Immunochemical quantitation of DNA adducts derived from the human bladder carcinogen 4-aminobiphenyl.

To assess target-tissue exposure to the human urinary bladder carcinogen 4-aminobiphenyl (ABP), we have developed a sensitive immunochemical method for measuring the major arylamine-DNA adduct formed, N-(guan-8-yl)-ABP (Gua-C8-ABP). High-affinity polyclonal antisera from rabbits immunized with N-(guanosin-8-yl)-ABP coupled to keyhole limpet hemocyanin were characterized and shown to have high specificity for antigenic determinants on the purine and biphenyl rings of Gua-C8-ABP and minimal cross-reactivity with ABP, deoxyguanosine, or hydrolyzed DNA. Assay standards containing ABP-modified DNA were prepared by reacting [3H]N-hydroxy-ABP with calf thymus DNA.

Specificity of mutagenesis by 4-aminobiphenyl. A possible role for N-(deoxyadenosin-8-yl)-4-aminobiphenyl as a premutational lesion.

Mutagenesis by N-acetoxy-N-trifluoroacetyl-4-aminobiphenyl, a reactive form of the human bladder carcinogen 4-aminobiphenyl (ABP), was studied in Escherichia coli virus M13mp10. N-acetoxy-N-trifluoroacetyl-4-ABP-treated DNA containing 140 lesions/duplex genome, when introduced into excision repair-competent cells induced for SOS mutagenic processing, resulted in a 40-fold increase in mutation frequency over background in the lacZ alpha gene fragment. DNA sequence changes were determined for 20 independent mutants.

Metabolism of the carcinogen [3H]6-nitrochrysene in the preweanling mouse: identification of 6-aminochrysene-1,2-dihydrodiol as the probable proximate carcinogenic metabolite.

6-Nitrochrysene (NC) is a potent lung and liver carcinogen when administered in multiple doses to preweanling mice. We have investigated both the in vitro metabolism of [3H]NC by 9000 g supernatants (S9) prepared from the livers of preweanling mice and the in vivo metabolism of [3H]NC in these animals. The in vitro covalent binding of primary metabolites of NC to DNA after further reductive and/or oxidative metabolism was then examined in an attempt to define the metabolic activation pathway responsible for the formation of carcinogen--DNA adducts in NC-treated preweanling mice.

Mechanism of formation and structural characterization of DNA adducts derived from peroxidative activation of benzidine.

Although the peroxidative metabolism of benzidine and other carcinogenic arylamines has been regarded as a significant pathway in extrahepatic tissues, the mechanism of peroxidase-mediated covalent binding to DNA has remained unclear. In this study, we have compared the metabolic activation of benzidine by prostaglandin H synthase, horseradish peroxidase, chloroperoxidase, and lactoperoxidase. All four peroxidases mediated the binding of benzidine to DNA and equimolar amounts of hydrogen peroxide (or arachidonic acid) and benzidine were required for the maximal binding in the system with either horseradish peroxidase or prostaglandin H synthase.

Comparative carcinogenicity of the food pyrolysis product, 2-amino-5-phenylpyridine, and the known human carcinogen, 4-aminobiphenyl, in the neonatal B6C3F1 mouse.

2-Amino-5-phenylpyridine (2-APP) is a mutagenic heterocyclic aromatic amine that is formed by pyrolysis of phenylalanine in proteins. Since this mutagen is structurally similar to the multipotent carcinogen, 4-aminobiphenyl (4-ABP), we compared their relative tumorigenic activity in the neonatal male B6C3F1 mouse. After determinations of the maximum tolerated dose (MTD), both aromatic amines were administered i.

N-methylation: potential mechanism for metabolic activation of carcinogenic primary arylamines.

Two amine N-methyltransferases isolated from rabbit liver catalyze S-adenosylmethionine-dependent N-methylation of benzidine and 4-aminobiphenyl but not of 4-aminoazobenzene or 2-aminobiphenyl. The enzymatic reaction products were analyzed and found to be identical to synthetic N-methylbenzidine and N-methyl-4-aminobiphenyl. N-Methylation may be a critical step in the metabolic activation of primary arylamines because N-methylarylamines, unlike primary arylamines, are readily N-oxygenated by the NADPH- and oxygen-dependent microsomal flavin-containing monooxygenase.

Cocarcinogenicity of saccharin and N-alkylnitrosoureas in cultured human diploid fibroblasts.

Previous attempts to transform human foreskin fibroblasts in vitro with N-methylnitrosourea (MNU) or N-ethylnitrosourea (ENU) have been unsuccessful, and concurrent treatment with cocarcinogens or tumor promotors and either MNU or ENU have also failed to produce a neoplastic response. The present study was undertaken to test the effect of sodium saccharin on MNU- or ENU-induced cell transformation. Saccharin alone was not effective in inducing the growth of colonies in soft agar (anchorage-independent growth).

Assessment of exposure and susceptibility to aromatic amine carcinogens.

As a consequence of human exposure to carcinogenic aromatic amines, biochemical approaches to risk assessment have emphasized metabolic determinants of individual susceptibility and quantification of arylamine-macromolecular adducts. A known genetic polymorphism in humans, hepatic arylamine acetyltransferase activity, has been associated with differences in individual susceptibility to urinary bladder (slow acetylators) and colorectal (rapid acetylators) cancers. Similarly, the high specificity of an inducible human cytochrome P450 towards the N-oxidation of 4-aminobiphenyl and other aromatic amines is consistent with metabolic differences that can be used to predict relative human risk.

The S-acetyl coenzyme A-dependent metabolic activation of the carcinogen N-hydroxy-2-aminofluorene by human liver cytosol and its relationship to the aromatic amine N-acetyltransferase phenotype.

A genetic polymorphism in the enzymatic N-acetylation of sulfamethazine and other drugs in humans is well known and has been related to differential susceptibility to drug toxicities. Carcinogenic aromatic amines such as 2-aminofluorene also undergo N-acetylation, and phenotypic slow acetylator individuals have been suggested to be at increased risk to arylamine-induced urinary bladder cancer. However, acetyltransferases have also been shown to catalyze a final metabolic activation step in the conversion of both hydroxamic acid (e.

Carcinogen-DNA adduct formation in the lungs and livers of preweanling CD-1 male mice following administration of [3H]-6-nitrochrysene, [3H]-6-aminochrysene, and [3H]-1,6-dinitropyrene.

6-Nitrochrysene (NC) and 6-aminochrysene (AC) have been shown to be potent lung and liver carcinogens when administered in multiple i.p. doses to preweanling mice.

Identification of C8-modified deoxyinosine and N2- and C8-modified deoxyguanosine as major products of the in vitro reaction of N-hydroxy-6-aminochrysene with DNA and the formation of these adducts in isolated rat hepatocytes treated with 6-nitrochrysene and 6-aminochrysene.

Since 6-nitrochrysene and 6-aminochrysene have shown activity in carcinogenicity bioassays, we have begun an investigation of their metabolic activation pathways and the nature of the carcinogen-DNA adducts that may be formed. N-Hydroxy-6-aminochrysene (N-hydroxy-AC), a candidate proximate or ultimate carcinogen and the highest polycyclic N-hydroxy arylamine homolog studied thus far, was prepared by direct chemical synthesis and characterized by 1H-n.m.

Circular dichroism of poly(dG-dC) modified by the carcinogens N-methyl-4-aminoazobenzene or 4-aminobiphenyl.

Poly(dG-dC) was modified to different extents by the carcinogens 4-aminobiphenyl (ABP) or N-methyl-4-aminoazobenzene (MAB). HPLC analysis of the enzymatically hydrolyzed modified polymers indicates that more than 90% of the ABP and 81% of the MAB modification occurs at the C8 position of guanine. The conformational changes of the unmodified and modified polymers were studied as a function of ethanol and magnesium ion concentrations by the use of circular dichroism (CD).

Mutagenicity of nitrofurans in Salmonella typhimurium TA98, TA98NR and TA98/1,8-DNP6.

8 representative 2-substituted 5-nitrofurans were assayed for mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6. The tested compounds were: 5-nitro-2-furanacrylic N-(5-nitro-2-furfurylidene)hydrazide (1); furazolidone (2); 5-nitro-2-furanacrolein (3); 5-nitro-2-furaldehyde semicarbazone (4); 5-nitro-2-furaldehyde (5); nitrofurantoin (6); 5-nitro-2-furaldehyde diacetate (7); and 5-nitro-2-furoic acid (8). These compounds exhibited markedly different mutagenic activities in TA98, and these mutagenicities were similar both in the presence and the absence of rat-liver hepatic S9 activation enzymes.

A probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site.

The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8-ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8-ABP residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoracetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG8-ABPpCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases.

Characterization and properties of the DNA adducts formed from N-methyl-4-aminoazobenzene in rats during a carcinogenic treatment regimen.

Chronic oral administration of the carcinogenic aminoazo dye N-methyl-4-aminoazobenzene (MAB) to rats is known to result in the induction of liver tumors. In order to assess the role of carcinogen-DNA adduct formation in MAB hepatocarcinogenesis, male rats were fed 0.06% [3'-3H]MAB in the diet for 1, 3 or 5 weeks.

The binding of an aminoazo dye carcinogen to a specific methionine residue in rat liver alcohol dehydrogenase in vivo.

On the administration of 3'-methyl-N,N-dimethyl-4-aminoazobenzene to rats pure aminoazo dye-bound alcohol dehydrogenase accounting for 45% of the total soluble protein bound aminoazo dye is isolated from the liver soluble supernatant. Tryptic digestion of that purified aminoazo dye-bound enzyme yields an aminoazo dye-bound nonapeptide which has a sequence identical to amino acids 301-309 in the known sequence of alcohol dehydrogenase (H. Jornvall and O.

The effects of saccharin on the development of neoplastic lesions initiated with N-methyl-N-nitrosourea in the rat urothelium.

Saccharin has been reported to induce urinary bladder tumors in multigeneration rat feeding studies and to promote bladder carcinogenesis in rats initiated with known bladder carcinogens. To examine the dose-dependent effects of saccharin on tumor promotion, sodium saccharin was administered at six levels in the diet (5.0, 2.

Role of aromatic amine acetyltransferase in human colorectal cancer.

Hepatic arylamine acetyltransferase phenotype has been suggested to be an important risk factor for urinary bladder carcinogenesis in individuals with known exposure to aromatic amines. This study was performed to evaluate the relative distribution of fast- and slow-acetylator phenotypes both in a population of men, 45 to 75 years of age, with a history of colorectal cancer and in a matched control group. Acetyltransferase activity was determined by administration of sulfamethazine and by subsequent analysis of blood and urine samples for N-acetylsulfamethazine and sulfamethazine using high-pressure liquid chromatography.

Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay.

Eight nitropolycyclic aromatic hydrocarbons (PAHs), including 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 7-nitrobenz[a]anthracene, 6-nitrochrysene and 6-nitrobenzo-[a]pyrene and their parent PAHs were tested fro tumorigenicity in the newborn mouse model by i.p. administration at 1, 8, and 15 days after birth.

Acetyl coenzyme A-dependent metabolic activation of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl and several carcinogenic N-hydroxy arylamines in relation to tissue and species differences, other acyl donors, and arylhydroxamic acid-dependent acyltransferases.

The metabolic activation of several carcinogenic N-hydroxy (N-OH)-arylamines by cytosolic S-acetyl coenzyme A (AcCoA)-dependent enzymes was examined in tissues and species susceptible to arylamine carcinogenesis. Comparisons of the AcCoA-dependent activity were also made with known cytosolic arylhydroxamic acid-dependent acyltransferases and with the ability of different acyl donors to mediate the binding of N-OH-arylamines to DNA. With rat hepatic cytosol, AcCoA-dependent DNA binding was demonstrated for several [3H]N-OH-arylamines, in the order: N-OH-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMABP), N-OH-2-aminofluorene (N-OH-AF) greater than N-OH-4-aminobiphenyl greater than N-OH-N'-acetylbenzidine greater than N-OH-2-naphthylamine; N-OH-N-methyl-4-amino-azobenzene was not a substrate.

Reactivity of benzidine diimine with DNA to form N-(deoxyguanosin-8-yl)-benzidine.

Benzidine diimine (BZDI), a reactive intermediate in the metabolic peroxidation of the carcinogen benzidine, has been reported to bind covalently to cellular proteins and nucleic acids. We have examined the nature of this interaction with DNA and have identified a major carcinogen-nucleoside adduct. BZDI (20-50 microM) reacted rapidly with DNA in vitro to give relatively high levels of covalently bound products (1-2 adducts/10(3) nucleotides; 30-45% yield).

Formation and persistence of arylamine DNA adducts in vivo.

Aromatic amines are urinary bladder carcinogens in man and induce tumors at a number of sites in experimental animals including the liver, mammary gland, intestine, and bladder. In this review, the particular pathways involved in the metabolic activation of aromatic amines are considered as well as the specific DNA adducts formed in target and nontarget tissue. Particular emphasis is placed on the following compounds: 1-naphthylamine, 2-naphthylamine, 4-aminobiphenyl, 4-acetylaminobiphenyl, 4-acetylamino-4'-fluorobiphenyl, 3,2'-dimethyl-4-aminobiphenyl, 2-acetylaminofluorene, benzidine, N-methyl-4-aminoazobenzene, 4-aminoazobenzene, and 2-acetylaminophenanthrene.

Identification of the major serum albumin adduct formed by 4-aminobiphenyl in vivo in rats.

Serum albumin was isolated from rats at 27 h after administration of the carcinogen [2,2'-3H]-4-aminobiphenyl. Pronase digestion of the purified albumin yielded a mixture of radiolabeled materials which was resolved into 5 major components by reverse-phase liquid chromatography. From detailed UV, 1H-NMR, and mass spectral analyses, four of these were determined to be 4-aminobiphenyl, 4'-hydroxy-4-acetylaminobiphenyl, and two other metabolites, all of which are presumed to be non-covalently associated with the serum albumin.

DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo.

The presence of relatively high levels of prostaglandin H synthase (PHS) in the dog urinary bladder and its ability to mediate the activation of carcinogenic arylamines to DNA-bound products in vitro suggests the involvement of this enzyme in arylamine-induced bladder carcinogenesis. Since the PHS-dependent metabolism of 2-naphthylamine (2-NA) had been shown to yield both ring- and N-oxidation products in vitro, we compared the reactivity of 3H-labeled N-hydroxy-2-naphthylamine (N-OH-2-NA), 2-nitrosonaphthalene, and 2-amino-1-naphthol (2-AN) toward DNA and protein. In the PHS-incubation system, all three derivatives bound at high levels to protein, but only N-OH-2-NA and 2-AN bound appreciably to DNA.