Role of N-acetyltransferase 2 acetylation polymorphism in 4, 4'-methylene bis (2-chloroaniline) biotransformation.

Arylamine N-acetyltransferase 1 (NAT1) and 2 (NAT2) catalyze the acetylation of arylamine carcinogens. Single nucleotide polymorphisms in the NAT2 coding exon present in NAT2 haplotypes encode allozymes with reduced N-acetyltransferase activity towards the N-acetylation of arylamine carcinogens and the O-acetylation of their N-hydroxylated metabolites. NAT2 acetylator phenotype modifies urinary bladder cancer risk following exposures to arylamine carcinogens such as 4-aminobiphenyl. 4, 4'-methylene bis (2-chloroaniline) (MOCA) is a Group 1 carcinogen for which a role of the NAT2 acetylation polymorphism on cancer risk is unknown. We investigated the role of NAT2 and the genetic acetylation polymorphism on both MOCA N-acetylation and N-hydroxy-MOCA O-acetylation. MOCA N-acetylation exhibited a robust gene dose response in rabbit liver cytosol and in cryopreserved human hepatocytes derived from individuals of rapid, intermediate and slow acetylator NAT2 genotype. MOCA exhibited about 4-fold higher affinity for recombinant human NAT2 than NAT1. Recombinant human NAT2*4 (reference) and 15 variant recombinant human NAT2 allozymes catalyzed both the N-acetylation of MOCA and the O-acetylation of N-hydroxy-MOCA. Human NAT2 5, NAT2 6, NAT2 7 and NAT2 14 allozymes catalyzed MOCA N-acetylation and N-hydroxy-O-acetylation at rates much lower than the reference NAT2 4 allozyme. In conclusion, our results show that NAT2 acetylator genotype has an important role in MOCA metabolism and suggest that risk assessments related to MOCA exposures consider accounting for NAT2 acetylator phenotype in the analysis.

A method for routine analysis of urinary 4,4'-methylenebis (2-chloroaniline) by gas chromatography-electron capture detection.

OBJECTIVES: The purpose of this study was to develop a simple and cost-effective method for the determination of urinary 4,4'-methylenebis (2-chloroaniline) (MBOCA) by gas chromatography-electron capture detection (GC-ECD) for biological monitoring of occupational exposure to MBOCA. METHODS: MBOCA was prepared by liquid-liquid extraction after alkaline hydrolysis, derivatized with N-methyl-bis (trifluoroacetamide) and then analyzed using GC-ECD. The proposed method was validated in accordance with the US Food and Drug Administration guidance. RESULTS: The calibration curve showed linearity in the range 1-100 µg/l, with a correlation coefficient of >0.999. The limits of detection and quantification were 0.3 µg/l and 1 µg/l, respectively. The recovery was 94-99%. Intraday accuracy, expressed as the deviation from the nominal value, was 90.5-100.3%, and intraday precision, expressed as the relative standard deviation, was 0.3-2.4%. Interday accuracy and precision were 87.8-100.2% and 0.3-4.1%, respectively. CONCLUSIONS: The proposed method is a simple and cost-effective method suitable for routine analyses and could be useful for biological monitoring of occupational exposure to MBOCA.

Rapid and intermediate N-acetylators are less susceptible to oxidative damage among 4,4'-methylenebis(2-chloroaniline) (MBOCA)-exposed workers.

OBJECTIVES: In this study, we explored the association between a marker of oxidative stress, 8-hydroxydeoxyguanosine (8-OHdG), and genetic polymorphism of the carcinogen-metabolizing enzyme N-acetyltransferase 2 (NAT2) among 4,4'-methylenebis(2-chloroaniline) (MBOCA)-exposed workers. METHODS: The study population was recruited from four MBOCA-producing factories, and included 57 MBOCA-exposed workers and 101 unexposed control workers. Personal characteristics were collected by questionnaire. Plasma 8-OHdG levels were measured by LC/MS/MS. NAT2 alleles were measured by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). RESULTS: NAT2 polymorphism influenced the plasma 8-OHdG levels of MBOCA-exposed workers, but not of non-exposed workers. No difference between exposed and control groups was found for the crude 8-OHdG levels among rapid, intermediate, and slow acetylators. After adjusting for gender, age, smoking, and alcohol consumption habit, the 8-OHdG concentration in the MBOCA-exposed workers was 0.18pg/ml (95% CI -1.80 to -0.12) lower than the control group among rapid and intermediate acetylators. However, the difference between exposed and control groups was not significant for slow acetylators. CONCLUSION: Gene-environment interactions could play a role in the carcinogenesis of occupational MBOCA exposure. We suggest that the impact of the NAT2 acetylator status is low, if at all, on the generation of the oxidative stress marker 8-OHdG in the investigated exposed group.

Simultaneous analysis of urinary 4,4'-methylenebis(2-chloroaniline) and N-acetyl 4,4'-methylenebis(2-chloroaniline) using solid-phase extraction and liquid chromatography/tandem mass spectrometry.

Analysis of 4,4'-methylenebis(2-cholroaniline) (MOCA) or its metabolites in urine has been considered as the appropriate method to assess MOCA exposures through inhalation and skin absorption. MOCA and its metabolite, N-acetyl 4,4'-methylenebis(2-chloroaniline) (acetyl-MOCA), are analyzed using methods either limited by sensitivity or sample preparation. Therefore, a solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to simultaneously analyze MOCA and acetyl-MOCA in urine to serve as biomarkers for MOCA exposure. Protein was precipitated by using acetonitrile, and SPE were applied to clean up samples to eliminate the matrix effect and to improve the recovery. The limit of quantitation of this method was at 1.0 ng/mL for MOCA and 0.03 ng/mL for acetyl-MOCA (signal-to-noise (S/N) ratio = 10). Urinary MOCA and acetyl-MOCA levels in MOCA-exposed workers were analyzed and quantitated to be 191.9 +/- 373.2 (mean +/- standard deviation (SD)) and 11.79 +/- 23.8 ng/mL (N = 54) with the median values 38.6 and 1.8 ng/mL, respectively. MOCA concentrations are significantly correlated with their corresponding acetyl-MOCA levels in urine (Spearman correlation coefficient r = 0.916, p < 0.001). These results show that this method has been successfully developed and provides high-throughput potential to analyze MOCA and acetyl-MOCA to serve as exposure biomarkers for future study of the potential health effects associated with MOCA exposures.

Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens.

Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovo turkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using (32)P-postlabeling for DNA adducts. In ovo exposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had (32)P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity.

Neoplastic transformation and DNA-binding of 4,4'-methylenebis(2-chloroaniline) in SV40-immortalized human uroepithelial cell lines.

The tumorigenic transformation of certain occupationally significant chemicals, such as N-hydroxy-4-4'-methylenebis[2-chloroaniline] (N-OH-MOCA), N-hydroxy-ortho-toluidine (N-OH-OT), 2-phenyl-1,4-benzoquinone (PBQ) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) were tested in vitro using the well established SV40-immortalized human uroepithelial cell line SV-HUC.PC. SV-HUC cells were exposed in vitro to varying concentrations of N-OH-MOCA, N-OH-OT, N-OH-ABP and PBQ that caused approximately 25% and 75% cytotoxicity. The carcinogen treated cells were propagated in culture for about six weeks and subsequently injected subcutaneously into athymic nude mice. Two of the fourteen different groups of SV-HUC.PC treated with different concentrations of N-OH-MOCA, and one of the three groups exposed to N-OH-ABP, formed carcinomas in athymic nude mice. 32P-postlabeling analyses of DNA isolated from SV-HUC.PC after exposure to N-OH-MOCA revealed one major and one minor adduct. The major adduct has been identified as the N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-amino-3-chlorob enz yl alcohol (pdAp-ACBA) and the minor adduct as N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-amino-3-chlorot oluene (pdApACT). Furthermore, SV-HUC.PC cytosols catalyzed the binding of N-OH-MOCA to DNA, in the presence of acetyl-CoA, to yield similar adducts. The same adducts were also formed by chemical interaction of N-OH-MOCA with calf thymus DNA, suggesting that the aryl nitrenium ion may be the ultimate reactive species responsible for DNA binding. The tumorigenic activity of N-OH-MOCA in this highly relevant in vitro transformation model, coupled with the findings that SV-HUC.PC cells formed DNA-adducts in vitro and contained enzyme systems that activated N-OH-MOCA to reactive electrophilic species that bound to DNA, strongly suggest that MOCA could be a human bladder carcinogen. These findings are consistent with the International Agency for Research on Cancer's classification of MOCA as a probable human carcinogen.

Determination of 4,4'-methylene-bis(2-chloroaniline)-DNA adduct formation in rat liver and human uroepithelial cells by the 32P postlabeling assay.

The probable human carcinogen 4,4'-methylene-bis(2-chloroaniline) (MOCA) was utilized to develop biomarkers of exposure to occupational carcinogens. The 32P postlabeling assay, utilizing the nuclease P1 enhancement procedure, was used to evaluate MOCA-DNA adduct formation in target tissues. Male Sprague-Dawley rats were treated with different dosing regimens of MOCA, and DNA was isolated from the liver. Additionally, a human uroepithelial cell (HUC) line was treated with N-hydroxy-MOCA for 24 hr, cells were harvested, and DNA was isolated. DNA was analyzed for MOCA-DNA adduct formation by the 32P postlabeling assay. Five MOCA adducts were detected in rat liver DNA. Adduct A, which corresponded to N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, was the major adduct in rat liver DNA appearing in all treatment groups. Levels of adduct A were higher when MOCA was administered by ip injection versus oral gavage. Phenobarbital pretreatment increased the amount of adduct A approximately 12-fold. The pathway leading to the formation of adduct A in DNA from HUC appeared to be saturated at the concentrations used: 2.5, 5, and 10 microM. However, an additional adduct (E) was observed at the 10 microM treatment level only. A major DNA adduct was detected in the target tissue of rats and target human cells for MOCA-induced carcinogenesis, thus making it useful as a biomarker of exposure. Other DNA adducts were also observed with the different doses and routes of exposure investigated.

Formation of 4,4'-methylene-bis(2-chloroaniline)-DNA adducts in yeast expressing recombinant cytochrome P450s.

N-Oxidation of 4,4'-methylene-bis(2-chloroaniline) (MBOCA) may lead to formation of DNA adducts. To determine if cytochrome P450s are involved in the formation of MBOCA derived-DNA adducts, yeast strains expressing rodent P450s were exposed to MBOCA, and 32P-postlabelling of nucleotides from yeast genomic DNA was done. Chromatographic analysis on PEI cellulose showed that, upon exposure to MBOCA for 1 h, nine DNA adducts were formed in yeast expressing phenobarbital-inducible rabbit P450 2B5. With a 4-h-exposure, all adducts increased in parallel. In cell-free experiments, the incubation of MBOCA with phenobarbital-induced rat microsomal fraction followed by incubation with thymus DNA, led to the formation of more than ten DNA adducts. When yeast expressing 3-methylcholanthrene-inducible rat P450 1A1 was exposed to MBOCA, one major and two minor adducts were formed. No adducts were detected in control yeast. These results show that recombinant rabbit P450 2B5 exhibits a potential activation of MBOCA and that rat P450 1A1 has some effect. The use of yeast expressing recombinant P450s and the technique of 32P-postlabelling facilitates a simple search for chemicals with carcinogenic potential.

32P-postlabelling analysis of DNA adducts of 4,4'-methylenebis(2-chloroaniline) in target and nontarget tissues in the dog and their implications for human risk assessment.

4,4'-Methylenebis(2-chloroaniline) (MOCA) has considerable human occupational exposure and it induces urinary bladder tumors in the dog, a species that has been often used as a model for aromatic amine-induced urinary bladder carcinogenesis in humans. Metabolic activation and formation of DNA adducts are considered to be critical steps in this process; and two major C8-adenine adducts have been shown to be formed in vitro by reaction with the proximate carcinogenic metabolite N-hydroxy-MOCA. MOCA-DNA adducts have also been detected in vivo in treated rats and in exfoliated urothelium of a worker accidentally exposed to MOCA. Thus, the aim of this study was to detect and quantify DNA adducts in the urinary bladder of dogs exposed to MOCA and thereby provide data that could be useful for risk assessment after human exposure to MOCA. Beagle dogs were treated with single and multiple doses of MOCA and DNA adduct levels were determined in liver and bladder epithelium. After a single dose, adduct levels in the liver were 1.5-fold higher than that in the bladder epithelium. Adduct levels in these two organs increased 3- to 5-fold after 10 doses and adducts in the liver were then 2.8-fold higher than that in the bladder epithelium. The levels found in these two organs after single exposures were compared, per unit exposure dose, with that reported for other carcinogenic aromatic amines. The comparison showed that MOCA was as effective in DNA adduct formation as most other potent urinary bladder carcinogens. These results suggest that MOCA may have high carcinogenic potential in humans and are consistent with the recent classification of MOCA as a probable human carcinogen.

Elucidation of catalytic specificities of human cytochrome P450 and glutathione S-transferase enzymes and relevance to molecular epidemiology.

A number of different approaches have been used to determine the catalytic selectivity of individual human enzymes toward procarcinogens. Studies with cytochrome P450 (P450) enzymes and glutathione S-transferases are summarized here, and recent work with pyrrolizidine alkaloids, aflatoxins, 4,4'-methylenebis(2-chloroaniline), and ethyl carbamate is discussed. In some cases a single enzyme can catalyze both activation and detoxication reactions of a chemical. The purification and characterization of human lung P4501A1 and the development of a noninvasive assay for human P4502E1 are also described.

Contributions of human liver cytochrome P450 enzymes to the N-oxidation of 4,4'-methylene-bis(2-chloroaniline).

4,4'-Methylene-bis(2-chloroaniline) (MOCA) can produce tumors in rodents and dogs and an increased incidence of bladder tumors has been reported in exposed workers. It is therefore of interest to identify the human cytochrome P450 (P450) enzymes involved in MOCA N-oxidation, the primary reaction involved in the formation of an electrophilic product. Human liver microsomes were fractionated and MOCA N-oxidation activity was monitored through the procedure. The most active enzyme fraction corresponded to P450 3A4, as determined by immunochemical assays and N-terminal amino acid sequence analysis. Yeast recombinant P450 3A4 also had MOCA N-oxidation activity. Purified human liver P450 2A6 showed catalytic activity; however, anti-P450 2A6 inhibited less than 20% of the microsomal activity while anti-P450 3A4 inhibited up to 75%. Levels of marker activities of both P450 3A4 (nifedipine oxidation) and P450 2A6 (coumarin 7-hydroxylation) were measured in a set of human liver microsomes and both were correlated with MOCA N-oxidation rates. Gestodene and troleandomycin inhibited up to half of the microsomal MOCA N-hydroxylation activity but 7,8-benzoflavone showed only slight inhibition. Anti-P450 3A4 inhibited (up to 80% of) the microsomal transformation of MOCA to a product genotoxic as judged by bacterial SOS response. The work indicates that P450 3A4 makes a major contribution to human liver microsomal MOCA N-oxidation, and P450 2A6 has a minor role. P450 1A2, which catalyzes the hydroxylation of many arylamines, does not contribute to a great extent.

Mutagenicity and effect on gap-junctional intercellular communication of 4,4'-methylenebis(2-chloroaniline) and its oxidized metabolites.

Oxidized metabolites of 4,4'-methylenebis(2-chloroaniline) (MBOCA) were tested for direct mutagenicity in a Salmonella typhimurium assay and for effects on gap-junctional communication of WB-F344 rat liver cells. The mutagenicities of the N-hydroxy, mononitroso and o-hydroxy (ring) metabolites of MBOCA were assayed without adding activating enzyme systems, using the frame-shift sensitive strain TA98 and the base pair substitution sensitive strain TA100. The mutagenicity of the hydroxylamine was demonstrated by a linear increase in the formation of mutant colonies in both strains, with a formation of two revertants/nmol by TA98 and 21 revertants/nmol by TA100. The mononitroso metabolite showed a slight positive effect on TA100, but effects were masked by its cytotoxicity towards this strain. This metabolite was neither mutagenic nor cytotoxic to TA98. The o-hydroxy and the dinitroso metabolites were negative for mutagenicity at concentrations up to 50 and 500 micrograms/plate, respectively. The effects of parent MBOCA and N-hydroxy, mononitroso and o-hydroxy metabolites on cell-cell communication were determined by a scrape loading/fluorescent dye transfer technique. Cytotoxicity was assessed by determination of colony-forming efficiency and lactate dehydrogenase release. MBOCA itself caused an inhibition of dye transfer at concentrations of 7.5, 11.3 and 15 nmol/ml, whereas measures of cytotoxicity were not seen until 15 and 30 nmol/ml for LDH release and plating efficiency, respectively. None of the oxidized metabolites were active in inhibiting dye transfer at non-cytotoxic concentrations.

Unlabeled hemoglobin adducts of 4,4'-methylenebis (2-chloroaniline) in rats and guinea pigs.

The capacity of N-oxidized metabolites of 4,4'-methylenebis(2-chloroaniline) (MBOCA) to form hemoglobin (Hb) adducts was determined in vitro, and the formation of Hb adducts following in vivo administration of MBOCA was assessed with or without prior induction of cytochrome P-450 enzymes with phenobarbital or beta-naphthoflavone. Hb adduct formation was determined by electron-capture GLC of MBOCA as the heptafluorobutyryl derivative following mild acid hydrolysis of protein-bound MBOCA. The method was confirmed by gas chromatography-mass spectrometry with selected ion monitoring. N-hydroxy- and mononitroso-MBOCA, but not MBOCA itself, formed adducts to rat and human Hb in vitro in a dose-related manner. Binding was inhibited by cysteine and glutathione but not oxidized glutathione or methionine. Intravenous administration of as little as 0.04 mumol/kg N-hydroxy-MBOCA to rats resulted in measurable formation of MBOCA-Hb adducts (0.9 ng/50 mg Hb). Intraperitoneal administration of 0.5-50 mg/kg MBOCA to rats, and subcutaneous administration of 5-500 mg/kg MBOCA to rats and 4-100 mg/kg to guinea pigs resulted in dose-related formation of Hb adducts. MBOCA-Hb remained elevated in blood for greater than 10 weeks following a single subcutaneous dose in guinea pigs. Pretreatment of rats with phenobarbital induced microsomal benzphetamine N-demethylase (BND) activity and resulted in a small increase in in vitro N- and ortho-hydroxylation of MBOCA, but did not increase in vivo Hb adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Review of the genotoxicity and carcinogenicity of 4,4'-methylene-dianiline and 4,4'-methylene-bis-2-chloroaniline.

4,4'-Methylene-dianiline (MDA) and 4,4'-methylene-bis-2-chloroaniline (MOCA) are polycyclic aromatic amines that are currently used in industry. Both compounds have been found to be bacterial mutagens and to be positive in a number of assays for genotoxicity. In animal studies, MDA has induced thyroid and liver neoplasms while exposure to MOCA resulted in a variety of tumors including those of the liver, mammary gland and bladder. Epidemiologic proof of human carcinogenicity of both compounds is lacking; however, there is evidence that MOCA can be metabolized to mutagenic products by human tissue. In this paper, the major finding concerning the biotransformation, genotoxicity and carcinogenicity of MDA and MOCA are reviewed.

Evidence that a beta-N-glucuronide of 4,4'-methylenebis (2-chloroaniline) (MbOCA) is a major urinary metabolite in man: implications for biological monitoring.

Urine samples from workers exposed to 4,4'-methylenebis (2-chloroaniline) (MbOCA) contain a labile metabolite(s) that, on hydrolysis, yields the parent compound at concentrations two to three times those of free MbOCA. Evidence has now been obtained that the major labile metabolite is an N-glucuronide of MbOCA. The N-glucuronide of MbOCA was synthesised chemically, characterised by thermospray mass spectrometry, and found to have a pseudomolecular (M + 1) ion at m/z 443/445. MbOCA and [14C] uridine diphosphoglucuronic acid [( 14C]UDPGA) were incubated with liver microsomes from rats induced with polychlorinated biphenyls. The stoichiometry of the reaction product was about 1:1 (MbOCA:UDPGA). This product, the chemically synthesised glucuronide, and the labile urinary metabolite had identical chromatographic and hydrolytic (heat and beta-glucuronidase) properties. These studies show that the major labile conjugate of MbOCA in the urine of workers exposed to this compound is probably the mono N-glucuronide. In view of the lability of this compound and the fact that its concentration in urine is two to three times that of free MbOCA, it is essential that any strategy for the biological monitoring of exposed workers takes into account the N-glucuronide.

4,4'-Methylene-bis(2-chloroaniline) (MOCA): comparison of macromolecular adduct formation after oral or dermal administration in the rat.

The macromolecular binding of 4,4'-methylenebis(2-chloroaniline) (MOCA), a suspect human carcinogen, was studied in the adult male Sprague-Dawley rat after both oral and dermal administration. Rats were euthanized 1, 3, 7, 10, 14, and 29 days after a single 281 mumol/kg body wt dose of [14C]MOCA (oral, 213 muCi/kg; dermal, 904 muCi/kg). DNA from various tissues and hemoglobin were isolated for determination of the time course of MOCA macromolecular binding. After oral administration adduct formation was rapid with maximum levels appearing at 24 hr. The 24-hr covalent binding associated with the globin was 7.84 pmol/mg globin (t1/2 = 14.3 days). More extensive 24-hr covalent binding was detected for liver DNA with 49.11 pmol/mg DNA (t1/2 = 11.1 days). After dermal administration of MOCA the major portion of the dose, 86.2%, remained at the application site throughout the study. For these rats the 24-hr covalent binding determined for liver DNA was 0.38 pmol/mg DNA (t1/2 = 15.6 days). Although lower levels were detected after dermal application, similar stability of MOCA-DNA adducts indicates that quantification of such MOCA adducts may be useful for the long-term industrial biomonitoring of MOCA exposure and for the evaluation of human DNA-MOCA adduct formation, a lesion thought to be associated with the production of cancer.

Quantification of haemoglobin binding of 4,4'-methylenebis(2-chloroaniline) (MOCA) in rats.

4,4'-Methylenebis(2-chloroaniline) (MOCA) is used as a curing agent in the production of polyurethane. MOCA is carcinogenic in experimental animals. Haemoglobin adducts have been proposed as dosimeters of aromatic amines for biological monitoring. A quantitative method to determine the adduct has now been worked out in female Wistar rats dosed per os with 3.82, 14.2 and 16.2 mumol/kg 14C-ring labeled MOCA or 0.25 and 0.50 mmol/kg unlabeled MOCA. MOCA bound in decreasing amounts to DNA, RNA and proteins of the lung, liver and kidney. Fractions of 0.19% and 0.026% of the dose were bound to the blood proteins haemoglobin and albumin, respectively. MOCA released by hydrolysis from haemoglobin was determined by HPLC with electrochemical detection or by GC-MS. Albumin did not form any hydrolysable adducts with MOCA.

Hydroxylation of 4,4'-methylenebis(2-chloroaniline) by canine, guinea pig, and rat liver microsomes.

An in vitro microsomal mixed function oxidase enzyme system was used to study the phase I metabolism of 4,4'-methylenebis(2-chloroaniline) (MBOCA) by dog, guinea pig, and rat liver. TLC with color development and autoradiography, and HPLC with detection by UV absorbance and radioactivity flow monitoring were utilized to isolate metabolites. Reference standards of the N-oxidized metabolites were prepared by oxidation of MBOCA with 3-chloroperoxybenzoic acid and structures confirmed by mass spectrometry and proton NMR. These were utilized to identify the N-hydroxy and nitroso metabolites of MBOCA isolated from the microsomal incubations by comparison of their HPLC retention times and mass spectra. The structure of the o-hydroxy metabolite (ring, ortho to the amine) isolated from the microsomal incubations was elucidated by mass spectrometry and proton NMR. N- and o-hydroxylations of MBOCA were shown to increase with incubation time, microsomal protein, substrate, and NADPH concentration, and were inhibited by 2,3-dichloro-6-phenylphenoxyethylamine, an inhibitor of the microsomal mixed function oxidase enzyme system. Guinea pig liver microsomes oxidized MBOCA to the N-hydroxy metabolite predominantly, whereas the dog liver formed predominantly the o-hydroxylated metabolite, with significant amounts of the hydroxylamine as well. The rat liver formed lesser amounts of the N- and o-hydroxylated metabolites, but larger numbers of other polar compounds.

Metabolic oxidation of the carcinogens 4-aminobiphenyl and 4,4'-methylene-bis(2-chloroaniline) by human hepatic microsomes and by purified rat hepatic cytochrome P-450 monooxygenases.

Metabolic N-oxidation and ring-oxidation of carcinogenic arylamines by hepatic cytochromes P-450 are generally regarded as critical activation and detoxification pathways, respectively. Two arylamines with known human exposure, 4-aminobiphenyl (ABP) and 4,4'-methylene-bis(2-chloroaniline) (MOCA), have been examined as substrates for 10 different purified rat hepatic cytochromes P-450 and for human liver microsomal preparations from 22 individuals. Metabolites were analyzed by high-performance liquid chromatography and flow scintillation techniques. As reported for certain other carcinogenic arylamines, the isosafrole-inducible isozyme, P-450ISF-G, had the highest catalytic activity for ABP N-oxidation (13.6 nmol/min/nmol P-450), but P-450BNF-B, P-450UT-A, P-450UT-F, and P-450PB-B also showed appreciable activity. Ring-oxidation of ABP occurred only to a minor extent. In contrast, N-oxidation of MOCA was preferentially catalyzed by the phenobarbital-inducible enzymes, P-450PB-B and P-450PB-D (9.0 and 6.6 nmol/min/nmol P-450, respectively). MOCA ring-oxidation and methylene carbon oxidation showed varied cytochrome P-450 selectivity and accounted for 14 to 79% of total oxidation products. There was a 44-fold variation in rates of ABP N-oxidation in the 22 human liver microsomal preparations, while rates of N-oxidation of MOCA varied only 8-fold. Ring/methylene carbon-oxidation of MOCA accounted for 6-19% of total oxidation products in the case of the human microsomal preparations, whereas ring-oxidation of ABP accounted for less than 7% of total oxidation. In addition, there was a strong correlation (R = 0.90) between rates of ABP N-oxidation and phenacetin O-deethylation, which is considered a human genetic polymorphism. Moreover, both the ABP N-oxidation and phenacetin O-deethylation activities of human liver microsomes showed a good correlation (R = 0.72) with the levels of cytochrome P-450 immunochemically related to rat P-450ISF-G. These data indicate that specific inducible and constitutive cytochromes P-450 are involved in the metabolic activation and detoxification of the carcinogens ABP and MOCA. Therefore, individual profiles of cytochromes P-450, affected by both environmental and genetic factors, may be significant determinants of individual susceptibility to arylamine carcinogenesis.