Effects of poly I: poly C on rat pulmonary and hepatic cytochromes P-450 and b5

Interferon inducing agents such as poly I:poly C have been shown to reduce the hepatic hemoproteins cytochromes P-450 and b5 along with the associated monooxygenase activities [el Azhary and Mannering, Molec. Pharmac. 15, 698 (1979)]. In a previous study [Kikkawa et al., Lab. Invest. 50, 62 (1984)], we demonstrated that the interferon inducing agent poly I:poly C reduces pulmonary microsomal hemoprotein by 50% when administered to rats. The current investigation was conducted to characterize these changes in more detail and compare them to analogous changes in the liver. Compared to controls, cytochrome P-450 in both the lungs and livers of poly I:poly C treated rats declined by 40% at 24 hr and 55% at 48 hr (P less than 0.01). By 72 hr the decline was only 25%. In contrast, cytochrome b5 levels declined by less than 30% of control values during the first 48 hr following poly I:poly C injection (P less than 0.01) and returned to control levels by 72 hr. These changes in both cytochrome P-450 and b5 were reflected in decreases in pulmonary microsomal hemoprotein. Benzphetamine-N-demethylase activity declined by 45% in lung microsomes at 48 hr (P less than 0.01) after injection of poly I:poly C, while 7-ethoxycoumarin-O-deethylase (P less than 0.05) and 7-ethoxyresorufin-O-deethylase activities declined by approximately 41%. In the liver from these same poly I:poly C treated groups, benzphetamine-N-demethylase declined by 66% (P less than 0.05), while 7-ethoxycoumarin-O-deethylase and 7-ethoxyresorufin-O-deethylase activities declined by 60% (P less than 0.02 and P less than 0.05 respectively).     

Effect of cytochrome P-450 and flavin-containing monooxygenase modifying factors on the in vitro metabolism of amiodarone by rat and rabbit

Experiments were conducted to affirm hepatic cytochrome P-450 involvement in the biotransformation of the class III antiarrhythmic agent, amiodarone (Am; Cordarone X) to its major metabolite, desethylamiodarone (DEA). Male Sprague-Dawley rats and male New Zealand white rabbits were treated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) (to induce cytochrome P-450 (PB-inducible cytochrome(s) P-450) or P-448 (MC-inducible cytochrome P-450). In vivo decreases in rat hepatic microsomal cytochrome P-450 were achieved either by a single ip dose of CCl4 or by a 2-day treatment with CoCl2. In vitro biotransformation of Am by hepatic microsomes from PB-induced and 3-MC-induced rats and PB-induced rabbits was significantly greater than that from noninduced animals. Conversely, in vitro DEA production was significantly decreased with hepatic microsomes from CCl4- and CoCl2-pretreated rats. The classic P-450 inhibitors, piperonyl butoxide, SKF 525A, n-octylamine, and CO provided a significant reduction in the in vitro formation of DEA by microsomes from induced animals. In vitro DEA formation by hepatic microsomes from PB- and 3-MC-induced rats was significantly decreased by 0.5 mM chloroquine (specific inhibitors of PB-inducible cytochrome(s) P-450) and 0.3 mM quinacrine (specific inhibitor of MC-inducible cytochrome(s) P-450), respectively. Further evidence for involvement of gut microsomal flavin-containing monooxygenase was provided by the inhibition of gut microsomal-mediated in vitro DEA formation in the presence of methimazole. Methimazole had no effect on hepatic microsomal DEA production in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of constitutive cytochrome P-450 gene expression in livers of rats undergoing an acute phase response to endotoxin

Administration of purified bacterial lipopolysaccharide (LPS) to male rats suppressed the constitutive hepatic expression of the male-specific cytochrome P-450 [AH, reduced flavoprotein:oxygen oxidoreductase (RH hydroxylating), E.C.1.14.14.1] isozyme P-450h (P450IIC11) to about 35% of control levels within 24 hr. The mRNA for P-450h was more rapidly and more profoundly suppressed than was the protein, indicating (a) that the decrease in the mRNA was responsible for the suppression of the protein and (b) that other mechanisms work to maintain expression of P-450h apoprotein in the face of repression of its mRNA. Suppression of P-450h expression was maximal at an endotoxin dose of 30-100 micrograms/kg, indicating that P-450 suppression is concomitant with the acute-phase response of hepatic secretory proteins. The female-specific cytochrome P-450 isozyme, P-450i (P450IIC12), was suppressed to 17% of control levels by LPS administration in female rats. Suppression of the P-450i apoprotein by LPS, and recovery of its expression, was more rapid than was suppression of P-450h in males. P-450i protein and mRNA levels were concomitantly suppressed by LPS, indicating that although there is a pretranslational component to the suppression, other mechanisms may also contribute. Calculations based on estimations of the microsomal contents of P-450h and P-450i relative to the total cytochrome P-450 in untreated rat livers indicate that suppression of these forms contributes significantly to the decreases in total microsomal P-450 after LPS treatment. In these studies, hepatic microsomal NADPH-cytochrome c reductase (TPNH2-cytochrome c reductase, E.C.1.6.2.4) activities and content of cytochrome b5 were decreased by LPS administration in both male and female rats. Like its effects on cytochrome P-450 expression, endotoxin suppression of NADPH-cytochrome c reductase activities and cytochrome b5 levels was more rapid in female rats than in males. The production of a local inflammatory response in male rats by subcutaneous injection of turpentine caused effects on cytochrome P-450, P-450h expression, and cytochrome b5 that were similar to those of endotoxin but were less rapidly achieved.     

Protective effect of glutathione on the in vitro inhibition of hepatic cytochrome P-450 by captan.

The in vitro effect of various concentrations of captan on hepatic microsomal cytochrome P-450 from pehnobarbital-pretreated rats was studied. The I-50 value, namely the concentration of the inhibitor necessary to produce 50% loss of cytochrome P-450 was determined from theplotted inhibition curve. The presence of ethylenediaminetetraacetic acid (EDTA) in microsomal incubations prior to the addition of captan failed to prevent the loss of cytochrome P-450 by captan. In contrast, reduced glutathione (0.5 mM) added to microsomal incubations before captan (0.1 mM) afforded almost complete protection of cytochrome P-450 from captan inhibition. These data indicate that the inhibitory effect of captan on vitally important drug-metabolizing enzyme system, of which cytochrome P-450 is a major component, can be prevented by prior presence of reduced glutathione (GSH) but not of EDTA.     

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.     

Effect of riboflavin deficiency on phenobarbital and 3-methylcholanthrene induction of microsomal drug-metabolizing enzymes of the rat

The effect of riboflavin deficiency on the induction of hepatic microsomal enzymes by phenobarbital and 3-methylcholanthrene has been investigated. A decrease in microsomal flavin levels of 56 per cent was associated with a decrease in NADPH cytochrome c reductase (52 per cent), azoreductase (71 per cent) and benzpyrene hydroxylase (74 per cent). Microsomal cytochrome P-450 content and aminopyrine demethylase were not significantly affected by flavin deficiency. Phenobarbital or 3-methylcholanthrene pretreatment did not affect hepatic microsomal flavin levels in normal or deficient animals. In flavin-deficient animals, phenobarbital pretreatment significantly increased cytochrome c reductase, cytochrome P-450 content, aminopyrine demethylase and azoreductase. Thus the carbon monoxide-sensitive pathway (cytochrome P-450 mediated) of azoreductase was essentially unaffected by flavin deficiency. In deficient animals, the carbon monoxide-insensitive microsomal azoreductase pathway (non-cytochrome P-450 mediated) normally induced by 3-methylcholanthrene was unaffected. Thus, induction of azoreductase by 3-methylcholanthrene was found to be flavin dependent. However, 3-methylcholanthrene did increase cytochrome P-450 content and benzpyrene hydroxylase in flavin-deficient animals. The induction of benzpyrene hydroxylase by 3-methylcholanthrene increased with increasing microsomal flavin content. Part of the mechanism of azoreductase induction by 3-methylcholanthrene was due to an induced change in the structure or composition of microsomal flavoprotein. This interpretation is supported by the findings that: (1) induction by 3-methylcholanthrene in riboflavin-deficient rats required a minimal flavin level, (2) increased enzyme activity was not compensated by an increase in microsomal flavin and (3) induction by 3-methylcholanthrene augmented FMN-stimulation of microsomal azoreductase in vitro.     

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 washing the hepatic microsomal fraction in sucrose solutions and in sucrose solution containing edta upon the metabolism of foreign compounds

Washing the hepatic microsomal fraction of the rat in 0·25 M sucrose containing 0·05 M Tris buffer (pH 7·4) resulted in the metabolism and binding to cytochrome P-450 of Type I substrates, although not of a Type II substrate. The levels of microsomal cytochrome b₅, cytochrome P-450 and NADPH-cytochrome c reductase was unchanged, whilst the activity of NADPH-cytochrome P-450 reductase was reduced. The inhibitors of lipid peroxidation EDTA and Mn²⁺ added to the washing medium prevented the decrease in the metabolism of Type I substrates. It is suggested that the effects of washing may be related to the increase in the level of peroxidised microsomal lipid which could lead to a selective destruction of the Type I binding site. EDTA added to the washing medium also produced an increase above control values, in the metabolism and binding of both Type I and Type II substrates, which may be related to the apparent increase in the amount of microsomal cytochrome P-450. Washing the microsomal fraction almost completely abolished the ability of acetone to enhance aniline hydroxylation. It is concluded that the effects of acetone are not due to the displacement of endogenous substrates bound to the microsomal fraction.     

Qualitative alteration in hepatic microsomal cytochrome P-450 apoproteins associated with bile duct ligation, and the administration of ethinyl estradiol, phenobarbital and 3-methylcholanthrene

We have investigated in rat liver whether different forms of cytochrome P-450 are altered in hepatic disorders associated with impaired drug metabolism. Total hepatic cytochrome P-450 is decreased after either bile duct ligation or the administration of ethinyl estradiol. In contrast, phenobarbital administered alone increases hepatic content of cytochrome P-450, and when administered with ethinyl estradiol the reduction in cytochrome P-450 was prevented. Microsomal ethylmorphine N-demethylase activities paralleled changes in cytochrome P-450 content, except in bile duct ligation. where it is diminished to a greater extent. Four forms of microsomal cytochrome P-450 apoproteins. ranging in molecular weight from 50,000 to 58,000, were tentatively identified in a sodium dodecyl sulfate (SDS)-6 M urea polyacrylamide gel electrophoresis system by their responsiveness to pharmacological agents, turnover and benzidine peroxidase staining. Phenobarbital administration increased primarily band IV (50,000 daltons); in contrast only band III (53,000 daltons) was responsive to 3-methyl-cholanthene. Bile duct ligation was associated with a marked reduction in bands I, III and IV while bands I and III were decreased with ethinyl estradiol administration. Simultaneous administration of phenobarbital and ethinyl estradiol demonstrated a return of band I and an increase in density of bands II and IV. The mechanism of this reversal by phenobarbital was determined by the double-isotope technique and demonstrates that phenobarbital increases the relative synthesis rates of P-450 apoproteins in ethinyl estradiol-treated rats. These sludies support the hypothesis that mulliple forms of cytochrome P-450 are present in liver microsomal membranes and that alterations in specific apoproteins may be associated with an increase or a decrease in the functional properties of cytochrome P-450.     

Identification of the hepatic cytochrome P-450 isozymes induced and decreased by picloram

Microsomes from male rats treated with picloram (100 mg/kg/day) for 7 days showed a 48% decrease in 16 alpha-hydroxylase activity when incubated with (4-14C) androstenedione. These data are consistent with the assertion that picloram decreases the titer of hepatic male specific cytochrome P-450h. Several lines of evidence suggested that picloram is an inducer of hepatic cytochrome P-450 in male rats. First, SDS polyacrylamide gel electrophoresis revealed an intensified hepatic microsomal polypeptide (MW 54,000) following picloram pretreatment. This polypeptide co-migrated with protein bands which were correspondingly intensified after pretreatment with known inducers of cytochrome P-450d (3-methylcholanthrene and isosafrole). Second, no increase in the binding of metyrapone to picloram treated microsomes was noted compared with controls, suggesting no increase in phenobarbital-inducible forms of cytochrome P-450. Third, hepatic microsomes from picloram treated rats activated 2-amino-3-methylimidazo [4,5-f] quinoline (a cytochrome P-450d mediated catalysis) causing a 5-fold increase in the number of induced Salmonella typhimurium TA98 revertant colonies formed compared with control microsomes. Fourth, the binding of n-octylamine to hepatic microsomes from picloram-treated rats showed, like microsomes from 3-methylcholanthrene-treated rats, an increase in the proportion of high-spin cytochrome P-450 present. Cytochrome P-450d is known to be a high spin haemoprotein.     

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats was examined with respect to the mixed function oxidase system, anaerobic dehalogenation activity of halothane, and the antioxidant system. The levels of cytochrome P-450 and NADPH-cytochrome P-450 reductase were significantly higher in guinea pigs than in rats. There was no difference between the two animals in anaerobic dehalogenation activity of halothane per cytochrome P-450 in microsomes. Microsomal alpha-tocopherol was significantly lower in guinea pigs than in rats, and was increased by multiple exposure to halothane in guinea pigs but remained lower than in rats. Microsomal alpha-tocopherol was decreased in rats by multiple exposure. The concentration of reduced glutathione and ascorbic acid was decreased significantly by multiple exposure to halothane in guinea pigs but not in rats. These results suggest that the higher level of halothane-induced hepatic microsomal lipid peroxidation in guinea pigs is due to the large production of radical metabolites resulting from the large amounts of cytochrome P-450, the high activity of NADPH-cytochrome P-450 reductase, and the low concentration of microsomal alpha-tocopherol.     

Reversible inhibition of aromatic hydroxylation of methamphetamine in rat liver microsomal preparations pretreated with methamphetamine

The effects of a single and repeated administration of methamphetamine (MP) in vivo in rats on its own metabolism in vitro were investigated. In both cases, the p-hydroxylation of MP to p-hydroxymethamphetamine by a microsomal fraction from rat liver was inhibited for a period of 16 hr after the last injection of MP. This inhibition was diminished by dialysis of the microsomal preparations. In contrast, the reduced level of cytochrome P-450 in hepatic microsomes from rats pretreated with the SKF 525-A did not revert to the control value after dialysis. When microsomes were preincubated with N-hydroxymethamphetamine, which is the metabolite of MP and a potent substrate for the formation of a metabolic intermediate (MI) complex with cytochrome P-450, the content of the MI was increased and the MP-hydroxylation activity decreased in direct proportion to the length of the preincubation. These results suggest that the inhibition of MP-hydroxylation may be due to reduction of the level of cytochrome P-450 that accompanies the formation of the MI complex. Furthermore, it appears that the complex can be dissociated by dialysis.     

One-electron reduction of mitomycin c by rat liver: role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of p-nitrobenzylpyridine (NBP) in the absence of O2 were taken as parameters. 2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems. 3. Under aerobic conditions the H2O2 production in the microsomal systems was dependent on NADPH, O2 and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A. 4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O2, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient. 5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450. 6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase. 7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O2 tension is more likely the haem site of cytochrome P-450.     

Interaction of endosulfan and dietary vitamin A on rat hepatic drug metabolising enzymes

The effect of interaction of Endosulfan, a chlorinated insecticide of the cyclodiene group, with dietary vitamin A on the hepatic mixed function oxidase system in rats has been studied. Endosulfan administration (ten days) significantly increased microsomal protein content and cytochrome P-450 levels, NADPH cytochrome C-reductase aminopyrine N-demethylase and aniline hydroxylase activities, with respect to control rats. Administration of vitamin A (10,000 I.U./100 g body weight) daily for ten days reduced the activity of the above mentioned enzymes, when vitamin A and endosulfan were given together, vitamin A reduced the endosulfan induced increase of microsomal proteins and cytochrome P-450 levels, the activity of NADPH cytochrome C-reductase, aminopyrine N-demethylase and aniline-hydroxylase.     

Effects of the interferon inducing agents tilorone and polyriboinosinic acid · polyribocytidylic acid (poly IC) on the hepatic monooxygenase systems of pregnant and fetal rats

Interferon inducing agents, including tilorone and polyriboinosinic acid polyribocytidylic acid (poiy IC), are known to depress hepatic cytochrome P-450-dependent monooxygenase systems and the induction of these systems by phenobarbital (PB) and 3-methylcholanthrene (MC) in mature male rats. The current study investigated the effects of tilorone and poly IC on the cytochrome P-450 systems of non-induced, PB-induced, MC-induced and pregnenolonecarbonitrile (PCN)-induced pregnant rats and their fetuses. Pregnant rats received either tilorone or poly IC and saline, PB, MC or PCN, and microsomes from their livers and those of their fetuses were examiued for cytochrome P-450 content, aminopyrine (AP) N-demethylase activity and benxo[a]pyrene (BP) hydroxylase activity. The generalixation can be made from these studies that, when the interferon inducing agents caused changes in cytochrome P-450 content or monooxygenase activities of either induced (PB, MC or PCN) or non-induced (saline) animals, decreases were seen in maternal livers and increases in fetal livers. Thus, in maternal livers tilorone depressed cytochrome P-450 and AP N-demethylase activity in non-induced and PB-, MC- and PCN-induced rats and BP hydroxylase activity in the induced animals; BP hydroxylase activity was not depressed in non-induced maternal livers. Poly IC depressed cytochrome P-450 and AP N-demethylase activity in non-induced and PB-induced rats but not in PCN-induced animals. BP hydroxylase was depressed by poly IC in both PB- and PCN-induced animals. Fetal hepatic cytochrome P-450 and monooxygenase activities were increased by tilorone in PB- and PCN-induced rats but not in non-induced or MC-induced animals. Poly IC increased cytochrome P-450 and both monooxygenase activities in PB- and PCN-induced fetal livers, whereas only BP hydroxylase activity was increased in the fetuses of non-induced rats. Several possible explanations are offered for the opposite effects produced by interferon inducing agents in maternal and fetal livers. Unlike maternally administered tilorone, which induced fetal cytocbrome P-450 and monooxygenase activities in the liver, intrauterine tilorone depressed cytochrome P-450 and had no effect on AP N-demethylase or BP hydroxylase activities in the fetal liver. Intrauterine poly IC was without effect on the cytochrome P-450 systems of the fetal liver. Treatment of pregnant rats with tilorone on days 17–20 of gestation inhibited normal maternal weight gain and produced overt signs of toxicity. A dose of 10 mg/kg of poly IC was very toxic in pregnant rats but produced no overt signs of toxicity in virgin female rats. Time courses of the depressant effects of a single injection of poly IC were observed in mid-term pregnant, late-term pregnant, lactating and adult virgin females. Maximum losses of cytochrome P-450 and ethyhnorphine (EM) N-demethylase activity were seen 48 hr after poly IC administration to pregnant and virgin rats, and recoveries were complete within 96 hr. Similar results were observed in lactating rats except that the nadir occurred at 24 rather than at 48 hr. The response of BP hydroxylase activity to poly IC was qualitatively similar except that this activity was not depressed in the mid-term pregnant rats.     

Protective Effect of Glutathione on the In Vitro Inhibition of Hepatic Cytochrome P-450 by Captan

ABSTRACT

The in vitro effect of various concentrations of captan on hepatic microsomal cytochrome P-450 from phenobarbital-pretreated rats was studied. The 1–50 value, namely the concentration of the inhibitor necessary to produce 50% loss of cytochrome P-450 was determined from the plotted inhibition curve. The presence of ethylenediaminetetraacetic acid (EDTA) in microsomal incubations prior to the addition of captan failed to prevent the loss of cytochrome P-450 by captan. In contrast, reduced glutathione (0.5 mM) added to microsomal incubations before captan (0.1 mM) afforded almost complete protection of cytochrome P-450 from captan inhibition. These data indicate that the inhibitory effect of captan on vitally important drug-metabolizing enzyme system, of which cytoehrome P-450 is a major component, can be prevented by prior presence of reduced glutathione (GSH) but not of EDTA.     

Effects of in vivo administration of detergents on the hepatic microsomal cytochrome P-450 system in rat

The influence of in vivo administration of detergents on the hepatic microsomal cytochrome P-450 system was studied in rat. Male Wistar rats were administered detergents, Emulgen 913 (50 mg or 100 mg kg-1 of body weight (B.W.], or sodium dodecylsulfate (SDS, 25 mg or 50 mg kg-1 of B.W.) intraperitoneally once a day for 3 days. Cytochrome P-450 content in liver microsomes was significantly decreased to 85% and 73% of control by the administration of 50 mg or 100 mg Emulgen, respectively, but the microsomal protein concentration was not changed by these administrations. The content of cytochrome P-450 was also reduced to 76% and 70% of control by the administration of 25 mg or 50 mg SDS/kg of B.W., respectively. The total hydroxylation activity (the sum of omega- and (omega-1)-hydroxylase activity) of laurate almost paralleled the decrease in cytochrome P-450 in detergent-treated rats. However, the omega/omega-1-hydroxylation ratio was not changed. These results suggest that the administration of these detergents lowered the level of cytochrome P-450 species catalyzing omega- and (omega-1)-hydroxylation of laurate to a similar extent. On the other hand, aminopyrine N- and p-nitroanisole O-demethylation activities in Emulgen 913-treated rats was decreased while those in SDS-treated rats did not change, though the content of cytochrome P-450 was decreased by both administrations. Thus, it was demonstrated that the livers of rats responded to exogenous detergents in different manners.     

The effect of galactosamine on rat liver cytochrome P-450 activities

The effect of galactosamine on hepatic drug metabolizing activities was examined in rats. In the microsomal fraction, the contents of cytochrome P-450 (P-450) and cytochrome b5 (b5) and the activity of NADPH-cytochrome c reductase (reductase) were examined for 7 days after galactosamine administration. In addition, substrate metabolizing activities in damaged microsomes were examined using four substrates: aminopyrine, aniline, benzo(a)pyrene (B(a)P) and 7-ethoxycoumarine (7-EC). The contents of P-450 and b5 and the activity of reductase showed a minimal value after 3 days of galactosamine administration and then gradually increased, reaching to the control level after 7 days. All four substrate metabolizing activities showed a similar response as the content of P-450, but the decrement among the four activities was not uniform. The activities of B(a)P hydroxylation and 7-EC deethylation were more impared than those of aminopyrine demethylation and aniline hydroxylation. This nonuniformity was clear on the activity based on P-450. This result suggested that galactosamine disturbed the population of multiple P-450 subtypes, and each P-450 subtype was damaged to the various extent by galactosamine administration.     

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.     

Biochemical basis for impaired drug metabolism in tumor-bearing rats: Evidence for altered regulation of hepatic microsomal hemeprotein synthesis

The pharmacologie effects of many drugs are enhanced in animals bearing tumors. This apparently stems from a decrease in the microsomal metabolism of these compounds in liver cells, owing to a decrease in either the activities of certain enzymes or in the content of cytochrome P-450 in microsomes, or both. Since impaired drug metabolism may have a direct bearing on the outcome of chemotherapy, a study of the biochemical basis for this alteration was begun. In female Sprague-Dawley rats bearing Walker 256 solid tumors i.m., pentobarbital metabolism was impaired as judged from prolonged sleeping-times. This effect was accompanied by a decrease in the microsomal content of cytochrome P-450. Analysis of hepatic microsomes by sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that a microsomal protein of ~53,000 daltons was diminished in livers of tumor-bearing animals, when compared to normal controls, as determined by staining with Coomassie Blue. When rats were injected with [³H]leucine, although the dpm/100 μg of microsomal protein were the same for both normal and tumor-bearing rats, ~40% less ³H was associated with the microsomal proteins in the cytochrome P-450 region (~43,000 to ~58,000 daltons) of tumor-bearing animals than with those of normal controls. This was attributed to a decrease in the rate of synthesis of these, but not of other, microsomal proteins. Either staining of microsomal proteins with 3,3',5,5'-tetramethyl-benzidine, or labelling of them with δ-[¹⁴C]aminolevulinic acid, revealed that the overall content of microsomal hemeproteins of tumor-bearing rats was reduced considerably, when compared to normal controls (~70% as judged by the incorporation of ¹⁴C). These observations prompted investigations of the apparent rate-limiting synthetic and degradative enzymes of heme. We found that in the livers of 7-day tumor-bearing rats, the activity of δ-aminolevulinic acid synthetase was only 16% of control activity; conversely, the activity of hepatic microsomal heme oxygenase in the tumor-bearing rats was nearly eight-times greater than that of the normal animals. Together, these data indicate that perturbations in heme- and hemeprotein-synthesis cause the reduced content of cytochrome P-450 seen in tumor-bearing rats, and they provide a partial explanation for diminished drug metabolism by hepatic microsomes in the presence of a growing, transplantable, non-hepatic tumor.