Metabolism of azo food dyes by bacterial members of the human gut microbiome.

OBJECTIVES: We set out to survey the capacities of bacterial isolates from the human gut microbiome to reduce common azo food dyes in vitro. METHODS: A total of 206 strains representative of 124 bacterial species and 6 phyla were screened in vitro using a simple azo dye decolorization assay. Strains which showed azoreductive activity were characterized by studies of azoreduction kinetics and bacterial growth. RESULTS: Several groups of gut bacteria, including ones not previously associated with azoreduction, reduced one or more of the four azo food dyes commonly used in Canada: Allura Red, Amaranth, Sunset Yellow, and Tartrazine. Strains within some species differed in their azoreductive capabilities. Some strains displayed evidence of effects on growth related to the presence of azo dyes and/or the products of their azoreduction. CONCLUSION: The continued widespread use of food azo dyes requires re-evaluation in light of the potential for disturbance of the gut microbial ecosystem resulting from azoreduction and the possibility of consequences for human health.

Structure of an Unusual Tetracyclic Deoxyguanosine Adduct: Implications for Frameshift Mutagenicity of ortho-Cyano Nitroanilines.

Nitroaromatic compounds represent a major class of industrial chemicals that are also found in nature. Polycyclic derivatives are regarded as potent mutagens and carcinogens following bioactivation to produce nitrenium electrophiles that covalently modify DNA to afford N-linked C8-2'-deoxyguanosine (C8-dG) lesions that can induce frameshift mutations, especially in CpG repeat sequences. In contrast, their monocyclic counterparts typically exhibit weak mutagenicity or a lack thereof, despite also undergoing bioactivation to afford N-linked C8-dG adducts. Recently, it has been reported that cyano substitution can greatly increase the mutagenicity of nitroaniline derivatives that are components of azo dyes. The basis of this "cyano effect" may be rooted in the formation of a novel polycyclic adduct arising from initial formation of the N-linked C8-dG adduct followed by a cyclization process involving N7 of dG and the ortho-CN group of the attached C8-aryl moiety to generate a quinazolinimine ring as part of a fused tetracyclic C8,N7-dG adduct structure. The present work structurally characterizes this novel cyclic adduct using a combination of optical spectroscopies, NMR analysis, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. Our data indicate that this highly fluorescent cyclic adduct adopts the promutagenic syn conformation and can stabilize the slipped mutagenic intermediate (SMI) within the CpG repeat of the NarI sequence, which is a hotspot for frameshift mutagenesis mediated by polycyclic N-linked C8-dG adducts. In contrast, the open para-CN (4-aminobenzontrile-derived) N-linked C8-dG adduct is less likely to disrupt the canonical B-form. Together, our results provide a rationale for the potent mutagenicity of cyano-substituted nitroaniline derivatives recently reported in frameshift-sensitive tester strains.

Acetylation of aromatic cysteine conjugates by recombinant human N-acetyltransferase 8.

1. The mercapturic acid (MA) pathway is a metabolic route for the processing of glutathione conjugates to MA (N-acetylcysteine conjugates). An N-acetyltransferase enzyme, NAT8, catalyzes the transfer of an acetyl group from acetyl-CoA to the cysteine amino group, producing a MA, which is excreted in the urine. We expressed human NAT8 in HEK293T cells and developed an HPLC-MS method for the quantitation of the S-aryl-substituted cysteine conjugates and their MA. 2. We measured the activity of the enzyme for acetylation of benzyl-, 4-nitrobenzyl-, and 1-menaphthylcysteine substrates. 3. NAT8 catalyzed the acetylation of all three cysteine conjugates with similar Michaelis-Menten kinetics.

Inhibition of human glutathione transferases by dinitronaphthalene derivatives.

Glutathione transferase (GST) enzymes catalyze the conjugation of glutathione with reactive functional groups of endogenous compounds and xenobiotics, including halonitroaromatics. 1-Chloro-2,4-dinitrobenzene (CDNB) is one of the most commonly used substrates for GST activity assays. We have studied the interactions of dinitronaphthalene analogues of CDNB with recombinant human GST enzymes (Alpha, Mu, and Pi classes) expressed in Escherichia coli. Dinitronaphthalene derivatives were found to be GST inhibitors. The highest potency of inhibition was observed towards Mu-class GSTs, M1-1 and M2-2; IC50 values for 1-methoxy- and 1-ethoxy-2,4-dinitronaphthalene were in the high nanomolar to low micromolar range. Inhibition accompanies the formation, at the enzyme active site, of very stable Meisenheimer complex intermediates.

Reductive metabolism of azo dyes and drugs: Toxicological implications.

Azo compounds are widely distributed synthetic chemicals in the modern world. Their most important applications are as dyes, but, in addition, several azo compounds are used as pharmaceuticals. Ingested azo compounds can be reduced by the action of bacteria in the gut, where the oxygen tension is low, and the development of microbiome science has allowed more precise delineation of the roles of specific bacteria in these processes. Reduction of the azo bond of an azo compound generates two distinct classes of aromatic amine metabolites: the starting material that was used in the synthesis of the azo compound and a product which is formed de novo by metabolism. Reductive metabolism of azo compounds can have toxic consequences, because many aromatic amines are toxic/genotoxic. In this review, we discuss aspects of the development and application of azo compounds in industry and medicine. Current understanding of the toxicology of azo compounds and their metabolites is illustrated with four specific examples - Disperse Dyes used for dyeing textiles; the drugs phenazopyridine and eltrombopag; and the ubiquitous food dye, tartrazine - and knowledge gaps are identified. SUBMISSION TO: FCT VSI: Toxicology of Dyes.

Preparation, analysis and toxicity characterisation of the redox metabolites of the azo food dye tartrazine.

Tartrazine (E102, FD&C Yellow 5) is a vibrant yellow azo dye added to many processed foods. The safety of this ubiquitous chemical has not been fully elucidated, and it has been linked to allergic reactions and ADHD in some individuals. In our study, bacterial species isolated from human stool decolourised tartrazine and, upon exposure to air, a purple compound formed. Tartrazine is known to undergo reduction in the gut to sulfanilic acid and 4-amino-3-carboxy-5-hydroxy-1-(4-sulfophenyl)pyrazole (SCAP). These metabolites and their derivatives are relevant to the toxicology of tartrazine. The toxicity of sulfanilic acid has been studied before, but the oxidative instability of SCAP has previously prevented full characterisation. We have verified the chemical identity of SCAP and confirmed that the purple-coloured oxidation derivative is 4-(3-carboxy-5-hydroxy-1-(4-sulfophenyl)-1H-pyrazol-4-yl)imino-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (purpurazoic acid, PPA), as proposed by Westöö in 1965. A yellow derivative of SCAP is proposed to be the hydrolysed oxidation product, 4,5-dioxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid. SCAP and PPA are moderately toxic to human cells (IC50 89 and 78 µM against HEK-293, respectively), but had no apparent effect on Escherichia coli and Bacillus subtilis bacteria. These results prompt further analyses of the toxicology of tartrazine and its derivatives.

Hydrolysis of S-aryl-cysteinylglycine conjugates catalyzed by porcine kidney cortex membrane dipeptidase.

Following conjugation with glutathione, xenobiotics are converted into cysteinylglycine conjugates, cysteine conjugates, and finally, mercapturic acids. The structural factors determining the activities of dipeptidases for the metabolism of toxicologically-relevant cysteinylglycine conjugates are not well understood. We purified porcine kidney cortex membrane dipeptidase (MDP) to homogeneity, via phosphatidylinositol-specific phospholipase C-mediated cleavage of the protein's membrane anchor and cilastatin affinity chromatography. The homodimeric structure of the MDP protein was confirmed by mass spectrometry. The cysteinylglycine conjugates of 1-(chloromethyl)naphthalene, 4-nitrobenzyl chloride, and 1-chloro-2,4-dinitrobenzene were synthesized and HPLC separation methods for their quantitation were developed. MDP catalyzed the hydrolysis of all three conjugates, but the rate of this activity was strongly dependent on the nature of the substituent on the cysteine sulfur atom.

Functional studies of single-nucleotide polymorphic variants of human glutathione transferase T1-1 involving residues in the dimer interface.

Glutathione transferase T1-1 catalyses detoxication and bioactivation processes in which glutathione conjugates are formed from endogenous and xenobiotic substrates, including alkylating agents and halogenated alkanes. Although the common null polymorphism of the human GSTT1 gene has been studied extensively, little is known about the consequences of GSTT1 single-nucleotide polymorphisms (SNPs). Here, we have examined the effects of two SNPs that alter amino acid residues in the dimer interface of the GST T1-1 protein and one that causes a conservative substitution in the core of the subunit. Variant proteins were expressed in an Escherichia coli strain in which the metabolism of ethylene dibromide to a glutathione conjugate leads to lacZ reversion mutations. We measured the kinetic properties of the enzymes with the characteristic substrate 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and determined the specific activities with several other substrates. Circular dichroism spectroscopy was used to measure protein thermal denaturation profiles. Variant T104P, which has been reported as inactive, showed weak but detectable activity with each substrate. Variant R76S was expressed at lower levels and showed much-reduced thermal stability. The results are interpreted in the context of the three-dimensional structure of human GST T1-1.

Single-nucleotide polymorphic variants of human glutathione transferase T1-1 differ in stability and functional properties.

We have previously expressed hexa-histidine-tagged human glutathione transferase GST T1-1 at very high levels in an Escherichia colilacZ mutagenicity assay strain. Ethylene dibromide (EDB), which is activated by GST T1-1, produces a potent response in the mutation assay. We have now constructed and expressed two SNP variants of wild-type GST T1-1:D141N and E173K. The EDB activation activities of both variant enzymes, as measured by the lacZ mutagenicity assay, are greatly reduced The D141N variant behaved similarly to the wild-type enzyme, in terms of expression level and specific activities for conjugation of glutathione with 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethylene diiodide (EDI), and 4-nitrobenzyl chloride (NBCl), and for peroxidative detoxication of cumene hydroperoxide (CuOOH). In contrast, variant E173K is poorly expressed, has no detectable activity with EPNP, NBCl, or CuOOH, and has EDI activity much lower than that of the wild-type enzyme. The circular dichroism (CD) thermal denaturation profiles of the wild-type protein and variant D141N show a sharp two-state transition between native and denatured states. Variant E173K showed a very different profile, consistent with improper or incomplete protein folding. Our results show that SNP variants can give rise to GSTT1-1 proteins with significantly altered properties.

Activation of aminoimidazole carcinogens by nitrosation: mutagenicity and nucleotide adducts.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) are heterocyclic amines (HCAs) derived from high temperature cooking of meat and thought to cause colon cancer in humans. Reactive nitrogen oxygen species, which are mediators of the inflammatory response, can convert these amines to the corresponding N-nitrosamines, N-NO-IQ and N-NO-MeIQx. This study was designed to evaluate whether these N-nitrosamines are genotoxic and could be responsible, in part, for the high incidence of colon cancer in individuals with colitis. Such an association would counsel reduced intake of well-done red meat by colitis patients. Mutagenicity was evaluated by reversion of a lacZ frameshift allele in three different E. coli strains. Strains DJ701 and DJ702 express recombinant(S. typhimurium) aromatic amine N-acetyltransferase (NAT); DJ702 also expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase; and DJ2002 served as an N-acetyltransferase negative control. In strain DJ701, N-NO-IQ and N-NO-MeIQx elicited dose-dependent mutagenicity,which was not further increased in DJ702. Neither nitrosamine was mutagenic in strain DJ2002. While both N-nitrosamines are stable for >4 h (pH 7.4, 37 degrees C), they react with DNA or 2'-deoxyguanosine 3'-monophosphate at lower pH (5.5) to form adducts. HOCl, a component of the inflammatory response,increased adduct formation, as measured by 32P-postlabeling. Following treatment with nuclease P1and separation by two-dimensional thin-layer chromatography and then HPLC, N-NO-IQ and N-NOMeIQxwere shown to form the same adducts as those formed by N-OH-MeIQx or N-OH-IQ, namely N-(deoxyguanosin-8-yl) adducts. In summary, these N-nitrosamines are genotoxic and might be alternatives to their hydroxylamine analogues as activated intermediates leading to initiation of colon cancer in individuals with colitis.

Ames test evaluation of two commercially available zero-valent nickel compounds.

Zero-valent nickel compounds are organometallic chemicals that are used in synthetic applications and may also occur as intermediates in nickel-catalyzed hydrogenation reactions used in food processing. Few studies have been performed on their possible genotoxic actions. We have tested two commercially available examples of this class of compounds. Solubility and stability were examined. Mutagenicity testing did not confirm a previous report that bis(1,5-cyclooctadiene)nickel is positive in the Ames assay. No stimulation of lipid peroxidation was observed in studies of bovine erythrocytes exposed in vitro. Our results do not indicate that zero-valent nickel compounds have genotoxic effects.

Structure-activity investigation of the potentiating effect of cyano substitution on nitroaniline mutagenicity in the ames test.

2,6-Dicyano-4-nitroaniline and 2-cyano-4-nitroaniline (CNNA; 2-amino-5-nitrobenzonitrile) are potent mutagens in the Ames test, even though unsubstituted nitroanilines (NAs) are no more than weak mutagens. These compounds are putative reduction products of many commercial azo dyes, including Disperse Blue 165, Disperse Blue 337, Disperse Red 73, Disperse Red 82, Disperse Violet 33, and Disperse Violet 63. We have examined the mutagenicity in strains TA98 and YG1024 of a series of commercially-available isomers of CNNA, and some related compounds, to probe the relationship between structure and genotoxic activity in this class of compounds. The potentiating effect of the cyano substituent is seen in many cases; e.g. 2-amino-4-nitrobenzonitrile is a much more potent mutagen than 3-NA. 2,4-Dinitrobenzonitrile is also highly mutagenic. Possible mechanisms for the "cyano effect" are considered, with respect to the likely structures of cyanonitroaniline-DNA adducts and the roles of the enzymes (nitroreductase and acetyl CoA:arylamine N-acetyltransferase) believed to be involved in the activation of nitroaromatic compounds. Environ. Mol. Mutagen. 59:114-122, 2018. © 2017 Wiley Periodicals, Inc.

Screening and characterization of variant Theta-class glutathione transferases catalyzing the activation of ethylene dibromide to a mutagen.

Ethylene dibromide (EDB) is a widespread environmental pollutant and mutagen/carcinogen. Certain Theta-class glutathione transferases (GSTs), enzymes that catalyze the reaction of reduced glutathione (GSH) with electrophiles, activate EDB to a mutagen. Previous studies have shown that human GST T1-1, but not rat GST T2-2, activates EDB. We have constructed an E. coli lacZ reversion mutagenicity assay system in which expression of recombinant GST supports activation of EDB to a mutagen. Hexa-histidine N-terminal tagging of GST T1-1 results in greatly enhanced expression of the recombinant enzyme and gives a lacZ strain that shows a mutagenic response to EDB at extremely low levels (approximately 1 ng EDB per plate). The hexa-histidine-tagged enzyme was purified in one step by Ni(2+)-affinity chromatography. We applied the lacZ mutagenicity assay to the rapid screening of a library of variant GST Theta enzymes. Sequence variants with altered catalytic activities were identified, purified, and characterized.

Myeloperoxidase genotype, fruit and vegetable consumption, and breast cancer risk.

Myeloperoxidase (MPO), an antimicrobial enzyme in the breast, generates reactive oxygen species (ROS) endogenously. An MPO G463A polymorphism exists in the promoter region, with the variant A allele conferring lower transcription activity than the common G allele. Because oxidative stress may play a role in breast carcinogenesis, we evaluated MPO genotypes in relation to breast cancer risk among 1,011 cases and 1,067 controls from the Long Island Breast Cancer Study Project (1996-1997). We also assessed the potential modifying effects of dietary antioxidants and hormonally related risk factors on these relationships. Women over 20 years with incident breast cancer who were residents of Nassau and Suffolk Counties, NY, were identified as potential cases. Population-based controls were frequency matched by 5-year age groups. Genotyping was performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) technology, and suspected breast cancer risk factors and usual dietary intake were assessed during an in-person interview. Unconditional logistic regression was used to estimate odds ratios and 95% confidence intervals. Having at least one A allele was associated with an overall 13% reduction in breast cancer risk. When consumption of fruits and vegetables and specific dietary antioxidants were dichotomized at the median, inverse associations with either GA or AA genotypes were most pronounced among women who consumed higher amounts of total fruits and vegetables (odds ratio, 0.75; 95% confidence interval, 0.58-0.97); this association was not noted among the low-consumption group (P for interaction = 0.04). Relationships were strongest among premenopausal women. Results from this first study of MPO genotypes and breast cancer risk indicate that MPO variants, related to reduced generation of ROS, are associated with decreased breast cancer risk, and emphasize the importance of fruit and vegetable consumption in reduction of breast cancer risk.

Potent mutagenicity in the Ames test of 2-cyano-4-nitroaniline and 2,6-dicyano-4-nitroaniline, components of disperse dyes.

Genotoxicity data on commercial azo dyes and their components remain sparse, despite their widespread use. We have tested the mutagenicity of 2-cyano-4-nitroaniline (CNNA) and 2,6-dicyano-4-nitroaniline (CNCNNA), components of azo dyes such as Disperse Blue 165 and Disperse Red 73, in Ames test strains. Both compounds are extraordinarily potent frameshift mutagens, with much greater activity than structurally similar dihalonitroanilines and halodinitroanilines. Analysis of the responses of strains over-expressing or deficient in bioactivation enzymes shows that bacterial nitroreductase and acetyl CoA: arylamine N-acetyltransferase are important mediators of the mutagenicity of CNNA and CNCNNA.

Screening and characterizing human NAT2 variants.

Acetyl CoA:arylamine N-acetyltransferase (NAT; E.C. 2.3.1.5) enzymes play a key role in the metabolic activation of aromatic amine and nitroaromatic mutagens to electrophilic reactive intermediates. We have developed a system in which the activation of mutagens by recombinant human NAT2, expressed in Escherichia coli, can be detected by the appearance of Lac+ revertants. The mutagenesis assay is based on the reversion of an E. coli lacZ frameshift allele; the host strain for the assay is devoid of endogenous NAT activity and a plasmid vector is used for expression of human NAT2. A high-throughput version of the assay facilitates rapid screening of pools of NAT2 variants generated (for example) by random mutagenesis. Along with the methods for these assays, we present selected results of a screening effort in which mutations along the length of the NAT2 sequence have been examined. Homology modeling and simulated annealing have been used to analyze the potential effects of these mutations on structural integrity and substrate binding.

Perspectives on the chemical etiology of breast cancer.

Multiple factors, known and unknown, contribute to human breast cancer. Hereditary, hormonal, and reproductive factors are associated with risk of breast cancer. Environmental agents, including chemical carcinogens, are modifiable risk factors to which over 70% of breast cancers have been attributed. Polymorphisms of drug-metabolizing enzymes may influence risk of breast cancer from environmental chemicals, dietary agents, and endogenous steroids. The environmental factors discussed in this review include pollutants, occupational exposures, tobacco smoke, alcohol, and diet. Aromatic amines are discussed as potential mammary carcinogens, with a focus on heterocyclic amine food pyrolysis products. These compounds are excreted into the urine after consumption of meals containing cooked meats and have recently been detected in the breast milk of lactating women.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis in cultured Big Blue rat mammary epithelial and fibroblast cells.

Epithelial cells are the primary site of carcinogenesis in most tissues, including the mammary gland. As an alternative to the study of mutation induction in whole tissues in vivo, we have established Big Blue transgenic rat cell lines from the mammary epithelium (BBR/ME) and the mammary stroma (BBR/MFib), to permit a comparison of their mutagenic responses to carcinogens. We previously demonstrated their responsiveness to the alkylating agent N-ethyl-N-nitrosourea (ENU) (McDiarmid H et al. [2001]: Mutat Res 497:39-47). Here, we examined the responses of cultured epithelial and stromal cells to the protein pyrolysis product and mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Rat hepatic S9 was used as a source of bioactivation enzymes. Mutant induction (cII locus) and clonogenic survival were measured as a function of PhIP concentration. PhIP mutagenicity was observed in the fibroblast cells, but the greater toxicity of PhIP to the epithelial cells prevented a definitive evaluation of mutagenicity. Since PhIP may be detoxified by conjugation with glutathione, we measured glutathione levels and glutathione-S-transferase expression and activities in both cell lines. The epithelial cells had higher glutathione-S-transferase enzyme activity and protein expression than did the fibroblast cell line. Because the epithelial cells were more sensitive to toxicity, glutathione conjugation evidently plays only a minor role in PhIP toxicity and mutagenicity in our cell lines.