Phytochemical induction of cell cycle arrest by glutathione oxidation and reversal by N-acetylcysteine in human colon carcinoma cells.

Cancer prevention by dietary phytochemicals has been shown to involve decreased cell proliferation and cell cycle arrest. However, there is limited understanding of the mechanisms involved. Previously, we have shown that a common effect of phytochemicals investigated is to oxidize the intracellular glutathione (GSH) pool. Therefore, the objective of this study was to evaluate whether changes in the glutathione redox potential in response to dietary phytochemicals was related to their induction of cell cycle arrest. Human colon carcinoma (HT29) cells were treated with benzyl isothiocyanate (BIT) (BIT), diallyl disulfide (DADS), dimethyl fumarate (DMF), lycopene (LYC) (LYC), sodium butyrate (NaB) or buthione sulfoxamine (BSO, a GSH synthesis inhibitor) at concentrations shown to cause oxidation of the GSH: glutathione disulfide pool. A decrease in cell proliferation, as measured by [(3)H]-thymidine incorporation, was observed that could be reversed by pretreatment with the GSH precursor and antioxidant N-acetylcysteine (NAC). Cell cycle analysis on cells isolated 16 h after treatment indicated an increase in the percentage (ranging from 75-30% for benzyl isothiocyanate and lycopene, respectively) of cells at G2/M arrest compared to control treatments (dimethylsulfoxide) in response to phytochemical concentrations that oxidized the GSH pool. Pretreatment for 6 h with N-acetylcysteine (NAC) resulted in a partial reversal of the G2/M arrest. As expected, the GSH oxidation from these phytochemical treatments was reversible by NAC. That both cell proliferation and G2/M arrest were also reversed by NAC leads to the conclusion that these phytochemical effects are also mediated, in part, by intracellular oxidation. Thus, one potential mechanism for cancer prevention by dietary phytochemicals is inhibition of the growth of cancer cells through modulation of their intracellular redox environment.

Detection of DNA adducts in developing CD4+ CD8+ thymocytes and splenocytes following in utero exposure to benzo[a]pyrene.

Environmental carcinogen exposure may play an important role in the incidence of cancer in children. In addition to environmental pollutants, maternal smoking during pregnancy may be a contributing factor. Major carcinogenic components of cigarette smoke and other combustion by-products in the environment include polycyclic aromatic hydrocarbons (PAH). Mouse offspring exposed during midpregnancy to the PAH, benzo[a]pyrene (B[a]P), show significant deficiencies in their immune functions, observed in late gestation which persist for at least 18 months. Tumor incidences in these progeny are 8 to 10-fold higher than in controls. We have demonstrated a significant reduction in thymocytes (CD4+ CD8+, CD4+ CD8+ Vbeta8+, CD4+ CD8+ Vgamma2+) from newborn and splenocytes (CD4+ CD8+) from 1-week-old mouse progeny exposed to B[a]P in utero. To investigate possible causes of the observed T cell reduction, we analyzed the thymocytes and splenocytes from progeny and maternal tissues for the presence of B[a]P-DNA adducts. Adducts were detected in maternal, placental and offspring lymphoid tissues at day 19 of gestation, at birth and 1-wk after birth. The presence of B[a]P-DNA adducts in immature T cells may, in part, explain the previously observed T cell immunosuppression and tumor susceptibility in mice exposed to B[a]P in utero. The effects of DNA lesions on progeny T cells may include interference with normal T-cell development. These results provide a possible explanation for the relationship between maternal smoking during pregnancy and childhood carcinogenesis.

Dietary compounds that induce cancer preventive phase 2 enzymes activate apoptosis at comparable doses in HT29 colon carcinoma cells.

Dietary agents that induce glutathione S-transferases and related detoxification systems (Phase 2 enzyme inducers) are thought to prevent cancer by enhancing elimination of chemical carcinogens. The present study shows that compounds of this group (benzyl isothiocyanate, allyl sulfide, dimethyl fumarate, butylated hydroxyanisole) activated apoptosis in human colon carcinoma (HT29) cells in culture over the same concentration ranges that elicited increases in enzyme activity (5-25, 25-100, 10-100, 15-60 micromol/L, respectively). Pretreatment of cells with sodium butyrate, an agent that induces HT29 cell differentiation, resulted in parallel increases in Phase 2 enzyme activities and induction of apoptosis in response to the inducers. Cell death characteristics included apoptotic morphological changes, appearance of cells at sub-G1 phase on flow cytometry, caspase activation, DNA fragmentation and TUNEL-positive staining. The results suggest that dietary Phase 2 inducers may protect against cancer by a mechanism distinct from and in addition to that associated with enhanced elimination of carcinogens. If this occurs in vivo, diets high in such compounds could eliminate precancerous cells by apoptosis at time points well after initial exposure to chemical mutagens and carcinogens.

Maternal exposure to benzo[a]pyrene alters development of T lymphocytes in offspring.

Childhood cancer has been increasing significantly over the past two decades in the United States, suggesting that environmental exposures may be playing a causative role. One such cause may be maternal smoking during pregnancy. Suspected carcinogens in cigarette smoke and environmental pollution include N-nitrosamines and polycyclic aromatic hydrocarbons, which may be several micrograms per exposure. Previously, we have shown that mouse progeny of mothers exposed to benzo[a]pyrene (B[a]P) during midpregnancy had abnormalities in their humoral and cell-mediated immune response. Immunodeficiency was detectable during gestation, at one week after birth and persisted for 18 months. Tumor incidences in progeny were eight to 10-fold higher than in controls. The present study compared frequencies of CD4+, CD8+, V gamma 2+, and V beta 8+ T cells in progeny following in utero exposure to B[a]P. The significant reduction in newborn CD4+CD8+, CD4+CD8+V beta 8+ thymocytes and CD4+ splenocytes from 1-week-old progeny, suggests that B[a]P induces abnormal changes in developing T cells. These early alterations may lead to postnatal T cell suppression, thus providing a more suitable environment for the growth of tumors later in life. These results suggest that developmental immunosuppression mediated by B[a]P may play a critical role in the relationship between maternal exposures and childhood carcinogenesis.

Glutathione redox potential in response to differentiation and enzyme inducers.

The reduced glutathione (GSH)/oxidized glutathione (GSSG) redox state is thought to function in signaling of detoxification gene expression, but also appears to be tightly regulated in cells under normal conditions. Thus it is not clear that the magnitude of change in response to physiologic stimuli is sufficient for a role in redox signaling under nontoxicologic conditions. The purpose of this study was to determine the change in 2GSH/GSSG redox during signaling of differentiation and increased detoxification enzyme activity in HT29 cells. We measured GSH, GSSG, cell volume, and cell pH, and we used the Nernst equation to determine the changes in redox potential Eh of the 2GSH/GSSG pool in response to the differentiating agent, sodium butyrate, and the detoxification enzyme inducer, benzyl isothiocyanate. Sodium butyrate caused a 60-mV oxidation (from -260 to -200 mV), an oxidation sufficient for a 100-fold change in protein dithiols:disulfide ratio. Benzyl isothiocyanate caused a 16-mV oxidation in control cells but a 40-mV oxidation (to -160 mV) in differentiated cells. Changes in GSH and mRNA for glutamate:cysteine ligase did not correlate with Eh; however, correlations were seen between Eh and glutathione S-transferase (GST) and nicotinamide adenine dinucleotide phosphate (NADPH):quinone reductase activities (N:QR). These results show that 2GSH/GSSG redox changes in response to physiologic stimuli such as differentiation and enzyme inducers are of a sufficient magnitude to control the activity of redox-sensitive proteins. This suggests that physiologic modulation of the 2GSH/GSSG redox poise could provide a fundamental parameter for the control of cell phenotype.

Human acetylator genotype: relationship to colorectal cancer incidence and arylamine N-acetyltransferase expression in colon cytosol.

Polymorphic expression of arylamine N-acetyltransferase (EC 2.3.1.5) may be a differential risk factor in metabolic activation of arylamine carcinogens and susceptibility to cancers related to arylamine exposures. Human epidemiological studies suggest that rapid acetylator phenotype may be associated with higher incidences of colorectal cancer. We used restriction fragment length polymorphism analysis to determine acetylator genotypes of 44 subjects with colorectal cancer and 28 non-cancer subjects of similar ethnic background (i.e., approximately 25% Black and 75% White). The polymorphic N-acetyltransferase gene (NAT2) was amplified by the polymerase chain reaction from DNA templates derived from human colons of colorectal and non-cancer subjects. No significant differences in NAT2 allelic frequencies (i.e., WT, M1, M2, M3 alleles) or in acetylator genotypes were found between the colorectal cancer and non-cancer groups. No significant differences in NAT2 allelic frequencies were observed between Whites and Blacks or between males and females. Cytosolic preparations from the human colons were tested for expression of arylamine N-acetyltransferase activity. Although N-acetyltransferase activity was expressed for each of the arylamines tested (i. e., p-aminobenzoic acid, 4-aminobiphenyl, 2-aminofluorene, beta-naphthylamine), no correlation was observed between acetylator genotype and expression of human colon arylamine N-acetyltransferase activity. Similarly, no correlation was observed between subject age and expression of human colon arylamine N-acetyltransferase activity. These results suggest that arylamine N-acetyltransferase activity expressed in human colon is catalyzed predominantly by NAT1, an arylamine N-acetyltransferase that is not regulated by NAT2 acetylator genotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemoglobin adduct and hepatic- and urinary bladder-DNA adduct levels in rapid and slow acetylator Syrian inbred hamsters administered 2-aminofluorene.

The levels of covalently bound arylamine-hemoglobin and DNA adduct formation were used as dosimeters to measure the effect of acetylator genotype and sex on the metabolic conversion of the carcinogen, 2-aminofluorene, to reactive intermediates. A single high dose of 2-aminofluorene (60 mg/kg b.wt. i.p.) was administered to male and female homozygous rapid (Patr/Patr) acetylator hamsters (MHA/SsLaK) and homozygous slow (Pats/Pats) acetylator hamsters (Bio. 82.73/H). By using 32P-postlabeling assay methodology, a sole nonacetylated DNA adduct, which cochromatographed with authentic N-(deoxyguanosin-8-yl)-2-aminofluorene was detected at 3, 6, 12, 18 or 24 hr postdosing in liver and urinary bladder DNA of both rapid and slow acetylator hamsters. The highest levels were detected at 18 hr post 2-aminofluorene injection at which time the average levels of hepatic 2-aminofluorene-DNA adducts were similar between male and female rapid and slow acetylators. By comparison, the levels of 2-aminofluorene-DNA adducts in the urinary bladder at 18 hr were about 4-fold lower than in the liver, and were significantly greater in homozygous rapid than in homozygous slow acetylator counterparts (P less than .01). In both the liver and urinary bladder, the levels of 2-aminofluorene-DNA adducts were independent of sex. In contrast to the DNA adduct data, the levels of 2-aminofluorene-hemoglobin adducts, evaluated by capillary gas chromatography-mass spectrometry, were significantly higher in the homozygous slow acetylators than in homozygous rapid acetylators. However, there again were no differences between males and females.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylator genotype-dependent expression of arylamine N-acetyltransferase in human colon cytosol from non-cancer and colorectal cancer patients.

Human epidemiological studies suggest an association between rapid acetylator phenotype and colorectal cancer. Acetylator genotype-dependent expression by the human colon of arylamine N-acetylation capacity, catalyzed by acetyl coenzyme A-dependent N-acetyltransferase(s) (EC 2.3.1.5) (NAT), may be an important risk factor in the initiation of colorectal cancer. Human colon cytosols from 48 fresh surgical samples were investigated for NAT activity toward p-aminobenzoic acid and the arylamine carcinogens 4-aminobiphenyl, 2-aminofluorene, and beta-naphthylamine. Apparent Vmax determinations of NAT activity toward these substrates indicated that 40 of these colons segregated into 3 distinct phenotypes. The distribution of the patients into rapid (5), intermediate (18), or slow (17) acetylators is a ratio that is not significantly different from the expected Hardy-Weinberg distribution of 3:16:21 (chi 2 = 2.206, P = 0.363). Significantly greater mean apparent Vmax levels were found in colons from rapid as compared to intermediate acetylators (1.5-3-fold) (P less than 0.001) and intermediate as compared to slow (2.5-3-fold) (P less than 0.005) acetylator phenotypes for the four arylamine substrates. Apparent Km determinations indicated that human colon NAT from rapid acetylators had a significantly lower affinity for the arylamine substrates (P less than 0.05) compared to intermediate or slow acetylator groups. No difference in apparent Km was detected for the cofactor acetyl coenzyme A between the three acetylator phenotypes. The colon samples were also tested for cytosolic N-hydroxy-2-acetylaminofluorene sulfotransferase activity and found to be monomorphically distributed for this enzyme activity. Of the 40 colon samples, 37 were from individuals of known pathology, 25 with colorectal cancer and 12 with no diagnosed neoplasia. Comparisons between mean apparent Vmax and mean apparent Km levels for each of the acetylator phenotypes indicated no significant differences between non-cancer and colorectal cancer patients. The distribution of rapid, intermediate, and slow acetylator phenotypes among the colon samples derived from colorectal cancer patients was precisely that predicted from published frequencies for the rapid and slow acetylator allele in Americans of African and European ancestry.

Purification of hepatic polymorphic arylamine N-acetyltransferase from homozygous rapid acetylator inbred hamster: identity with polymorphic N-hydroxyarylamine-O-acetyltransferase.

The polymorphic acetyltransferase isozyme expressed in homozygous rapid acetylator inbred hamster liver cytosol was purified over 2000-fold by sequential Q-Sepharose fast-flow anion-exchange chromatography, Sephacryl S-200 high-resolution size-exclusion chromatography, Mono Q anion-exchange fast-protein liquid chromatography, and preparative polyacrylamide gel electrophoresis. The isozyme migrated as a single homogeneous monomer following both preparative and sodium dodecyl sulfate-polyacrylamide electrophoresis. The molecular weight was estimated at 34,170 following elution via size-exclusion chromatography and 35,467 following migration via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The homogeneous polymorphic acetyltransferase exhibited a broad substrate specificity; it catalyzed the acetyl coenzyme A-dependent N-acetylation of p-aminobenzoic acid, carbocyclic arylamine carcinogens such as 2-aminofluorene, 4-aminobiphenyl and beta-naphthylamine, and heterocyclic arylamine carcinogens such as 2-aminodipyrido[1,2-a:3'2'd]imidazole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole. It also readily catalyzed the acetyl coenzyme A-dependent metabolic activation (via O-acetylation) of N-hydroxy-2-aminofluorene to DNA adducts but not the metabolic activation (via intramolecular, N,O-acetyltransfer) of N-hydroxy-2-acetylaminofluorene or N-hydroxy-4-acetylaminobiphenyl to DNA adducts. Conversely, the partially purified monomorphic acetyltransferase isozyme from the same hamsters readily catalyzed the metabolic activation of N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylaminobiphenyl, and rates of metabolic activation of these substrates did not differ between homozygous rapid and slow acetylator liver, intestine, kidney, and lung cytosols. Heat inactivation rates for the purified polymorphic acetyltransferase isozyme were first order and indistinguishable for the acetyl coenzyme A-dependent N-acetylation and O-acetylation activities. The results strongly suggest the expression of a single polymorphic acetyltransferase product of the hamster polymorphic acetyltransferase gene that catalyzes both acetyl coenzyme A-dependent N-acetylation and O-acetylation of arylamine and N-hydroxyarylamine carcinogens but not the metabolic activation of N-hydroxy-N-acetylarylamines (arylhydroxamic acids) via intramolecular N,O-acetyltransfer. Consequently, acetylator genotype-dependent metabolic activation of N-hydroxyarylamines to a DNA adduct in hamster is catalyzed by direct O-acetylation of the hydroxyl group and not via sequential N-acetylation followed by N,O-acetyltransfer.

Acetylator genotype-dependent expression of arylamine N-acetyltransferase and N-hydroxyarylamine O-acetyltransferase in Syrian inbred hamster intestine and colon. Identity with the hepatic acetylation polymorphism.

Human epidemiological studies suggest an association between rapid acetylator phenotype and the incidence of colorectal cancer. Genetic regulation of acetyl coenzyme A-dependent N-acetyltransferase (NAT) and O-acetyltransferase (OAT) enzymatic activities may play a role in the metabolic activation of arylamine chemicals in the intestine and colon. In this study, the inheritance of acetyltransferase activity in the intestine and colon was investigated in the Syrian inbred hamster model. Relatively high levels of both arylamine NAT and N-hydroxyarylamine OAT activities were expressed in hamster intestine and colon cytosols, at levels similar to those in the liver. Acetylator genotype-dependent levels of NAT activity were expressed towards p-aminobenzoic acid and the carbocyclic arylamine carcinogens 2-aminofluorene (AF), 4-aminobiphenyl, and beta-naphthylamine. However, acetylator genotype-independent activity was found with the heterocyclic arylamine carcinogens 2-aminodipyrido[1,2-a:3',2'd]imidazole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, and 2-amino-9H-pyrido-[2,3,b]indole. F1 hybrid heterozygous acetylator progeny expressed unimodal levels of acetyltransferase activity intermediate between the homozygous rapid and slow acetylator parental strains. F2 generation progeny segregated into three modes (low, intermediate, and high) in a ratio of 1/2/1, and both sets of backcrosses yielded bimodal distributions of low and intermediate or high and intermediate in equal ratios. The genetic data is consistent with simple autosomal Mendelian inheritance of two codominant alleles (rapid and slow) at a single genetic locus, the polymorphic acetyltransferase gene. Levels of N-hydroxy-2-aminofluorene OAT activity were acetylator genotype-dependent in liver, intestine, and colon cytosols, which correlated well with AF NAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of Syrian inbred hamster acetylator genotype to the mutagenic activation of 2-aminofluorene.

The genetic constitution of mammalian enzymes involved in the metabolism of xenobiotics is one of the important factors responsible for large inter-individual differences in the rate of biotransformation and consequently the magnitude of genotoxic effects exerted in target tissues. The present study examines the mutagenic activation of 2-aminofluorene (AF) with hepatic post-mitochondrial (S9) preparations derived from homozygous rapid (Patr/Patr) acetylator and homozygous slow (Pats/Pats) acetylator Syrian inbred hamsters and its relationship to acetylator genotype. These hamster strains differ in their capacities for acetyl coenzyme A (AcCoA)-dependent, N-acetylation and O-acetylation of carcinogenic arylamines and their N-hydroxyarylamine metabolites. AF N-acetyltransferase activities determined in hepatic S9 fractions were 72.2 +/- 4.2 nmol/min/mg in rapid acetylator hamsters and 6.65 +/- 0.37 nmol/min/mg in slow acetylators, and were unaffected by the presence of 0.1 mM paraoxon. Mutagenic activation of AF was measured by reversion to histidine prototrophy in Salmonella typhimurium strain TA98. The metabolic activation of AF utilizing standard hepatic S9 preparations exhibited typical saturation kinetics that did not differ between acetylator genotypes. However, the addition of AcCoA to the standard S9 mix resulted in a dose-dependent reduction in the number of histidine revertants. In dose-response studies in which the concentrations of AF, AcCoA or S9 protein were varied, higher numbers of revertants were consistently generated with hepatic S9 derived from the slow acetylator compared to the rapid acetylator hamsters. These results indicate an acetylator genotype-dependent modulation of arylamine genotoxicity was reflected as a reduction in the levels of mutagenic metabolites generated in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and kinetic characterization of acetylator genotype-dependent and -independent arylamine carcinogen N-acetyltransferases in hamster bladder cytosol.

Recent studies from our laboratory have shown relatively high levels of polymorphic N-acetyltransferase (NAT)(EC 2.3.1.5) activity toward carcinogenic arylamines in urinary bladder cytosol of humans and in the inbred hamster model of the N-acetylation polymorphism. The expression of this polymorphism is of interest because of the higher incidence of bladder cancer among human slow acetylators with documented exposures to arylamine bladder carcinogens. In this study, arylamine NAT activity was partially purified and characterized in inbred hamster urinary bladder cytosols of defined acetylator genotype. Acetylator gene-dose response relationships were observed for the N-acetylation of p-aminobenzoic acid, p-aminosalicyclic acid, and the arylamine carcinogens 2-aminofluorene, 4-aminobiphenyl, and beta-naphthylamine in hamster bladder cytosol. Partial purification of hamster bladder cytosol by anion-exchange fast protein liquid chromatography yielded two NAT isozymes that catalyzed the N-acetylation of each of the arylamine substrates. The catalytic activity of the first isozyme was acetylator genotype-dependent (polymorphic), whereas the second isozyme appeared to be acetylator genotype-independent (monomorphic). Catalytic activities between homozygous rapid, heterozygous, and homozygous slow acetylator genotypes were compared with respect to both initial rates and apparent maximum velocities. Comparison of homozygous rapid and slow acetylator bladder cytosol showed that the apparent Vmax for 2-aminofluorene NAT activity was significantly higher in rapid than slow acetylators (6-fold in cytosol, 50-fold in the polymorphic NAT isozyme). These results suggest a key role for a polymorphic NAT isozyme, regulated by the acetylator genotype and expressed in urinary bladder cytosol, in the initiation of bladder cancer via arylamine carcinogens.

Kinetic characterization of acetylator genotype-dependent and -independent N-acetyltransferase isozymes in homozygous rapid and slow acetylator inbred hamster liver cytosol.

Acetyl-coenzyme A (AcCoA)-dependent arylamine N-acetyltransferase (NAT) activity (EC 2.3.1.5) was examined in liver cytosol derived from homozygous rapid acetylator (Bio. 87.20) and homozygous slow acetylator (Bio. 82.73/H) Syrian inbred hamsters. Expression of NAT activity toward p-aminobenzoic acid (PABA), 2-aminofluorene (AF), and 4-aminobiphenyl (ABP) was acetylator genotype-dependent, whereas N-acetyltransferase activity toward isoniazid was acetylator genotype-independent. Two isozymes of NAT activity were partially purified by anion exchange fast protein liquid chromatography from the hepatic cytosol of both homozygous rapid and homozygous slow acetylator hamsters. The first eluting NAT isozyme exhibited a polymorphic expression toward AF, ABP, and PABA although the second eluting NAT isozyme exhibited a monomorphic expression across acetylator genotypes toward the same substrates. Determination of Michaelis-Menten kinetic constants in hepatic cytosol of homozygous rapid and slow acetylator hamsters suggests that PABA, AF, and ABP NAT activities were acetylator genotype-dependent because of catalysis by polymorphic NAT isozyme that is both an apparent Km and Vmax variant, whereas, the acetylator genotype-independent expression of isoniazid NAT activity appeared to result from catalysis via a common monomorphic NAT isozyme in both acetylator genotypes. Additional kinetic studies on the partially purified NAT isozymes of homozygous rapid and slow acetylator hamster liver confirmed that the polymorphic NAT isozyme exhibited a substantially higher apparent maximum velocity in homozygous rapid acetylators than slow acetylators toward PABA, AF, and ABP as well as acetylator genotype-related differences in the apparent Km toward each of these substrates. In contrast, the monomorphic NAT isozyme of both acetylator genotypes showed apparent Vmax levels of NAT activity that did not vary with acetylator genotype. Furthermore, the monomorphic NAT isozyme did not show acetylator genotype-related variations in apparent Km toward the arylamine carcinogens AF and ABP, although differences were noted for PABA and AcCoA. These results suggest that the acetylator genotype-dependent expression of AcCoA-dependent NAT activity in hamster hepatic cytosol toward arylamines is primarily accountable by structural variants (allozymes) of polymorphic NAT under the genetic regulation of the acetylator gene locus. The acetylator genotype-independent expression of isoniazid NAT activity is attributable to a common monomorphic NAT isozyme in both acetylator genotypes.

Polymorphic expression of acetyl coenzyme A-dependent arylamine N-acetyltransferase and acetyl coenzyme A-dependent O-acetyltransferase-mediated activation of N-hydroxyarylamines by human bladder cytosol.

Human epidemiological studies suggest a genetic predisposition to bladder cancer among slow N-acetylators. The capacity of human bladder to N-acetylate arylamines, catalyzed by acetyl coenzyme A-dependent N-acetyltransferase(s) (EC 2.3.1.5) (NAT), may be an important step in the activation and/or deactivation of arylamines in the pathways leading to the initiation of bladder cancer. Another possible activation step is the direct O-acetylation of N-hydroxyarylamines via O-acetyltransferase(s) (OAT) to DNA-binding electrophiles. Human bladder cytosol from nine fresh autopsy specimens were investigated for NAT activity towards p-aminobenzoic acid, and the arylamine carcinogens 4-aminobiphenyl, 2-aminofluorene, and beta-naphthylamine. Apparent Km determinations indicated little difference in NAT affinity (100-300 microM) for any of the substrates between the nine individual bladders. However, the apparent Vmax determinations indicated that the bladders could be classified into rapid or slow acetylator phenotypes based on their NAT activity towards 4-aminobiphenyl, 2-aminofluorene, and beta-naphthylamine. Four of the bladder cytosols had mean activities significantly (P less than 0.01) higher (approximately 10-fold) than the mean NAT activities of the other five bladder cytosols towards each arylamine carcinogen. However, no significant difference was detected in their NAT activities using p-aminobenzoic acid as a substrate. The human bladder cytosols were also tested for their capacity to activate N-hydroxy-3,2'-dimethyl-4-aminobiphenyl to a DNA-binding electrophile through a direct OAT-mediated catalysis. The N-hydroxyarylamine OAT activity also discriminated between two levels of activation, being significantly (P = 0.0002) higher (about twofold) in the rapid N-acetylator bladder cytosols, that correlated (r = 0.94) with the measured levels of NAT activity in each bladder cytosol. These results suggest that NAT activity and OAT activity of the human bladder vary concordantly with N-acetylator phenotype. The polymorphic expression of these acetylation activities may be important risk factors in human susceptibility to bladder cancer from arylamine carcinogens.

Genetic control of acetyl coenzyme A-dependent arylamine N-acetyltransferase, hydrazine N-acetyltransferase, and N-hydroxy-arylamine O-acetyltransferase enzymes in C57BL/6J, A/J, AC57F1, and the rapid and slow acetylator A.B6 and B6.A congenic inbred mouse.

Acetyl coenzyme A-dependent N-acetyltransferase and O-acetyltransferase activities were examined in liver cytosols derived from homozygous rapid acetylator C57BL/6J and A.B6 congenic inbred mouse strains, from homozygous slow acetylator A/J and B6.A congenic inbred mouse strains, and from the (C57BL/6J x A/J)F1 heterozygous acetylator hybrid mouse strain. Acetylator genotype-dependent N-acetyltransferase activity was exhibited for the N-acetylation of p-aminobenzoic acid, 2-aminofluorene, and 4-aminobiphenyl. In contrast, levels of isoniazid N-acetyltransferase and N-hydroxy-3,2'-dimethyl-4-aminobiphenyl O-acetyltransferase activities in mouse liver cytosol appeared to be independent of the arylamine Nat acetylator gene. Although cytosolic N-acetyltransferase activities differed about 2-fold between the parental C57BL/6J and A/J strains for p-aminobenzoic acid, 2-aminofluorene, and 4-aminobiphenyl, the same N-acetyltransferase activities differed about 6-7-fold between the homozygous rapid acetylator A.B6 and the homozygous slow acetylator B6.A congenic inbred strains. Partial purification of acetyl coenzyme A-dependent arylamine N-acetyltransferase activity in the five inbred mouse strains showed one major paraoxon-resistant enzyme in liver cytosol in each of the rapid and slow acetylator mouse strains examined. Levels of partially purified 2-aminofluorene and 4-aminobiphenyl N-acetyltransferase activity were about 7-fold higher in the A.B6 than the B6.A congenic inbred strain. Partial purification of acetyl coenzyme A-dependent isoniazid N-acetyltransferase activity showed catalysis by a paraoxon-resistant enzyme(s) distinct from the major arylamine N-acetyltransferase enzyme(s). These results suggest that isoniazid N-acetyltransferase(s) in mouse liver cytosol is a product of a separate gene that segregates independently of the arylamine Nat gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylator genotype-dependent metabolic activation of carcinogenic N-hydroxyarylamines by S-acetyl coenzyme A-dependent enzymes of inbred hamster tissue cytosols: relationship to arylamine N-acetyltransferase.

A genetic polymorphism in S-acetyl coenzyme A (AcCoA)-dependent N-acetyltransferase has been associated with a differential risk for certain cancers in humans. In this study, several tissues from the inbred Syrian hamster with a genetically defined AcCoA-dependent N-acetyltransferase polymorphism (homozygous rapid acetylator, Bio. 87.20; homozygous slow acetylator, Bio. 82.73/H; and heterozygous acetylator, Bio. 87.20 X Bio. 82.73/H F1), were investigated for the relationship of arylamine N-acetyltransferase to the AcCoA-dependent metabolic activation of carcinogenic N-hydroxy (N-OH)-arylamines to bind to DNA (O-acetyltransferase). The levels of both 2-aminofluorene (AF) N-acetyltransferase and N-OH-AF O-acetyltransferase activity reflected the N-acetylator genotype in liver, intestine, kidney and lung cytosols. A significant acetylator gene--dose response for AF N-acetyltransferase and N-OH-AF O-acetyltransferase activities was observed in liver and lung cytosols. In contrast, acetylator genotype was not consistently expressed for the AcCoA-dependent N-acetylation of 4-aminobiphenyl (ABP), nor for the AcCoA-dependent metabolic activation of N-OH-ABP and N-OH-3,2'-dimethyl-4-aminobiphenyl in these same tissue cytosols. Two peaks of acetyltransferase activity were partially purified by ion exchange FPLC chromatography from the hepatic cytosol of both the homozygous rapid and homozygous slow acetylator hamster. In contrast to unfractionated cytosol, the isozyme(s) eluting first clearly demonstrated levels of AcCoA-dependent arylamine N-acetyltransferase and N-OH-arylamine O-acetyltransferase activities that were consistent with N-acetylator genotype (polymorphic) for all substrates tested. In contrast, the slower eluting isozyme(s) in each acetylator cytosol showed levels of AcCoA-dependent N- and O-acetyltransferase activities that did not vary with N-acetylator genotype (monomorphic). The AcCoA-dependent O-acetyltransferase activity of both the monomorphic and polymorphic peaks was paraoxon resistant. These studies demonstrate acetylator genotype-dependent control of AcCoA-dependent metabolic activation of N-OH-arylamines(O-acetylation) by polymorphic isozyme(s) similar to that for AcCoA-dependent N-acetylation of arylamines in the hamster. The polymorphic genetic control of N-OH-arylamine O-acetyltransferase may be an important risk factor for arylamine-induced cancer, in those species and tissues expressing appreciable levels of O-acetyltransferase activity.

Inheritance of acetylator genotype-dependent arylamine N-acetyltransferase in hamster bladder cytosol.

Acetyl coenzyme A-dependent arylamine N-acetyltransferase (EC 2.3.1.5) was examined in bladder cytosol derived from inbred Syrian hamsters. Expression of N-acetyltransferase activity towards p-aminobenzoic acid, p-aminosalicylic acid and 2-aminofluorene was acetylator genotype-dependent. Highest levels of bladder N-acetyltransferase activity were expressed in homozygous rapid acetylator hamsters (Bio. 87.20), lowest levels in homozygous slow acetylator hamsters (Bio. 82.73/H), and intermediate levels in Bio. 87.20 X Bio. 82.73/H F1 generation progeny. The N-acetyltransferase activity was acetylator genotype-dependent in both epithelial and non-epithelial bladder tissue. Genetic crosses using p-aminobenzoic acid and p-aminosalicylic acid as substrates indicated that bladder N-acetyltransferase activity is controlled via simple autosomal Mendelian inheritance of two codominant alleles at a single genetic locus. Acetylator genotype as assessed by bladder N-acetyltransferase activity was completely concordant with acetylator genotype as assessed by liver N-acetyltransferase activity. N-Acetyltransferase in slow acetylator bladder cytosol was both an apparent Km and Vmax variant compared to N-acetyltransferase in rapid acetylator bladder cytosol. These results suggest that genetic control of arylamine N-acetyltransferase in bladder urothelium may be a factor in hereditary predisposition to arylamine-induced bladder cancer.

Extrahepatic expression of N-acetylator genotype in the inbred hamster.

The genetic control of S-acetylcoenzyme A (AcCoA)-dependent N-acetyltransferase activity (EC 2.3.1.5) was investigated in liver, intestine, kidney, and lung cytosols derived from homozygous rapid acetylator (Bio. 87.20), heterozygous acetylator (Bio. 87.20 X 82.73/H F1), and homozygous slow acetylator (Bio. 82.73/H) Syrian inbred hamsters. AcCoA-dependent N-acetyltransferase activity was highest in hepatic cytosol, followed by intestine, kidney, and lung cytosol. In each of these tissues, cytosolic N-acetyltransferase exhibited an acetylator genotype-dependent activity with highest levels in homozygous rapid, intermediate levels in heterozygous F1 progeny, and lowest levels in homozygous slow acetylators. The ratio of N-acetyltransferase activity between acetylator genotypes was in general substrate dependent but not tissue dependent. Acetylator genotype-dependent N-acetyltransferase activity differences were highest for p-aminobenzoic acid, followed by p-aminosalicylic acid, 2-aminofluorene, and beta-naphthylamine. Expression of isoniazid N-acetyltransferase activity in each tissue was acetylator genotype independent. Determination of Michaelis-Menten kinetic constants in each tissue suggested that p-aminobenzoic acid N-acetyltransferase activity was acetylator genotype-dependent because of catalysis by an isozyme(s) that is both an apparent Km and a Vmax variant. In contrast, the acetylator genotype-independent expression of isoniazid N-acetyltransferase activity in each tissue appeared to result from a common isozyme(s) present in each tissue with equivalent kinetic constants in the two phenotypes. These data suggest that acetylator genotype-dependent expression of AcCoA-dependent N-acetyltransferase activity in extrahepatic tissues may play an important role in hereditary predisposition to toxicity and/or carcinogenesis in extrahepatic organs following exposure to arylamine drugs and foreign chemicals.

Identification and inheritance of inbred hamster N-acetyltransferase isozymes in peripheral blood.

Acetyl CoA-dependent p-aminobenzoic acid and p-aminosalicylic acid N-acetyltransferase (NAT) activity was determined in peripheral blood and blood cells from homozygous rapid (RR) acetylator (Bio. 87.20) and homozygous slow (rr) acetylator (Bio. 82.73/H) inbred hamsters and in their F1, F2 and backcross progeny. NAT activity was localized primarily in erythrocytes and was acetylator genotype dependent, as highest levels were expressed in homozygous rapid acetylator hamsters, intermediate levels in heterozygous acetylator hamsters and lowest levels in homozygous slow acetylator hamsters. Bio. 87.20 X Bio. 82.73/H F1 progeny expressed a unimodal nonoverlapping distribution of NAT activity intermediate between the RR and rr parentals. F2 generation progeny segregated into three modes (low, intermediate and high) of 21, 42 and 11, which is not significantly different from 1, 2 and 1. Bio. 82.73/H X F1 backcross progeny segregated into two modes (low and intermediate) of 18 and 16, whereas Bio. 87.20 X F1 backcross progeny segregated into two modes (intermediate and high) of 17 and 14, neither of which is significantly different from 1 and 1. These data are consistent with simple autosomal Mendelian inheritance of blood NAT activity by two codominant alleles at a single genetic locus. Partial purification of peripheral blood NAT activity by ion-exchange chromatography yielded separation of two isozymes that both exhibited acetylator genotype-dependent expression with highest activity in RR, intermediate activity in Rr and nondetectable activity in rr genotypes, respectively.