Gerald N. Levy

Binding of carbon tetrachloride metabolites to rat hepatic mitochondrial DNA

Radioactivity from [14C]CCl4 was bound to highly purified mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) prepared from livers of rats after a single dose of [14C]CCl4. At a low, non-necrotizing dose as well as at an acutely toxic dose, mtDNA bound 20-50-fold more radioactivity per mg than did nDNA. Extensive enzymatic digestion and purification of mtDNA did not remove radioactivity. Binding of radioactivity to mtDNA could also be demonstrated after anaerobic incubation of isolated mitochondria with [14C]CCl4, NADPH, ADP, and succinate. Our results suggest that CCl4 can be activated by rat hepatic mitochondrial enzymes to metabolites which bind covalently to mtDNA.

Kinetics of arylamine N-acetyltransferase in tissues from human breast cancer

N-Acetyltransferase activity and Michaelis-Menten kinetic constants were determined in cancerous and non-cancerous breast tissues from 30 female patients with breast cancer. The results derived from tissue cytosol showed that 12 rapid, ten intermediate and eight slow acetylators based on p-aminobenzoic acid and 2-aminofluorene for substrates. The mean apparent Km values for the monomorphic substrate p-aminobenzoic acid and polymorphic substrate 2-aminofluorene were: 55.0 +/- 18.7, 114.0 +/- 30.0, and 137.0 +/- 37.2 microM; and 62.5 +/- 23.7, 166.0 +/- 67.0, and 239.0 +/- 76.6 microM for the slow, intermediate, and rapid enzymes, respectively. Compared to the enzymes from slow acetylators, the rapid acetylators exhibited mean apparent Vmax values eight- and ten-fold greater for p-aminobenzoic acid and 2-aminofluorene, respectively. A similar trend was obtained from the blood cytosols of cancerous patients and healthy volunteers. N-Acetyltransferase activity of breast cancerous and non-cancerous tissues were 1.5- and 2.2-fold different between rapid and slow acetylator with p-aminobenzoic acid and 2-aminofluorene as substrates, respectively. In breast cancerous tissues, 75% and 70% of the cytosolic N-acetyltransferase activity were inhibited under 2 mM of tamoxifen as substrates of 2-aminofluorene and p-aminobenzoic acid, respectively. Similar results were also found in non-cancerous tissues and blood samples from breast cancer patients and healthy volunteers. The effect of 1 mM tamoxifen on the N-acetyltransferase activity from breast cancerous tissues with positive estrogen receptor was 1.6-fold higher than that of negative estrogen receptor. This is the first demonstration to show that anti-estrogen drug can affect N-acetyltransferase activity in breast cancerous tissues. Therefore, this finding may provide a clue to the use of tamoxifen in prevention of human breast cancer.

Evidence for arylamine N-acetyltransferase in the nematode Anisakis simplex

N-Acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene were determined in Anisakis simplex, a nematode found in the intestine of the salt water fish Trichiurus lepturus. The N-acetyltransferase activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activity from a number of Anisakis simplex whole tissue homogenizations was found to be 2.89 +/- 0.52 nmol/min per mg for 2-aminofluorene and 2.54 +/- 0.45 nmol/min per mg for p-aminobenzoic acid. The K(m) and Vmax values obtained were 1.06 +/- 0.69 mM and 9.34 +/- 1.94 nmol/min per mg for 2-aminofluorene, and 2.25 +/- 0.10 mM and 14.44 +/- 0.7 nmol/min per mg for p-aminobenzoic acid. The optimal pH value for the enzyme activity was pH 8.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The N-acetyltransferase activity was inhibited by iodoacetamide: at 0.25 mM iodoacetamide, activity was reduced 50% and 1.0 mM iodoacetamide inhibits activity more than 90%. Among a series of divalent cations and salts, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. This is the first demonstration of acetyl CoA/arylamine N-acetyltransferase activity in a nematode and extends the number of phyla in which this activity has been found.

Activation of heterocyclic amines by combinations of prostaglandin H synthase-1 and -2 with N-acetyltransferase 1 and 2

Cooking of meats produces several heterocyclic amines which are mutagenic and potentially carcinogenic. We found that metabolic activation of one of these heterocyclic amines, the quinoline derivative 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), can be catalyzed by prostaglandin H synthase (PHS) as well as by CYP1A2. N-Acetyltransferase (NAT) increased IQ-DNA adduct formation by either of these pathways. In sonicate from transiently transfected COS cells, NAT1 increased CYP1A2 catalyzed adduct formation 4-fold while NAT2 increased adduct formation 12-fold. Both expressed human and purified ovine PHS-1 and PHS-2 catalyzed IQ-DNA adduct formation. The presence of NAT1 and NAT2 increased PHS-1 catalyzed adduct formation 2.5- and 4-fold, respectively. PHS-2 catalyzed IQ adduct formation was also enhanced by either NAT. The pyridine derivative, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, also produced by protein pyrolysis, did not form detectable DNA adducts during incubation with PHS. These results indicate that IQ is a substrate for both PHS-1 and PHS-2 and that NAT increases the ability of the resulting IQ metabolites to cause DNA damage. PHS activity, constitutive and induced, as well as NAT polymorphisms should be considered as factors in environmental carcinogenesis.

Arylamine N-acetyltransferase 1 (NAT1) genotypes in a Lebanese population.

The frequency distributions of human N-acetyltransferase 1 (NAT1*) alleles in various ethnic groups are largely unknown. This lack of information is in contrast to the many studies of ethnic differences in NAT2* alleles and phenotypes. Increasing interest in NAT1 due to its potential roles in carcinogen metabolism and cancer risk makes it desirable to know the distribution of NAT1* alleles in various populations. Using a polymerase chain reaction-restriction fragment length polymorphism genotyping assay, the frequency of NAT1* alleles in a Lebanese population was determined. Of 84 NAT1* alleles assayed, 56% were NAT1*4. Alleles NAT1*3, *10, and *14 were found at frequencies of 0.036, 0.107, and 0.238, respectively. Five additional alleles (6%) differed from previously reported alleles. Nearly 50% of the population were heterozygous for a NAT1*14 allele. The unusually high frequency of NAT1*14 alleles in Lebanese may be useful for epidemiological studies of the effects of the NAT1 polymorphism in this population.

Metabolic, molecular genetic and toxicological aspects of the acetylation polymorphism in inbred mice

Over the past 10 years, much fascinating information has been obtained concerning the biochemistry, genetics, toxicological implications and molecular genetics of the N-acetylation polymorphism in mice. Using C57BL/6J (B6) mice as representative of rapid acetylation and A/J (A) mice as representing slow acetylation, it has been shown that the polymorphism observed in N-acetyltransferase (NAT) activity in liver also occurs in kidney, bladder, blood, and other tissues. The development of congenic acetylator mouse lines derived from B6 and A, have provided the necessary tools to study the role of the acetylation polymorphism, on either the B6 or A genetic background, free of nearly all other genetic differences between these strains. Eliminating genes which modify and complicate the differences due to the acetylator genes make the congenic lines very useful in toxicology studies, particularly those involving carcinogenesis. The molecular genetic basis of the acetylator polymorphism in B6 and A mice involves two Nat genes. Nat-1 encodes a protein termed NAT1 which is identical in rapid and slow acetylator strains. Nat-2, however, differs between rapid and slow strains by a single nucleotide change in the coding region. The corresponding NAT2 proteins differ by a single change at amino acid 99: an hydrophilic asparagine in rapid acetylator NAT2 to an hydrophobic isoleucine in NAT2 from slow acetylators. The mechanistic basis for the differences between rapid and slow acetylation in mice appears to be that NAT2 from the rapid B6 strain is 15-fold more stable at 37 degrees C and is transcribed/translated with a maximal efficiency twice that of the enzyme from slow acetylator A mice. Results discussed in this review indicate that mice provide an excellent system for studying the N-acetyltransferase polymorphism and also are useful for modelling several aspects of the human N-acetyltransferase polymorphism.

Induction of human prostaglandin endoperoxide H synthase-2 (PHS-2) mRNA by TCDD.

Numerous transcription response elements (e.g. AP-1, AP-2, GRE, CREB, as well as DRE) have been identified in the transcription regulation region of the PHS-2 gene in both mouse and human. The discovery of a DRE in the region raised the possibility that PHS-2 could be induced by TCDD, a dioxin compound. The time course and dose dependence of TCDD induction of PHS-2 mRNA expression were observed in HUVEC, primary human epithelial cells. In the observed time range (0-24 hours) the steady-state mRNA expression levels of PHS-2, as well as of mRNA for CYP1A1, increased with time at a TCDD dose of 20 nM. At the 24 hour time point, TCDD-treated cells displayed significant dose-dependent elevation of PHS-2 over the range of 0-40 nM TCDD. The increases in PHS-2 mRNA in both the time course and dose dependence experiments were consistent with that of CYP1A1. In contrast, mRNA for PHS-1, the constitutively expressed isoform of PHS, did not show significant changes under the conditions tested. These results are the first to indicate that TCDD can elevate PHS-2 mRNA level in a time and dose dependent manner. Further work needs to be done to learn the molecular mechanism of activation of PHS-2 by TCDD and the relation of TCDD action with other regulatory factors in the control of PHS-2 expression.

Polymorphic N-acetylation of 2-aminofluorene by cell-free colon extracts from inbred mice

The increased risk of rapid acetylator humans for the development of colorectal cancer has created interest in experimental animal models to study the relationship of N-acetyltransferase phenotype to colon cancer. Colon cytosols from inbred mouse lines were assayed for the ability to N-acetylate 2-aminofluorene to determine if the mouse model of the N-acetyltransferase polymorphism could be used to study this relationship. The results indicate that the colon acetylcoenzyme A: 2-aminofluorene-N-acetyltransferase activity parallels that of the liver. Colon activity from slow acetylator (A and B6.A) mouse lines is significantly lower than that of rapid acetylator (B6, B6.D, and A.B6) lines. p-Aminobenzoic acid N-acetyltransferase activity also differed between colon cytosols from rapid and slow acetylator strains. Isoniazid acetylation in colon and in liver did not differ between phenotypes. Northern blot analysis demonstrated the presence of mRNA for both NAT-1 and NAT-2 in mouse colon as well as in mouse liver. These results indicate that the N-acetyltransferase polymorphism is expressed in mouse colon when 2-aminofluorene or p-aminobenzoic acid is used as substrate and therefore the mouse may be a model for study of the effect of acetylator phenotype on development of colorectal cancer in humans.