Metabolism Of Aniline Research Articles

Characterization of a Pseudomonas sp. Capable of Aniline Degradation in the Presence of Secondary Carbon Sources

Pseudomonas strain K1 is a gram-negative rod which grows aerobically on minimal media containing aniline with a doubling time of 2 h at 30 degrees C. The half-saturation parameter for aniline metabolism by aniline-grown cells was 3.8 mumol . liter. Concentrations of aniline as low as 50 nM were metabolized. Neither substituted anilines nor other aromatic compounds (other than aromatic amino acids) supported growth. Cells grew as fast on aniline as on nonaromatic substrates such as lactate. The aromatic ring was cleaved via the meta pathway. Catechol 2,3-oxygenase activity was induced by aniline, even in cultures containing alternative carbon sources such as lactate. Cultures grown on a mixture of aniline and lactate mineralized aniline in the presence of the second substrate. Lactate-grown cultures lacked catechol oxygenase activity, and resting cells from these cultures did not respire aniline. Resting cells from aniline-grown cultures exhibited high respiratory activity upon the addition of aniline or catechol, some activity with toluidine, and no activity after addition of a wide variety of other aromatic compounds, including dihydroxybenzylamine, chloroanilines, ethylanilines, aminophenols, aminobenzoates, and dihydroxybenzoates. Although substituted anilines were not metabolized, 3-or 4-chloroaniline did induce the enzymes for aniline oxidation.

Effect of endotoxin to differentially affect cytochrome P-450 monooxygenase activities of untreated rats and animals induced with phenobarbital or 3-methylcholanthrene

The effect of endotoxin in decreasing the cytochrome P-450-dependent metabolism of aniline, aminopyrine and ethoxycoumarin was examined in untreated rats, and in rats pretreated with either phenobarbital or 3-methylcholanthrene. Ethoxycoumarin metabolism was determined at two substrate concentrations (5 μM and 500 μM) to determine the effect of endotoxin on the high and low affinity enzyme activities. In untreated animals, endotoxin depressed both aniline and ethoxycoumarin metabolism by the high and low affinity enzymes by approximately by 70%, but aminopyrine was decreased by only 47%. In phenobarbital pretreated rats, endotoxin decreased enzyme activities less than in untreated animals. Aniline metabolism and low affinity ethoxycoumarin metabolism were decreased by only 24%, and aminopyrine metabolism was decreased by 35%. The high affinity ethoxycoumarin metabolism was least affected, being decreased by only 12%. In 3-methylcholanthrene pretreated rats, aniline and ethoxycoumarin (500 μM) metabolism were decreased by approximately 45%, but aminopyrine metabolism was only decreased by 20%. In these animals, endotoxin did not significantly affect the activity of ethoxycoumarin metabolism assayed with the low substrate concentration. Endotoxin decreased total cytichrome P-450 level of untreated rats by 32%, of phenobarbital pretreated rats by 39%, and in 3-methylcholanthrene pretreated animals the decrease was only 21%. Heme oxygenase activity of untreated animals was induced most by endotoxin administration and least in phenobarbital treated rats. The data suggests that endotoxin may differentially affect the various isozymes of cytochrome P-450 associated with the metabolism of aniline, aminopyrine and ethoxycoumarin. The results also suggest that the isozymes associated with these activities in untreated, phenobarbital or 3-methylcholanthrene pretreated rats may differ in their sensitivity to the effect of endotoxin.

Effects of sulphydryl reagents on the formation of the aniline metabolite 4-aminophenol and its sulphate and glucuronide conjugates in primary cultures of rat hepatocytes.

1. The effects of various sulphydryl-blocking reagents on aniline biotransformation and cytochrome P-450 levels were studied in cultured rat hepatocytes. 2. Exposure of aniline-metabolizing hepatocytes to p-chloro-mercuribenzoate (PCMB) or p-chloromercuribenzenesulphonic acid (PCMBS) resulted in decreased levels of cytochrome P-450, decreased glucuronidation of 4-aminophenol and increased levels of free 4-aminophenol. 3. Incubation of aniline-metabolizing hepatocytes with disulfiram resulted in decreased formation of 4-aminophenol, but this was not associated with impaired glucuronidation or cytochrome P-450 levels. 4. Exposure of aniline-metabolizing hepatocytes to mersalyl, 2,2'-dithiodipyridine (DTP), 6,6'-carboxydipyridine disulphide (CPDS) or N-ethylmaleimide did not affect the biotransformation of aniline or cytochrome P-450 levels. 5. Metyrapone prevented degradation of cytochrome P-450. Exposure of cells to SKF-525 A inhibited aniline biotransformation without altering cytochrome P-450 levels. 6. PCMB and PCMBS inhibited aniline metabolism, probably by binding to a cysteinyl-SH residue in cytochrome P-450 apoenzyme and 'active sites' of UDP-glucuronyl transferases. Disulfiram inhibited aniline biotransformation, probably indirectly by diminishing NADPH.

Interaction between phencyclidine and its pyrolysis product, 1-phenylcyclohexene

The interaction between phencyclidine (PCP) and its pyrolysis product, 1-phenylcyclohexene (PC), at metabolic level was evaluated in Swiss male mice (21–24 g). PC (1.1, 2.2 and 4.4 mmol/kg/day for 4 days, IP, in corn oil) treatment to mice induced the in vitro metabolism ( p<0.05) of amidoryrine (17%), aniline (12%), phenacetin (62–100%), pentobarbital (20–26%), PCP (25–80%) and benzo[ a]pyrene (81– 147%) in the 9000 g liver fraction and the hepatic microsomal contents of cytochrome P-450 (18–42%). The induction of the mixed function oxygenase (MFO) system was consistent with the decreases in the concentrations of IP administered pentobarbital (0.27 mmol/kg, in saline) and PCP (16.4, 32.8 and 65.6 μmol/kg, in saline) in the serum, brain, liver and kidneys of PC pretreated mice. At 1 hr after the above doses of PC, the in vitro metabolism of amidopyrine, aniline, or phenacetin was not inhibited. However, the biotransformation of benzo[ a]pyrene was inhibited by 33 to 45%. Though PC after a single dose did not alter the tissue concentrations of PCP, it increased the pentobarbital concentrations of PCP, it increased the the pentobarbital concentrations in the tissues studied ( p<0.05). These results indicate that PC has a potential to induce the MFO system after the 4-day treatment. This property of PC plays an important role in the reduction of the action of PCP by enhancing its metabolism, thereby decreasing its tissue levels.

Identification of P-450 ALC in microsomes from alcohol dehydrogenase-deficient deermice: Contribution to ethanol elimination in vivo

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450 ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450 ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH −) and -positive (ADH +) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH − than from naive ADH + animals. Ethanol treatment induced the protein about 3-fold in the ADH + strain and about 4-fold in the ADH − strain. The antibody to rabbit P-450 ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450 ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450 ALC homolog, increased about 3-fold upon ethanol treatment in the ADH + strain and about 4-fold in the ADH − strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH + strain and 1.9-fold and 3.3-fold, respectively, in the ADH − strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450 ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450 ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH + and ADH − strains, respectively. After chronic ethanol treatment, P-450 ALC could account maximally for 8% of the total ethanol elimination in the ADH + strain and 22% in the ADH − strain. Further, cytochrome P-450 ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450 ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant. Peroxisomal β-oxidation capacity was increased 40% over control values by ethanol treatment, consistent with the hypothesis that the increase in ethanol elimination in the ADH-negative deermouse is mediated predominantly via catalase-H 2O 2.

Biochemical and immunochemical evidence for the induction of an ethanol-inducible cytochrome p-450 isozyme in male syrian golden hamsters

The effects of ethanol and of phenobarbital pretreatment on hamster microsomal metabolism of aniline and p-nitrophenol have been investigated. Hydroxylation of both compounds was increased over 2-fold by ethanol pretreatment, whereas phenobarbital pretreatment had little effect on either activity. Ethanol pretreatment had no effect on the specific content of total cytochrome P-450, while phenobarbital pretreatment increased the specific content 1.6-fold. Comparison of the specific activities for aniline hydroxylation and p-nitrophenol hydroxylation of individual microsomal samples from control, ethanol-pretreated and phenobarbital-pretreated animals showed a high degree of correlation r 2 = 0.98) consistent with the involvement of the same site for catalysis of these two compounds. Antibody to rabbit ethanol-inducible cytochrome P-450 (isozyme 3a) inhibited over 80% of the aniline (high affinity) and p-nitrophenol hydroxylase activities of microsomes from ethanol-treated hamsters. A comparison of the antibody-inhibitable rates for both hydroxylase activities with microsomes from untreated, ethanol- or phenobarbital-pretreated hamsters suggested that an isozyme homologous to rabbit isozyme 3a (hamster cytochrome P-450alc) was induced in hamsters about 3.5-fold by ethanol and was unaffected by phenobarbital. The induction of hamster cytochrome P-450alc was confirmed by immunoblot analysis of hamster microsomes. A single protein with a molecular weight of approximately 54,000 was recognized by antibody to the rabbit isozyme. Quantification of the immunoblots demonstrated that the hamster isozyme was increased about 3-fold, in good agreement with the induction determined by a comparison of the antibody-inhibitable rates. The results indicated that, although there was no change in the total spectrally observable cytochrome P-450, there was a marked change in the distribution of the isozymes of cytochrome P-450, with an increase in the alcohol-inducible form after 28-day ethanol consumption by chow-fed hamsters. This isozyme can be readily monitored by either high-affinity aniline or p-nitrophenol hydroxylation or by Western immunoblot analysis and appears to be the ethanol-inducible form of cytochrome P-450 in hamsters.

Age- and sex-related changes in the components of the hepatic microsomal mixed function oxidase system in Sprague-Dawley rats.

1. A study of the sex- and age- (4 to 103 weeks) related changes in liver microsomal mixed-function oxidase components of Sprague-Dawley rats, showed that total cytochrome P-450 ranged from 12.0 to 34.0 nmol/g, peaking between weeks 26 and 39; males had higher levels than females at weeks 4 to 51. Cytochrome b5 ranged from 5.0 to 15.0 nmol/g with inconsistent age- and sex-related differences. 2. NADPH:cytochrome C reductase ranged from 2.6 to 4.8 mumol/min per g, with maximal activity at week 39; males had greater activity at weeks 12 to 78. NADH:cytochrome C reductase ranged from 5.2 to 26.9 mumol/min per g, peaking between weeks 51 and 78; females had greater activity at weeks 12 to 51. 3. This age- and sex-related pattern of changes in these components is specific to the Sprague-Dawley strain. There are slight but significant differences between sexes only in rats less than 1 year old. There was no significant correlation between rates of p-nitroanisole or aniline metabolism and cytochrome P-450 concentrations.

Purification and characterization of ethanol-inducible human hepatic cytochrome P-450HLj

Human hepatic cytochrome P-450HLj was purified in a catalytically active state from microsomes obtained from an ethanol-intoxicated man. The electrophoretically homogeneous preparation of HLj was compared to rat P-450j and found to have a slightly different apparent molecular mass (54 vs 51.5 kDa) but highly similar immunochemical, spectral, and catalytic properties. Purified HLj exhibited high activity toward N-nitrosodimethylamine (NDMA) and aniline metabolism, low but measurable activity toward benzphetamine and 7-ethoxycoumarin, and no detectable activity toward benzo[ a]pyrene, testosterone, and progesterone. Antibody against rat P-450j reacted with HLj in immunoblot analyses and, when added directly to HLj before reconstitution with NADPH-cytochrome P-450 reductase and lipid, the antibody inhibited (96%)NDMA metabolism by HLj almost completely. However, if HLj was reconstituted with the other components before the addition of the anti- P-450j IgG, the ability of the antibody to inhibit the metabolism of NDMA was greatly diminished. This suggests that the interactions between reductase and HLj are similar to those previously observed between rat P-450j and reductase, and appear to prevent the complete access of anti- P-450j to HLj. The addition of cytochrome b 5 to reconstitution systems containing HLj resulted in a small increase in the V max for NDMA demethylation accompanied by a decrease in K m,app (1.3 to 0.3 m m) as has been observed in reconstitution systems with rat P-450j. Therefore, in reconstituted systems, cytochrome b 5 appears to play an important role in the biotransformations mediated by HLj and P-450j. In conclusion, this study demonstrates that HLj is functionally related to ethanol-inducible rat P-450j and rabbit LM3a.

The metabolism of N-benzyl-4-substituted anilines: factors influencing in vitro C- and N-oxidation.

N-Benzyl-4-substituted anilines are metabolized by N-debenzylation, aniline-ring hydroxylation and N-oxidation. Factors affecting metabolism of these secondary anilines have been studied in vitro. Enzymes responsible for C- and N-oxidation are present mainly in the liver, but also occur in lung and kidney and are localized in the endoplasmic reticulum. Quantitative and qualitative species differences were observed in microsomal N-benzylaniline metabolism. In hamsters and mice N-debenzylation is the major route of metabolism. In guinea pig and rabbit, ring hydroxylation is the major pathway whereas the rat uses both pathways to equal extents. The hamster has particularly high N-oxidase activity. A sex difference in microsomal N-benzylaniline metabolism is evident in rats but not mice. Kinetic constants for C- and N-oxidation of N-benzylanilines are reported.

Monoclonal antibodies to ethanol induced cytochrome P-450 that inhibit aniline and nitrosamine metabolism

Monoclonal antibodies to ethanol induced cytochrome P-450 that inhibit aniline and nitrosamine metabolism

Inhibition of hepatic microsomal drug-metabolizing enzymes by imperatorin

The effect of imperatorin on hepatic microsomal mixed function oxidases (MFO) was investigated. On acute treatment, imperatorin (30 mg/kg, i.p.) caused a significant reduction in activities of hepatic aminopyrine N-demethylase, hexobarbital hydroxylase and aniline hydroxylase as well as cytochrome p-450 content in rats and mice. Kinetic studies on rat liver enzymes revealed that imperatorin appeared to be a competitive inhibitor of aminopyrine N-demethylase (Ki, 0.007 mM), whereas a non-competitive inhibitor of hexobarbital hydroxylase (Ki, 0.0148mM). Imperatorin also inhibited non-competitively aniline metabolism (Ki, 0.2mM). Imperatorin binds to phenobarbital-induced cytochrome p-450 to give a typical type 1 binding spectrum (max. 388nm, min 422nm). Multiple administrations of imperatorin (30 mg/kg, i.p. daily for 7 days) to mice shortened markedly the duration of hexobarbital narcosis and increased activities of hepatic aminopyrine N-demethylase and hexobarbital hydroxylase and the level of cytochrome p-450 whereas aniline hydroxylase activity was unaffected.

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH 2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17–18, 37–46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH 2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (>90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.

Effects of alkyl phosphorothioates on the hepatic microsomal mixed-function oxidase system in rats: Inhibition of drug-metabolizing enzyme activity and selective increase of NADPH-cytochrome c reductase activity

Four series of alkyl phosphorothioates were administered to adult male rats by intraperitoneal injection, and their influences on the drug-metabolizing enzyme system in hepatic microsomes were examined. Among the alkyl phosphorothioates tested, O, O, O-trialkyl phosphorothioates (I) and O, O, S-trialkyl phosphorodithioates (II) significantly decreased hepatic microsomal cytochrome P-450 content and the metabolism of aniline and aminopyrine. Six hours after administration, triethyl compounds were the most effective of the trialkyl esters tested. In experiments on rats pretreated with phenobarbital or 3-methylcholanthrene, the inhibitory effects of triethyl esters were increased strongly by phenobarbital pretreatment and decreased by 3-methylcholanthrene. After the administration of I, a selective increase of NADPH-cytochrome c reductase activity was also observed. In the phenobarbital-pretreated rats, no further increase of NADPH-cytochrome c reductase activity was observed as a result of the administration of I. Except for the two dibutyl esters, O, O-dialkyl phosphorothioates (III) and O, O-dialkyl phosphorodithioates (IV) caused no significant inhibitory effect on the drug-metabolizing enzyme system under the same conditions.

Mechanism of potentiation of BPMC toxicity by fenthion pretreatment in mice.

The mechanism of potentiation of 2-sec-butylphenyl N-methylcarbamate (BPMC) toxicity by O,O-dimethyl O-(3-methyl-4-methylthiophenyl) phosphorothioate (fenthion) in mice was investigated in relation to BPMC metabolism in the liver. Simultaneous administration of BPMC and either one of the thiophosphates (fenthion, its sulfoxide and sulfone) in a dose of 1/40 of its LD50 resulted in a 2- to 3-fold potentiation. On the other hand, one hour pretreatment with these thiophosphates in the same dose resulted in a 7- to 9-fold potentiation of BPMC toxicity, but that with fenthion oxon resulted in only a 2-fold potentiation. Plasma levels of BPMC were significantly increased by pretreatment with the thiophosphates, but not by the oxon. In an in vitro study, an inhibition of hepatic microsomal metabolism of BPMC and a decrease of cytochrome P-450 content by thiophosphates were observed at the concentration of 10 microM, but not by 25 microM of oxon. In an in vivo study, an inhibition of hepatic microsomal metabolism of BPMC, aminopyrine and aniline by the thiophosphates pretreatment were observed in doses of 1/40 of their LD50's, but not by the oxon in doses of up to 4/10 of its LD50. Cytochrome P-450 content was decreased by the thiophosphates in doses of 4/10 of their LD50's, but not by the oxon. These results suggested that the inhibition of BPMC metabolism might be, at least in part, the mechanism for the fenthion-induced potentiation of BPMC toxicity and that desulfuration of fenthion might be responsible for the inhibition of BPMC metabolism.

Studies on the mechanism of nabam- and zineb-induced inhibition of the hepatic microsomal monooxygenases of the male rat

In vitro effects of the ethylene bis-dithiocarbamate fungicides, nabam and zineb, on the hepatic microsomal monooxygenases of male rats were examined. Incubation of nabam and zineb with hepatic microsomes, without NADPH, leads to an inhibition of the metabolism of aminopyrine and aniline and to a denaturation of cytochrome P-450 into cytochrome P-450; in addition nabam causes the destruction of cytochrome P-450. Addition of NADPH into the incubation medium increases the inhibition of the monooxygenases, principally the inhibition of the metabolism of aniline induced by nabam. We studied the in vitro effects of three of the chief breakdown products of these fungicides: ethylene bis-isothiocyanate sulfide (EBIS), ethylene thiourea (ETU), and carbon disulfide (CS 2). EBIS appears to be the only metabolite affecting directly (without NADPH) the hepatic monooxygenases activity. EBIS accounted partly for nabam-induced inhibition of the hepatic microsomal monooxygenases. The data suggest that the decrease of monooxygenases activity seen on incubation of nabam with hepatic microsomes may be due to the denaturation and destruction of cytochrome P-450 resulting from covalent binding of the compounds with cysteine sulfhydryl groups in cytochrome P-450. Inhibition of monooxygenase activity induced by zineb seems to be due to the reaction with the sulfhydryl groups of cytochrome P-450 and to another mechanism, probably related to its lipophilic character.

Amelioration of bromobenzene hepatotoxicity in the male rat by zinc

Experiments were conducted to examine the role of zinc in the prevention of bromobenzene hepatoxicity in male rats. Bromobenzene (BB) (7.5 mmol/kg, ip) produced a marked hepatotoxicity as evidenced by increases in plasma alanine aminotransferase (ALT) and aspartate aminotransferse (AST) activities and a marked depression in hepatic glutathione (GSH) content 24 hr after administration. The administration of zinc (92 μmol Zn/kg, ip, at 48 and 24 hr prior to the bromobenzene) ameliorated the bromobenzene elevations in plasma AST (25%) and plasma ALT (50%) but did not alter the decreases in hepatic GSH. Following administration of [ 14C]BB, the radioactive label was distributed primarily in the cytosolic and lipid fractions derived from liver homogenates. Furthermore, the subcellular distribution of [ 14C]BB was not altered by zinc pretreatment. The extent of covalent binding of [ 14C]BB metabolites to hepatic tissue was significantly depressed in zinc-treated rats. Zinc induced the hepatic levels of metallothionein but [ 14C]BB did not bind to this sulfhydryl rich protein. Further experiments showed that zinc treatment depressed cytochrome P-450 content, the activity of NADPH cytochrome c reductase, and the metabolism of aniline, but not that of ethylmorphine. These studies suggest that the hepatoprotective effect of zinc against bromobenzene toxicity does not involve altered binding of the reactive toxic metabolite to glutathione or metallothionein, but it may be mediated by the inhibitory effect of zinc on the microsomal cytochrome P-450-dependent drug metabolizing system.

Effects of chronic ethanol consumption on male syrian hamster hepatic, microsomal mixed-function oxidases

Chronic alcohol consumption significantly increases the risk of drug interactions. We have described its effects on hamster microsomal monooxygenases. Male Syrian hamsters (85 g) were given 10% ethanol in water and food ad lib for up to 6 weeks. Microsomal electron transport components and metabolism of ethylmorphine, benzphetamine, aniline, and acetaminophen were measured. At 4 weeks, SDS-PAGE of ethanol microsomes showed an induced band with an Mr of 53,900 daltons and there was a 2–3 fold stimulation of aniline and acetaminophen metabolism. Cytochrome P-450 increase was not significant. For the six week period, Caloric intake (3 weeks, p<0.001), liquid consumption (3 weeks, p<0.05) and body weights (6 weeks, p<0.05) of ethanol animals were significantly greater than controls; kidney weights were significantly less ( p<0.05). Ethanol consumption increased from 20% of the daily caloric intake (week 1) to 31% (week 6). Induction of specific substrate metabolism without apparent deleterious physiological changes establishes hamsters fed 10% ethanol in drinking water as a biochemical model for the study of chronic alcohol consumption and specific drug interactions.

Effect of manganese on the hepatic microsomal mixed function oxidase enzyme system in the rat

Experiments were conducted to examine the effect of manganese on the hepatic mixed function oxidase system in the rat. Acute treatment with manganese chloride (1–10 mg Mn/kg, ip) produced a significant prolongation of hexobarbital hypnosis in male rats on Days 2 and 3 following metal administration. The threshold dose of manganese to produce this alteration in response was 5 mg Mn/kg and the altered response returned to control values by Day 5. The prolonged hexobarbital hypnosis resulted from Mn inhibition of the hepatic microsomal mixed function oxidase system, the activity of which was assessed using aniline (23%), ethylmorphine (26%), and hexobarbital (27%) as substrates. Manganese treatment also produced significantly reduced levels of cytochrome P-450 (23%) and b 5 (21%), but the substrate-induced spectral binding of all three substrates was not altered significantly by Mn when expressed as Δ A per nanomole of cytochrome P-450. The activity of NADPH cytochrome c reductase was also significantly decreased (25%) by Mn treatment. Following the in vitro addition of Mn in concentrations ranging from 1 × 10 −6 to 1 × 10 −3 m Mn to microsomes derived from naive rats, there was no decrease in the metabolism of aniline or hexobarbital or cytochrome P-450 levels. Significant inhibition in ethylmorphine metabolism was observed with Mn concentrations of 1 × 10 −4 m and greater. These experiments indicate that acute Mn treatment can alter drug response as the result of decreased hepatic biotransformation which occurs by an indirect mechanism.

Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation

Antibodies to cytochrome P-450 isozyme 3a, the ethanol-inducible isozyme in rabbit liver, were used to determine the role of this enzyme in the microsomal oxidation of alcohols and the p-hydroxylation of aniline. P-450 isozymes, 2, 3b, 3c, 4, and 6 did not crossreact with anti-3a IgG as judged by Ouchterlony double diffusion, and radioimmunoassays indicated a crossreactivity of less than 1%. Greater than 90% of the activity of purified form 3a toward aniline, ethanol, n-butanol, and n-pentanol was inhibited by the antibody in the reconstituted system. The catalytic activity of liver microsomes from control or ethanol-treated rabbits was unaffected by the addition of either desferrioxamine (up to 1.0 m m) or EDTA (0.1 m m), suggesting that reactions involving the production of hydroxyl radicals from H 2O 2 and any contaminating iron in the system did not make a significant contribution to the microsomal activity. The addition of anti-3a IgG to hepatic microsomes from ethanol-treated rabbits inhibited the metabolism of ethanol, n-butanol, n-pentanol, and aniline by about 75, 70, 80, and 60%, respectively, while the inhibition of the activity of microsomes from control animals was only about one-half as great. The rate of microsomal H 2O 2 formation was inhibited to a lesser extent than the formation of acetaldehyde, thus suggesting that the antibody was acting to prevent the direct oxidation of ethanol by form 3a. Under conditions where purified NADPH-cytochrome P-450 reductase-catalyzed substrate oxidations was minimal, the P-450 isozymes other than 3a had low but significant activity toward the four substrates examined. The residual activity at maximal concentrations of the antibody most likely represents the sum of the activities of P-450 isozymes other than 3a present in the microsomal preparations. The results thus indicate that the enhanced monooxygenase activity of liver microsomes from ethanol-treated animals represents catalysis by P-450 isozyme 3a.

Novel Pseudomonas Plasmid Involved in Aniline Degradation

A plasmid of ca. 100 kilobases was detected in a Pseudomonas species which was isolated from soil by growth on aniline as the sole carbon and energy source. The plasmid was shown to be involved in aniline metabolism.