Modifications of drug metabolism by disulfiram and diethyldithiocarbamate. I. Mixed-function oxygenase.

Disulfiram and diethyldithiocarbamate were administered to rats for 4 days alone (300 mg/kg, daily, per os) or in combination with phenobarbital (80 mg/kg, daily, i.p.), in order to observe the effects of these compounds on the microsomal membrane components and on the mixed-function oxygenase system. Both disulfiram and diethyldithiocarbamate increased the liver to body weight ratio, and the total hepatic protein content. Disulfiram significantly increased also the microsomal protein and phospholipid contents. Diethyldithiocarbamate and disulfiram partially prevented the increase of microsomal protein and phospholipid contents caused by phenobarbital. Disulfiram and diethyldithiocarbamate decreased the amount of cytochrome P-450 and P-420, and the activity of p-nitroanisole O-demethylase. These changes were more pronounced after diethyldithiocarbamate than after disulfiram treatment. On the contrary, the activity of NADPH-cytochrome c reductase was enhanced only by disulfiram. The induction by phenobarbital of cytochrome P-450 and p-nitrosanisole O-demethylase was partially prevented on concomitant treatment with disulfiram and diethyldithiocarbamate. These compounds. however, had an additive effect with phenobarbital in enhancing the microsomal NADPH-cytochrome c reductase activity.     

Drug metabolism in adult white leghorn hens--response to enzyme inducers

1. Adult hens were given a mixture of polychlorinated biphenyls (0.5 and 2 g/kg Aroclor 1254), 3-methylcholanthrene (80 mg/kg) or beta-napthoflavone (80 mg/kg). 2. beta-Napthoflavone elevated activities of both microsomal and nonmicrosomal enzymes 48 hr after dosing, with cytochrome P-450 p-nitroanisole O-demethylase, aniline hydroxylase, and glutathione S-epoxytransferase at 319% +/- 25, 157% +/- 12, 410% +/- 26 and 120% +/- 3 of control values, respectively. 3. Aroclor 1254 and 3-methylcholanthrane also elevated microsomal enzyme activities, but did not increase the activity of glutathione S-epoxytransferase. 4. Drug metabolizing capability in control and experimental hens differed from that in rats and mice.     

In vivo effects of 3-methylcholanthrene, phenobarbital, pyrethrum and 2,4,5-T isooctylester on liver, lung and kidney microsomal mixed-function oxidase system of guinea-pig: a comparative study

The optimum conditions (pH, microsomal protein amount and substrate concentration) of guinea-pig liver, lung and kidney microsomal aniline 4-hydroxylase, ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities were determined. Male guinea-pigs weighing 500-700 g were administered 3-methylcholanthrene (25 mg/kg, i.p. 3 days), phenobarbital (75 mg/kg, i.p. 3 days), pyrethrum (120 mg/kg, i.p. 2 days) and 2,4,5-T isooctylester (200 mg/kg, i.p. 3 days). 3-Methylcholanthrene treatment caused significant increases in liver microsomal benzo[a]pyrene hydroxylase and kidney microsomal aniline 4-hydroxylase activities. However, with phenobarbital treatment the only significant increase was observed in liver microsomal ethylmorphine N-demethylase activity. Pyrethrum treatment decreased kidney microsomal ethylmorphine N-demethylase activity significantly. 2,4,5-T isooctylester treatment increased liver microsomal aniline 4-hydroxylase and lung microsomal ethylmorphine N-demethylase activities significantly. Liver microsomal NADPH-cytochrome c reductase activity was increased significantly by phenobarbital and pyrethrum treatment. The other treatments did not cause any significant changes in microsomal NADPH-cytochrome c reductase activities of liver, lung and kidney. Cytochrome P-450 content of guinea-pig liver microsomes were increased significantly about 2.5-fold and 2-fold by treatment with 3-methylcholanthrene and phenobarbital, respectively. 3-Methylcholanthrene also caused 1 nm spectral shift in the absorption maxima of CO difference spectrum of the dithionite-reduced liver microsomal cytochrome P-450, forming P-449.     

Regulation of hepatic drug metabolism by elaidic and linoleic acids in rats.

Elaidic and linoleic acids were administered at doses of 40 and 200 mg/kg i.p. every second day for 4 weeks to rats fed a fat-free diet. The fatty acids had only a slight effect on the weight gain of the animals. The amount of microsomal protein was slightly decreased with the higher dose of linoleic acid. The higher dose level of both fatty acids decreased the microsomal phospholipid content. The relative amounts of microsomal phospholipid fatty acids were also altered due to fatty acid administration. The activity of microsomal NADPH cytochrome c reductase and microsomal cytochrome P-450 contents were decreased by the higher dose of linoleic acid. The hepatic aryl hydrocarbon hydroxylase and p-nitroanisole O-demethylase activities decreased in fatty acid-treated rats. The UDP-glucuronosyltransferase activity was also lowered after the fatty acid administration. The results suggest that fatty acid-induced changes in the activities of drug-metabolizing enzymes may be due to the microenvironmental changes of membrane-bound enzymes.     

Effect of a single oral dose of methanol, ethanol and propan-2-ol on the hepatic microsomal metabolism of foreign compounds in the rat.

Methanol and ethanol administered to rats as a single oral dose increased aniline hydroxylation by the hepatic microsomal fraction by a maximum of 169 and 66% respectively, whereas aminopyrine demethylation was inhibited by 51 and 61%. The concentration of microsomal cytochrome P-450, and the activities of NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were unchanged. Propan-2-ol, administered as a single oral dose, increased microsomal aniline hydroxylation by 165% and increased aminopyrine demethylation by 83%. The concentration of cytochrome P-450 was unchanged whereas NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were both increased by 38%. Methanol, ethanol and propan-2-ol administration resulted in a decreased type I spectral change but had no effect on the reverse type I spectral change. Methanol administration decreased the type II spectral change whereas ethanol and propan-2-ol had no effect. Cycloheximide blocked the increases in aniline hydroxylation and aminopyrine demethylation but could not completely prevent the decreases in aminopyrine demethylation. The increases in aniline hydroxylation were due to an increase in V, but Km was unchanged. The ability of acetone to enhance and compound SKF 525A to inhibit microsomal aniline hydroxylation was decreased by the administration of all three alcohols. The decrease in the metabolism of aminopyrine may result from a decrease in the binding to the type I site with a consequent failure of aminopyrine to stimulate the reduction of cytochrome P-450. Methanol administration may lead to an increase in aniline hydroxylation because of a failure of aniline to inhibit cytochrome P-450 reduction.     

Induction of cytochrome P-450 in rat liver microsomes by perfluorodecalin

Perfluorodecalin was incorporated into phospholipid liposomes and injected intraperitoneally in various dozes. The maximal cytochrome P-450 induction is reached 48 hours after perfluorodecalin injection. Cytochrome P-450 content increases 4 times after perfluorodecalin injection in dose of 0.6 ml/kg in homogenate, and 6 times after perfluorodecalin injection in a dose of 0.4 ml/kg in microsomes. Phenobarbital and perfluorodecalin induce several cytochrome P-450 isozymes and cause the appearance of a new isozyme with mass 56 kD absent in microsomes of intact CBA mice. Perfluorodecalin induction strongly increased the rate of NADPH-dependent aminopyrine nN-demethylation (6-7 times per mg of microsomal protein and 1.5 times per nmol cytochrome P-450). The rate of NADPH-dependent hydroxylation of aniline was not affected by perfluorodecalin induction.     

Tetraphenylporphyrin-Sn4+ induction of cytochrome P-450

TPP-Sn4+ was administered intraperitoneally (25 mg/kg body weight). The study was performed for 1-30 days. A day after administration the increase in the hemoprotein level 1.4 times was observed, as well as an increase in the level of p-hydroxylation of aniline. On 7-14 days the greatest increase in cytochrome P-450 content was observed. To clarity the mechanism of TPP-Sn4+ effect on cytochrome P-450, we studied its effect on the activity of heme oxygenase and LP rate. This compound is an inhibitor of heme oxygenase activity and reduces the rate of LP in the microsomes which regulates porphyrin metabolism in the organism.     

Time-course of effects of toluene on microsomal enzymes in rat liver, kidney and lung during and after inhalation exposure

Inhalation of toluene vapour of 2000 ppm increased the activities of aniline hydroxylase, aminopyrine N-demethylase, aryl hydrocarbon hydroxylase and NADPH-cytochrome c reductase and the concentrations of cytochromes P-450 and b5 in liver microsomes of adult male rats after an exposure period of 1 day or less. Repeated treatments, 8 h daily for 1-16 days, had only a slight further effect. In lung microsomes, the activities of monooxygenases and the concentration of cytochrome P-450 decreased after 6-24 h toluene exposure, but those of cytochrome b5 and NADPH-cytochrome c reductase did not change. In kidney microsomes the changes were mostly insignificant. After discontinuation of exposure the activities of enzymes and the concentrations of cytochromes returned to the control level in 1-4 days. The results obtained resemble the time-courses for the induction of monooxygenases by other inducers. The tissue differences suggest the unequal distribution of various cytochrome P-450 forms and their individual responsiveness to induction in liver, kidneys and lungs.     

Destruction of hepatic mixed-function oxygenase parameters by CCl4 in rats following acute treatment with chlordecone, Mirex, and phenobarbital

Previous work has established the marked potentiation of CCl4 hepatoxicity by prior exposure to chlordecone (CD). This study was conducted to determine if prior exposure to CD results in enhancement of CCl4-induced destruction of the hepatic microsomal mixed-function oxygenase (MFO) system. Male Sprague-Dawley rats received a single oral dose of CD (10 mg/kg) or corn oil vehicle alone (1 ml/kg) 24 hr prior to a single ip injection of CCl4 (0-100 microliter/kg). Mirex (M; 10 mg/kg) and phenobarbital (PB; 80 mg/kg/day for two days) were used as negative and positive controls respectively for the potentiation of CCl4 hepatotoxicity. Hepatotoxicity was evaluated 24 hrs after CCl4 administration by elevations of three serum enzymes (GPT, GOT, and ICD). The key hepatic microsomal MFO parameters measured were microsomal protein, cytochrome P-450 content, glucose-6-phosphatase (G-6-Pase), and aminopyrine demethylase (APD). As previously demonstrated using a subchronic dietary pretreatment protocol, CD potentiated CCl4 hepatotoxicity over a range of CCl4 doses to a greater extent than PB or M, as judged by elevations in serum enzymes. PB caused the greatest increase in total P-450 content and the greatest increase in CCl4-mediated destruction of microsomal protein and APD activity. M caused the least destruction of total hepatic cytochrome P-450, despite the same level of cytochrome P-450 as in the PB group. CD treatment caused the greatest decrease in G-6-Pase activity in comparison to PB or M pretreatments and a similar degree of P-450 destruction as observed with the PB group. These findings suggest that in general, CCl4-induced destruction of hepatic MFO parameters measured in this study is disproportional to the known degree of potentiated hepatotoxicity by the pretreatments and does not accurately reflect the potentiation of CCl4 hepatotoxicity by CD.     

CCl4-induced alterations in Ca++ homeostasis in chlordecone and phenobarbital pretreated animals

Male S-D rats were maintained on normal powdered diet or on the same diet containing 10 ppm chlordecone or 225 ppm phenobarbital for 15 days. On day 15, all the animals received a single ip injection of either corn oil or a subtoxic dose of CCl4 (25-200 microliter/kg) in corn oil vehicle (1 ml/kg). The animals were sacrificed 12 hrs later. Liver microsomal cytochrome P-450 and Ca++ levels in whole liver, mitochondria, microsomes and cytosol were determined. Cytochrome P-450 induction was greater with phenobarbital pretreatment than with chlordecone but the CCl4 induced destruction of cytochrome P-450 was almost similar in both groups and progressive with the dose of CCl4. CCl4 given to animals on normal diet in a dose range of 25-200 microliter/kg did not significantly alter the cytochrome P-450 levels. These findings are consistent with greater bioactivation of CCl4 after the above two pretreatments. There was a massive accumulation of Ca++ in chlordecone and phenobarbital pretreated animals after CCl4 administration. Cytosolic Ca++ levels remained high despite the mitochondrial and microsomal sequestration. This perturbation of hepatocellular Ca++ homeostasis might lead to hepatic lesion and hepatic failure. Chlordecone or phenobarbital alone do not alter hepatic Ca++ levels. These findings suggest that excessive accumulation of Ca++ may be causally related to the progression of hepatotoxic response due to CCl4 in chlordecone treated animals.     

Biphasic effect of methadone on hepatic drug metabolism

When methadone HCl (30 mg/kg, po) was given acutely to mice, it was found to inhibit drug metabolism as evidenced by a prolongation of hexobarbital sleeping time and zoxazolamine paralysis time. Pharmacokinetic studies revealed that this acute dose of the narcotic analgesic could also prolong the plasma half-life of aminopyrine without any change in its volume of distribution. When added to the incubation mixture containing 10,000 g mouse liver supernatant fraction and a complete system for measuring aminopyrine N-demethylase or aniline hydroxylase, methadone showed a dose-dependent inhibition of the enzymes; the former enzyme was inhibited to a greater extent than the latter one. However, subacute treatment of mice with methadone HCl (30 mg/kg, po, twice daily for 3 days) resulted in increases in liver weight, microsomal protein, and cytochrome P-450 content in consonant with the increased activities of four hepatic drug-metabolizing enzymes: aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole, O-demethylase, and benzphetamine N-demethylase. Moreover, both hexobarbital sleeping time and zoxazolamine paralysis time were shortened. The plasma half-life of aminopyrine was decreased. These changes were prevented by simultaneous administration of puromycin diHCl (80 mg/kg, ip). Methadone thus seems to act in a manner very similar to that of propoxyphene or SKF-525A, acting as a potent inhibitor of hepatic drug metabolism when given acutely and as an inducer when given subacutely.     

Effect of 1,10-phenanthroline on acute liver injury induced by dimethylnitrosamine in the rat

Acute liver damage was induced in rats by intragastric doses of dimethylnitrosamine (DMN, 3 mg/100 g body weight) and measured 24 hours later by morphological and biochemical methods. 1, 10-Phenanthroline (1, 10-P, 2 mg/100 g) administered simultaneously with DMN prevented the development of the characteristic morphological picture of liver injury. At the same time, the amount and synthesis of total liver proteins, the activity and distribution of liver β-glucuronidase, and the level of seromucoid and isocitric dehydrogenase (ICDH) activity in the serum, significantly changed by DMN, was within the range of control values when 1, 10-P was simultaneously administered. The protective effect of 1, 10-P against acute DMN hepatotoxicity paralleled the inhibition of some liver microsomal drug-metabolizing enzymes (aniline hydroxylase, morphine demethylase, content of cytochrome P-450). At higher doses of DMN (6 mg and 10 mg/100 g), the administration of 1, 10-P was no longer protective, although the content of cytochrome P-450 was only 20% of the value for normal liver microsomes. Therefore, in acute administration within a certain range of concentration of DMN, 1, 10-P might inhibit the microsomal drug-oxidizing enzymes, thus inhibiting the metabolism of the drug to a more toxic product.     

The Protective Effects of Eugenol on Carbon Tetrachloride induced Hepatotoxicity in Rats

Our earlier studies in vitro have shown that eugenol inhibits liver microsomal monooxygenase activities and carbon tetrachloride (CCl₄)-induced lipid peroxidation (Free Rad. Res. 20,253-266,1994). The objective of the present investigation was to study the in vivo protective effect of eugenol against CCI₄ toxicity. Eugenol (5 or 25 mg/kg body wt) given orally for 3 consecutive days did not alter the levels of serum glutamic oxalacetic transaminase (SGOTJ, microsomal enzymes such as cytochrome P450 reductase, glucose-6-phosphatase (G-6-Pase) xenobiotic-metabolizing enzymes (aminopyrine-N-demethylase, N-nitrosodimethylamine-demethylase and ethoxyresorufin-O-deethylase) and liver histology. Doses of eugenol (5 or 25 mg/kg) administered intragastrically to each rat on three consecutive days i.e. 48 hr, 24 hr and 30 min before a single oral dose of CCU (2.5 ml/kg body wt) prevented the rise in SGOT level without appreciable improvement in morphological changes in liver. Eugenol pretreatment also did not influence the decrease in microsomal cytochrome P₄₅₀ content, G-6-Pase and xenobiotic-metabolizing enzymes brought about by CCI₄. Since eugenol is metabolized and cleared rapidly from the body, the dose schedule was modified in another experiment. Eugenol (0.2,1.0,5.0 or 25 mg/kg) when given thrice orally i.e. prior to (-1 hr) along with (0 hr) and after (+ 3 hr) the i.p. administration of CCI₄ (0.4 ml/kg) prevented significantly the rise in SGOT activity as well as liver necrosis. The protective effect was more evident at 1 mg and 5 mg eugenol doses. However, the decrease in microsomal G-6-Pase activity by CCI₄ treatment was not prevented by eugenol suggesting that the damage to endoplasmic reticulum is not protected. The protective effect of eugenol against CC1₄ induced hepatotoxicity is more evident when it is given concurrently or soon after rather than much before CCU treatment.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

The responses of hepatic monooxygenases of guinea pig to cadmium and nickel

When Cd (3.58 mg CdCl₂·H₂O/kg, ip) was administered to male guinea pigs 72 h prior to sacrifice, the metal significantly inhibited the aniline 4-hydroxylase (AH) (16%), ethylmorphoneN-demethylase (EMND) (26%), and aminopyrineN-demethylase (AMND) (18%) activities and cytochrome P-450 (12%) and cytochrome b₅ (10%) levels. Cd did not alter the hepatic microsomal heme level. Cd, however, significantly increased the hepatic microsomalp-nitroanisoleO-demethylase (p-NAOD) (53%) activity. When Ni (59.5 mg NiCl₂·6H₂O/kg, sc) was administered to the guinea pigs 16 h prior to sacrifice, the metal significantly depressed AH (49%),p-NAOD (66%), EMND (47%), and AMND (37%) activities, and cytochrome P-450 (15%), cytochrome b₅ (24%), and microsomal heme (28%) levels. For the combined treatment, animals received the single dose of Ni 56 h after the single dose of Cd and then were killed 16 h later. In these animals, significant inhibitions were noted in AH (51%), EMND (47%), and AMND (30%) activities, and cytochrome P-450 (15%), cytochrome b₅ (26%), and microsomal heme (30%) compared to those of controls. In the case ofp-NAOD activity, the influence was in favor of Ni, i.e, the inhibition was about 61% by the combined treatment. These results reveal that:

Effects of phenobarbitone and short-term exposure to heat on microsomal cytochrome P-450 and associated monooxygenases in rat liver

Short-term exposure of the control and phenobarbitone-treated rats to high ambient temperature caused a different response of the hepatic microsomal cytochrome P-450-dependent monooxygenase system participating in the oxidation of aniline, aminopyrine and p-nitroanisole. The highest differences of the enzyme activities were observed in rats exposed to 28 degrees C, as compared with animals exposed to 21 degrees C or 37 degrees C.     

Cysteamine in combination with N-acetylcysteine prevents acetaminophen-induced hepatotoxicity.

N-Acetylcysteine (NAC) is protective against acetaminophen-induced hepatotoxicity primarily by providing precursor for the glutathione synthetase pathway, while cysteamine has been demonstrated to alter the cytochrome P-450 dependent formation of toxic acetaminophen metabolite. Mice administered acetaminophen (500 mg/kg) had elevations of serum alanine aminotransferase (ALT) to 273.0 +/- 37.5 and 555.8 +/- 193.4 U/mL at 12 and 24 h, respectively, after injection. Administration of cysteamine (100 mg/kg) or NAC (500 mg/kg) significantly reduced serum ALT activity (p less than 0.001). Reducing the dose of NAC or cysteamine by 50% greatly reduced their hepatoprotective effect while the co-administration of the reduced doses of NAC (250 mg/kg) and cysteamine (50 mg/kg) following acetaminophen overdose prevented elevation of serum ALT activity (39.2 +/- 1.17 and 32.5 +/- 5.63 U/mL at 12 and 24 h post-injection, p less than 0.001) and preserved normal mouse hepatic histology. Neither NAC (500 mg/kg), cysteamine (100 mg/kg), or the lower doses in combination of both agents were found to alter the half-life or peak levels of acetaminophen. Liver microsomal aryl hydrocarbon hydroxylase activity measured 24 h after drug administration was not significantly different between treatment groups and controls receiving only saline. These results indicate a possible role for the concomitant use of NAC and cysteamine in the prevention of hepatic necrosis following toxic doses of acetaminophen. Neither decrease in plasma acetaminophen levels nor depression of cytochrome P-450 enzyme activity appears to be the mechanism of protection when these doses of NAC, cysteamine, or both drugs together are administered with a toxic dose of acetaminophen in mice.     

Induction of cytochrome P-450 isozymes by mirex and chlordecone

The effect of the insecticides, mirex and chordecone (Kepone), on the cytochrome P-450 monooxygenase system in C57BL/6N mouse liver microsomes was studied. Mice were treated intraperitoneally with low (6 mg/kg) and high (30 mg/kg) doses of mirex and chlordecone in corn oil for 2 days. For comparison, mice were also treated with either phenobarbital (PB) or 3-methylcholanthrene (3-MC). All treatments significantly increased the hepatic microsomal P-450 content over that of controls. Benzphetamine N-demethylase, ethoxyresorufin O-deethylase, benzo[a]pyrene hydroxylase, and acetanilide hydroxylase activities were also determined. Mirex and chlordecone resembled phenobarbital with respect to the induction of monooxygenase activities. Immunoquantitation with antibodies to purified P-450 IIB1 (Pb-induced P-450) and P-450 IA1 (3-MC-induced P-450) indicated that mirex and chlordecone induced P-450 IIB1 in a dose-dependent manner. The high dose of mirex also induced a small amount of a protein cross reacting with the antibody to IA1. The induction of this isozyme did not, however, contribute significantly to the monooxygenase activities measured.     

Microsomal monooxygenase system in Morris hepatoma: purification and characterization of cytochromes P-450 from Morris hepatoma 5123D of 3-methylcholanthrene-treated rats

Two forms of cytochrome P-450 (hepatoma P-450MCI and P-450MCII) were purified from hepatoma 5123D microsomes of tumor-bearing rats treated with 3-methylcholanthrene. Hepatoma P-450MCI had a specific content of 18.4 nmol/mg protein and showed a main protein band with a minimum molecular weight of 56,000 on sodium dodecyl sulfate-polyacrylamide gel. Hepatoma P-450MCII had a specific content of 7.38 nmol/mg protein and a minimum molecular weight of 50,000. The carbon monoxide-reduced difference spectral peak of hepatoma P-450MCI was at 446.5 nm, whereas the peak of hepatoma P-450MCII was at 451 nm. In the reconstituted system, hepatoma P-450MCI catalyzed 3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin, but showed low activities for N-demethylation of benzphetamine and aminopyrine, O-demethylation of p-nitroanisole, and p-hydroxylation of aniline. On the other hand, hepatoma P-450MCII did not catalyze hydroxylation of any of the substrates tested. By Ouchterlony double-diffusion analysis, hepatoma P-450MCI was immunologically indistinguishable from rat liver cytochrome P-450c, but hepatoma P-450MCII was distinct from hepatoma P-450MCI and rat liver cytochrome P-450c. Peptide maps of hepatoma P-450MCI and rat liver cytochrome P-450c after proteolysis with Staphylococcus aureus V8 protease demonstrated the similarity of the two cytochromes P-450.     

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid

Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins.