N-nitrosodimethylamine Demethylation Research Articles

Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes

To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl 4 to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl 4 supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme.

Metabolism of methyl tert-butyl ether and other gasoline ethers in mouse liver microsomes lacking cytochrome P450 2E1

To reduce the production of pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE) and other ethers such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Metabolism of these gasoline ethers is catalyzed by cytochrome P450 (P450) enzymes. P450 2E1, which metabolizes diethyl ether, was suggested to be an enzyme involved. The present study used 2E1 knock-out mice (2E1 −/−) to assess the contribution of 2E1 to the metabolism of MTBE, ETBE and TAME. Liver microsomes prepared from the 2E1 knock-out mice lacked 2E1 activity (assayed as N-nitrosodimethylamine demethylation), but were still active in metabolizing all three gasoline ethers. The levels of ether-metabolizing activity (nmol/min per mg) in the liver microsomes from 7 week old female 2E1 knock-out mice were 0.54±0.17 for MTBE, 0.51±0.24 for ETBE and 1.14±0.25 for TAME at a 1 mM substrate concentration. These activity levels were not significantly different from those of the sex- and age-matched C57BL/6N and 129/Sv mice, which are the parental lineage strains of the 2E1 knock-out mice and are both 2E1 +/+. Our results clearly demonstrate that 2E1 plays a negligible role in the metabolism of MTBE, ETBE and TAME in mouse livers.

Peroxidases from Tulip Bulbs (Tulipa fosteriana, L.) Oxidize Xenobiotics NNitrosodimethylamine and N-Nitroso-N-methylaniline in vitro

Tulip bulbs (Tulipa fosteriana, L.) contain peroxidases catalyzing the oxidation of the xenobiotics N-nitrosodimethylamine (NDMA) and N-nitroso-N-methylaniline (NMA). Three anionic (A1, A2, A3) and four cationic (B, C, D, E) peroxidases were purified from this tissue, partially characterized and used for kinetic studies. Demethylation of NDMA and NMA producing formaldehyde is catalyzed by one anionic (A1) and three cationic (C, D, E) peroxidases. The oxidation of NDMA by tulip peroxidases exhibits the Michaelis-Menten kinetics. The apparent Michaelis constant and the maximal velocity values for this substrate were determined. On the other hand, non-Michaelian kinetics for the NMA oxidation were observed with tulip peroxidases. The most abundant cationic peroxidase (peroxidase C) was used for detailed enzymatic studies. In addition to formation of formaldehyde, methylaniline, aniline, 4-aminophenol and phenol were found to be metabolites formed from NMA. Phenol was formed presumably by N-demethylation via a benzenediazonium ion, while methylaniline, aniline and 4-aminophenol were products of denitrosation of the substrate. The efficiencies of plant peroxidases to oxidize NDMA and NMA in vitro are compared with those of cytochromes P450 and discussed.

Induction and suppression of renal and hepatic cytochrome P450-dependent monooxygenases by acute and chronic streptozotocin diabetes in hamsters.

The acute and chronic effects of streptozotocin diabetes on kidney and liver microsomal monooxygenases were studied using hamsters 2 days and 6 weeks following treatment with the diabetogen, respectively. Acute diabetes increased aniline hydroxylation and N-nitrosodimethylamine demethylation, decreased pentoxyresorufin O-dealkylation, without affecting benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation in kidney and liver microsomes. The effects of chronic diabetes on the microsomal monooxygenases were similar to the effects of acute diabetes, except that the chronic diabetic condition markedly decreased benzo(a)pyrene and 7-ethoxycoumarin oxidations in kidney microsomes. Total cytochrome P450 content and NADPH-cytochrome P450 reductase activity in kidney and liver microsomes of the diabetic hamsters were similar to the controls. Gel electrophoresis of microsomes from control and streptozoptocin treated hamster tissues revealed that diabetes enhanced the intensity of protein band(s) in the P450 molecular weight region. Immunoblotting of microsomal proteins showed that acute and chronic streptozotocin diabetes induced proteins immunorelated to P450s 2E1 and 1A in kidney and liver. In marked contrast, the acute and chronic diabetic conditions decreased the level of a P450 2B-immunorelated protein(s) in kidney and liver. The present study demonstrates that acute and chronic streptozotocin diabetes has the ability to induce P450 2E1 and 1A and suppress P450 2B in hamster kidney and liver and that the hamster monooxygenase responds to diabetes differently from the rat enzyme.

Validation of the (ω-l)-hydroxylation of lauric acid as an in vitro substrate probe for human liver CYP2E1

The (ω-1)-hydroxylation of lauric acid (11-OH-LA), a model substrate of fatty acids, was previously shown to be due to CYP2E1 in rat liver microsomes. The present study examined changes in hepatic CYP2E1 content and 11-OH-LA in a panel of 29 human liver microsomes. The 11-OH-LA activity was strongly correlated with the CYP2E1 content, quantitated by immunoblot ( r = 0.75) and with four monooxygenase activities known to be mediated by CYP2E1: chlorzoxazone-6-hydroxylation ( r = 0.73), 4-nitrophenol hydroxylation ( r = 0.84), N-nitrosodimethylamine demethylation ( r = 0.79) and n-butanol oxidation ( r = 0.73). The (ω-1)-hydroxylation of lauric acid was inhibited by ethanol ( K i = 3.5 mM), acetone (IC 50 = 10 mM), dimethylsulfoxide, chlorzoxazone (competitive inhibitors of CYP2E1), diethyldithiocarbamate, and diallylsulfide (both selective mechanism-based inactivators of CYP2E1). The weak value of ethanol K i on the (ω-1)-hydroxylation of lauric acid suggested that low levels of alcohol could modify fatty acid metabolism in the liver. Furafylline and gestodene, suicide substrates of CYP1A and CYP3A4, respectively, did not modify the 11-hydroxylation of lauric acid. Polyclonal antibody directed against rat CYP2E1 inhibited the formation of 11-OH-LA without affecting 12-OH-LA activity. Taken together, these results suggest that CYP2E1 is involved in the (ω-1)-hydroxylation of lauric acid in human liver microsomes, and ω-hydroxylation is mediated by another enzyme. Finally, the use of yeasts and mammalian cells genetically engineered for expression of 9 human P450s demonstrated that CYP2E1 was the one enzyme involved in the (ω-1)-hydroxylation of lauric acid.

Induction and Purification of Cytochrome-P450 1A1 from 3-Methylcholanthrene-Treated Tilapia, Oreochromis niloticus × Oreochromis aureus

Pretreatments of freshwater fish tilapia, Oreochromis niloticus × O. aureus, with 3-methylcholanthrene (3-MC) and polychlorinated biphenyls (PCBs) increased liver microsomal cytochromes P450 (P450) and b5 contents, benzo[a]pyrene hydroxylation, and 7-ethoxyresorufin and 7-ethoxycoumarin O-dealkylations. The pretreatments also increased gill microsomal benzo[a]pyrene and 7-ethoxyresorufin oxidations. Immunoblot analysis of liver and gill microsomes revealed that 3-MC and PCBs induced a protein recognized by the mouse monoclonal antibody (MAb) 1-12-3 against scup P450 1A1. Northern analysis of liver and gill RNA showed that 3-MC and PCBs increased the intensity of 2.9-kb- and 1.5-kb-sized mRNA bands hybridizable to a trout P450 1A1 cDNA probe. Pretreatment with phenobarbital was without effects on the monooxygenase activity or protein or mRNA levels in liver and gill. A 3-MC-inducible P450 hemoprotein (Mr = 59,000) and a NADPH-cytochrome P450 reductase flavoprotein (Mr = 74,000) were purified from liver microsomes. The tilapia P450 hemoprotein showed an absorption maximum at 447 nm in CO-difference spectrum and a strong immunoreactivity with MAb 1-12-3. A reconstituted tilapia monooxygenase system consisting of P450 and NADPH-cytochrome P450 reductase was effective in the catalysis of 7-ethoxyresorufin, benzo[a]pyrene, and 7-ethoxycoumarin oxidations, but not in N-nitrosodimethylamine demethylation. These results show that 3-MC and PCBs can induce P450 1A1 in tilapia liver and gill and the tilapia P450 is highly similar to other teleost P450 1A1 with respect to spectral, immunochemical, and catalytic properties.

Probing the Active Sites of Rat and Human Cytochrome-P450 2E1 with Alcohols and Carboxylic Acids

Cytochrome P450 2E1 (P450 2E1) catalyzes the biotransformation of many low-molecular-weight compounds including industrial solvents, indoor pollutants, alcohol, and drugs. In order to understand the nature of the P450 2E1 active site, we studied the competitive inhibition of the P450 2E1-catalyzed N-nitrosodimethylamine demethylation by alcohols and carboxylic acids with different alkyl chain lengths. Using microsomes from acetone-treated rats as the enzyme source of P450 2E1, the K i value for each compound was measured. With primary alcohols and secondary alcohols, the K i decreased with the increase in the carbon chain length until the carbon number reached 6 or 7; the free energy increment of binding was 0.28 kcal/mol CH 2 group. Similar inhibitory effects were also observed with human P450 2E1 heterologously expressed in Hep G2 cells. These results suggest that both rat and human P450 2E1 contain a pocket with hydrophobicity that serves as a binding site for low-molecular-weight substrates. Among a series of carboxylic acids and ω-hydroxycarboxylic acids investigated, dodecanoic acid was the strongest inhibitor ( K i = 22 μM), and 12-hydroxydodecanoic acid had the lowest K i (320 μM) within the series of ω-hydroxycarboxylic acids. The free energy increment of binding for carboxylic acids was 0.35 kcal/mol CH2 group. 1,10-Decanedicarboxylic acid, which contains a carboxylic group at each end, showed a K i > 20 mM. We suggest that for optimal interaction of a carboxylic acid moiety with the active site of P450 2E1, the hydrocarbon end of the molecule binds to the substrate binding site, leaving the carboxylic acid group outside of a proposed substrate access channel. The length of optimal substrates such as dodecanoic acid may reflect the length of the substrate access channel and the size of the active site. We estimate that the distance from the opening of the access channel to the oxygenation site is about 15 Å. Based on the structural features of P450 2E1 substrates and competitive inhibitors, we propose a conceptual model to illustrate the binding of these molecules in the active site of P450 2E1.

Hepatoprotective mechanism of silymarin: No evidence for involvement of cytochrome P450 2E1

The involvement of the alcohol-inducible cytochrome P450 2E1 in the hepatoprotective mechanism of the plant flavonoid extract silymarin, and its main active component silybin, was investigated in isolated hepatocytes. Allyl alcohol toxicity, associated lipid peroxidation and GSH depletion was efficiently counteracted by silymarin (0.01–0.5 mM), and at higher concentrations by silybin. Cell damage by t-butyl hydroperoxide was also prevented by silymarin and silybin, but less efficiently. However, the covalent binding of the acetaminophen intermediate, formed via P450 2E1, was unaffected by addition of the flavonoids. Silybin did not influence microsomal 2E1-catalyzed demethylation of N-nitrosodimethylamine. Neither did demethylation of N-nitrosodimethylamine or aminopyrine by isolated microsomes affect the in vivo administration of silybin. Addition of silymarin or silybin to primary cultures of isolated hepatocytes did not prevent cell damage induced by exposure to the P450 2E1 substrate CCl 4. In contrast, the mere presence of low concentrations (25–50 μM) of these compounds was found to inhibit cell attachment to the matrix and eventually resulted in cell damage. We conclude that contrary to earlier reports we found no evidence for an interaction of silymarin or silybin with cytochrome P450 2E1. This suggests that the antioxidant and free radical scavenging properties may account for most of the therapeutic effect of these compounds. The untoward effect of silymarin on cultured cells may have consequences when considering long-term prescription of this therapeutic agent.

The effect of cytosol on liver microsomal metabolic activation and demethylation of N-nitrosodimethylamine.

The role of rat liver cytosol in the demethylation and metabolic activation of N-nitrosodimethylamine (NDMA) was examined. Addition of cytosol to liver microsomes from pyridine-pretreated rats enhanced DNA alkylation by NDMA 10- to 14-fold over microsomes alone, while cytosol alone had little DNA alkylating activity. The cytosolic activity responsible for the enhancement of DNA alkylation was heat labile, required NADPH, and was not a general protein effect. Addition of cytosol to purified rabbit liver cytochrome P450 2E1 in a reconstituted system consisting of NADPH-cytochrome P450 reductase, 2E1, and phospholipid produced an 18-fold increase in DNA alkylation over that observed with the reconstituted system alone. The cytosolic activity responsible for the enhancement of DNA alkylation did not work by inhibition of lipid peroxidation, nor did the addition of cytosol affect the level of NADPH present in the reaction mixtures. Attempts to identify the cytosolic component(s) responsible for the DNA alkylation enhancing activity demonstrated no evidence for the involvement of sulfhydryl-dependent enzymes, a flavoprotein, or conjugating enzymes. Studies with semicarbazide and phenylhydrazine suggest that carbonyl groups may be involved in the cytosolic activity. Measurements of NDMA demethylation demonstrated that cytosol addition led to a significant decrease in formaldehyde production, indicating that cytosol was not enhancing the activation of NDMA to a DNA alkylating species by facilitating the cytochrome P450-catalyzed demethylation reaction, and suggested that a cytosolic reaction might be occurring at the expense of formaldehyde formation.

Induction of Cytochrome P450-Dependent Monooxygenases in Hamster Tissues by Fasting

The effects of fasting on liver, kidney, and lung monooxygenases were studied using hamsters starved for 4 days. Fasting treatment increased microsomal cytochrome P450 content and NADPH-cytochrome P450 reductase activity in kidney and lung. The treatment caused significant increases of aniline hydroxylation, N-nitrosodimethylamine demethylation, and 7-ethoxycoumarin 0-deethylation activities in the liver, kidney, and lung. Fasting caused a threefold increase of benzphetamine N-demethylation activity in lung and a 25% increase in liver and had no effect in kidney. Benzo[a]pyrene hydroxylation activities in the fasted hamster liver, kidney, and lung were higher, lower, and similar to the controls, respectively. Gel electrophoresis of tissue microsomes from control and fasted hamsters revealed that fasting enhanced the intensity of protein band(s) in the P450 molecular weight region. Immunoblotting of the microsomal proteins showed that fasting induced a protein cross-reactive with rabbit antibody raised against human P450 2E1 in hamster liver, kidney, and lung. Immunoblotting analysis using mouse monoclonal antibody 2-66-3 raised against rat P450 2B1 revealed that fasting induced an immunorelated protein preferentially in hamster lung, with minimal effects on liver and kidney. Protein blots probed with mouse monoclonal antibody 1-12-3 indicated that fasting induced a protein related to P450 1A1 in hamster liver, kidney, and lung. These results demonstrate that fasting causes a variety of inductive effects on the enzyme components and catalytic activities of monooxygenase systems as well as on the P450s 2E, 2B, and 1A in the hamster tissues.

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 b 5 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 K m and V max values for NDMA demethylation were 22 μ m and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK 143 cells, which do not contain b 5, displayed K m and V max values of 31 μ m and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b 5 into TK −143 microsomes increased the V max 2.2-fold and decreased the K m to 22 μ m. Addition of b 5 to Hep G2 microsomes resulted in a 1.6-fold increase in V max, but showed no effect on the K m . P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a K m of 47 μ m and V max of 213 pmol/min/mg microsomal protein. Addition of b 5 lowered the K m to 27 μ m, but had no effect on V max. These results demonstrate conclusively that P450 2E1 is responsible for the low K m forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b 5.

Differences in hepatic microsomal cytochrome P-450 isoenzyme induction by pyrazole, chronic ethanol, 3-methylcholanthrene, and phenobarbital in high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats.

High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Deuterium isotope effect on the metabolism of N-nitrosodimethylamine and related compounds by cytochrome P4502E1.

1. Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity, and produces an isotope effect on its metabolism in vivo. Consistent with these results are the observations that deuteration caused a 5-fold increase in the apparent Km, but not the Vmax for the demethylation and denitrozation of NDMA in acetone-induced rat liver microsomes. These microsomes are a good source of cytochrome P4502E1. 2. For demethylation of Z-[2H3]NDMA and E-[2H3]NDMA, the Km values were indistinguishable, and were between the values for those of NDMA and [2H6]NDMA. Almost all the formaldehyde formed was derived from the non-deuterated methyl group, indicating a lack of stereoselectivity in the demethylation of NDMA. 3. NDMA and [2H6]NDMA displayed apparent Ki values of 59 and 441 microM, respectively, for N-nitrosodiethylamine deethylase, showing an apparent isotope effect of 0.13, and displayed an isotope effect of 0.21 in the Ki values for p-nitrophenol hydroxylase. 4. With acetone and deuterated acetone as inhibitors for p-nitrophenol hydroxylase, the isotope effect on the Ki was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase. 5. In the microsomal oxidation of ethanol, a deuterium isotope effect of about five was observed in the Vmax/Km when carbon-1 was deuterated, but was not observed in the Vmax. 6. Results illustrate a unique deuterium isotope effect on the Km values of reactions catalysed by P4502E1.

Effects of ethanol consumption on bioactivation and hepatotoxicity of N-nitrosodimethylamine in rats

To study the effects of ethanol on the hepatotoxicity of N-nitrosodimethylamine (NDMA), 5 mg NDMA/kg body weight was injected intraperitoneally 3 times a week for 6 weeks into rats pairfed liquid diets containing 36% of energy either as ethanol or as additional carbohydrates. Another group of rats was pair-fed with the same diets but injected with saline instead of NDMA. Co-administration of ethanol and NDMA produced much higher elevations of serum alanine and aspartate aminotransferase and glutamic dehydrogenase activities than the administration of either agent alone. The combined treatment also slightly increased focal necrosis, whereas other liver lesions (steatosis and fibrosis) and the functional impairment of mitochondrial respiration were not affected significantly. Microsomal low K m NDMA demethylation, as well as NDMA denitrosation, were inhibited markedly by incubation with an antibody against P450IIE1, suggesting the involvement of this alcohol-inducible P450 in both NDMA bioactivation reactions. The addition of ethanol inhibited P450-dependent demethylation and denitrosation of NDMA in liver microsomes, whereas both activities were enhanced markedly by chronic ethanol administration. At ethanol concentrations similar to those prevailing in the blood of alcohol-fed animals at the time of NDMA administration, hepatic microsomal demethylation and denitrosation remained significantly higher in ethanol-fed rats given NDMA than in controls. Our results suggest that bioactivation plays a critical role in the hepatotoxicity of NDMA and its aggravation by chronic alcohol consumption.

Deuterium isotope effect in the interaction of N-nitrosodimethylamine, ethanol, and related compounds with cytochrome P-450IIE1

Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity and produces an isotope effect on its metabolism. Our previous results showed that deuteration causes a 5-fold increase in the apparent Km, but not the Vmax, for the demethylation and denitrosation of NDMA in microsomes. In the present work, we studied the nature of this deuterium isotope effect with several compounds using acetone-induced microsomes as a source of cytochrome P-450IIE1. In the microsomal N-nitrosodiethylamine deethylase reaction, NDMA and [2H6]NDMA were competitive inhibitors and displayed apparent Ki values of 59 and 441 mM, respectively, showing an isotope effect of 0.13. Similarly, in the p-nitrophenol hydroxylase reaction, a deuterium isotope effect of 0.21 on the Ki was observed. With acetone as an inhibitor for p-nitrophenol hydroxylase, the isotope effect on the Ki was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase. When the oxidation of ethanol, [1,1-2H2]ethanol, [2,2,2-2H3]ethanol, and [2H6]ethanol was compared, an isotope effect of about 5 was found in the Vmax/Km due to the deuteration of the methylene group (carbon 1) but not due to the methyl group. However, the Vmax was not affected. A corresponding deuterium isotope effect was observed in the Ki when these compounds were used as competitive inhibitors for the NDMA demethylase reaction. The results demonstrate that deuteration of NDMA, ethanol, and related compounds results in an increase in the Km or Ki with little change in the Vmax of P-450IIE1-catalyzed reactions. The molecular basis of this isotope effect is discussed.

Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrene-pretreated rats: Stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA

1. The effect of various cytochrome P-450 inducers, namely acetone, phenobarbital (PB) and 3-methylcholanthrene (MC), on the pharmacokinetics of styrene metabolism was studied. 2. Styrene metabolism in vivo was studied measuring phenylglyoxylic acid (PGA), the enantiomers of mandelic acid (MA), and total thioethers excreted in the urine during a 24 h period of airborne exposure to styrene at 500 cm3/m3 (2100 mg/m3). In acetone-pretreated rats, PGA and MA and thioether formation were elevated 30-50%. The R/S ratio of MA enantiomers was about two in all styrene-exposed groups except PB-pretreated rats, which showed a ratio of four. 3. Styrene metabolism in liver microsomes measured in vitro was increased by styrene 140%, acetone plus styrene by 190%, methylcholanthrene plus styrene by 180% and phenobarbital plus styrene by 250%. 4. N-Nitrosodimethylamine demethylation (NDMAD) and 7-pentoxyresorufin dealkylation (PROD) in liver microsomes were enhanced 100-150% by styrene inhalation. The metabolism of 7-ethoxyresorufin was not significantly enhanced. 5. Monoclonal antibodies to P-450 IA1, IA2, IIB1 and IIE1 were utilized to identify cytochrome P-450s by Western blot analysis. These studies showed clearly that styrene inhalation induced principally cytochrome P450IE1, whereas styrene given by gavage at a high narcotic dosage induced both P450IIE1 (NDMAD, 60%) and P450IIB (PROD, 3000%). 6. Our conclusions are that styrene metabolism in vivo in both autoinduced and induced by other foreign compounds, that cytochrome P450IIE1 induction has a major impact on styrene metabolism and that P450IIB1 induction yields an altered MA metabolite enantiomer ratio.

Regulation of rat hepatic cytochrome P450IIE1 in primary monolayer hepatocyte culture

1. Rat hepatic cytochrome P450IIE1 is an ethanol-inducible enzyme which catalyses ethanol oxidation and activation of the procarcinogen, N-nitrosodimethylamine (NDMA) to its carcinogenic metabolite. 2. Initial studies in adult rat indicated that the regulation of cytochrome P450IIE1 is complex, therefore we strove to identify a central regulatory mechanism, using primary monolayer hepatocyte culture. These studies examined the effect of a range of agents (i.e. inducers, hormones, sodium butyrate and 5-aminolaevulinic acid) on amounts of cytochrome P450IIE1 protein and mRNA expression in rat hepatocytes maintained in serum-free medium on both Vitrogen and Matrigel, a laminin-rich basement membrane. 3. At time 0, immunoreactive cytochrome P450IIE1 protein was easily detectable in control cultures, yet decreased rapidly with time in culture to nearly undetectable levels at 120 h. Addition of inducers (notably, pyrazole) to the culture medium increased cytochrome P450IIE1 above that of untreated cultures at similar time points, yet did not elevate cytochrome P450IIE1 or NDMA demethylation above their levels at time 0. 4. Cytochrome P450IIE1 hybridizable mRNA also rapidly declined in culture. The decline in mRNA was not significantly altered in cultures exposed to pyrazole or any other agent. Thus, post-transcriptional factors appear to play an important role in the regulation of hepatic cytochrome P450IIE1, with protein stabilization being the most probable mechanism.

Effect of Dietary Protein Depletion and Ethanol on Demethylation and Denitrosation of N-nitrosodimethylamine by Rabbit Liver Microsomes.

To study how the metabolism of N-nitrosodimethylamine (NDMA) is affected by dietary protein level and chronic ethanol feeding, 24 rabbits at 18 weeks of age, assigned to four groups, each with 6 rabbits, were fed for 8 weeks on the following 4 different diets; 21% protein content containing 21.1% casein and water, 21% protein content containing 21.1% casein and 7.5% ethanol, 7% protein content containing 4.8% casein and water, 7% protein content containing 4.8% casein and 7.5% ethanol. Body weights, liver weights and microsomal protein contents in the liver were significantly smaller in 7% protein diet groups than in 21% protein diet groups. Microsomal P-450 contents per protein content were less in 7% protein diet groups than in 21% protein diet groups. NDMA demethylation activity due to non-arachidonic acid stimulated system (NASS) and NDMA denitrosation activity were greater in 21% protein diet groups than in 7% diet groups. Chronic ethanol feeding increased NDMA denitrosation activity due to arachidonic acid stimulated system in both dietary protein levels. The ratio of NDMA demethylation activity (carcinogenic action) to NDMA denitrosation activity (detoxication action) was greater in 7% dietary protein groups than 21% dietary protein groups. The value of this ratio was decreased by chronic ethanol feeding in 7% dietary protein groups. Cytotoxic and carcinogenic actions of NDMA might be enhanced by low protein diet due to inhibition of denitrosation of NDMA in liver microsome and chronic 7.5% ethanol feeding alleviates metabolic activation of NDMA to carcinogenic compounds.

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 N-nitrosodimethylamine metabolism in rat liver and lung by ethanol

The metabolic activation of N-nitrosodimethylamine (NDMA) to an active metabolite is important in the manifestation of its carcinogenic effect. The lung and liver were compared for their responses to the induction of NDMA demethylation by 10% ethanol in the drinking water and by repeated bolus injections. Ethanol in the drinking water increased NDMA metabolism several-fold in both the liver and the lung. Repeated ip injections with 0.6 and 3.0 ml ethanol/kg for 7 days also enhanced this activity in a dose-dependent fashion. These results suggest that in the lung, as in the liver, ethanol may influence the metabolic activation of this nitrosamine.