Guanethidine N-oxidation in human liver microsomes.

The capacity of human liver microsomes to N-oxidize guanethidine from 25 subjects has been assessed. Guanethidine N-oxidation was optimal at pH 8.5 and proceeded at only 16% of the maximal rate at pH 7.4. The mean rates of guanethidine N-oxidation at pH 8.5 and 7.4 were 2.46 +/- 0.89 (mean +/- s.d., n = 25) and 0.38 +/- 0.22 (mean +/- s.d., n = 22), respectively. Interindividual differences in the rate of guanethidine N-oxidation at pH 8.5 and 7.4 were 17- and 11-fold, respectively. The cytochrome P450 inhibitors, proadifen and 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), at both pH 8.5 and 7.4 caused less than 20% reduction in the rate of guanethidine N-oxidation by human liver microsomes. These data indicate that guanethidine N-oxidation can be used as a measure of flavin-containing monooxygenase activity in human liver.

Structural studies and two-dimensional gel electrophoresis of gamma-glutamyl transpeptidase.

The heterodimeric enzyme gamma-glutamyl transpeptidase (EC 2.3.2.2) was isolated from adult rat kidney and purified to homogeneity for structural studies using papain solubilization and multiple chromatographies. Two-dimensional gel electrophoresis was found to resolve the active papain-purified enzyme into at least 18 components. Seven components with apparent molecular masses of 23,000-26,000 and isoelectric point range of 5.4-7.0 constitute the light subunit, and 11 components with apparent molecular mass of 51,000-53,000 and isoelectric point range of 5.8-7.1 constitute the heavy subunit. Immunoblot analysis of two-dimensional gels showed that all of these components are immunoreactive with a mixture of the two antibodies generated separately against the light and heavy subunits. Preparative subunit separation was achieved using reverse-phase HPLC under acidic but nonreducing conditions. N-Terminal amino acid sequencing of the separated subunits of the papain-purified enzyme yielded sequence information for the first 32 residues of the heavy chain with the N-terminal starting sequence Gly-Lys-Pro-Asp-His-Val-Tyr-Ser-Arg-Ala, and for the first 36 residues of the light subunit with the N-terminal starting sequence Thr-Ala-His-Leu-Ser-Val-Val-Ser-Glu-Asp.

Amino-terminal sequence and structure of monoclonal antibody immunopurified cytochromes P-450.

Six hepatic cytochromes P-450 were isolated from 3-methylcholanthrene-treated animals by immunopurification with monoclonal antibodies. The purified cytochromes P-450 include 57- and 56-kDa polypeptides from Sprague-Dawley rats, 57- and 56-kDa polypeptides from C57BL/6 mice, a 56-kDa polypeptide from DBA/2 mice, and a 53-kDa polypeptide from guinea pigs. These isozymes were structurally compared by peptide mapping using both sodium dodecyl sulfate--polyacrylamide gel electrophoresis and high-pressure liquid chromatography and by amino acid and NH2-terminal sequence analyses. The 57-kDa polypeptides from rats and mice have similar but nonidentical peptide maps and amino acid compositions and are about 80% homologous in their NH2-terminal amino acid sequence. The 56-kDa polypeptides from rats and both mice strains have very similar peptide maps and amino acid compositions and identical NH2-terminal sequences. The NH2-terminal sequence of the mice 56-kDa polypeptides corresponds to that reported for the mouse P1-450 isozyme except that we identified two additional residues, proline and serine, at the NH2 terminus in the 57-kDa polypeptide from C57BL/6 mice that were not deduced from the cDNA sequence of the mouse P1-450 isozyme. The guinea pig 53-kDa polypeptide has a distinct peptide map relative to the other polypeptides studied and an NH2-terminal sequence with only partial homology to the 56- and 57-kDa polypeptides from rats and mice. This report shows the varying degree of structural relatedness among the isozymes examined and demonstrates the suitability and advantage of immunopurified cytochromes P-450 for sequencing and structural studies.

Effect of microsomal enzyme inducers on the urinary excretion pattern of mutagenic metabolites of the carcinogen 2,4-toluenediamine.

2,4-Toluenediamine [(TDA) CAS: 95-80-7] was administered to rats pretreated with the microsomal enzyme inducers phenobarbital (PB), beta-naphthoflavone (beta NF), or 3-methylcholanthrene (MCA). The 24-hour urines of male F344 rats were examined for their mutagenic potency by means of the Salmonella assay, with the Aroclor 1254-pretreated rat liver S-9 fraction as an activating system. No revertants were found with TDA or its urinary metabolites in the absence of the S-9 fraction. In the presence of S-9, the number of revertants increased as the concentration of TDA or its urinary metabolites increased. The urinary metabolites, generated after the microsomal enzyme inducers (PB, beta NF, MCA), had increased mutagenic activity as compared with the controls (saline, corn oil). In the presence of beta-glucuronidase (beta G), increased numbers of TA98 revertants were noted in the urine of rats pretreated with PB, saline, or corn oil. Addition of sulfatase did not alter the number of TA98 revertants. Conversely, beta G treatment of urine from rats pretreated with MCA or beta NF led to a decrease in the number of TA98 revertants as compared to levels in urine without beta G. Addition of known urinary metabolites of TDA, such as 4-acetylamino-2-aminobenzoic acid or 2,4-diacetylaminobenzoic acid, to beta NF-pretreated rat urine had no inhibitory effect on the mutagenicity in the absence of beta G. However, in the presence of beta G, the inhibitory effect was similar to that noted with beta NF-pretreated rat urine. Upon separation of urinary metabolites (beta NF-pretreated rat urine) into free, conjugated, and water-soluble forms, the maximum number of TA98 revertants was associated with the free ethyl acetate-extractable fraction, which accounted for the total mutagenic activity associated with the original volume of urine. Conjugated metabolites showed much less mutagenic activity, and an inhibitory principle was associated with the water-soluble fraction.

Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo.

Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.

Monoclonal antibody directed isolation and amino-terminal sequence analysis of phenobarbital induced rat liver cytochromes P-450.

Cytochrome P-450 was isolated from liver microsomes of phenobarbital treated rats by an essentially single step immunopurification with a monoclonal antibody (MAb). The amino terminal sequence of the isolated cytochrome P-450 displayed a microheterogeneity of isozymes related to previously identified phenobarbital induced forms, indicating that each of these isozymes possess the MAb-specific epitope. This monoclonal antibody-based approach to isolation and subsequent identification of cytochrome P-450 may serve to classify different isozymes by their content of epitopes that bind to different MAbs.

Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene.

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N-OAc-AAF) have previously been shown to induce dose-dependent DNA strand breaks in primary hepatocytes from mice and rats. In an attempt to determine the relationship between the extent of DNA strand breaks and the formation of specific DNA-carcinogen bound adducts in murine liver, the capability of N-OH-AAF and N-OAc-AAF to induce both DNA single strand breaks and adduct formation in in vivo and in primary hepatocytes was measured. N-OH-AAF induced a low level of DNA damage in F344 rats (10 mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment. The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene (Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene (Gua-C8-AF) 34% versus 67% and 3-(guanin-N2-yl)-2-acetylaminofluorene (Gua-N2-AAF) 11% versus 10%, respectively, for rat and mouse liver. An additional unknown adduct (12%) was detected in mouse liver. Dose dependent DNA binding and formation of individual DNA adducts were observed in rat and mouse primary hepatocytes following 1 h exposure to [ring-3H]-N-OH-AAF (0.1-20 microM) and [ring-3H]-N-OAc-AAF (5-20 microM). The patterns of DNA adducts in mouse and rat primary hepatocytes exposed to N-OH-AAF and N-OAc-AF were similar to those obtained in liver following in vivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon (10(-4) M) completely inhibited DNA damage induced by N-OH-AAF in mouse and partially in rat hepatocytes while DNA damage caused by N-OAc-AAF was only partially inhibited by paraoxon (10(-4) M) in both species. Parallel experiments showed that paraoxon, at low concentration (10(-6) M), did not alter either the level of DNA binding or the pattern of adduct formation in rat hepatocytes treated with N-OH-AAF (20 microM). However, at 10(-4) M paraoxon partially blocked DNA binding (60%) and the formation of Gua-C8-AAF (95%) and Gua-N2-AAF (80%) while Gua-C8-AF was increased two-fold. In mouse hepatocytes paraoxon pretreatment (10(-4) M) inhibited the formation of Gua-C8-AF by 70% following exposure to N-OH-AAF (20 microM). Gua-C8-AAF and Gua-N2-AAF were also inhibited but only at 10(-4) M paraoxon.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino-terminal sequence analysis of six cytochrome P-450 isozymes purified by monoclonal antibody directed immunopurification.

Six hepatic microsomal cytochromes P-450 were isolated from 3-methylcholanthrene induced animals by immunopurification using two monoclonal antibodies. Two forms of cytochromes P-450 (MW 56K and 57K) were from Sprague-Dawley rats, two from C57BL/6 mice (56K and 57K), one form from DBA/2 mice (56K) and one form from guinea pigs (53K). NH2-terminal sequences of the first ten amino acids of these cytochromes P-450 were determined by automated Edman degradation. The 56K polypeptides from rats, C57BL/6 mice, and DBA/2 mice were shown to have identical NH2-terminal sequences. The 57K polypeptides from rats and C57BL/6 mice are homologous to each other but exhibit no homology to 56K polypeptides. The 53K polypeptide from guinea pigs has a unique NH2-terminal sequence with no apparent homology to the other five cytochromes P-450.

Guanethidine N-oxide formation as a measure of cellular flavin-containing monooxygenase activity.

The usefulness of guanethidine N-oxide formation as a measure of cellular flavin-containing monooxygenase activity was assessed using the purified hog liver enzyme, rat liver microsomes and hepatocytes. The apparent Km and Vmax for this reaction in hepatocytes were 0.30 +/- 0.20 mM and 0.81 +/- 0.36 nmole per 10(6) cells min-1 respectively. The Km for the purified enzyme was 0.31 mM and the Vmax was 0.56 nmole per microgram enzyme min-1. 2-Diethylaminoethyl-2,2-diphenyl valerate (SKF-525A) at a concentration of 0.5 mM had no effect on guanethidine N-oxide formation by either rat liver microsomes or the purified enzyme. In contrast 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) at the same concentration caused greater than a 100% increase in the microsomal production of guanethidine N-oxide. The tertiary amines imipramine, chloropromazine and methylpyrilene inhibited N-oxide formation by both hepatocytes and the purified enzyme. These data indicate that guanethidine N-oxide formation can be used as a measure of cellular flavin-containing monooxygenase activity.

The metabolic basis for inhibitory effects in chemical carcinogenesis by arylamines.

The inhibition of the carcinogenicity of N-2-fluorenylacetamide (2-FAA) by acetanilide (AA), p-hydroxyacetanilide (p-OH-AA), butylated hydroxytoluene (BHT), and chloramphenicol is reviewed. The mechanisms of action by which inhibition may occur are as follows: 1) inhibition of the binding of the activated metabolite of FAA to cellular macromolecules (DNA, RNA, and proteins), 2) changes in the amount of the N-hydroxylated metabolite, believed to be the first step of activation of FAA, formed and excreted in the urine, 3) induction of the enzyme glucuronyl transferase which increases the formation of glucosiduronic acid that results in a rapid excretion of the carcinogens, 4) depletion of sulfate by p-OH-AA, the major metabolite of AA. The sulfate ion is required for the second activation step, i.e., the formation of the sulfate ester of N-OH-FAA. The data show several of the inhibitors may operate by one or more of the above mechanisms of inhibition.

Mutagenicity of urine from rats after administration of 2,4-diaminoanisole: the effect of microsomal enzyme inducers.

Ring 14C-labelled 2,4-diaminoanisole disulfate was administered to rats pretreated with the microsomal inducers phenobarbital (PB), beta-naphthoflavone (BNF), 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The 24-h urine from rats pretreated with PB showed a 2-fold increase in revertant rate over the corresponding control as measured by the Ames Salmonella test system. Pretreatment of rats with BNF, MC or TCDD decreased the mutagenicity of urine by about 70% when an activating system was used. However, in the absence of an activating system, the urine from rats induced with BNF, MC or TCDD showed a significant (P < 0.001) degree of mutagenicity compared with urine from controls or urine from phenobarbital-induced rats. Release of conjugates by beta-glucuronidase increased the mutagenicity of urine even in the absence of an activating system, but the number of revertants was almost doubled in the presence of an activating system. The urine from rats treated only with the 4 inducers did not show any mutagenicity. 2,4-Diaminoanisole itself was mutagenic only in the presence of an activating system. alpha-Naphthoflavone (ANF) (0.1 mM) inhibited by 85--90% the in vitro mutagenicity of urine, mediated by Aroclor 1254, MC or TCDD induced rat-liver microsomes. The mutagenicity mediated through PB-induced rat-liver microsomes was, however, inhibited only by 16%. Similarly, 0.1 mM metyrapone (MP) inhibited the mutagenicity of urine by Aroclor 1254, MC or TCDD induced rat-liver microsomes by 13--18%. For the same MP concentration a 50% inhibition of the mutagenicity mediated through PB-induced rat-liver microsomes was observed. The mutagenicity pattern for urine in vitro was shown to be similar with liver S9 from rats induced either with Aroclor 1254 or with MC.

Comparison of the metabolism of 2,4-toluenediamine in rats and mice.

The excretion, distribution, and metabolism of 2,4-toluenediamine (TDA) have been compared in rats and mice. The elimination of TDA metabolites into urine was faster and more complete in mice than in rats. However, the feces of rats accounted for a greater percentage of the dose in rats than in mice. The distribution of metabolites in tissues were considerably lower in mice than in rats. The major urinary metabolites observed in the rat were 4-acetylamino-2-aminotoluene, 2,4-diacetylaminotoluene, 4-acetylamino-2-aminobenzoic acid. The major metabolites in the mice were 4-acetylamino-2-aminobenzoic acid, 4-acetylamino-2-aminotoluene and 2,4-diacetylaminobenzoic acid.

Metabolism of the dyestuff intermediate 2,4-diaminoanisole in the rat.

1. 2,4-Diamino[ring-U-14C]anisole.2HCl administered intraperitoneally to rats is excreted chiefly via the urine (79 and 85% of the dose in 24 and 48 h, respectively). The isotope in the faeces was 2.1 and 8.9% of the dose at 24 and 48 h. 2. The major metabolic pathway was acetylation of the amine groups(s), resulting in 4-acetylamino-2-aminoanisole and 2,4-diacetylaminoanisole. 3. Oxidate pathways yielded 2,4-diacetylaminophenol (O-demethylation), 5-hydroxy-2,4-diacetylaminoanisole (ring hydroxylation), and 2-methoxy-5-(glycol-amido)acetanilide or its isomer (omega-oxidation). 4. These major metabolites were excreted in the urine both as free and glucuronic acid conjugates.

The effects of a marginally lipotrope-deficient diet on the hepatic levels of S-adenosylmethionine and on the urinary metabolites of 2-acetylaminofluorene in rats.

Hepatic levels of S-adenosylmethionine (AdoMet), of glutathione, and of the microsomal enzymes p-nitroanisole demethylase and benzo(a)pyrene hydroxylase were measured in male and female rats fed a diet marginally deficient in choline and methionine and void of folic acid (lipotrope deficient) or an adequate diet for 0 to 14 weeks with and without added 2-acetylaminofluorene (AAF). The urinary metabolites of AAF were determined throughout the experimental period. After 2 to 4 weeks of dietary administration, the hepatic AdoMet levels were 43% lower in male rats fed the lipotrope-deficient diet than in male rats fed the lipotrope-adequate diet; no differences were found in hepatic AdoMet of females fed the lipotrope-deficient or lipotrope-adequate diets for 2 to 14 weeks. Administration of AAF to lipotrope-deficient female rats for 2 weeks led to a transient decrease in hepatic levels of AdoMet. The administration of AAF for 2 to 14 weeks did not significantly affect hepatic AdoMet in female rats fed the lipotrope-adequate diet or in male rats fed either diet. Female rats fed the lipotrope-deficient diet and treated with AAF excreted decreased proportions of N-hydroxy-2-acetylaminofluorene and increased proportions of 5-hydroxy-2-acetylaminofluorene in their urine. However, the urine of lipotrope-deficient male rats treated with AAF contained increased proportions of N-hydroxy-2-acetylaminofluorene and decreased levels of 5-hydroxy-2-acetylaminofluorene. The urinary excretion of 7-hydroxy-2-acetylaminofluorene by male and female lipotrope-deficient rats treated with AAF was generally similar to that in lipotrope-adequate rats. The lipotrope-deficient diet did not appear to alter the hepatic levels of glutathione, p-nitroanisole demethylase, or benzo(a)pyrene hydroxylase activity was lower in the livers of lipotrope-deficient male rats treated with AAF for 8 to 14 weeks than in the livers of lipotrope-deficient rats not receiving the carcinogen. The altered metabolism of AAF correlated well with the previously reported effects of a marginal lipotrope deficiency on AAF carcinogenesis.

Mechanisms of the inhibitory action of p-hydroxyacetanilide on carcinogenesis by N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide.

The metabolism of N-2-fluorenylacetamide (FAA) and N-hydroxy-2-fluorenylacetamide (N-OH-FAA) was studied in groups of rats that had been prefed the protective agent p-hydroxyacetanilide (p-OH-AA) alone or in combination with each of the carcinogens for 4 weeks. Compared with controls, pretreatment increased the percentage of metabolites in the urine, chiefly as glucuronic acid conjugates, whereas the fecal excretion of FAA metabolites was lowered. The levels of total and tissue-bound material in the liver and blood plasma were also lower after prefeeding. Liver aryl hydrocarbon hydroxylase and liver deacetylase were not affected by p-OH-AA pretreatment. However, liver glucuronyl transferase was increased by either prefeeding with p-OH-AA and/or the carcinogen. The protective effect of p-OH-AA against liver tumor induction with FAA or N-OH-FAA may in part result from a combination of the decreased binding of carcinogen to hepatic cellular macromolecules and the increased excretion as the glucuronide conjugates.

Metabolism of N-2-fluorenylacetamide in X/Gf mice: lack of correlation between biochemical interaction and carcinogenicity.

A comparative study of the metabolism of the carcinogen N-2-fluorenylacetamide by mice of the X/Gf strain (resistant to the tumorigenic action) and the NiH Swiss strain (susceptible to the tumorigenic action) showed slight but not outstanding differences in metabolic patterns. The activated metabolite N-hydroxy-N-2-fluorenylacetamide was excreted by both strains, and the levels of carcinogens or metabolites bound to liver macromolecular constituents were comparable in the X/Gf and NiH Swiss mice.