Mechanisms of inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone bioactivation in mouse by dietary phenethyl isothiocyanate.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), induces lung tumors in mice, rats, and hamsters. Phenethyl isothiocyanate (PEITC), which occurs as gluconasturtiin in cruciferous vegetables, is a potent inhibitor of NNK-induced carcinogenesis. The present study investigated the enzymatic basis for the bioactivation of NNK and the mechanisms of the inhibition of this process by dietary PEITC in mice. The apparent Km for the formation of keto aldehyde, keto alcohol, and NNK-N-oxide in lung microsomes was 4.9, 2.6, and 1.8 microM and, in liver microsomes, 5.5, 5.1, and 8.8 microM, respectively. Immunoinhibition studies suggested that cytochrome P450s (P450s) 2A1 and 2B1 or related forms are the major enzymes involved in the oxidative metabolism of NNK in mouse lung microsomes. When female A/J mice were fed diets containing 0, 1, or 3 mumol of PEITC/g of diet for 4 wk, the dietary PEITC had no significant effects on the food consumption and body weight of the mice. NNK oxidation in the lung microsomes of mice consuming the 1 or 3 mumol of PEITC/g of diet was decreased by 13 to 27% or 30 to 50%, respectively. In liver microsomes, whose NNK oxidative metabolism rates were about twice those of lung microsomes on a per mg of protein basis, the activities were decreased by 14 to 31% by the 3 mumol of PEITC/g of diet. The apparent Km remained unchanged, and the apparent Vmax decreased in the lung and liver microsomes of PEITC-fed mice, suggesting a noncompetitive nature of the inhibition. When added to the incubation mixture, PEITC decreased NNK metabolism in a concentration-dependent manner and exhibited a competitive inhibition with apparent Ki values of 51 to 93 nM. Dietary PEITC decreased the hepatic P450 content by 25%, but increased (2-fold) the O-dealkylase activities of 7-pentoxyresorufin (indicative of P450 2B1) and 7-ethoxyresorufin (indicative of P450 1A) in the liver microsomes of mice consuming the 3 mumol of PEITC/g of diet. The P450 2B level was increased in liver microsomes but slightly decreased in the lung microsomes. The p450 2E1 level was increased by dietary PEITC by 1.2- and 1.6-fold in the liver and lung microsomes, respectively. The activities of glutathione S-transferase and NAD(P)H-quinone oxidoreductase in liver and lung microsomes were not affected appreciably by the dietary PEITC treatment. The results suggest that chronic consumption of PEITC decreases the rate of metabolic activation of NNK by chemical inactivation and competitive inhibition of the enzyme(s) responsible for NNK oxidation.

Transcriptional activation of cytochrome P450 2B1/2 genes in rat liver by diallyl sulfide, a compound derived from garlic.

Previous work in our laboratory demonstrated that cytochrome P450 2B1 in rat liver was induced, but P450 2E1 was inhibited and inactivated, by diallyl sulfide (DAS), a compound derived from garlic. Such a selective effect on P450 enzymes is of considerable interest toward the understanding of dietary effects on xenobiotic metabolism. In the present study, the mechanism of P450 2B1 induction by DAS was investigated. Following a single dose of DAS (200 mg/kg body weight, ig), liver microsomal pentoxyresorufin dealkylase (PORd) activity, a representative activity of P450 2B1, was induced 3-, 16-, 26-, and 43-fold at 6, 12, 18, and 24 h after the treatment, respectively. A corresponding increase in the level of P450 2B1/2 protein was observed by immunoblot analysis. The level of P450 2B1/2 mRNA in rat liver also increased markedly, reaching a maximum at 12 h after the DAS treatment. Hybridization with the isozyme-specific oligonucleotide probes revealed that the mRNA levels of both P450s 2B1 and 2B2 were induced. In contrast, the level of P450 2E1 mRNA in the liver of DAS-treated rats was not changed. The results of nuclear run-on assay revealed that the transcriptional rate of P450 2B1/2 genes in the rat liver increased 13-fold at 6 h after DAS administration and returned to the control value at 24 h. The transcription of P450 2B1/2 genes was blocked completely by alpha-amanitin, an inhibitor of RNA polymerase II. These results clearly demonstrate that the induction of P450 2B1/2 in rat liver by DAS is mainly due to transcriptional activation. In the DAS-treated rats, P450 2B1/2 mRNA was also markedly induced in the stomach and duodenum. The maximal induction was found at 12 h after the treatment while the levels of P450 2B1/2 mRNA increased 66-fold in the duodenum and 23-fold in the stomach. DAS treatment, however, did not change the levels of P450 2B1/2 mRNA in the lung and nasal mucosa.

Induction of liver microsomal cytochrome P-450 2B1 by dimethyl diphenyl bicarboxylate in rats.

Dimethyl diphenyl bicarboxylate (dimethyl-4,4'-dimethyloxy-5,6,5',6'-dimethylene-dioxy-di phe nyl-2,2'- bicarboxylate, DDB), a synthetic mimic of the natural product schizandrin C, is used in China as a hepatoprotective agent to improve the liver functions of patients with hepatitis or under cancer chemotherapy. In this study, we investigated the effects of DDB on liver microsomal drug-metabolizing enzymes. When male Sprague-Dawley rats were treated with a daily intragastric dose of DDB (200 mg.kg-1) for 3 d, the microsomal pentoxyresorufin dealkylase activity and P-450 2B1 protein levels were markedly increased. The fold increase was lower than that by phenobarbital (75 mg.kg-1, ip once daily x 3 d). The level of P-450 2B1 mRNA was elevated by DDB but the magnitude of the elevation was much less than that caused by phenobarbital. DDB also increased the rates of testosterone hydroxylation at positions 16 beta, 16 alpha, 6 beta, and 2 beta as well as the rate of ethoxyresorufin dealkylation, suggesting moderate increases in the levels of P-450 3A and P-450 1A1 in addition to the huge increase in P-450 2B1. The level of glutathione S-transferase was also slightly increased, but the levels of P-450 2E1 and NAD(P)H: quinone oxidoreductase were not changed. The results indicate that DDB is an inducer of P-450 2B1.

Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells.

A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.

Kinetics and enzyme involvement in the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in microsomes of rat lung and nasal mucosa.

The rat lung and nasal cavity are two target organs for carcinogenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In order to characterize further the enzymes involved in the bioactivation of NNK, detailed kinetic and inhibitory studies were conducted with rat lung and nasal mucosa microsomes, and the results were compared with previous studies. The enzymes in rat lung microsomes catalyzed the alpha-hydroxylation, pyridine N-oxidation and carbonyl reduction of NNK. The apparent Km for the formation of the NNK-derived keto aldehyde, NNK-N-oxide, the NNK-derived keto alcohol and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were 28.8, 10.4, 7.0 and 178.1 microM respectively. In rat nasal microsomes, alpha-hydroxylation was the predominant pathway and the rate was approximately 200 times higher than that in lung microsomes. The apparent Kms for keto aldehyde and keto alcohol formation in rat nasal microsomes were 9.6 and 10.1 microM respectively. The cytochrome P450 inhibitors metyrapone and carbon monoxide markedly inhibited the metabolism of NNK in both rat lung and nasal microsomes. In rat lung microsomes, alpha-naphthoflavone and monospecific antibodies against P450s 1A2, 2A1 and 2B1 inhibited the formation of keto aldehyde by 39, 46, 64 and 23% respectively. In rat nasal microsomes, alpha-naphthoflavone and antibodies against P450s 1A2, 2A1 and 3A inhibited the metabolism of NNK by 80, 35, 20 and 14% respectively. The results indicate that cytochromes P450 play a major role in the metabolic activation of NNK in rat lung and nasal microsomes, and that there are tissue-related differences in NNK metabolism.

Modulation of the levels of cytochromes P450 in rat liver and lung by dietary lipid.

This study was undertaken to investigate the effect of dietary lipid on the regulation of several constitutive P450 isozymes. Male Sprague-Dawley rats with body weights of 130-140 g were fed either a 20% corn oil (CO) diet or a fat-free (FF) diet for 4 days following 2 days of fasting. Using liver microsomes, the catalytic activities and immunochemically detectable protein levels of P450s 1A1 and 2, 2A1, 2B1 and 2, 2C11, 2E1, and 3A were determined. The microsomes from rats fed the 20% CO diet exhibited 2-fold higher levels in N-nitrosodimethylamine demethylase activity and P450 2E1 protein than those from rats fed the FF diet. The CO group also showed 2.5-fold higher levels in 6 beta-hydroxylation of testosterone and P450 3A protein than the FF group. In contrast, the CO diet did not affect the immunodetectable level of P450 2C11 protein and its catalytic activities such as benzphetamine demethylase activity and 2 alpha-hydroxylation of testosterone. P450 1A1 was not detectable in either group, but 1A2 was 2.5-fold higher in the CO group than in the FF group. In the liver, the P450 2B1 level was very low in both groups as measured by pentoxyresorufin dealkylase activity and the protein level, whereas 2B2 was 2.5-fold higher in the CO diet group. In lung microsomes from rats fed different amounts of CO, an inverse relationship was observed between the P450 2B1 level and the dietary CO level. The results suggest that the constitutive levels of P450 isozymes are modulated by dietary lipid in a selective manner; the levels of hepatic P450s 1A2, 2B2, 2E1, and 3A were regulated positively but the level of pulmonary P450 2B1 was suppressed by dietary lipid.

Regulation of hepatic microsomal cytochrome P450IIE1 level by dietary lipids and carbohydrates in rats.

The present work tests the hypothesis that high fat/low carbohydrate diets elevate the level of liver microsomal cytochrome P450IIE1. Male Sprague-Dawley rats were fed liquid diets containing varied ratios of corn oil/carbohydrate for 4 d. Rats fed diets with higher fat/carbohydrate ratios produced higher serum acetone levels and higher hepatic microsomal P450IIE1 content and N-nitrosodimethylamine demethylase activity than those fed diets with lower fat/carbohydrate ratios. This dietary fat/carbohydrate effect on P450IIE1 also was observed with modified semipurified AIN-76A diets. In addition, both the quantity and the extent of unsaturation of dietary lipids affected P450IIE1 regulation. At moderate fat levels (5 and 20% diet), rats fed corn oil and menhaden oil diets produced higher P450IIE1 activity than those fed lard and olive oil diets. Rats fed a diet containing 20% corn oil or an amount of linoleic acid equivalent to the 20% corn oil diet showed twofold to threefold increases in the level of P450IIE1 over those fed a fat-free diet. Rats fed a 25% corn oil diet showed twofold higher enflurane metabolism in vivo than those fed a 0.5% corn oil diet. The present results suggest that the constitutive P450 enzyme level is regulated by dietary fat/carbohydrate ratios.

Effects of disulfiram on hepatic P450IIE1, other microsomal enzymes, and hepatotoxicity in rats.

Disulfiram, widely used in avoidance therapy for alcohol abuse, has been shown to have protective effects against chemically induced toxicity and carcinogenesis. The purpose of this work was to elucidate the biochemical mechanisms of this protective action by examining its effects on cytochrome P450IIE1 and other related microsomal enzyme activities. When a dose of disulfiram was given intragastrically to rats, a very rapid decrease of N-nitrosodimethylamine (NDMA) demethylase activity, possibly due to the inactivation of P450IIE1, was seen. The loss of P450IIE1 protein from the microsomal membrane was observed at 18 hr after receiving disulfiram, but not within the first 5 hr after the treatment. P450IIB1, on the other hand, was induced markedly between 15 and 72 hr after the disulfiram treatment. The treatment, however, caused only moderate changes in some other P450 isozymes. Carbon disulfide, a putative metabolite of disulfiram, produced similar effects on P450IIE1, but with shorter duration. Carbon disulfide, however, did not induce P450IIB1. Diethyldithiocarbamate, a reductive product of disulfiram, was an inhibitor of P450IIE1 activity in vitro, and upon preincubation with microsomes, it produced an NADPH-dependent inactivation of NDMA demethylase activity. The results suggest that this or other metabolites of disulfiram are inhibitors of P450IIE1 and are responsible for the inactivation of P450IIE1 in vivo. Hepatotoxicity of NDMA or CCI4 in rats was blocked by pretreatment with disulfiram. The present work demonstrates that P450IIE1 was inhibited and inactivated by disulfiram, and this mechanism can account for many of the reported inhibitory actions of disulfiram against chemically induced toxicity and carcinogenesis.

Modulation of rat hepatic microsomal monooxygenase enzymes and cytotoxicity by diallyl sulfide.

Diallyl sulfide (DAS) and other organosulfur compounds inhibit chemically induced carcinogenic and toxic responses in rodent model systems. A possible mechanism of action is the inhibition of the hepatic cytochrome P450IIE1-dependent bioactivation of the procarcinogens and protoxicants. Previous work showed competitive inhibition by DAS of N-nitrosodimethylamine (NDMA) demethylase activity in vitro, and a reduction in the microsomal level of P450IIE1 after in vivo treatment with DAS. The present studies demonstrated a time- and dose-dependent decrease of hepatic microsomal P450IIE1 activity, induction of P450IIB1 and pentoxyresorufin dealkylase activity, and moderate induction of ethoxyresorufin dealkylase activity by oral DAS treatment. DAS treatment elevated P450IIB1 mRNA but had no effect on P450IIE1 mRNA. Treatment with putative metabolites of DAS, diallyl sulfoxide and diallyl sulfone, led to similar modulations in monooxygenase activities, but the decrease of P450IIE1 activity by the sulfone occurred more rapidly. In studies in vitro, diallyl sulfone caused a metabolism-dependent inactivation of P450IIE1, but such inactivation was not observed with DAS or diallyl sulfoxide. The profile of microsomal testosterone metabolism after DAS treatment indicated an enhancement of P450IIB1-dependent 16 beta-hydroxylase activity, and a decrease in 6 beta-hydroxytestosterone production possibly related to a lower level of P450IIIA1 or IIIA2. When rats were subjected to a 48-hr fast and DAS treatment, the starvation-induced microsomal P450IIE1 level was decreased by DAS. Inhibition of hepatotoxicity due to exposure to P450IIE1 substrates, CCl4 and NDMA, by DAS was observed under a variety of treatment schedules.

Metabolism of carcinogenic nitrosamines by rat nasal mucosa and the effect of diallyl sulfide.

Rat nasal cavity is one of the target organs for carcinogenesis induced by N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The present work investigated the metabolism of these nitrosamines by rat nasal microsomes, as well as the possible modulating factors. Microsomes prepared from rat nasal mucosa were efficient in metabolizing these nitrosamines. In general, the metabolism of the nitrosamines was slightly higher in 9-week-old rats than in 4-week-old animals, and there was no sex-related difference. Fasting of rats for 48 h, which is known to induce hepatic cytochrome P450IIE1 and NDMA metabolism, did not increase the nasal metabolism of NDMA, NDEA, or NNK. Pretreatment of rats with acetone, another inducer of hepatic P450IIE1, did not increase the metabolism of NDMA. Furthermore, it decreased the nasal metabolism of NDEA and NNK. Immunoinhibition studies suggest that, in the nasal mucosa, P450IIE1 is only partially responsible for the oxidation of NDMA and other P450 isozymes are responsible for the metabolism of NDEA. A single p.o. pretreatment of male rats with diallyl sulfide (DAS), a component of garlic oil, caused a significant decrease in the oxidative metabolism of NDEA and NNK in rat nasal mucosa. Whereas the nasal metabolism of NDMA was reduced by DAS pretreatment, there was no change in the amount of the nasal microsomal proteins immunoreactive with the antibodies against P450IIE1. The inhibitory effect of DAS on the nasal oxidative metabolism of NDMA, NDEA, and NNK was also observed in experiments in vitro. The results demonstrate the ability of nasal mucosa to metabolically activate these nitrosamines and the inhibition of this process by DAS, suggesting that DAS may be effective in inhibiting the related nasal tumorigenesis.

Roles of dietary corn oil in the regulation of cytochromes P450 and glutathione S-transferases in rat liver.

To study the molecular mechanisms by which dietary lipids affect the levels of cytochrome P450 (P450) isozymes, male Sprague-Dawley rats were fed either fat-free (FF) or 20% corn oil (CO) diet in combination with one of the following three treatments: no inducer, phenobarbital (PB) and acetone. Dietary CO did not affect the constitutive level of P450IIB (PB-inducible), but it affected the induction of P450IIB by PB treatment. The induction of P450IIB by PB in the CO group as determined by 7-pentoxy-resorufin O-dealkylase activity and immunochemically detected protein level was twofold higher than that in the FF group, and this difference was also reflected in the level of mRNA for this enzyme. In contrast, dietary CO increased the constitutive level of P450IIE (ethanol-inducible) twofold as indicated by N-nitrosodimethylamine demethylase activity and immunochemically detectable protein, but it had no effect on the induction of P450IIE by acetone. The induced level of P450IIE by acetone in the CO group did not differ from that in the FF group as measured by the enzyme activity and protein level. It was demonstrated that dietary CO affects P450IIB and IIE activities by altering the concentration of the isozymes rather than by modulating their catalytic activities. In addition, dietary CO increased the microsomal testosterone 6 beta-hydroxylase activity but not 7 alpha- and 2 alpha-hydroxylase activities, suggesting an increase in P450IIIA and/or IIC13 but not in IIA1 and IIC11, respectively. Dietary CO also affected the constitutive and induced levels of glutathione S-transferase (GST) isozymes in a different manner: it increased the constitutive level of GST-B but not that of GST-A. Nevertheless, it was important for the induction of both GST-A and GST-B by PB treatment. The results suggest that lipid nutrition affects xenobiotic metabolism activities by altering constitutive and inducible levels of certain P450 and GST isozymes.

Roles of pituitary hormones in the regulation of hepatic cytochrome P450IIE1 in rats and mice.

Cytochrome P450IIE1 (P450IIE1) is responsible for the metabolic activation of N-nitrosodimethylamine (NDMA), a potent environmental carcinogen. This P450 enzyme displays a high-affinity NDMA demethylase (NDMAd) activity and is known to be induced by fasting and acetone administration. In the present work, the effects of pituitary hormones on the regulation of P450IIE1 in the liver were investigated and compared in rats and mice. There was no difference in liver microsomal NDMAd activity (nmol/min/mg protein) in rats in the intact (0.38 +/- 0.12), sham-operated (0.44 +/- 0.06), and hypophysectomized (0.52 +/- 0.04) groups. However, hypophysectomy caused a 2-fold increase in hepatic P450IIE1 protein levels as determined by immunoblot analysis. The P450IIE1 mRNA level in hypophysectomized rat was also significantly increased. The levels of blood ketone bodies (acetone, acetoacetate, and beta-hydroxybutyrate) were not different in the intact, sham-operated, and hypophysectomized groups, suggesting that ketone bodies are not involved in the induction of P450IIE1 protein and its mRNA by hypophysectomy. The discrepancy between the NDMAd activity and the increased P450IIE1 protein in rat liver by hypophysectomy can be partially explained by the lower hepatic NADPH-P450 reductase activity (50% that of the control) in the hypophysectomized rats. Upon the induction of liver NDMAd activity by fasting and acetone, hypophysectomy attenuated the effect of acetone but abolished the effect of fasting completely. Nevertheless, fasting still caused a 3-fold increase in the liver P450IIE1 mRNA level. An involvement of pituitary hormones in the regulation of liver microsomal P450IIE1 in mouse, however, was not observed. There was no difference in constitutive NDMAd activity between genetically growth hormone-deficient (lit/lit) mice and their phenotypically normal heterozygotes (lit/+). Fasting for 48 h caused 1.5- to 2-fold induction and acetone caused 2- to 3-fold induction, in both groups. The above changes in enzyme activity were due to the changes of P450IIE1 levels as verified by the immunoblot analysis. In male BALB/c mice, neither the hepatic NDMAd activity nor the P450IIE1 protein level was altered by hypophysectomy. The effects of acetone on the liver NDMAd activity were also similar in hypophysectomized and sham-operated mice. The results suggest that pituitary hormones are important in the regulation of the expression and activity of hepatic P450IIE1 in rats but not in the mouse strains investigated.

Effect of phenethyl isothiocyanate on microsomal N-nitrosodimethylamine metabolism and other monooxygenase activities.

1. Phenethyl isothiocyanate (PEITC), a dietary compound derived from cruciferous vegetables, has previously been shown to decrease N-nitrosodimethylamine (NDMA)-induced methylation of hepatic DNA, apparently by inhibition of microsomal activation of the procarcinogen. 2. Using hepatic microsomes from acetone-treated rats, PEITC exhibited competitive inhibition of NDMA demethylase activity with an apparent Ki of 1 microM. In studies using a two-stage incubation protocol, the inhibition by PEITC was time- and metabolism-dependent. 3. Using control rat liver microsomes, PEITC selectively inhibited P450 IIE1-mediated NDMA-demethylase activity as compared to the demethylation of benzphetamine and ethylmorphine. 4. Pretreatment of rats with a single oral dose of PEITC (1 mmol/kg body wt) 24 h before killing caused a marked decrease in hepatic NDMA demethylase activity, but an 11-fold increase in 7-pentoxyresorufin O-dealkylase activity. These trends agreed with immunoblot analysis which indicated that PEITC was a suppressor of P450 IIE1 but an inducer of P450 IIB1. 5. The selective inhibition of P450 IIE1 activity and suppression of its level in microsomes indicates a role for PEITC as a chemopreventive agent against toxic or carcinogenic metabolites of this isozyme.

Effects of thiamine deficiency on hepatic cytochromes P450 and drug-metabolizing enzyme activities.

To elucidate the mechanisms by which thiamine deficiency affects hepatic microsomal monooxygenase activities, the effect of thiamine deficiency on two constitutive cytochrome P450 isozymes, P450IIE1 and P450IIC11, was investigated, using weanling male Sprague-Dawley rats. The clinical signs of thiamine deficiency were apparent after feeding a thiamine-deficient diet for 3 weeks. Thiamine deficiency caused an increase in P450IIE1, which was determined by N-nitrosodimethylamine demethylase assay and immunoquantitation of P450IIE1. This increase in the P450IIE1 level was mainly attributed to thiamine deficiency per se but not to dietary restriction. Ketone bodies were not elevated in thiamine-deficient rats, whereas ketone bodies were elevated and may have served as inducing factors in calorically restricted pair-fed animals. Injections of pyruvate or pyrithiamine in addition to thiamine deficiency did not potentiate the induction effect. On the other hand, thiamine deficiency did not affect the level of P450IIC11 during the 3 weeks of feeding the thiamine-deficient diet. In addition, thiamine deficiency increased cytosolic glutathione S-transferase activity but not steroid isomerase activity. The present study demonstrates the specificity of thiamine deficiency per se in the induction of P450IIE1 which does not involve an increase in the ketone body level.

Metabolism of methyl tertiary-butyl ether by rat hepatic microsomes.

Exposure to methyl tertiary-butyl ether (MTBE), a commonly used octane booster in gasoline, has previously been shown to alter various muscle, kidney, and liver metabolic activities. In the present study, the metabolism of MTBE by liver microsomes from acetone- or phenobarbital-treated Sprague-Dawley rats was studied at concentrations of up to 5 mM MTBE. Equimolar amounts of tertiary-butanol, as measured by head-space gas chromatography, and formaldehyde were formed. The Vmax for the demethylation increased by 4-fold and 5.5-fold after acetone and phenobarbital treatments, respectively. The apparent Km value of 0.70 mM using control microsomes was decreased slightly after acetone treatment, but was increased by 2-fold after phenobarbital treatment. The metabolism of MTBE (1 mM) was inhibited by 35% by monoclonal antibodies against P450IIE1, the acetone/ethanol inducible form of cytochrome P450, suggesting a partial contribution by this isozyme. A single 18-h pretreatment of rats with 1 or 5 ml/kg MTBE (i.p.) resulted in a 50-fold induction of liver microsomal pentoxyresorufin dealkylase activity but no change in N-nitrosodimethylamine demethylase activity. These trends in activity agreed with immunoblot analysis which showed an elevation in P450IIB1 but no change in P450IIE1 levels.

Alteration of rat liver microsomal monooxygenase activities by gasoline treatment.

Previous work has shown an increase in rat liver enzyme activities after chronic exposure to gasoline vapor. In the present study, male Sprague-Dawley rats were pretreated with unleaded gasoline at 1 and 5 ml/kg, i.p., and selected hepatic microsomal monooxygenase activities were determined at 18, 48, and 72 h. At 18 h, moderate increases were observed in P450 content (1.3-fold), cytochrome c-reductase activity (1.25-fold), and in N-nitrosodimethylamine demethylation rate (1.25- to 1.6-fold). Pentoxyresorufin dealkylase activity (an activity displayed primarily by P450IIB1) was significantly elevated at 18 and 48 h (30- to 60-fold), and ethoxyresorufin dealkylase activity (an activity displayed by P450 IA1) was elevated (2- to 4-fold). Immunoblot analysis revealed no change in P450IIE1 at these time points, but an elevation in P450IIB1 in agreement with the pentoxyresorufin dealkylase activity measurements.

Induction of cytochromes P450IIE1 and P450IIB1 by secondary ketones and the role of P450IIE1 in chloroform metabolism.

It has been shown previously that the potentiation of chloroform-induced hepatotoxicity by linear secondary ketones increases with the carbon-chain length. The present work examines the possibility that this potentiation is due to the induction of P450IIE1. The metabolism of chloroform, as measured using headspace gas chromatography, in the presence of microsomes from acetone-treated rats was elevated threefold compared to controls. Inclusion of monoclonal antibody against P450IIE1 inhibited the metabolism by 81%. Alternate substrates of P450IIE1 were also inhibitory. Chloroform metabolism was observed using purified, reconstituted P450IIE1 plus cytochrome b5, but was not detected using P450IIB1. The inductive effect of 18-hr oral pretreatment (15 mmol/kg body wt) with each of three secondary ketones on two isozymes of rat liver microsomal cytochrome P450, P450IIE1, and P450IIB1 was studied. The content of total microsomal P450 and NADPH-dependent cytochrome c reductase, the rates of oxidation of N-nitrosodimethylamine, benzphetamine, and pentoxyresorufin, as well as levels of immunoreactive protein for both of the isozymes were elevated by the pretreatments in the rank order of acetone less than or equal to 2-butanone less than 2-hexanone, in agreement with other trends noted by previous investigators. The results provide further evidence for the role of P450IIE1 induction in the potentiation phenomenon.

Molecular basis for the sex-related difference in renal N-nitrosodimethylamine demethylase in C3H/HeJ mice.

Previous work with rat and rabbit liver enzymes has demonstrated that cytochrome P450IIE1 is responsible for the metabolism of N-nitrosodimethylamine (NDMA), a widely occurring carcinogen. The present study demonstrated that a similar enzyme also exists in the mouse kidney and is regulated by testosterone. These results can account for the reported sex-related difference in the renal metabolism of NDMA in mouse strains such as C3H/HeJ. NDMA demethylase activities (expressed as pmol/min/mg protein) in kidney microsomes of female and male C3H/HeJ mice were 3.0 +/- 0.7 and 51.9 +/- 11.2, respectively. After testosterone treatment (500 mg/kg b.w. in olive oil, s.c.) for 2 days, the renal NDMA demethylase activity of the female mice was elevated 17-fold. The difference and change in NDMA demethylase activity were accompanied by corresponding differences and changes in P450IIE1 as quantified by immunoblot analysis (using antibodies prepared against rat P450IIE1) as well as in the mRNA level for P450IIE1 as determined by Northern and slot blot analyses (using a cDNA probe containing the coding sequence of rat P450IIE1 gene). Based on gel electrophoresis, the molecular weight of mouse renal P450IIE1 was 52,000 and the size of mouse renal P450IIE1 mRNA was approximately 1.8 kilobases; both were similar to those found in rat liver and kidney. Renal P450IIE1 mRNA levels in female, male, and testosterone-treated female mice were at a ratio of 1:22:20. On the other hand, this testosterone-related difference was not observed in hepatic P450IIE1. In liver microsomes, there were no significant differences in NDMA demethylase activity, P450IIE1 content, and P450IIE1 mRNA level between male and female mice or between untreated and testosterone-treated female mice. The apparent Km value of NDMA demethylase in mouse kidney microsomes (22 to 27 microM NDMA) were similar to that in rat liver microsomes. Renal NDMA demethylase activity was inhibited by a monoclonal antibody prepared against rat P450IIE1. These results suggest that mouse renal P450IIE1 is similar to rat P450IIE1 and is responsible for the low Km form of NDMA demethylase activity. Nevertheless, only the mouse renal enzyme is regulated by testosterone.

Regulation of N-nitrosodimethylamine demethylase in rat liver and kidney.

In previous work, the low Km form of N-nitrosodimethylamine (NDMA) demethylase has been demonstrated to be due to a specific form of cytochrome P-450 (designated as P-450ac) and to be the enzyme required for the metabolic activation of NDMA. The present work deals with the regulation of P-450ac in rat liver during development as well as the mechanism of induction of P-450ac in rat liver and kidney by inducers. NDMA demethylase activity was almost undetectable in the liver of newborn rats, increased after day 4, and remained elevated throughout the first 17 days of the neonatal period. The enhancement of NDMA demethylase activity during development was accompanied by corresponding increases of P-450ac content and P-450ac mRNA levels as determined by Western and slot blot analyses, respectively. No sex differences with respect to this enzyme were observed in the developing rats. Acetone treatment on late-term pregnant rats for 2 days resulted in transplacental inductions of P-450ac and P-450ac mRNA in the newborn rats. Pretreatment of young male rats and adult female rats with acetone or isopropyl alcohol caused increases of NDMA demethylase activity and P-450ac content in the liver but no significant change in the P-450ac mRNA level. These facts suggest the possible existence of a posttranscription regulatory mechanism under these induction conditions. The presence of P-450ac in rat kidney was demonstrated by Western and Northern blot analyses. The renal form of P-450ac seemed to be regulated in a fashion similar to the hepatic P-450ac regarding its response to inducing factors such as fasting and acetone treatment.

Metabolism and activation of N-nitrosodimethylamine by hamster and rat microsomes: comparative study with weanling and adult animals.

It has been reported that hamster liver preparations are more effective for the metabolic activation of N-nitrosodimethylamine (NDMA) to a mutagen than rat liver preparations. The enzymatic basis for this phenomenon, however, has not been clearly elucidated. The present study was undertaken to examine the enzymology of NDMA metabolism by different hepatic subcellular fractions prepared from hamsters and rats of two different ages, and to investigate the correlation between the metabolism and the activation of NDMA to a mutagen for Chinese hamster V79 cells. The content of cytochrome P-450 was approximately 1.5-fold higher in hamster microsomes than in rat microsomes from both ages (1.19-1.38 versus 0.73-0.83 nmol P-450/mg protein). Weanling hamster microsomes exhibited multiple apparent Km values for NDMA metabolism as did weanling rat microsomes. The apparent Km I value of NDMA demethylase (NDMAd) in hamster microsomes was about one-half that in rat microsomes (36 versus 83 microM) with corresponding Vmax values of 2.09 and 2.57 nmol/min/nmol P-450. The Km I values for denitrosation did not differ from the corresponding values for NDMAd with Vmax values of 0.17 and 0.22 nmol/min/nmol P-450 for hamster and rat microsomes, respectively. These apparent Km values were affected neither by sonication nor by the presence of cytosolic proteins in S9 fractions. Adult rat liver microsomes showed less than one-half the NDMAd activity in weanling rat liver microsomes, whereas such age difference was not observed in hamster liver microsomes. This result was confirmed by Western blotting showing the levels of P-450ac (an acetone-inducible form of P-450) of these microsomes at comparable levels to their NDMAd activities. NDMAd was highly correlated to the metabolic activation of NDMA to a mutagen for V79 cells in an activation system mediated by microsomes prepared from hamsters and rats of different ages. The results from this study clearly demonstrate the enzymatic basis for the more effective metabolism of NDMA in adult hamsters than in adult rats.