Antonio M. Bonin

Detection of mutagenic activity in human urine following fried pork or bacon meals

Mutagenic activity has been demonstrated in urine of human subjects after ingestion of fried pork or bacon. Activity was detected with Salmonella strains TA1538 and TA98 particularly, in the presence of liver homogenate S9, and was not enhanced by prior incubation of urine with beta-glucuronidase. Chemical and biological characteristics of the urine activity closely resemble those found in extracts of fried pork and bacon which also increase the frequency of sex-linked recessive mutations in Drosophila melanogaster. Microwave-cooked meat neither contained extractable mutagenic activity, nor contributed to urinary mutagenicity, possible due to the paucity of browning reactions in meat cooked under these conditions. If the urine and meat factors are chemically identical, then approximately one-third of the food activity is recovered from the urine. These results show that mutagenic factors, generated during cooking of pork and bacon, are ingested and absorbed and are subject to urinary clearance in biologically detectable quantities. It is possible that the potential for genetic toxicity in humans of these and related factors has been underestimated.

Mutagenicity of arylmethane dyes in Salmonella

22 arylmethane dyes which have been used as food colours, commercial dyes, laboratory stains and pH indicators were tested in the Salmonella/mammalian microsome mutagenicity assay. 8 mutagenic dyes were identified, including 5 food colours and 3 common laboratory strains; none of the 11 indicator dyes tested was mutagenic. The commercial and laboratory dyes Methyl Violet 2B C.I. 42535 and Crystal Violet C.I. 42555 were mutagenic in base-pair substitution mutation detector strain TA1535 in the absence of metabolic activation. By contrast, the food colours Benzyl Violet 4B C.I. 42640, Guinea Green B.C.I. 42085, Light Green SF C.I. 42095, Lissamine Green B C.I. 44090 and Violet BNP C.I. 42581 and the bacteriological stain, Basic Fuchsin C.I. 42500-42510, were all mutagenic in frameshift mutation detector strains TA98 and/or TA1538 and required metabolic activation. Most of these compounds gave weak mutagenic responses with Salmonella and were positive only within narrow dose ranges. Since conflicting results were obtained using dyes from different sources, minor dye components may have been responsible for their mutagenicity. This suggests the need to improve knowledge about impurities in arylmethane colours still used in food and to review the toxicological role of such impurities.

DNA Interactions and Bacterial Mutagenicity of Some Chromium(III) Imine Complexes and their Chromium(V) Analogues. Evidence for Chromium(V) Intermediates in the Genotoxicity of Chromium(III)

The in vitro DNA interactions and bacterial mutagenicities of cis-[CrIII(phen)2(OH2)2]3+ and trans-[CrIII(salen)(OH2)2]+ and their CrV analogues are reported. At pH 3.3, cis-[Cr(phen)2(OH2)2]3+ (0.02–2.0 mM) causes negatively supercoiled pUC9 DNA to smear on agarose gels, with substantial precipitation in the well at ≥1.0 mM. Much weaker interactions between CrIII and DNA were apparent at pH 7.4. The interactions between DNA and CrV phen complexes (0.5 mM total Cr, pH 3.3) generated by oxidation of cis-[Cr(phen)2(OH2)2]3+ (for 10–30 min) resulted in almost complete nicking of form I DNA to forms II and III DNA. Nicking of form I DNA (≥80%) was also apparent at pH 7.4 following reaction of DNA with PbO2-oxidized [Cr(phen)2(OH2)2]3+ (2 mM Cr). Interactions between trans-[CrIII(salen)(OH2)2]+ and DNA were weaker than those of the CrIII phen complex at both pH 3.3 and 7.4. The CrV salen derivative (0.5 mM total Cr) caused the disappearance of form I DNA at oxidation times of ≥10 min and at pH 3.3 with substantial cleavage. While oxidation of [Cr(salen)(OH2)2]+ by PbO2 was not observed at pH 7.4, the complex was oxidized by iodosobenzene to produce short-lived [CrO(salen)]+ that caused DNA smearing on the agarose gel. In bacterial mutagenicity assays, the CrIII imine complexes and their CrV analogues produced similar mutagenic responses, which were believed to be due to the instabilities of the CrV species in the bacterial growth medium. While the spectrum of the mutagenic activities differed between the chromium phen and salen complexes, both exhibited greatest mutagenicity in Salmonella typhimurium TA102. These data suggest that CrV species, generated in vivo by cellular oxidative enzymes, may be responsible for CrIII-induced mutagenesis.

Comparison of rat and guinea pig as sources of the S9 fraction in the salmonella/mammalian microsome mutagenicity test

A highly significant enhancement of mutagenicity occurs with 11 polycyclic aromatic hydrocarbons when 3-methylcholanthrene-induced guinea pig liver S9 is substituted for Aroclor-induced rat liver S9 in the Ames test. The use of MC-induced guinea pig liver S9 is particularly valuable for detecting the weak mutagenicity of benz[c]acridine, which is barely positive in a standard Ames assay. However, anthracene and phenanthrene, which are generally considered not to be carcinogens, remain non-mutagenic for strain TA100. This enhancement of mutagenicity does not correlate with arylhydrocarbon hydroxylase activities of the various liver preparations and does not apply to certain other non-PAH mutagens, including beta-naphthylamine, aflatoxin B1 and 4-dimethylaminoazobenzene.

Mutagenicity of electrophilic N-acyloxy-N-alkoxyamides

N-acyloxy-N-alkoxybenzamides are mutagenic in TA100 without the need for metabolic activation with S9. Electronic effects of substituents on both the benzamide ring in N-acetoxy-N-butoxybenzamides or the benzyloxy ring in N-acetoxy-N-benzyloxybenzamides do not influence mutagenicity levels. For N-benzoyloxy-N-benzyloxybenzamides, mutagenicity levels are inversely related to the electron-withdrawing effect of substituents on the benzoyloxy leaving group. Since reactivities increase with increasing electron-withdrawing effects, mutagenicity correlates with stability rather than reactivity of these mutagens. Hydrophobicity is the dominant factor controlling mutagenicity levels and data for all mutagens correlate with computed logP values with a lower dependence (h=0.22) than that recorded for indirect mutagens (h=1.0), except where a sterically demanding p-tert-butyl substituent or a naphthyl group is present. N-acetoxy-N-butoxynaphthamide exhibits a much higher level of mutagenicity than predicted by its logP value and activity may be ascribed to an intercalative binding process with DNA rather than straightforward hydrophobic binding in the major or minor groove. Since these are direct-acting mutagens, structural factors influence binding and reactivity towards DNA.

Mutagenicity and DNA damage studies of N-acyloxy-N-alkoxyamides — the role of electrophilic nitrogen

N-Acyloxy-N-alkoxyamides are anomeric amides that are direct-acting mutagens. They have been shown to damage DNA in the major and the minor grooves in a pH and sequence-selective manner. In acidic media, they damage adenines at N3 in the minor groove but above neutral pH, only guanine is damaged at N7 in the major groove. Both the acyloxy leaving group and the alkoxy group at the amide nitrogen are responsible for their electrophilicity and Salmonella mutagenicities in TA 100 and DNA damage data confirm that the mutagens react with DNA in an intact form, rather than by solvolysis to electrophilic nitrenium ions in the cytosol, or in vitro, prior to reacting with DNA. Hydrophobicity plays a role in both mutagenicity and DNA damage.

Mutagenic N-acyloxy-N-alkoxyamides: Probes for drug-DNA interactions

N-acyloxy-N-alkoxyamides are direct-acting mutagens in Salmonella typhimurium TA100 and react with DNA at N7 of guanine and N3 of adenine. From extensive mutagenicity data a quantitative structure–activity relationship (QSAR) has been derived that predicts activity to be dependent upon hydrophobicity (represented by log P) and stability to chemical reactions (through the pKA value of the leaving carboxylic acid group). Sterically bulky substituents (represented by Taft ES parameters) reduce activity. Deviations from this QSAR highlight structural features that can enhance or impede association of small molecules with DNA. Naphthyl, pyrenyl, and fluorenyl substituents raise activity significantly, possibly through an intercalative effect. The influence of a single sterically bulky tert-butyl group remote from the reactive nitrogen is adequately modelled by the QSAR. However, substrates with two or more such groups show radically reduced activity, most likely through a groove exclusion process. Branching close to the reactive centre strongly reduces activity in line with a steric inhibition of reaction with DNA.

Reactive intermediates from the solvolysis of mutagenic O-Alkyl N-acetoxybenzohydroxamates

Mutagenic O-(para-substituted benzyl)N-acetoxybenzohydroxamates undergo acid-catalysed solvolysis in aqueous acetonitrile but there is a change in mechanism from AA11 to E1 on going from para electron-withdrawing substituents to para+M electron-donating groups. The former permit the formation of a discrete nitrenium ion intermediate whereas the latter promote a concerted elimination of a resonance stabilized benzyl carbocation.

Comparative mutagenicity of two triarylmethane food dyes in salmonella, saccharomyces and drosophila

Abstract The dye Violet BNP C.I. 42581 (C.I. Food Violet 3), as used in food, was shown to induce point mutations in Salmonella typhimurium , mitotic convertants and recombinants in Saccharomyces cerevisiae and recessive X-linked lethal mutants in Drosophila melanogaster . One or more constituents of Violet BNP may have been genotoxic, as the sample of dye used was shown to be an impure mixture, consisting of at least seven different chemical components. A second dye, Lissamine Green B C.I. 44090 (Wool Green 5, Acid Green 50, Green 5) was weakly mutagenic when tested with Salmonella. However, no conclusive evidence of genetic activity in yeast was found, and in two experiments no lethal mutants were detected in Drosophila.

Metabolism of the carcinogen 7-methylbenz[c]-acridine in the rat

The biliary excretion of radioactivity by adult Wistar rats given i.v. 7-methyl-[7-14C]benz[c]acridine(14C-7-MBAC) and [methyl-3H]-7-methylbenz[c]acridine (3H-7-MBAC) (2 mg/kg) was 61% and 48%, respectively, in males in six hours. Females excreted 33% of a 2 mg/kg dose of 3H-7-MBAC in the same time-period. For male rats, the urinary and faecal excretions were about 10% and 61% of the dose of 14C-7-MBAC, respectively, in seven days. No enterohepatic circulation could be demonstrated in control male rats. The biliary excretion of radioactivity by phenobarbital- and 3-methylcholanthrene-induced male rats given 14C-7-MBAC was similar to or greater than that of control male rats. The organo-soluble biliary metabolites after beta-glucuronidase/arylsulphatase hydrolysis were separated by h.p.l.c., and quantitative metabolite distributions were obtained for induced and control rats by comparison with metabolite standards. The mutagenicity of bile from carcinogen-dosed control rats was greater than that of equivalent bile from carcinogen-dosed 3-methylcholanthrene-pretreated animals.