Combinations of cytochrome P-450 genotypes and risk of early-onset lung cancer in Caucasians and African Americans: a population-based study.

Polymorphisms in CYP1A1 and CYP1B1 genes in humans are associated with reduction of enzymatic activity towards several substrates, including those found in tobacco smoke. To investigate the potential role these polymorphisms have as modulators of early-onset lung cancer risk, a population-based case-control study involving early-onset lung cancer cases was performed. Biological samples were available for 383 individuals diagnosed prior to 50 years of age identified from the metropolitan Detroit Surveillance, Epidemiology and End Results (SEER) program and 449 age, race and sex-matched controls ascertained through random digit dialing. Genotype frequencies varied significantly by race for CYP1A1 Ile(462)Val and CYP1B1 Leu(432)Val genotypes, so all analyses were stratified by race. No association was seen between lung cancer risk and polymorphisms in CYP1A1 Msp1 or CYP1B1 Leu(432)Val for Caucasians or African Americans, after adjusting for age at diagnosis, sex, pack years of smoking and family history of lung cancer. In Caucasians, those with the IIe/Val genotype at CYP1A1 Ile(462)Val locus were at decreased risk of having lung cancer compared to those with the lle/lle genotype, after adjusting for age at diagnosis, sex, pack years of smoking and family history of cancer (OR=0.41 95% Cl 0.19-0.90). These results were not replicated among the African American population, nor were they modified by amount of smoking.

CYP1A1 and CYP1B1 polymorphisms and risk of lung cancer among never smokers: a population-based study.

The cytochrome P450 (CYP) superfamily of enzymes catalyse one of the first steps in the metabolism of carcinogens such as polycyclic aromatic hydrocarbons, nitroaromatics and arylamines. Polymorphisms within the CYP1A1 gene have been shown to be associated with lung cancer risk, predominantly among Asian populations. Despite functional evidence of a possible role of CYP1B1 in lung cancer susceptibility, only a few studies have evaluated polymorphisms in this gene in relation to lung cancer susceptibility. This population-based study evaluates polymorphisms in both of these CYP genes within never smokers, most of whom had environmental tobacco smoke (ETS) exposure. Cases (n = 160) were identified through the metropolitan Detroit Surveillance, Epidemiology and End Results program, and age, sex and race-matched population-based controls (n = 181) were identified using random digit dialing. Neither CYP1A1 MspI nor CYP1A1 Ile(462)Val was associated with lung cancer susceptibility among Caucasians or African-Americans. Among Caucasians, however, CYP1B1 Leu(432)Val was significantly associated with lung cancer susceptibility odds ratio (OR) for at least one valine allele = 2.87 [95% confidence interval (CI) 1.63-5.07]. Combinations of this Phase I enzyme polymorphism along with selected Phase II enzyme polymorphisms (GSTM1 null, GSTP1 Ile(105)Val and NQO1 C(609)T) were evaluated. The combination of CYP1B1 Leu(432)Val and NQO1 C(609)T appeared to be associated with the highest risk of lung cancer (OR = 4.14, 95% CI 1.60-10.74), although no combinations differed significantly from the risk associated with CYP1B1 Leu(432)Val alone. When individuals were stratified by household ETS exposure (yes/no), CYP1B1 Leu(432)Val alone and in combination with Phase II enzyme polymorphisms was more strongly associated with increased lung cancer susceptibility among those with at least some household ETS exposure. Additional studies will be required to further validate these findings among never smokers and to evaluate the effects of this polymorphism among smoking populations as well.

Combinations of glutathione S-transferase genotypes and risk of early-onset lung cancer in Caucasians and African Americans: a population-based study.

Polymorphisms in GSTM1, GSTT1 and GSTP1 genes in humans are associated with the reduction of enzymatic activity toward several substrates, including those in tobacco smoke. To investigate the potential role these polymorphisms have, as modulators of early-onset lung cancer risk, a population-based case-control study involving early-onset lung cancer cases was performed. Biological samples were available for 350 individuals diagnosed <50 years of age identified from the metropolitan Detroit Surveillance, Epidemiology and End Results (SEER) program and 410 cases of age, race and sex-matched controls ascertained through random digit dialing. African Americans carrying at least one G allele at the GSTP1 locus were 2.9-fold more likely to have lung cancer compared with African Americans without a G allele after adjustment for age, sex, pack years of smoking and history of lung cancer in a first-degree relative (95% CI 1.29-6.20). African Americans with either one or two risk genotypes at the GSTM1 and GSTP1 loci were at increased risk of having lung cancer compared with those having fully functional GSTM1 and GSTP1 genes (OR = 2.8, 95% CI 1.1-7.2 and OR = 4.0, 95% CI 1.3-12.2, respectively). No significant single gene associations between GSTM1, GSTT1 or GSTP1 and early-onset lung cancer were identified in Caucasians, after adjusting for age, sex, pack years and family history of lung cancer. However, our results suggest that specific combinations of glutathione S-transferase polymorphisms increase the risk of early-onset of lung cancer. Joint analysis of these genotypes may identify individuals who are at a higher risk of developing early-onset lung cancer with a greater certainty than single gene studies.

NQO1 T allele associated with decreased risk of later age at diagnosis lung cancer among never smokers: results from a population-based study.

The NAD(P)H:quinone oxidoreductase 1 gene, NQO1, contains a C to T transition at amino acid codon 187, which results in very low enzymatic activity. Previous studies of the association between NQO1 genotype and lung cancer have had mixed findings. This population-based case control study examines the association between NQO1 genotype and lung cancer in the largest sample of never smokers (<100 cigarettes, lifetime) to date. Cases (n = 161) were identified through the metropolitan Detroit Surveillance, Epidemiology and End Results (SEER) program, and 5-year age- and race-matched population-based controls (n = 173) were identified using random digit dialing. Allele frequencies of C and T, respectively, were 0.79 and 0.21 in Caucasians, and 0.84 and 0.16 in African Americans. Among those diagnosed aged >/=50 years, C/T and T/T genotyped individuals had 0.48 times lower lung cancer risk than individuals with C/C genotype (95% CI: 0.27-0.87). There was a non-significant suggestion of a protective effect associated with the T allele among those with a history of environmental tobacco smoke exposure (OR = 0.57, 95% CI: 0.32-1.03) but not among those without (OR = 0.98, 95% CI: 0.41-2.38). Sex, race, family history of lung cancer and histologic type did not modify the effect of NQO1 genotype on lung cancer risk. The observed risk reductions may be attributable to the greatly reduced procarcinogen activating of NAD(P)H:quinone oxidoreductase 1 in individuals with at least one copy of the T allele.

GSTM1, GSTT1 and GSTP1 polymorphisms, environmental tobacco smoke exposure and risk of lung cancer among never smokers: a population-based study.

Glutathione S-transferases detoxify polycyclic aromatic hydrocarbons found in tobacco smoke by glutathione conjugation. Polymorphisms within the GSTM1, GSTT1 and GSTP1 genes, coding for enzymes with deficient or reduced activity, have been studied as potential modifiers of lung cancer risk. It is hypothesized that risk associated with potential susceptibility gene polymorphisms might be most evident at low levels of exposure. Never smokers developing lung cancer represent a highly susceptible subset of the population, exposed to tobacco carcinogens only through environmental tobacco smoke. This population-based case-control study examines the association between GSTM1, GSTT1 and GSTP1 genotypes and lung cancer in one of the largest samples of never smokers to date. Cases (n = 166) were identified through the metropolitan Detroit Surveillance, Epidemiology and End Results (SEER) program and age- and race-matched population-based controls (n = 181) were identified using random digit dialing. Overall, there was no significant association between single or combinations of genotypes at GSTM1, GSTT1 or GSTP1 and lung cancer risk after adjustment for age, race, sex and household ETS exposure in years. However, in never smokers exposed to 20 or more years of household ETS, carrying the GSTM1 null genotype was associated with a 2.3-fold increase in risk [95% confidence interval (CI) 1.05-5.13]. Individuals in this high ETS exposure category carrying the GSTM1 null and the GSTP1 Val allele were at over 4-fold increased risk of developing lung cancer (OR = 4.56, 95% CI: 1.21-17.21). These findings suggest that in the presence of ETS, the GSTM1 genotype both alone and in combination with the GSTP1 genotype alters the risk of developing lung cancer among never smokers.

Genotoxic effects of heterocyclic aromatic amines in human derived hepatoma (HepG2) cells.

In order to study the mutagenic effects of heterocyclic aromatic amines (HAAs) in cells of human origin, five compounds, namely 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-3, 4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3, 8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), the pyridoimidazo derivative 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), were tested in micronucleus (MN) assays with a human derived hepatoma (HepG2) cell line. All HAAs caused significant, dose-dependent effects. The activities of IQ, MeIQ, MeIQx and PhIP were similar (lowest effective concentrations 25-50 microM), whereas Trp-P-1 was effective at a dose of >/=2.1 microM. In addition, the HAAs were tested in MN assays with Chinese hamster ovary (CHO) cells and in Salmonella strain YG1024 using HepG2 cell homogenates as an activation mix. In the CHO experiments, positive results were obtained with Trp-P-1 and PhIP, whereas the other compounds were devoid of activity under all experimental conditions. The discrepancy in the responsivity of the two cell lines is probably due to differences in their acetylation capacity: enzyme measurements with 2-aminofluorene as a substrate revealed that the cytosolic acetyltransferase activity in the HepG2 cells is approximately 40-fold higher than that of the CHO cells. In the bacterial assays all five HAAs gave positive results but the ranking order was completely different from that seen in the HepG2/MN experiments (IQ > MeIQ > Trp-P-1 >/= MeIQx >> PhIP) and the mutagenic potencies of the various compounds varied over several orders of magnitude. The order obtained in bacterial tests with rat liver S9 mix was more or less identical to that seen in the tests with HepG2 cell homogenates but the concentrations of the amines required to give positive results were in general substantially lower (10(-5)-10(-1) microM). Overall, the results of the present study indicate that MN/HepG2 tests might reflect the mutagenic effects of HAAs more adequately than other in vitro mammalian cell systems due to the presence of enzymes involved in the metabolic conversion of the amines.

Histological localization of acetyltransferases in human tissue.

The localization of human arylamine acetyltransferases (NAT) transcripts was performed by non-isotopic in situ hybridization, utilizing a combination of six NAT1 or NAT2 specific antisense oligonucleotide probes, in order to identify those tissues and organs that might be susceptible to the carcinogenic effects of aromatic amines. The intratissue differences in the level of NAT mRNA were observed: the most abundant NAT2 transcripts were found in hepatocytes, while NAT1 ones dominated in the urothelium and in the colon epithelial cells. The specific NAT1 and NAT2 mRNAs were present also in the epithelial lining of the lung bronchi, the mammary gland and the small intestine epithelial cells, the outer layer of placenta syncytiotrophoblast cells, the kidney tubules, and the pineal gland. Qualitative differences in the sites of mRNA of these two enzymes were seen only in the kidney specimens, in which NAT2 was expressed in both proximal and distal tubules, and the NAT1 was detected only in the former ones.

Role of acetyltransferases in the metabolism and carcinogenicity of aromatic amines.

The genotoxicity of N-substituted aryl compounds is dependent on their conversion to reactive metabolites, frequently through the production of reactive N-acetoxyarylamines. This activation is accomplished by acetyltransferases that are widely distributed. In the rat, the production of N-acetoxyarylamines has been most clearly related to the induction of tumors in the mammary gland, but this pathway also appears to be an important factor in the production of tumors in the liver, Zymbal gland and gastrointestinal tract. Expression of rat acetyltransferases responsible for acetylation of the nitrogen and the oxygen of arylamine derivatives (i.e., acetyltransferases 1 and 2) in bacterial cells has now permitted experiments which demonstrate that these enzymes exhibit good affinities for and N-acetylation of the endogenous arylalkylamines derived from tryptophan, i.e., tryptamine, 5-hydroxytryptamine (serotonin) and 5-methoxytryptamine, the immediate metabolic precursor of melatonin. Evidence that these reactions are likely to reflect real biological potentials is bolstered by histological localization of acetyltransferase mRNAs with synthetic antisense oligodeoxynucleotide probes. The results of these studies in rat indicate that the expression of acetyltransferase in tissues of the central nervous, gastrointestinal, urinary and reproductive systems is highly regulated, as it is in other organs commonly associated with aromatic amine carcinogenicity. Similar experimental approaches have been successful with human liver, mammary gland, kidney and bladder preparations. These observations give evidence that genotoxic N-acetoxyarylamines are produced by acetyltransferases that can metabolize, and possibly modulate, the hormonal and neurotransmitter effects of endogenous arylalkylamines. These relationships may help explain the occasional induction of tumors in organs not usually considered as targets of aromatic amines, as well as raise the possibility that the production of N-oxidized endogenous substrates may represent a mechanism for tumor induction in the absence of exogenous carcinogens.

Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids.

Genes for the 290 amino acid, 33-34 kDa cytosolic acetyltransferases (NAT1* and NAT2*) from rat and hamster were cloned and expressed in Escherichia coli. Active clones were selected by a simple visual test for their ability to decolorize 4-aminoazobenzene in bacterial medium by acetylation. These recombinant acetyltransferases were analyzed for: (i) N-acetyltransferase, which was assayed by the rate of acetyl coenzyme A-dependent N-acetylation of 2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamic acid acyltransferase, assayed by N,O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene. Both NAT2s showed first order increases in N-acetylation rates with increasing 2-AF or AAB concentrations between 5 and 100 microM, with apparent K(m) values of 22-32 and 62-138 microM respectively. Although under the same conditions the N-acetylation rates for the two NAT1s declined by > 50%, below 5 microM 2-AF or AAB, the NAT rate data fit Michaelis-Menten kinetics, and the apparent K(m) values were 0.2-0.9 microM. For N,O-acyltransferase, the apparent K(m) values of the NAT1s were approximately 6 microM, while the K(m) values of the NAT2s were approximately 20- to 70-fold higher. SDS-PAGE/Western blot analysis of the recombinant acetyltransferases gave apparent relative molecular weights (MWr) of approximately 31 kDa for both NAT1s and rat NAT2 and approximately 29 kDa for hamster NAT2. Comparable MWr values were observed for native hamster liver NAT1 and NAT2 and for rat NAT1 under the same conditions. Although we did not detect NAT2-like activity in rat liver cytosol previously, the present data show that the rat NAT2* gene does code for a functional acetyltransferase, with properties similar to those of hamster liver NAT2. The data also indicate that at low substrate concentrations, NAT1 would apparently play the predominant role in vivo in N-acetylation and N,O-acyltransfer of aromatic amine derivatives, including their metabolic activation to DNA-reactive agents.

Genetic analysis of two rat acetyltransferases.

Single copies of two closely related acetyltransferase genes were detected in Sprague-Dawley derived rat DNA by Southern blot analysis using gene-specific hybridization probes for the 3' end of the acetyltransferase coding regions. Sequence analysis of the two acetyltransferase genes showed that both had intronless, 870 bp coding regions and coded for 290 amino acid protein sequences that were approximately 85% homologous to one another. The calculated molecular weights were 33.4 and 33.9 kDa and the calculated isoelectric points 4.98 and 5.21 for AT1 and AT2, respectively. The inferred amino acid sequence of both the genes and cDNAs indicated that both rat acetyltransferases have cysteines at positions 44, 68 and 223 which have been conserved in all known vertebrate acetyltransferases. Transcripts for both AT1 and AT2 were detected in brain, colon, esophagus, heart, kidney, liver, lung, mammary gland, dorsal prostate, ventral prostate, salivary gland, seminal vesicles, small intestine, spleen, stomach, testes, urinary bladder and uterus of Sprague-Dawley rats by both Northern blot and RT-PCR analysis. A third gene with >80% sequence homology to codons 118-158 of acetyltransferase was also detected.

Histological localization of messenger RNAs for rat acetyltransferases that acetylate serotonin and genotoxic arylamines.

Rat acetyltransferases (ATs) can acetylate the endogenous arylalkylamines tryptamine, 5-hydroxytryptamine (serotonin), and 5-methoxytryptamine, the immediate precursor of melatonin. The same enzymes also acetylate and activate exogenous, carcinogenic arylamines, thereby being immediately responsible for the generation of DNA adducts. Localization of AT transcripts in the pineal gland and in specific cells of the intestine, cerebral cortex, pituitary, and lung identifies cells that may be important to the neurotransmitter and hormonal roles of the tryptamine derivatives. Transcript localization i liver, mammary gland, Zymbal gland, kidney, forestomach, and bladder, as well as intestine and lung, identifies cells that may be at increased carcinogenic risk because they can convert N-hydroxylated arylamines to genotoxic metabolites. Highly specific expression is also observed in the reproductive organs of both the male and female, including the testes, epididymis, uterus, ovary, and fallopian tube. In addition to these diverse organs, which are consistent with possible roles of the enzyme in carcinogen metabolism, neurotransmission, or hormonal regulation, specific cells of the cornea, cilliary process of the eye, olfactory process, adrenal gland, exorbital lacrimal gland, and skin also exhibit highly specific expression of AT mRNAs for which one can only speculate as to their function. In virtually every case, the extent of labeling suggested that AT1 was expressed at levels that were orders of magnitude higher than those of AT2. Qualitative differences in the sites of mRNA of these two enzymes were seen only in the olfactory process, in which AT1 was expressed in both respiratory and olfactory epithelia as well as Bowman's cells, and AT2 was detected only in the latter cells. The available data support the conclusion that the ATs are likely to be involved both in the metabolic activation of exogenous carcinogenic amines as well as the metabolism of endogenous arylalkylamines that play important hormonal and neurotransmitter roles.

Carcinogenicity of nitropyrenes in the newborn female rat.

The carcinogenicities of 1-nitropyrene (1-NP), 4-nitropyrene (4-NP), 1,3-dinitropyrene (1,3-DNP), 1,6-dinitropyrene (1,6-DNP), 1,8-dinitropyrene (1,8-DNP), 3-hydroxy-1-nitropyrene (3-OH-1-NP) and a mixture of 6- and 8-hydroxy-1-nitropyrene (6/8-OH-1-NP) were investigated in newborn female rats. Newborn female CD rats were treated s.c. eight times at weekly intervals with a total dose of 6.3 mumol 1-NP,1,3-DNP,1,6-DNP or 1,8-DNP; control animals received only dimethylsulfoxide (DMSO). The experiment was terminated at 67 weeks. With the exception of 1,6-DNP- and 1,8-DNP-treated animals, which had average survival periods of 149 and 164 days respectively, the animals administered the other compounds did not show decreased survival. Malignant fibrous histiocytomas were observed in 12%, 100% and 100% of the rats treated with 1,3-, 1,6- and 1,8-DNP respectively. Leukemia was found in 20% and 22% of the animals treated with 1,6- and 1,8-DNP respectively. No control rats developed these tumors. Additionally, mammary tumors were induced in rats treated with 1-NP. Newborn female CD rats were similarly treated with 1-NP, 4-NP, 3-OH-1-NP, 6/8-OH-1-NP or DMSO and newborn female F344 rats were treated with 1-NP or DMSO. The experiment was terminated at 86 weeks, 1-NP and 4-NP produced mammary adenocarcinoma in CD rats. Although 1-NP did not produce mammary adenocarcinoma in F344 rats, it induced leukemia. 4-NP also induced malignant fibrous histiocytomas in CD rats. This study demonstrates that 4-NP is more carcinogenic than 1-NP and that CD rats are more susceptible than F344 rats to mammary carcinogenesis by 1-NP. Additionally, 1,6- and 1,8-DNP are more potent than 1-NP in inducing malignant fibrous histiocytomas and leukemia.

Characterization of rat hepatic acetyltransferase.

Rat liver cytosol is capable of N-acetylation (NAT) of arylamines, O-acetylation (OAT) of arylhydroxylamines, and N,O-acetyltransfer (AHAT) of arylhydroxamic acids. Physical, enzymatic, and immunochemical techniques now support the conclusion that a single 32 kDa protein accounts for all of these activities. Of the five immunoglobulin (IgG1) mouse monoclonal antibodies (mAb) produced against this protein, each affected one or more of these acetylation activities. When mixed with rat hepatic cytosol and then chromatographed on a gel filtration column, mAbs 1F2 and 5F8 increased the apparent size of all enzymes capable of acetylation from 32 kDa to the exclusion volume. Each of the mAbs reacted with only a single 32 kDa protein on SDS-PAGE/Western blots, regardless of the state of purity of the enzyme. This enzyme is unstable in low salt solutions, as reflected by a relative loss in NAT versus AHAT activity, but it does not result in changes in either molecular weight or isoelectric point (pl). A second form of instability is shown by the formation of more basic peptides with pls as high as 6, again without change in molecular weight. Although NAT activity is retained in acetyltransferase (AT) that has a minimally modified pl, further increases in pl result in total loss of enzyme activity. The differential effects of the mAbs on AT suggest that the ratios of NAT, OAT, and AHAT may be highly dependent on the conformation of the enzyme and, consequently, provide insight as to why the abilities of ATs from different species exhibit such dissimilar potentials for the activation of aromatic amines by OAT and AHAT.

Biochemical and genetic analysis of two acetyltransferases from hamster tissues that can metabolize aromatic amine derivatives.

Cytosolic activities in liver, stomach, small intestine, colon, lung, kidney, brain and spleen of hamster have been shown to be capable of N-acetylation, O-acetylation and N,O-acetyltransfer utilizing aromatic amine derivatives as substrates. These activities, which have been implicated in the metabolic activation and carcinogenicity of these compounds, were highest in the liver and small intestine. N-Acetylation could be demonstrated with either acetyl CoA or N-hydroxy-N-acetylaminoarenes serving as acetyl donors. Each of these tissues gave two immunoreactive bands (approximately 29 and 31 kDa) on Western blots using anti-rat AT monoclonal antibodies for detection. Single copies of two distinct acetyltransferase genes, designated AT1 and AT2, were detected in hamster DNA by Southern blot analysis using gene-specific hybridization probes for the 3' end of the AT coding regions. A third gene with > 80% sequence similarity to codons 118-158 of AT2 was also detected. Sequence analysis of the two AT genes showed that both had intronless, 0.87 kb coding regions. One was identical with the AT1 reported by Abu-Zeid et al. (Abu-Zeid,M., Nagata, K., Miyata,M., Ozawa,S., Fukuhara,M. and Yamazoe,Y., 1991, An arylamine acetyltransferase (AT-1) from Syrian golden hamster liver: cloning, complete nucleotide sequence, and expression in mammalian cells. Mol. Carcinogen., 4, 81-88). The second, AT2 (GenBank accession number L24912), coded for a 290 amino acid sequence that was 79% homologous with hamster AT1 and had a calculated molecular weight of 33.8 kDa, a theoretical pI of 5.96 and the three cysteines that have been conserved in all known vertebrate ATs at positions 44, 68 and 223. Northern blot hybridization using gene-specific AT probes detected mRNAs for both AT1 and AT2 in each of the eight tissues analyzed. Two AT1 transcripts, approximately 1.7 and 2.3 kb, were found in approximately equal ratios in all eight tissues. Three transcripts for AT2, approximately 1.9, 2.1 and 2.4 kb, were present in approximately equal ratios in the brain, small intestine, lung and colon. The liver, stomach, kidney and spleen had primarily the smaller two AT2 mRNAs. The overall abundance of AT mRNAs was highest in liver, small intestine and colon. The coding sequences of all AT mRNAs analyzed were identical to their corresponding genes, although the lengths of both the 5' and 3' untranslated transcript regions varied.(ABSTRACT TRUNCATED AT 400 WORDS)

Characteristics of a purified dog hepatic microsomal N,O-acyltransferase.

Dog liver microsomes have at least three different enzymes that are capable of the deacylation of amides, N-arylhydroxamic acids and carboxylesters, the acyltransfer of N-arylhydroxamic acids and the N-acetylation of arylamines. As judged by SDS-PAGE stained with silver nitrate, one of these enzymes was purified to homogeneity by sequential treatment with Triton X-100, ion-exchange column chromatography, gel filtration and chromatofocusing. The protein was a glycoprotein trimer with a subunit weight of approximately 60 kDa. It showed microheterogeneity on analytical isoelectric focusing (IEF) in polyacrylamide with pls of 5.4-5.6. Following digestion with endoglycosidase H, its subunit weight was reduced to approximately 58 kDa, and it appeared to be homogeneous on IEF with a pl of approximately 5.6. A monoclonal antibody prepared against this enzyme also reacted with the pl 6.0 carboxylesterase of rat liver microsomes, but did not react with the other two dog hepatic acyltransferases. Conversely, a polyclonal antibody raised against the rat esterase reacted with the dog enzyme. The N-terminal sequence of the enzyme was Y-P-S-L-P-P-V-V-D-T-V-Q-G-K-V-, which was homologous to the form 1 carboxylesterase of rabbit liver and the pl 6.0 carboxylesterase of rat liver. Immunohistochemical analyses showed the presence of this enzyme in the epithelium of dog liver and urinary bladder, human liver and rat liver, esophagus, forestomach, glandular stomach, small and large intestines, renal tubules, trachea and prostate and alveolar cells of lung. Since this enzyme is present in the urothelium, it may be important for the activation of urinary metabolites of carcinogenic arylamines for the initiation of bladder carcinogenesis in the dog.

Purification and characterization of a rat hepatic acetyltransferase that can metabolize aromatic amine derivatives.

Rat liver cytosol is capable of N-acetylation of arylamines, O-acetylation of arylhydroxylamines and N,O-acyltransfer of arylhydroxamic acids. The objective of this study was to characterize the enzyme(s) responsible for these reactions. A partially purified acetyltransferase preparation from rat liver cytosol was used to produce five mouse monoclonal IgG1S that bound to acetyltransferase on Western blots and affected one or more of the acetylation reactions. Two immunoaffinity columns were prepared by covalently cross-linking monoclonal antibodies to protein A-Sepharose. The first column permitted recovery of a single, immunoreactive 32 kDa protein that was capable of catalyzing all three reactions, while the second removed all three acetylation activities from a partially purified enzyme preparation and yielded a single, immunoreactive 32 kDa protein on elution. The harsh conditions necessary for elution from the latter column precluded recovery of an active enzyme. Although Western blots from SDS-PAGE at all stages of purification showed a single 32 kDa protein, purification was associated with the production of multiple, immunochemically reactive peptides with higher pIs. Direct enzymatic assays of these immunochemically reactive components after isoelectric focusing on polyacrylamide gels demonstrated that a single 32 kDa, pI 4.5 protein is capable of all three cytosolic acetylation activities. A second 32 kDa protein, pI 4.8, was able to carry out N-acetylation but not N,O-acetyltransfer. Immunoreactive components with pIs > 4.8 that were formed during purification were catalytically inactive. However, isoelectric focusing in solution of cytosolic preparations that had been subjected only to gel filtration gave a single 32 kDa immunoreactive peptide that was capable of all three acetylation reactions. Buffer concentration differentially affected the enzymatic activities of the enzyme, i.e. as a pH 7.4 buffer was decreased from 50 mM sodium pyrophosphate to 2 mM, the ability to N-acetylate arylamines was lowered while the abilities for O-acetylation and N,O-acetyltransfer were unaffected. It has been shown that a single 32 kDa protein carries out all of the acetylation reactions in rat liver cytosol. Although it cannot be ruled out that other similarly sized and closely related enzymes that share antigenic sites are also capable of these acetylation reactions, these studies suggest that instabilities of the major peptide responsible for these activities, as reflected in changes in isoelectric point, may be responsible for changes in the enzymatic potentials of this peptide.

Acetylation of 2-aminofluorene derivatives by dog hepatic microsomes.

Dog urinary bladder is a target organ of carcinogenic arylamines. However, dog hepatic and urothelial cytosols lack acetylation enzymes that are capable of activating N-hydroxy metabolites of arylamines, suggesting that other enzymes may be involved. In the present study, we found that dog liver microsomes were capable of N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), and that these activities were inhibited by paraoxon. The 0.25% Triton X-100 extractable fraction of microsomes was resolved on an ion-exchange column into three different proteins that retained these activities. Two of these proteins, designated as enzyme I and enzyme II, were further chromatographed on a Sephacryl S-300 column. As judged from the gel filtration profile, the mol. wt of enzyme I was approximately 180 kDa and that of enzyme II was greater than 700 kDa. SDS-PAGE analysis showed that the subunit weight of enzyme II was approximately 150 kDa. In addition to N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-OH-AAF, these three enzymes were capable of the deacetylation of 2-acetylaminofluorene, N-OH-AAF and 4-nitrophenyl acetate. The ability of these microsomal enzymes to activate N-hydroxylated aromatic amines and the presence of these enzymes in urothelial cells, reported previously, suggests that they may play an etiological role in the carcinogenicity of these agents in the dog.

Metabolism of aromatic amines: relationships of N-acetylation, O-acetylation, N,O-acetyltransfer and deacetylation in human liver and urinary bladder.

N-Acetoxyarylamines are reactive metabolites that are implicated in the initiation of the carcinogenic process by some N-substituted aryl compounds. The objective of this study was to explore the relationship between the production of these reactive species and N-acetylation (NAT), a reaction previously demonstrated to be polymorphic in the human. Human liver and urinary bladder mucosa samples were frozen within 4-8 h post mortem. These tissues were assayed for the (i) O-acetylation (OAT) of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMABP) by acetyl CoA, (ii) intramolecular N,O-acetyltransfer (AHAT) of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), (iii) NAT of 2-aminofluorene (2-AF) and p-aminobenzoic acid (PABA) by acetyl CoA and (iv) deacetylation of N-OH-AAF. Cytosolic AHAT and OAT showed partial inhibition by paraoxon. The ratio of paraoxon insensitive AHAT to OAT to NAT of PABA to NAT of 2-AF appears to be 1:2:11:22 using freshly made cytosols from frozen livers. Freezing of the cytosol resulted in extensive loss of activities. All four of these cytosolic enzyme activities exhibited a similar polymorphic response. Microsomal deacetylation showed a monomorphic response. Similar to the liver, urinary bladder epithelial cells also catalyzed the same reactions. However, the OAT and AHAT activities were detected mainly in microsomes. These data suggest that phenotypically rapid acetylators have a greater biochemical potential for the metabolic activation of aromatic amines by pathways that involve O-acetylation.