Tetrachloroethylene metabolism by the hepatic microsomal cytochrome P-450 system

The interaction of tetrachloroethylene with hepatic microsomal cytochromes P-450 has been investigated using male Long-Evans rats. The spectral binding of tetrachloroethylene to cytochromes P-450 in hepatic microsomes from uninduced rats was characterized by a K_s of 0.4 mM. The K_s was not affected by phenobarbital induction, but was increased following pregnenolone-16α-carbonitrile induction. The K_M of 1.1 mM, calculated for the conversion of tetrachloroethylene to total chlorinated metabolites by the hepatic microsomal cytochrome P-450 system, was decreased by phenobarbital induction and increased by pregnenolone-16α-carbonitrile induction. The maximum extents of binding (ΔA_max) and metabolism (V_max) of tetrachloroethylene were increased by both phenobarbital and pregnenolone-16α-carbonitrile induction. Induction with β-naphthoflavone was without effect on any of the above parameters. The effects of the inducing agents on tetrachloroethylene-stimulated CO-inhibitable hepatic microsomal NADPH oxidation followed the same trend as their effects on V_max for the metabolism of tetrachloroethylene, although in all cases the extent of NADPH oxidation was 5- to 25-fold greater than the extent of metabolite production. The inhibitors of cytochromes P-450, viz. metyrapone, SKF 525-A, and CO, inhibited the hepatic microsomal binding and metabolism of tetrachloroethylene. Free trichloroacetic acid was found to be the major metabolite of tetrachloroethylene from the hepatic microsomal cytochrome P-450 system. Neither 2.2,2-trichloroethanol nor chloral hydrate was produced in measurable amounts from tetrachloroethylene. A minor but significant metabolite of tetrachloroethylene by cytochrome P-450 was the trichloroacetyl moiety covalently bound to components of the hepatic microsomes. Incubation of tetrachloroethylene. an NADPH-generating system. EDTA and hepatic microsomes was without effect on the levels of microsomal cytochromes P-450, cytochrome b₅, beme, and NADPH-cytochrome c reductase. It is concluded that hepatic microsomal cytochromes P-450 bind and metabolize tetrachloroethylene. The major product of this interaction is trichloroacetic acid, which is also the major urinary metabolite of tetrachloroethylene in vivo. The forms of cytochrome P-450 that bind and metabolize tetrachloroethylene include those induced by pregnenolone-16α-carbonitrile and by phenobarbital. Cytochrome P-448. which was induced in rat liver by β-naphthoflavone, does not appear to spectrally bind or metabolize tetrachloroethylene. The metabolism and toxicity of tetrachloroethylene are considered in relation to other chlorinated ethylenes.     

Differential effects of cyclosporin on hepatic and renal heme, cytochrome P-450 and drug metabolism. Possible role in nephrotoxicity of the drug

Treatment of rats with 25 or 50 mg/kg cyclosporin A for 6 days elicited vastly different responses in hepatic and renal heme and drug metabolism activities. In the liver, cytochrome P-450 concentration was decreased significantly (to 70-75% of the control). This was accompanied by a marked reduction in benzo[a]pyrene hydroxylase activity (to 20-28% of the control). Aniline hydroxylation was also decreased, but to a lesser extent (to 77% of the control). In contrast, in the kidney cytochrome P-450 concentration was significantly increased to (145-170% of the control), along with a modest decrease in benzo[a]pyrene hydroxylation activity. In this organ, the concentration of porphyrins was severely decreased (to 30% of the control). Also, the activities of delta-aminolevulinate (ALA) synthetase and ALA dehydratase, as well as that of heme oxygenase, were inhibited. It is suggested that in the kidney the inhibition of degradation, rather than an enhanced rate of synthesis of the heme molecule, contributes to the observed increase in cytochrome P-450 concentration. In the liver, the decrease in the cytochrome concentration could not be explained in terms of an alteration in the rate of heme biosynthesis or degradation. Therefore, the observed decrease in cytochrome P-450 concentration could reflect the direct inactivation of the hemoprotein or regulation of apoprotein production by cyclosporin and/or its metabolite(s). The possible relevance of the observations to cyclosporin nephrotoxicity is discussed.     

Mixed-function oxidase system induction and propylene hepatotoxicity

Propylene is hepatotoxic to male Charles River COBS Sprague-Dawley rats pretreated with polychlorinated biphenyls (PCB: Aroclor 1254). Four-hour inhalation exposure to 50,000 ppm propylene increased liver weight/body weight ratios and elevated serum enzyme activities in PCB-pretreated animals. Hepatic microsomal cytochrome P-450 content of PCB-pretreated rats dropped profoundly during propylene exposure and remained depressed for at least 24 h. In addition, PCB-pretreated, propylene-exposed rats exhibited a decrease in the specific activity of hepatic microsomal aniline hydroxylase. However, there was no change in activities of either hepatic microsomal aminopyrine demethylase or glucose-6-phosphatase. Propylene exposure of rats pretreated with beta-naphthoflavone (BNF), phenobarbital (PB), or a mixture of BNF and PB was not hepatotoxic. However, there was, in these animals, a substantial decline in hepatic microsomal cytochrome P-450 levels 24 h after the start of propylene exposure. Hence, the propylene-dependent process resulting in hepatic cytochrome P-450 destruction is qualitatively or quantitatively different from the process that causes acute hepatotoxicity. Preexposure fasting had no effect on the hepatotoxicity resulting from a 4-h exposure of PCB-pretreated rats to 50,000 ppm propylene. Administration of SKF-525A to PCB-pretreated rats immediately prior to propylene exposure completely prevented elevations in serum enzyme activities and liver weight/body weight ratios. In vitro incubation of hepatic microsomes prepared from either BNF-, PB-, or PCB-pretreated rats with an atmosphere of 20% propylene/80% air produced in NADPH-dependent decrease in cytochrome P-450 content. These results suggest that PCB pretreatment is a prerequisite for propylene hepatotoxicity in the rat. Cytochrome P-450-dependent bioactivation of propylene is associated with this hepatotoxicity, but further studies are needed to characterize the mechanism of the PCB-propylene interaction.     

Effects of pentachlorophenol on hepatic drug-metabolizing enzymes and porphyria related to contamination with chlorinated dibenzo-p-dioxins and dibenzofurans

The hepatic effects of technical and pure grade pentachlorophenol were investigated in female rats fed 20, 100 and 500 ppm of each for 8 months. Technical pentachlorophenol was contaminated with 8 ppm hexa-, 520 ppm hepta-, and 1380 ppm octachlorodibenzodioxtns and with 4 ppm tetra-, 42 ppm penta-, 90 ppm hexa-, 1500 ppm hepta- and 200 ppm octachlorodibenzofurans; pure pentachlorophenol contained less than 0.1 ppm of each of these contaminants. Technical pentachlorophenol produced hepatic porphyria and increased hepatic aryl hydrocarbon hydroxylase activity, glucuronyl transferase activity, liver weight, cytochrome P-450 and microsomal heme, but not N-demethylase activity. The peak of the CO-difference spectrum of cytochrome P-450 was shifted to 448 nm, and there was a dramatic increase in the 455-430 ratios of the ethyl isocyanide difference spectrum. The enzyme changes were observed at 20 ppm of technical pentachlorophenol. Porphyria occurred at 100 and 500 ppm. Pure pentachlorophenol had no significant effect on aryl hydrocarbon hydroxylase activity, liver weight, cytochrome P-450, microsomal heme, the ethyl isocyanide difference spectrum or N-demethylase activity at any dose level, but did increase glucuronyl transferase at 500 ppm. In contrast, both pure and technical pentachlorophenol decreased body weight gain comparably at 500 ppm. It is concluded that technical pentachlorophenol produces a number of liver changes which cannot be attributed to pentachlorophenol itself, but are consistent with the effects of biologically active chlorinated dibenzo-p-dioxins and dibenzofurans.     

Induction of hepatic mixed-function oxidase enzymes in adult and neonatal mice by kepone and mirex.

The effects of 1, 10, and 50 ppm dietary kepone on hepatic mixed-function oxidases of adult male mice were measured. After 14 days dose-dependent increases in liver weight, hepatic microsomal protein, cytochrome P-450, Type I and Type II ligand binding to cytochrome P-450 and O- and N-demethylation of p-nitroanisole and aminopyrine were observed. Induction was approximately two-fold for all parameters measured at the 50-ppm level. The results obtained suggest that kepone is a nonspecific inducer, the no-effect level for induction based on O-demethylase activity in mice being less than 10 ppm. Body weight gain decreased in mice treated with 50 ppm kepone for 14 days. The effects of kepone and mirex fed to the mother after parturition on hepatic tissue of male and female neonatal mice were also measured. It is suggested that the increases observed in O- and N-demethylation of p-nitroanisole and aminopyrine in hepatic tissue from neonates result from the transfer of kepone and mirex through the milk. Induction in neonates was greater than that observed in adult male mice treated with kepone.     

Alterations in hepatic heme and cytochrome P-450 metabolism in murphy-sturm lymphosarcoma-bearing rats implications for drug metabolism

Previous studies have shown that tumor-bearing rats have significantly decreased hepatic microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity with, consequently, significantly decreased capacity for microsomal oxidative drug metabolism. Subsequent investigations have revealed that the rates of hepatic cytochrome P-450 apo-protein synthesis and degradation are decreased significantly and hepatic microsomal heme oxygenase activity is increased significantly in rats bearing an extra-hepatic tumor. Further studies have been done to attempt to clarify the pathogenesis and significance of these observations. Hepatic delta-aminolevulinic acid (ALA) synthetase activity in male Wistar rats declined to a nadir of 162 ± 34 (S.E.) pmoles ALA per mg protein per 30 min 6 days following i.m. transplantation of Murphy-Sturm lymphosarcoma (vs control = 218 ± 36 pmoles per mg per 30 min). Turnover of ³H-labeled heme in microsomal CO-binding particles (i.e. cytochrome P-450 heme) was increased significantly 8 days following i.m. transplantation of Murphy-Sturm lymphosarcoma with a T_{$\text{1}\text{2}$} of 5.5 hr for the fast phase of hepatic cytochrome P-450 heme disappearance in tumor-bearing rats as compared with a T_{$\text{1}\text{2}$} of 7 hr in control rats. Hepatic cytochrome P-450 apo-protein concentration was slightly, but not significantly, increased in Murphy-Sturm lymphosarcoma-bearing rats as compared with control rats up to 10 days following tumor transplantation. These results suggest that, in Murphy-Sturm lymphosarcoma-bearing rats, decreased microsomal cytochrome P-450 concentration is the result of both decreased cytochrome P-450 apo-protein synthesis and increased cytochrome P-450 heme turnover. Apo-cytochrome P-450 concentration was not appreciably altered because increased cytochrome P-450 heme turnover and decreased cytochrome P-450 apo-protein degradation were balanced by decreased cytochrome P-450 apo-protein synthesis. Because of their effects on cytochrome P-450 concentration and action, these alterations in heme and hemoprotein metabolism may be of importance in regulating oxidative drug metabolism in the tumor-bearing state.     

Effect of acute and repeated selenium treatment on hepatic monooxygenase enzyme activity in male rats

The effect of sodium selenite administered acutely or repeatedly on the biochemical components of the hepatic microsomal monooxygenase enzyme system was examined in male rats. 72 h following acute administration of selenium (2.4 mg Se/kg, i.p.), there was a significant decrease in ethylmorphine-N-demethylase activity and cytochrome P-450 levels but no change in aniline hydroxylase or NADPH cytochrome c reductase activity. Following repeated administration of selenite in the drinking water (1, 2, or 4 ppm Se) for 30 days, there was no alteration in any of the parameters measured. Following the in vitro additions of selenite to microsomes obtained from untreated rats, ethylmorphine-N-demethylase and aniline hydroxylase activities were inhibited at selenium concentrations of 10(-4) M or greater, but the inhibition achieved was less than 50%. No alterations in cytochrome P-450 levels were observed. These results indicate that selenium is a rather weak, indirect, and substrate-specific inhibitor of the hepatic monooxygenase enzyme system.     

Regulation of renal cytochrome P-450. Effects of two-thirds hepatectomy, cholestasis, biliary cirrhosis and post-necrotic cirrhosis on hepatic and renal microsomal enzymes

The possibility of a relationship between hepatic and renal cytochrome P-450 contents was assessed in rats with liver disease. In rats killed 3 days after two-thirds hepatectomy (a model for hepatocellular insufficiency), the total microsomal cytochrome P-450 content of the whole liver was decreased by 60% as compared to that in control rats; renal cytochrome P-450 was increased by 30% while the 7-ethoxycoumarin deethylase activity of kidney microsomes was increased by 80%. In rats killed 7 days after bile duct ligation (a model for cholestasis) or 35 days after bile duct ligation (a model for biliary cirrhosis), hepatic cytochrome P-450 was decreased by 60% and 45%, respectively, while renal cytochrome P-450 content was increased by 50% and 150%, respectively. In contrast, in rats killed 15 days after the last dose of carbon tetrachloride, 1.3 ml/kg twice weekly for 3 months (a model for post-necrotic cirrhosis), both hepatic and renal cytochrome P-450 contents remained unchanged. Phenobarbital (80 mg/kg daily for 3 days) was a poor inducer of renal cytochrome P-450 in sham-operated rats but became a potent inducer of renal cytochrome P-450 in rats with two-thirds hepatectomy. We conclude that renal cytochrome P-450 is increased in three models in which hepatic cytochrome P-450 contents are decreased (two-thirds hepatectomy, cholestasis and biliary cirrhosis), but remains unchanged in a model of severe liver pathology, in which hepatic cytochrome P-450 content is not modified (late, post-necrotic cirrhosis). The hypothetical role of endogenous inducer(s) is discussed.     

Influence of estradiol and testosterone on cytochrome P-450 and monooxygenase activity in immature brook trout,Salvelinus fontinalis

Levels of hepatic microsomal cytochrome P-450 were depressed by administration of estradiol-17β and were elevated by administration of testosterone in both male and female juvenile brook trout (Salvelinus fontinalis). Treatment-associated changes in the levels of other microsomal electron transfer components in liver did not reflect the changes in cytochrome P-450 content and were also distinct from the changes in these components in kidney. Electrophoretic analysis of hepatic microsomes revealed that estradiol treatment reduced the amounts of several proteins including some heme-staining protein at 56,000 daltons, possibly containing cytochrome P-450. Hepatic microsomal benzo[a]pyrene hydroxylase and the response to 7,8-benzoflavone in vitro were affected little by steroid treatment, and ethoxyresorufin O-deethylase activity could not be detected in any of the samples. Hepatic microsomes metabolized testosterone to a suite of products including 6β-hydroxytestosterone (the major metabolite) and 16β-hydroxytestosterone, plus as many as eleven unknown metabolites. Estradiol-17β treatment depressed the rates of testosterone metabolism and particularly the rates of 6β-hydroxylase activity but did not affect 16β-hydroxylase activity. Both activities were largely unaffected by testosterone. The results are consistent with the idea that both androgens and estrogens regulate the levels of hepatic cytochrome P-450 in brook trout and that the effect, at least of estradiol-17β, involves regulation of forms that function in specific hydroxylation of testosterone. The significance of these effects and whether factors additional to steroids are involved in this regulation of hepatic cytochromes P-450 in fish remain to be established.     

Hepatic drug metabolism and haem biosynthesis in lead-poisoned rats

1 Pretreatment of rats with intraperitoneal injections of lead was shown to result in a depression of the microsomal mixed function oxidase system, as assessed by a decrease in hepatic microsomal P-450 and b5 content and by a decrease in the activity of the enzymes aniline hydroxylase and aminopyrine demethylase. Lead had a more marked effect on cytochrome P-450 than b5. 2 The activity of the rate-limiting enzyme of haem biosynthesis, delta-aminolaevulinic acid synthase, was inversely correlated with the microsomal cytochrome P-450 content. 3 The activity of the haem biosynthetic enzymes delta-aminolaevulinic acid dehydratase, coproporphyrinogen oxidase and ferrochelatase were decreased by increasing lead pretreatment. 4 The activity of the haem catabolic enzyme, haem oxygenase, was increased by lead pretreatment.     

MULTIGENERATIONAL STUDY OF THE EFFECTS OF CONSUMPTION OF PCB-CONTAMINATED CARP FROM SAGINAW BAY,LAKE HURON,ON MINK. 2. LIVER PCB CONCENTRATION AND INDUCTION OF HEPATIC CYTOCHROME P-450 ACTIVITY AS A POTENTIAL BIOMARKER FOR PCB EXPOSURE

This study examined the effect of polychlorinated biphenyls (PCBs) from Saginaw Bay (Lake Huron) carp on the hepatic cytochrome P-450 activity in mink (Mustela vison). Hepatic cytochrome P-450 activities are of interest for their possible use as biomarkers to indicate consumption and biological effects of PCBs in the environment. Adult mink were fed diets containing ocean fish (control diet, 0.0 ppm) or Saginaw Bay carp to provide 0.25, 0.5, or 1.0 ppm PCBs. Mink were bred after 3 mo of exposure, and half of the parental mink (P1) and kits (F1-1) previously consuming diets containing Saginaw Bay carp were switched to control diet at weaning of the F1-1 kits. P1 and F1-1 mink were then bred within their age and dietary groups after 15 mo of exposure, to produce the second-year F1(F1-2) and F2 kits. Mink were killed when the new kits were weaned. Transfer of half the animals to the control diet examined whether the effects of the PCB containing diet on hepatic cytochrome P-450 activity were permanent. Continual exposure to diets containing PCBs from Saginaw Bay carp induced cytochrome P-450 activity in a generally dose-dependent manner. Cytochrome P-450 activity was not different from untreated controls in animals switched to the control diet from the PCB-containing diet. The response of cytochrome P-450IA1 (EROD) activity in a dose-dependent manner and the lack of induction after transfer to noncontaminated diets suggest that this hepatic enzyme activity is a potential biomarker for current exposure to PCBs and other similar cytochrome P-450 inducers.     

Effects of model traumatic injury on hepatic drug metabolism in the rat. VI. Major detoxification/toxification pathways

A previously validated small mammal trauma model, hindlimb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on two of the major hepatic enzymes of detoxification, glutathione S-transferase and epoxide hydrolase. Hepatic cytosolic glutathione S-transferase activity toward a variety of substrates showed a 26-34% decrease at 24 hr after model injury. Hepatic microsomal epoxide hydrolase activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane was diminished by 53% after model trauma. Both enzymatic activities toward styrene oxide were similarly depressed. The toxicological sequelae of these derangements were illustrated by administering a dose of styrene oxide (300 mg/kg, ip) which was below the threshold dose (350 mg/kg, ip) necessary to produce hepatotoxicity in control animals. Model trauma dramatically enhanced the hepatotoxic effects of the subthreshold dose, as well as the covalent binding of labeled styrene oxide to liver proteins. These findings indicate that traumatic injury renders the animal more susceptible to agents which are detoxified by glutathione S-transferase and epoxide hydrolase. Conversely, model trauma provided almost complete protection from the hepatotoxic effects of a standard dose (200 mg/kg, ip) of bromobenzene. This protection appeared to derive from a post-traumatic alteration of cytochrome P-450 subpopulations that decreased the formation of the potentially toxic 3,4-epoxide metabolite, despite an increase in the cytochrome P-448-mediated generation of the nontoxic 2,3-epoxide. For bromobenzene, the change in cytochrome P-450-mediated activation appeared quantitatively more significant in overall toxicity than the post-traumatic depression of detoxification pathways described above, leading to decreased toxicity in vivo. For other compounds, the combination of post-traumatic influences on cytochrome P-450/P-448 activity and epoxide hydrolase/glutathione S-transferase activities could lead to markedly enhanced toxicity.     

Specific binding of polyhalogenated aromatic hydrocarbon inducers of cytochrome P-450d to the cytochrome and inhibition of its estradiol 2-hydroxylase activity

Treatment of male Sprague-Dawley rats with 3,4,5,3',4',5'-hexabromobiphenyl (HBB) at 10 mumol/kg followed by purification of hepatic microsomal cytochrome P-450d revealed that HBB remained specifically bound to P-450d throughout purification. Binding was noncovalent since HBB was removed by extraction with dichloromethane. Although HBB induced both cytochrome P-450c and P-450d, specific immunoprecipitation of these isozymes from HBB-treated rats showed that HBB was associated only with cytochrome P-450d. Quantitation of HBB and cytochrome P-450d in microsomes from HBB-treated rats suggested a 0.9:1 ratio of HBB to cytochrome P-450d. Five other halogenated aromatic hydrocarbon inducers of cytochrome P-450d, bearing steric similarity to HBB (including 2,3,7,8-tetrachlorodibenzo-p-dioxin), were associated with cytochrome P-450d when used to induce cytochrome P-450d in rats. HBB inhibited estradiol 2-hydroxylase activity of purified cytochrome P-450d in a noncompetitive manner with an I50 of 38 nM for 50 nM P-450d whereas its noncoplanar isomer, 2,4,5,2',4',5'-hexabromobiphenyl, had an I50 over 700-fold higher. Thus certain polyhalogenated aromatic hydrocarbons, with the capacity to induce cytochrome P-450d also bind to the cytochrome when used as inducing agents and inhibit catalytic activity of the cytochrome.     

The effect of ethylene exposure on ethylene oxide in blood and on hepatic cytochrome p450 in Fischer rats.

Ethylene (74-85-1) is an important petrochemical and is produced endogenously. It is metabolized to ethylene oxide (EO) by cytochrome P450. We studied the inhibition of cytochrome P450 activity during exposure to ethylene, and verified that this inhibition was reflected in the concentration of EO in the blood. Male F344 rats were exposed to 1000, 600, and 300 ppm ethylene by nose-only inhalation for up to 6 h. Blood samples were obtained during exposure. On exposure to 600 ppm ethylene, blood EO concentration increased during the first hour of exposure and then decreased to approximately half of the peak blood concentration. A less pronounced decrease was observed at 300 ppm, and at 1000 ppm little change was observed between 10 min and 6 h of exposure. For the analysis of cytochrome P450 and isozyme-specific substrate activities, groups of four male F344 rats were removed for the collection of liver at various times after exposure to 300, 600, or 1000 ppm ethylene. At all concentrations, liver microsomal cytochrome P450 decreased during exposure. Of the various monooxygenase activities measured, 4-nitrophenol hydroxylase was the one most consistently altered, with maximal inhibition (approximately 50%) at 2 h of exposure to 1000 ppm ethylene, 4 h at 600 ppm, and 6 h at 300 ppm. Activity recovered to control levels by 6 h after exposure. Cytochrome P450 2E1 appears to be the major isoform of cytochrome P450 inhibited by exposure to ethylene, and this may explain in part the observed alteration in EO blood kinetics.     

Role of 3,5-dichlorophenyl methyl sulfone, a metabolite of m-dichlorobenzene, in the induction of hepatic microsomal drug-metabolizing enzymes by m-dichlorobenzene in rats

The increases in the hepatic microsomal aminopyrine N-demethylase activity and in the content of cytochrome P-450 produced by m-dichlorobenzene (m-DCB) occurred after increases in the hepatic concentration of 3,5-dichlorophenyl methyl sulfone, a minor metabolite. The extent of increases in aminopyrine N-demethylase activity and in the content of cytochrome P-450 at 48 hr after po administration of 200 mg/kg (1.36 mmol/kg) of m-DCB was almost equal to that 72 hr after the ip administration of 25 mumol/kg of the sulfone (Kimura et al., 1983). m-DCB in liver was not detectable at that time, and the concentration of sulfone was 63 to 70% of that 48 to 72 hr after the ip administration of 50 mumol/kg of sulfone. Administration of m-DCB (200 mg/kg) produced a significant reduction in hexobarbital sleeping time, but this reduction was less than that produced by administration of the sulfone (50 mumol/kg). The protein band patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microsomes from rats treated with the sulfone and m-DCB were similar to those of phenobarbital-treated rats but were different from those of 3-methylcholanthrene-treated rats. The sulfone showed type I interaction with the cytochrome P-450 (Ks, 0.17 mM). The sulfone was formed from the sulfide but reduction of the sulfone was not observed when it was incubated in a hepatic microsomal preparation. The pattern of induction by the sulfone and m-DCB was similar to that by phenobarbital and differed from that by 3-methylcholanthrene. From these results, 3,5-dichlorophenyl methyl sulfone is considered to be a major contributing factor of the inducing activity of m-DCB and to be a potent phenobarbital-like inducer.     

Comparison of xenobiotic biotransformation enzymes in kidney and liver of rainbow trout (Salmo gairdneri)

The microsomal cytochrome P-450 content in kidney of rainbow trout (Salmo gairdneri) was approximately 5-fold lower than the content in liver. The renal ethoxycoumarin- and ethoxyresorufin-O-deethylase activities calculated on a per-cytochrome P-450 basis were, however, found to be about 10-fold higher than the hepatic activities. The patterns of time-dependent increase and subsequent decrease of microsomal cytochrome P-450-dependent monooxygenase activities after a single injection of beta-naphthoflavone (BNF) were similar in the kidney and liver. The microsomal ethoxyresorufin- and ethoxycoumarin-O-deethylase activities were maximally induced in liver (120- and 10-fold, respectively) by a single BNF injection (50 mg/kg body wt), whereas in kidney the maximal levels of induction (135- and 21-fold, respectively) were reached after three injections with BNF. The induction of cytochrome P-450 systems was associated with synthesis of a new microsomal protein of 58,000 Da in both kidney and liver. UDP-glucuronosyl transferase activity toward p-nitrophenol was about 8-fold lower in kidney than in liver. A significant 2.5-fold elevation in microsomal UDP-glucuronosyltransferase activity was found in the kidney 14 days after a single injection with BNF (50 mg/kg). In the liver, a 2-fold increase of this activity was seen 3 days after the treatment. The results indicate that the rainbow trout kidney in addition to the liver is of great importance in biotransformation of lipophilic xenobiotics.     

Age-dependent toxicity of acorn extract in young and old male rats

Intraperitoneal administration of acorn extract of dosage levels of 200, 400 and 600 mg/kg body weight did not produce significant change in the hepatic microsomal cytochrome P-450 levels and the activities of NADPH-cytochrome c reductase, benzphetamine N-demethylase and aniline hydroxylase in young, adult rats (weighing 200-250 g), with the exception of the activity of benzphetamine N-demethylase at the 600 mg/kg dose which was decreased significantly. On the other hand, a dose of only 100 mg/kg body weight ip to old rats (weighing 400-450 g) caused significant decreases in the microsomal cytochrome P-450, benzphetamine N-demethylase and NADPH-cytochrome c reductase activities. However, there was no significant change in the activity of aniline hydroxylase in these rats, indicating selective inhibition of the microsomal enzymes and higher susceptibility of old rats than young ones to acorn toxicants. When the serum samples from the treated young rats were analyzed for sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities as markers of liver toxicity, these activities were significantly higher in the treated rats than the corresponding control values. Similar changes were noted for old rats receiving a dose of 100 mg/kg body weight of acorn extract. The results indicate that acorn extract affects old rats more than young rats as measured by its effect on liver and liver microsomal enzymes.     

Effects of inhibitors of RNA and protein synthesis on hepatic microsomal cytochrome P-450 concentration in fasted and fed rats

A number of sex-related differences in the metabolism of drugs, steroids and xenobiotics have been reported in studies on rats. Generally, male rats tend to metabolize these compounds more efficiently than females. In the studies presented here, male and female rats were fasted for 24–48 hr, and the effects of fasting on total hepatic microsomal cytochrome P-450 were examined. Hepatic cytochrome P-450, as determined by CO difference spectra, was increased significantly as a percentage of control in microsomes from fasted female rats when compared to fasted male rats. Cytochrome P-450 concentration increased from 0.57 ± 0.07 nmole/mg protein to 0.99 ± 0.08 nmole/mg protein following a 24-hr fast. In male rats, cytochrome P-450 levels were essentially unaffected by the 24-hr fast. Cytochrome b₅ concentration was not altered by fasting. When female rats were fasted for 24 hr and refed, cytochrome P-450 levels were not significantly different from cytochrome P-450 levels in continuously fed animals. Treatment of fasted female rats with the protein synthesis inhibitor ethionine, or the RNA synthesis inhibitor actinomycin D, prevented the induction of cytochrome P-450 in the fasting animal. Cytochrome P-450 concentration in fed animals was not affected significantly by either inhibitor. Induction of cytochrome P-450 by phenobarbital (PB) and 3-methylcholanthrene (MC) under fed and fasted conditions was also investigated in male and female rats. Xenobiotic-induced cytochrome P-450 concentration was significantly higher in fasted female hepatic microsomes when compared to microsomes from fed female rats. Fasting did not significantly affect xenobiotic-induced cytochrome P-450 in male rats. Our results suggest that fasting in female rats results in an increase in cytochrome P-450 which is dependent upon synthesis of RNA and protein.     

Inhalation of isopropanol: induction of activating and deactivating enzymes in rat kidney and liver. Increased microsomal metabolism of n-hexane

Rats were exposed to isopropanol by inhalation of 200,2000 and 8000 ppm for 2 weeks with a daily exposure of 6 h. A pilot group exposed to 8000 ppm was given a recovery period of 4 weeks. Kidney and liver microsomal metabolism of n-hexane was investigated in vitro concomitant with activities of cytochrome P-450 and GSH enzymes and blood concentration of isopropanol and its metabolite acetone. A dose dependent increase was observed in the formation of all metabolites of n-hexane in both organs. Of special interest was the 9%, 80% and 198% increase of the preneurotoxic metabolite 2-hexanol in kidney microsomes. Cytochrome P-450 was increased 14%, 40% and 43% in kidney after 200, 2000 and 8000 ppm, respectively, and 10% and 19% in liver at 2000 and 8000 ppm. The activity of glutathione S-transferase was unaffected in kidney but elevated in liver, while GSH levels were elevated in both organs. The elevated level of kidney cytochrome P-450 did not return to normal during the 4-week-recovery period in contrast to liver cytochrome P-450. It is thus indicated that cytochrome P-450 and associated microsomal enzymes are more easily inducible and the changes more persistent in kidney than in liver. Our observations suggest that cytochrome P-450-mediated metabolic activation of n-hexane in the kidneys may have toxicological relevance in addition to liver metabolism, and that coexposure to isopropanol and n-hexane may represent an enhancement of the health hazard from n-hexane, possibly due to the isopropanol metabolite acetone.     

Regulatory effect of copper on rat adrenal cytochrome P-450 and steroid metabolism

Accumulation of Cu2+ in rat adrenal glands produced a biphasic response in concentrations of the mitochondrial cytochrome P-450 and heme which were, in turn, reflected in abnormal steroid biosynthesis and output. In the mitochondria, 1 day after Cu2+ treatment, when the concentration of the metal ion was increased by 2- to 3-fold over the control value, a significant increase in cytochrome P-450-dependent steroid 11 beta-hydroxylase activity was observed. These effects were accompanied by a nearly 85% increase in concentrations of cytochrome P-450 and heme. In addition, the activity of delta-aminolevulinate synthetase was increased by 3-fold. In those animals lipid peroxidation, assessed by measuring concentrations of conjugated dienes, was reduced to approximately 50% of the control value. However, after 7 days of Cu2+ treatment (via a mini-osmotic pump), a significantly lowered rate of 11 beta-hydroxylase activity was noted, and the plasma concentration of corticosterone was also reduced significantly. Also, in the mitochondria, the concentrations of cytochrome P-450 and heme were decreased in comparison with the control values. These decreases were accompanied by elevated levels of the mitochondrial lipid peroxidation and a further increase in adrenal Cu2+ content (5-fold). At this time, delta-aminolevulinate synthetase activity remained elevated but to a lower extent than that observed after 1 day of Cu2+ treatment. In contrast to 11 beta-hydroxylase activity, the reduction in cytochrome P-450 content was not reflected in a decrease in the rate of cholesterol side-chain cleavage; rather this activity was increased in Cu2+-treated animals. Adrenal heme oxygenase activity was unaffected by either Cu2+ treatment, as was the specific content of cytochrome P-450 in the microsomal fraction. The present findings suggest that the Cu2+-mediated regulation of cytochrome P-450-dependent steroidogenic activity in the adrenal mitochondria is predominantly a reflection of the metal ion affecting heme biosynthesis and lipid peroxidation in this organ. Moreover, these actions appear to differentially affect the mitochondrial cytochrome P-450 species catalyzing different hydroxylation reactions in the adrenal steroidogenesis pathway.