppm; this percentage decrease was consistent with the decreased cytochrome P-450 content induced by administration of 100 microliters CCl4. In 200 microliters CCl4-pretreated rats, where CCl4 metabolizing enzyme activity was completely inhibited, the Cart-Cinh curve gave a single line with a shallower slope than that of the steeper part of the curve for naive rats, reflecting a loss of cytochrome P-450 content during exposure. The curves of calculated uptake rate showed continued uptake in completely inhibited rats, representing the contribution of fat loading only. The rate of metabolism was approximated by the uptake rate for naive rats minus that for 200 microliters CCl4-pretreated rats, and decreased gradually with increasing Cinh over the range of saturable metabolism. From this rate curve, Vmax and Km for naive rats were 2.7 mg/kg/hr and of the order of 0.3 mg/liter, respectively. The gradual decrease in the rate of metabolism could be interpreted in terms of the rapid loss of cytochrome P-450 content. The Vmax for 100 microliters CCl4-pretreated rats decreased by about 57%, which was in good agreement with the decrease of cytochrome P-450 content. These experiments suggest the usefulness and validity of this approach for studying metabolism of a volatile compound.     

Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a

The hydroxylation of p-nitrophenol to 4-nitrocatechol was investigated using rabbit hepatic microsomes and six purified isozymes of cytochrome P-450. The microsomal activity was maximal at pH 6.8 and at 100 microM p-nitrophenol. At higher substrate concentrations inhibition was observed. At pH 6.8 and 100 microM p-nitrophenol, isozyme 3a exhibited the highest activity of the purified isozymes: 3.4-fold more active than isozyme 6, and 8-fold more active than isozymes 2 and 4. The isozyme 3a-catalyzed hydroxylation reaction was stimulated 2.4-fold by the addition of a 4:1 ratio of cytochrome b5/P-450. At optimal concentrations of cytochrome b5, isozyme 3a was 8- to 9-fold more active than isozymes 2 and 6 and 20-fold more active than isozyme 4. Under the same conditions, isozyme 3a-catalyzed butanol oxidation was inhibited 40%. Antibodies to isozyme 3a inhibited greater than 95% of the p-nitrophenol hydroxylase activity of microsomes from untreated or from ethanol- or acetone-treated rabbits. The microsomal hydroxylase activity was linearly correlated with the microsomal concentration of isozyme 3a (correlation coefficient of 0.94) and had an intercept near zero. The results from reconstitution, antibody inhibition, and correlation experiments indicate that isozyme 3a is the principal catalyst of rabbit microsomal p-nitrophenol hydroxylation. The ability of the ethanol-inducible isozyme to catalyze catechol formation may be important in the ethanol-enhanced toxicity of aromatic compounds such as benzene.     

Selective induction of renal microsomal cytochrome P-450-linked monooxygenases by 1,1-dichloroethylene in mice

Intraperitoneal administration of a single dose of 1,1-dichloroethylene (DCE) to C57 B1/6N mice (125 mg/kg) caused a selective 6- to 10-fold increase in renal microsomal 7-ethoxyresorufin O-deethylase ( EROD ) and 7-ethoxycoumarin O-deethylase ( ECOD ), without affecting benzo[a]pyrene hydroxylase activity (AHH) or total microsomal cytochrome P-450 content. The observed increases did not result from in vitro activation of the enzymes or from any analytical artifact. Moreover, studies with actinomycin D and cycloheximide demonstrated that the increases resulted from de novo enzyme synthesis. Maximal enzyme induction was observed after a DCE dose of approximately 125 mg/kg, and the induced enzyme decayed rapidly, returning to control levels in about 3 days. Compared to female mice, male mice had higher basal levels of renal EROD and ECOD and were more responsive to the inductive effects of DCE; this correlated with corresponding differences in microsomal cytochrome P-450 levels. Starvation of mice for 24 or 48 hr increased renal EROD and ECOD activities in both male and female mice, but not the extent observed after DCE. The present results support the view of multiple renal cytochrome P-450 isozymes.     

Inflammation, an inducer of metallothionein, inhibits carbon-tetrachloride-induced hepatotoxicity in rats.

Inflammation, induced by turpentine (0.1 ml i.m.), protected against carbon tetrachloride (CCl4)-induced hepatotoxicity based on serum activities of sorbitol dehydrogenase. Inflammation was confirmed by elevated serum ceruloplasmin activities, and was associated with high hepatic levels of metallothionein, a zinc protein proposed to protect against CCl4-induced injury. Inflammation suppressed cytochrome P-450 activities, but this was not associated with protection against CCl4-promoted liver microsomal injury as assessed by glucose-6-phosphatase activity loss. Thus, protection against plasma membrane injury did not result primarily from depressed microsomal activation of CCl4. Each effect of inflammation reported here resembled effects of zinc injections. This similarity strengthens the hypothesis that metallothionein protects against CCl4-induced hepatic plasma membrane injury.     

Imipramine prevention of carbon tetrachloride-induced liver necrosis at late states of the intoxication process

Imipramine administration (50 mg kg-1, i.p.) to Sprague-Dawley male rats (240-290 g) 6 or 10 h after CCl4 (1 ml kg-1, i.p.) partially prevents liver necrosis induced by the hepatotoxin. When imipramine is given 30 min before CCl4, it inhibits in part the CCl4-induced lipid peroxidation and the covalent interactions of reactive metabolites with microsomal lipids or proteins and partially prevents CCl4-induced cytochrome P-450 destruction, but not glucose 6 phosphatase activity depression. Imipramine administration prior to CCl4 does not modify levels of the hepatotoxin reaching the liver or the body temperature of CCl4 treated animals. Early preventive effects of imipramine on cytochrome P-450, might be attributed to inhibition of covalent interactions of reactive metabolites. The hypothesis that imipramine exerted late preventive effects by interfering with calcium deleterious effects or by modulation of protein and phospholipid synthesis or degradation is analyzed.     

Early destruction of cytochrome P-450 in testis of carbon tetrachloride poisoned rats

There is 20--36 percent decrease in the microsomal cytochrome P-450 (P450) content of the testes 3 or 6 h after CCl4 administration to Sprague--Dawley male rats. Irreversible binding of CCl4 metabolites to testicular microsomal lipids is observed as early as 3 h while CCl4 induced lipid peroxidation does not occur within the first 6 h of poisoning. Results suggest that reactive metabolites rather than lipid peroxidation is involved in P-450 destruction in the testes.     

The oxidation of acrolein by rat liver aldehyde dehydrogenases. Relation to allyl alcohol hepatotoxicity

The oxidation of acrolein by aldehyde dehydrogenase was studied in several subcellular fractions of rat liver by measuring acrolein-dependent production of NADH from NAD+. Mitochondrial and cytosolic fractions each contained two aldehyde dehydrogenase activities with Km values for acrolein of 0.4-0.7 mM and 0.015-0.025 mM. Microsomes demonstrated only a high Km (1.5 mM) activity. The low Km activities of mitochondria and cytosol differed in their sensitivity to inhibition by chloral hydrate and in their response to 1 mM MgCl2 (activation vs. inhibition). The metabolism of acrolein by low Km aldehyde dehydrogenase activities was markedly depressed in mitochondrial or cytosolic fractions from rats pretreated with cyanamide (2 mg/kg for 1 hr) or disulfiram (100 mg/kg for 24 hr). The effect of aldehyde dehydrogenase inhibition on allyl alcohol toxicity was determined by pretreating rats with cyanamide or disulfiram prior to treatment with allyl alcohol. Hepatotoxicity was assessed on the basis of elevated serum alanine aminotransferase and sorbitol dehydrogenase activities and the loss of microsomal cytochrome P-450. Pretreatment with the aldehyde dehydrogenase inhibitors enhanced the hepatotoxicity of allyl alcohol in both male and female rats. The results suggest that acrolein metabolism by rat liver aldehyde dehydrogenase isozymes is important for the inactivation of allyl alcohol-derived acrolein.     

Tolbutamide 4-hydroxylase activity of human liver microsomes: effect of inhibitors.

Eight samples of human liver have been characterised for microsomal protein content, cytochrome P-450 content, tolbutamide 4-hydroxylase and ethinyloestradiol 2-hydroxylase activities. Cytochrome P-450 content correlated significantly with ethinyloestradiol 2-hydroxylase activity but not with tolbutamide 4-hydroxylase activity. There was no significant correlation between ethinyloestradiol 2-hydroxylase and tolbutamide 4-hydroxylase activities. The maximum tolbutamide 4-hydroxylase activity was 0.45 nmol min-1 mg-1 microsomal protein, with a Km value of 74 microM. A number of compounds were tested for their ability to inhibit tolbutamide metabolism. All the compounds showing inhibition were either non-competitive or mixed non-competitive inhibitors of tolbutamide 4-hydroxylation. These studies suggest that tolbutamide is metabolised by an isozyme of cytochrome P-450 which appears to be distinct from those isozymes metabolising many other drugs.     

The cytochrome P-450 monooxygenase system of rabbit lung enzyme components, activities, and induction in the nonciliated bronchiolar epithelial (Clara) cell, alveolar type II cell, and alveolar macrophage

Enzyme components and activities of the cytochrome P-450 monooxygenase system in microsomal preparations from the Clara cell, alveolar type II cell, and alveolar macrophage fractions isolated from lungs of untreated rabbits and rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin were examined. Results are compared to those obtained with microsomal preparations from whole lung. Concentrations of cytochrome P-450 isozymes 2 and 5 and NADPH-cytochrome P-450 reductase activities were higher in preparations from Clara cell fractions than in preparations from type II cell fractions or whole lung. For the most part, however, differences among these preparations were 2-fold or less. Microsomal preparations from the macrophage fraction contained low or undetectable levels of cytochrome P-450 isozymes but relatively high levels of cytochrome P-450 reductase activity. The concentration of cytochrome P-450 isozyme 6, in contrast to those of isozymes 2 and 5, was found to be highest in microsomal preparations from whole lung. Treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin increased the concentrations of isozyme 6 in preparations from the Clara and type II cell fractions and from whole lung about 20-fold. In contrast, the content of isozyme 6 in preparations from the macrophage fraction increased greater than 90-fold. In all cases, induction of isozyme 6 resulted in substantial increases in the O-deethylation of 7-ethoxyresorufin and only minor increases in the hydroxylation of benzo(a)pyrene. Activities per unit of isozyme 6, following induction, were similar in all preparations, and we estimate that less than 20% of the potential activity of isozyme 6 is expressed with benzo(a)pyrene and greater than 40% with 7-ethoxyresorufin. These similarities exist in spite of significant differences among the preparations from different fractions in the ratios of isozyme 6 to NADPH-cytochrome P-450 reductase.     

Human hepatic cytochrome P-450 composition as probed by in vitro microsomal metabolism of warfarin

Human liver microsomal fractions from 27 renal donors (tissue obtained post mortem) and from six cancer patients (tissue obtained during surgery) were used to investigate human hepatic cytochrome P-450 isozyme compositions. In vitro microsomal metabolism of the R and S enantiomers of warfarin to dehydrowarfarin and 4'-, 6-, 7-, 8-, and 10-hydroxywarfarin is catalyzed by cytochrome P-450 isozymes and was used as the basis for evaluating similarities and differences between human cytochrome P-450 isozyme compositions. The mean hepatic cytochrome P-450 concentration from postmortem samples was not significantly different from that of surgical patients (0.51 +/- 0.16 vs. 0.35 +/- 0.14 nmol/mg protein), but the NADPH-cytochrome P-450 reductase activity of the former was significantly higher than that of the latter (141 +/- 56 vs. 29 +/- 6 nmol cytochrome c reduced/min/mg protein). In general, the microsomal preparations were overall stereoselective for R warfarin metabolism. The stereoselectivities for formation of the individual metabolites of the R enantiomer were 6-, 8-, and 10-hydroxywarfarin and the S enantiomer were 4'- and 7-hydroxywarfarin. Of the 33 microsomal preparations, 21 exhibited qualitatively similar warfarin metabolite profiles with 6R- and 7S-hydroxywarfarin having the highest formation rates. Some of the preparations exhibited markedly different metabolite profiles, the most notable having 10R-hydroxywarfarin as the major metabolite. Based on the known warfarin metabolite profiles of five purified cytochrome P-450 isozymes, the isozyme composition of the microsomes can be estimated. The majority of the microsomal preparations apparently had similar isozyme compositions but some preparations were markedly different.     

Trichloroethylene inhibits aldehyde dehydrogenase only for aliphatic aldehydes of short chains in rats

The effects of trichloroethylene (TCE) administration on aldehyde dehydrogenase (ALDH) and cytochrome P450 isozymes were studied in rats and compared with those of methanol. Intragastric administration of TCE to rats at 0.05 or 0.2 ml/kg for 1 week significantly inhibited ALDH activity for aliphatic aldehydes of short chains in the mitochondrial and cytosolic fractions of rat liver, respectively, but had no effect on the activity for long chain aliphatic aldehydes. ALDH activity catalyzing the metabolism of some aromatic aldehydes was even induced by TCE. Microsomal ALDH activity was not decreased by TCE treatment. A kinetic study showed that the low-Km isozyme of ALDH for propionaldehyde in mitochondrial and cytosolic fractions was inhibited by TCE treatment. Addition of TCE, trichloroethanol or trichloroacetic acid to the in vitro assay system did not affect the activity for acetaldehyde, but chloral hydrate at 0.02 mM decreased the activity by 42 and 35% in cytosol and the 700 x g supernatant, respectively. Methanol treatment, on the other hand, had no effect on any ALDH activity. Both TCE and methanol significantly induced CYP2E1 in rat liver. The combined effects of TCE on ALDH and cytochrome P450 may account for the degreasers' flush. Exposure to TCE and methanol may result in a change in the metabolism and toxicity of other chemicals.