Cytotoxicity and genotoxicity of the carcinogen aristolochic acid I (AA-I) in human bladder RT4 cells.

Aristolochic acid (AA-I) induces upper urothelial tract cancer (UUTC) and bladder cancer (BC) in humans. AA-I forms the 7-(2'-deoxyadenosin-N6-yl)aristolactam I (dA-AL-I) adduct, which induces multiple A:T-to-T:A transversion mutations in TP53 of AA-I exposed UTUC patients. This mutation is rarely reported in TP53 of other transitional cell carcinomas and thus recognized as an AA-I mutational signature. A:T-to-T:A transversion mutations were recently detected in bladder tumors of patients in Asia with known AA-I-exposure, implying that AA-I contributes to BC. Mechanistic studies on AA-I genotoxicity have not been reported in human bladder. In this study, we examined AA-I DNA adduct formation and mechanisms of toxicity in the human RT4 bladder cell line. The biological potencies of AA-I were compared to 4-aminobiphenyl, a recognized human bladder carcinogen, and several structurally related carcinogenic heterocyclic aromatic amines (HAA), which are present in urine of smokers and omnivores. AA-I (0.05-10 µM) induced a concentration- and time-dependent cytotoxicity. AA-I (100 nM) DNA adduct formation occurred at over a thousand higher levels than the principal DNA adducts formed with 4-ABP or HAAs (1 µM). dA-AL-I adduct formation was detected down to a 1 nM concentration. Studies with selective chemical inhibitors provided evidence that NQO1 is the major enzyme involved in AA-I bio-activation in RT4 cells, whereas CYP1A1, another enzyme implicated in AA-I toxicity, had a lesser role in bio-activation or detoxification of AA-I. AA-I DNA damage also induced genotoxic stress leading to p53-dependent apoptosis. These biochemical data support the human mutation data and a role for AA-I in BC.

Mutagenicity of N-hydroxy-4-aminobiphenyl in human TP53 knock-in (Hupki) mouse embryo fibroblasts.

TP53 harbors somatic mutations in more than half of human tumors with some showing characteristic mutation spectra that have been linked to environmental exposures. In bladder cancer, a unique distribution of mutations amongst several codons of TP53 has been hypothesized to be caused by environmental carcinogens including 4-aminobiphenyl (4-ABP). 4-ABP undergoes metabolic activation to N-hydroxy-4-aminobiphenyl (N-OH-4-ABP) and forms pre-mutagenic adducts in DNA, of which N-(deoxyguanosin-8-yl)-4-ABP (dG-C8-4-ABP) is the major one. Human TP53 knock-in mouse embryo fibroblasts (HUFs) are a useful model to study the influence of environmental carcinogens on TP53-mutagenesis. By performing the HUF immortalization assay (HIMA) TP53-mutant HUFs are generated and mutations can be identified by sequencing. Here we studied the induction of mutations in human TP53 after treatment of primary HUFs with N-OH-4-ABP. In addition, mutagenicity in the bacterial lacZ reporter gene and the formation of dG-C8-4-ABP, measured by 32 P-postlabelling analysis, were determined in N-OH-4-ABP-treated primary HUFs. A total of 6% TP53-mutants were identified after treatment with 40 µM N-OH-4-ABP for 24 hr (n = 150) with G>C/C>G transversion being the main mutation type. The mutation spectrum found in the TP53 gene of immortalized N-OH-4-ABP-treated HUFs was unlike the one found in human bladder cancer. DNA adduct formation (~40 adducts/108 nucleotides) was detected after 24 hr treatment with 40 µM N-OH-4-ABP, but lacZ mutagenicity was not observed. Adduct levels decreased substantially (sixfold) after a 24 hr recovery period indicating that primary HUFs can efficiently repair the dG-C8-4-ABP adduct possibly before mutations are fixed. In conclusion, the observed difference in the N-OH-4-ABP-induced TP53 mutation spectrum to that observed in human bladder tumors do not support a role of 4-ABP in human bladder cancer development.

4-Aminobiphenyl suppresses homologous recombination repair by a reactive oxygen species-dependent p53/miR-513a-5p/p53 loop.

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, can cause oxidative DNA damage and induce miR-513a-5p. However, the interplay between miR-513a-5p and DNA damage remains unclear. In our result of ChIP assay, we speculated that p53 as transcription factor could regulate miR-513a-5p expression. In addition, we found that miR-513a-5p-induced by 4-ABP could suppress p53 expression and HR repair activity. On the other hand, the levels of p53, miR-513a-5p, and γH2AX were attenuated by 5 mM N-acetyl-l-cysteine (NAC) pretreatment, indicating that the reactive oxygen species (ROS)-dependent p53-miR-513a-5p was involved in DSB repair in 4-ABP-treated cells. These findings indicated that the ROS/p53/miR-513a-5p/p53 loop axis plays a relevant role in regulating HR repair which may facilitate our understanding of molecular mechanisms regarding how miR-513a-5p impacts DSB repair in 4-ABP-treated cells.

The impact of sex on hepatotoxic, inflammatory and proliferative responses in mouse models of liver carcinogenesis.

Liver cancer is the third most common cause of cancer-related death but is almost 4-fold more prevalent in men than in women. Increased risk in men may be due in part to elevated chronic inflammation, which is a crucial driving force for many cancers. Male mice also have a greater incidence of liver cancer than females after postnatal exposure to procarcinogens such as 4-aminobiphenyl (ABP) or diethylnitrosamine (DEN), or in mice that transgenically express hepatitis B virus (HBV) proteins. Liver damage, inflammation and proliferation are central to liver cancer development, and previous studies have shown that hepatocellular damage, inflammation and proliferation are acutely elevated to a greater extent in adult male mice than in females after high-dose exposure to DEN. In contrast, postnatal exposure of mice to tumor-inducing doses of either DEN or ABP produces no such acute responses. However, it is not known whether sex differences in responses to postnatal carcinogen exposure or to HBV protein expression may develop over time following sexual maturation. We conducted an extended time course study to compare markers of liver damage, inflammation and proliferation between male and female mice exposed postnatally to 600 nmol ABP or 10 mg/kg DEN, and also in HBV transgenic (HBVTg) mice, over the duration of time that mice are normally maintained for standard liver tumor development protocols. Postnatal exposure to either ABP or DEN produced no evidence of either acute or chronic hepatocyte damage, liver inflammation or proliferation in either male or female mice. In contrast, HBVTg mice showed increased liver damage, inflammation and proliferation with age, but with no observed sex difference. These findings suggest that although chronic liver damage, inflammation and proliferation may be drivers for liver cancer development, they are unlikely to contribute directly to observed sex differences in liver tumor risk.

Role of Human N-Acetyltransferase 2 Genetic Polymorphism on Aromatic Amine Carcinogen-Induced DNA Damage and Mutagenicity in a Chinese Hamster Ovary Cell Mutation Assay.

Carcinogenic aromatic amines such as 4-aminobiphenyl (ABP) and 2-aminofluorene (AF) require metabolic activation to form electrophilic intermediates that mutate DNA leading to carcinogenesis. Bioactivation of these carcinogens includes N-hydroxylation catalyzed by CYP1A2 followed by O-acetylation catalyzed by arylamine N-acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in ABP- and AF-induced mutagenesis and DNA damage, nucleotide excision repair-deficient (UV5) Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. ABP and AF both caused significantly (P < 0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the UV5/CYP1A2/NAT2*4 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT2*5B cell lines. ABP- and AF-induced hprt mutant cDNAs were sequenced and over 80% of the single-base substitutions were at G:C base pairs. DNA damage also was quantified by γH2AX in-cell western assays and by identification and quantification of the two predominant DNA adducts, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) by liquid chromatography-mass spectrometry. DNA damage and adduct levels were dose-dependent, correlated highly with levels of hprt mutants, and were significantly (P < 0.0001) greater in the UV5/CYP1A2/NAT2*4 rapid acetylator cell line following treatment with ABP or AF as compared to all other cell lines. Our findings provide further clarity on the importance of O-acetylation in CHO mutagenesis assays for aromatic amines. They provide evidence that NAT2 genetic polymorphism modifies aromatic amine-induced DNA damage and mutagenesis that should be considered in human risk assessments following aromatic amine exposures. Environ. Mol. Mutagen. 61:235-245, 2020. © 2019 Wiley Periodicals, Inc.

Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells.

Occupational and tobacco exposure to aromatic amines (AAs) including 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are associated with bladder cancer (BC) risk. Several epidemiological studies have also reported a possible role for structurally related heterocyclic aromatic amines (HAAs) formed in tobacco smoke or cooked meats with BC risk. We had screened for DNA adducts of 4-ABP, 2-NA, and several prominent HAAs formed in tobacco smoke or grilled meats including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) in the bladder DNA of BC patients, using liquid chromatography/mass spectrometry. We detected DNA adducts of 4-ABP, but not adducts of the other carcinogens. In this study, we have examined the capacity of RT4 cells, an epithelial human bladder cell line, to bioactivate AAs and HAAs to DNA damaging agents, which may contribute to BC. 4-ABP and AαC formed DNA adducts, but DNA adducts of 2-NA, PhIP, and MeIQx were not detected. 4-ABP DNA adducts were formed at tenfold higher levels than AαC adducts. Pretreatment of RT4 cells with α-naphthoflavone (1-10 µM), a specific cytochrome P450 1 (CYP1) inhibitor, decreased AαC adduct formation by 50% but did not affect the level of 4-ABP adducts. However, cell pretreatment with 8-methoxypsoralen (0.1-1 µM), a potent inhibitor of CYP2A, resulted in a 90% decrease of 4-ABP DNA adducts levels. These data signify that CYP2A and CYP1A isoforms expressed in the target urothelium bioactivate 4-ABP and AαC, respectively, and may be a critical feature of aromatic amine-induced urinary bladder carcinogenesis. The bioactivation of other tobacco and environmental AAs by bladder CYPs and their ensuing bladder DNA damage warrants further study.

Primary aromatic amines and cancer: Novel mechanistic insights using 4-aminobiphenyl as a model carcinogen.

Aromatic amines are an important class of human carcinogens found ubiquitously in our environment. It is estimated that 1 in 8 of all known or suspected human carcinogens is or can be converted into an aromatic amine, making the elucidation of their mechanisms of toxicity a top public health priority. Decades of research into aromatic amine carcinogenesis revealed a complex bioactivation process where Phase I and Phase II drug metabolizing enzymes catalyze N-oxidation and subsequent conjugation reactions generating the highly electrophilic nitrenium intermediate that reacts with and forms adducts on cellular macromolecules. Although aromatic amine-DNA adducts were believed to be the main driver of cancer formation, several studies have reported a lack of correlation between levels of DNA adducts and tumors. Using genetically modified mouse models, our laboratory and others observed several instances where levels of conventionally measured DNA adducts failed to correlate with liver tumor incidence following exposure to the model aromatic amine procarcinogen 4-aminobiphenyl. In this review we first provide a historical overview of the studies that led to a proposed mechanism of carcinogenesis caused by aromatic amines, where their bioactivation to form DNA adducts represents the central driver of this process. We then highlight recent mechanistic studies using 4-aminobiphenyl that are inconsistent with this mechanism which suggest novel drivers of aromatic amine carcinogenesis.

Probing the Effect of Bulky Lesion-Induced Replication Fork Conformational Heterogeneity Using 4-Aminobiphenyl-Modified DNA.

Bulky organic carcinogens are activated in vivo and subsequently react with nucleobases of cellular DNA to produce adducts. Some of these DNA adducts exist in multiple conformations that are slowly interconverted to one another. Different conformations have been implicated in different mutagenic and repair outcomes. However, studies on the conformation-specific inhibition of replication, which is more relevant to cell survival, are scarce, presumably due to the structural dynamics of DNA lesions at the replication fork. It is difficult to capture the exact nature of replication inhibition by existing end-point assays, which usually detect either the ensemble of consequences of all the conformers or the culmination of all cellular behaviors, such as mutagenicity or survival rate. We previously reported very unusual sequence-dependent conformational heterogeneities involving FABP-modified DNA under different sequence contexts (TG1*G2T [67%B:33%S] and TG1G2*T [100%B], G*, N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl) (Cai et al. Nucleic Acids Research, 46, 6356-6370 (2018)). In the present study, we attempted to correlate the in vitro inhibition of polymerase activity to different conformations from a single FABP-modified DNA lesion. We utilized a combination of surface plasmon resonance (SPR) and HPLC-based steady-state kinetics to reveal the differences in terms of binding affinity and inhibition with polymerase between these two conformers (67%B:33%S and 100%B).

Influence of sex and developmental stage on acute hepatotoxic and inflammatory responses to liver procarcinogens in the mouse.

The incidence of liver cancer is higher in men than in women. This sex difference is also observed in murine tumor induction models that result in the appearance of liver tumors in adult mice following their exposure on postnatal days 8 and/or 15 to carcinogens such as 4-aminobiphenyl (ABP) or diethylnitrosamine (DEN). Previous studies performed in adult mice showed that acute hepatotoxic and inflammatory responses to high-dose DEN exposure were greater in males than in females, leading to the suggestion that these responses could account for the sex difference in tumor development. We also recently observed that female but not male mice exposed postnatally to ABP had slightly increased expression of the antioxidant defense genes Nqo1 and Ggt1, which are regulated by the oxidative stress response protein nuclear factor erythroid 2-related factor 2 (NRF2), while expression of Hmox1 was increased in both sexes. The goal of the present study was therefore to compare selected acute hepatotoxic, inflammatory and oxidative stress defense responses to ABP, DEN, or the prototype hepatotoxicant carbon tetrachloride (CCl4), in male and female mice exposed to these chemicals either postnatally or as adults. Exposure of adult mice to ABP, DEN or CCl4 produced a 2-fold greater acute elevation in serum levels of the hepatotoxicity biomarker alanine aminotransferase (ALT) in males than in females, while levels of the inflammatory biomarker interleukin-6 (IL-6) showed no sex difference. However, treatment of immature mice with either ABP or DEN using standard tumor-inducing postnatal exposure protocols produced no increase in serum ALT or IL-6 levels in either males or females, while CCl4 produced a 40-fold ALT elevation but with no sex difference. Basal expression of the NRF2-responsive gene Nqo1 was higher in adult females than in males, but there was no sex difference in basal expression of Ggt1 or Hmox1. Sexually immature animals showed no sex difference in basal expression of any of the three genes. Postnatal DEN exposure modestly increased the expression of Ggt1 only in male mice and Nqo1 in both sexes, while CCl4 slightly increased expression of Ggt1 in both males and females and Nqo1 only in females. Taken together, our results make it unlikely that acute hepatotoxic, inflammatory or NRF2-activated gene responses account for the male predominance in liver tumor growth following postnatal carcinogen exposure in mice. Our findings also suggest that acute toxicity studies performed in adult mice should be interpreted with caution when extrapolating potential mechanisms to liver carcinogenesis models that commonly use postnatally exposed mice.

CurveP Method for Rendering High-Throughput Screening Dose-Response Data into Digital Fingerprints.

The nature of high-throughput screening (HTS) puts certain limits on optimal test conditions for each particular sample, therefore, on top of usual data normalization, additional parsing is often needed to account for incomplete read outs or various artifacts that arise from signal interferences.CurveP is a heuristic, user-tunable, curve-cleaning algorithm that attempts to find a minimum set of corrections, which would give a monotonic dose-response curve. After applying the corrections, the algorithm proceeds to calculate a set of numeric features, which can be used as a fingerprint characterizing the sample, or as a vector of independent variables (e.g., molecular descriptors in case of chemical substances testing). The resulting output can be a part of HTS data analysis or can be used as input for a broad spectrum of computational applications, such as Quantitative Structure-Activity Relationship (QSAR) modeling, computational toxicology, bio- and cheminformatics.

Quantitative analysis of the relative mutagenicity of five chemical constituents of tobacco smoke in the mouse lymphoma assay.

Quantifying health-related biological effects, like genotoxicity, could provide a way of distinguishing between tobacco products. In order to develop tools for using genotoxicty data to quantitatively evaluate the risk of tobacco products, we tested five carcinogens found in cigarette smoke, 4-aminobiphenyl (4-ABP), benzo[a]pyrene (BaP), cadmium (in the form of CdCl2), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in the mouse lymphoma assay (MLA). The resulting mutagenicity dose responses were analyzed by various quantitative approaches and their strengths and weaknesses for distinguishing responses in the MLA were evaluated. L5178Y/Tk (+/-) 3.7.2C mouse lymphoma cells were treated with four to seven concentrations of each chemical for 4h. Only CdCl2 produced a positive response without metabolic activation (S9); all five chemicals produced dose-dependent increases in cytotoxicity and mutagenicity with S9. The lowest dose exceeding the global evaluation factor, the benchmark dose producing a 10%, 50%, 100% or 200% increase in the background frequency (BMD10, BMD50, BMD100 and BMD200), the no observed genotoxic effect level (NOGEL), the lowest observed genotoxic effect level (LOGEL) and the mutagenic potency expressed as a mutant frequency per micromole of chemical, were calculated for all the positive responses. All the quantitative metrics had similar rank orders for the agents' ability to induce mutation, from the most to least potent as CdCl2(-S9) > BaP(+S9) > CdCl2(+S9) > MeIQ(+S9) > 4-ABP(+S9) > NNK(+S9). However, the metric values for the different chemical responses (i.e. the ratio of the greatest value to the least value) for the different chemicals ranged from 16-fold (BMD10) to 572-fold (mutagenic potency). These results suggest that data from the MLA are capable of discriminating the mutagenicity of various constituents of cigarette smoke, and that quantitative analyses are available that can be useful in distinguishing between the exposure responses.

The inverse relationship between bladder and liver in 4-aminobiphenyl-induced DNA damage.

Bladder cancer risk is significantly higher in men than in women. 4-Aminobiphenyl (ABP) is a major human bladder carcinogen from tobacco smoke and other sources. In mice, male bladder is more susceptible to ABP-induced carcinogenesis than female bladder, but ABP is more carcinogenic in the livers of female mice than of male mice. Here, we show that castration causes male mice to acquire female phenotype regarding susceptibility of bladder and liver to ABP. However, spaying has little impact on organ susceptibility to ABP. Liver UDP-glucuronosyltransferases (UGTs) are believed to protect liver against but sensitize bladder to ABP, as glucuronidation of ABP and its metabolites generally reduces their toxicity and promotes their elimination via urine, but the metabolites are labile in urine, delivering carcinogenic species to the bladder. Indeed, liver expression of ABP-metabolizing human UGT1A3 transgene in mice increases bladder susceptibility to ABP. However, ABP-specific liver UGT activity is significantly higher in wild-type female mice than in their male counterparts, and castration also significantly increases ABP-specific UGT activity in the liver. Taken together, our data suggest that androgen increases bladder susceptibility to ABP via liver, likely by modulating an ABP-metabolizing liver enzyme, but exclude UGT as an important mediator.

Activation of MAPK pathways and downstream transcription factors in 2-aminobiphenyl-induced apoptosis.

2-Aminobiphenyls (2-ABP) induces oxidative DNA damage and leads to apoptosis. The precise signaling pathways of inducing apoptosis in vitro are still unknown. This study provides insight into the relationship between 2-ABP-induced apoptosis and the activation of MAPK and downstream transcription factors using pharmacological inhibitors of ERK, p38, and JNK pathways. Results showed that 2-ABP induced the activation of ERK and JNK but not p38. The ERK/JNK pathways downstream transcription factors, c-Jun and ATF-2, were also activated by 2-ABP. The inhibitory effects of ERK inhibitor, U0126, on 2-ABP-induced caspase-3 activity were not detected. However, JNK inhibitor, SP600125, significantly attenuated the caspase-3 activity induced by 2-ABP. The expression of the transcription factors c-Jun and ATF-2 were decreased in 2-ABP treated cells in the presence of ERK/JNK inhibitors, suggesting that the expression of ERK/JNK pathways leads to the downstream activation of c-Jun and ATF-2. N-acetylcysteine, an ROS scavenger, inhibited 2-ABP-induced activation of ERK and JNK, the cell death and caspase-3 activity, which suggested that oxidative stress plays a crucial role in apoptosis through activation of caspase-3 in a ROS/JNK-dependent signaling cascade.

Activation of the aryl hydrocarbon receptor by carcinogenic aromatic amines and modulatory effects of their N-acetylated metabolites.

Aromatic amines (AAs) are an important class of chemicals which account for 12 % of known carcinogens. The biological effects of AAs depend mainly on their biotransformation into reactive metabolites or into N-acetylated metabolites which are generally considered as less toxic. Although the activation of the aryl hydrocarbon receptor (AhR) pathway by certain carcinogenic AAs has been reported, the effects of their N-acetylated metabolites on the AhR have not been addressed. Here, we investigated whether carcinogenic AAs and their N-acetylated metabolites may activate/modulate the AhR pathway in the absence and/or the presence of a bona fide AhR ligand (benzo[a]pyrene/B(a)P]. In agreement with previous studies, we found that certain AAs activated the AhR in human liver and lung cells as assessed by an increase in cytochrome P450 1A1 (CYP1A1) expression and activity. Altogether, we report for the first time that these properties can be modulated by the N-acetylation status of the AA. Whereas 2-naphthylamine significantly activated the AhR and induced CYP1A1 expression, its N-acetylated metabolite was less efficient. In contrast, the N-acetylated metabolite of 2-aminofluorene was able to significantly activate AhR, whereas the parent AA, 2-aminofluorene, did not. In the presence of B(a)P, activation of AhR or antagonist effects were observed depending on the AA or its N-acetylated metabolite. Activation and/or modulation of the AhR pathway by AAs and their N-acetylated metabolites may represent a novel mechanism contributing to the toxicological effects of AAs. More broadly, our data suggest biological interactions between AAs and other classes of xenobiotics through the AhR pathway.

Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells.

Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS.

MicroRNA regulation of DNA repair gene expression in 4-aminobiphenyl-treated HepG2 cells.

We examined the role of miRNAs in DNA damage response in HepG2 cells following exposure to 4-aminobiphenyl (4-ABP). The arylamine 4-ABP is a human carcinogen. Using the Comet assay, we showed that 4-ABP (18.75-300µM) induces DNA damage in HepG2 cells after 24h. DNA damage signaling pathway-based PCR arrays were used to investigate expression changes in genes involved in DNA damage response. Results showed down-regulation of 16 DNA repair-related genes in 4-ABP-treated cells. Among them, the expression of selected six genes (UNG, LIG1, EXO1, XRCC2, PCNA, and FANCG) from different DNA repair pathways was decreased with quantitative real-time PCR (qRT-PCR). In parallel, using the miRNA array, we reported that the expression of 27 miRNAs in 4-ABP-treated cells was at least 3-fold higher than that in the control group. Of these differential 27 miRNAs, the most significant expression of miRNA-513a-5p and miRNA-630 was further validated by qRT-PCR, and was predicted to be implicated in the deregulation of FANCG and RAD18 genes, respectively, via bioinformatic analysis. Both FANCG and RAD18 proteins were found to be down-regulated in 4-ABP-treated cells. In addition, overexpression and knockdown of miRNA-513a-5p and miRNA-630 reduced and increased the expression of FANCG and RAD18 proteins, respectively. Based on the above results, we indicated that miRNA-513a-5p and miRNA-630 could play a role in the suppression of DNA repair genes, and eventually lead to DNA damage.

Human DNA damage by the synergistic action of 4-aminobiphenyl and nitric oxide: an immunochemical study.

4-Aminobiphenyl (4-ABP), an aromatic amine is a major environmental carcinogen found mainly in cigarette smoke. It has been vastly implicated in mutagenesis and cancer development. In this study, commercially available human placental DNA was exposed to 4-ABP (1.3 mM) in presence of sodium nitroprusside (SNP; 8 mM) at 37°C for 3 h. The 4-ABP + SNP-mediated structural changes in human DNA were studied by ultraviolet, circular dichroism and fluorescence spectroscopy, thermal melting profile, agarose gel electrophoresis, and nuclease S1 digestibility assay. Spectroscopical analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. This might be due to generation of single-stranded regions and destabilization of hydrogen bonds. Modification was also visualized in agarose gel electrophoresis. Furthermore, nuclease S1 digestibility confirmed the generation of single strand breaks. Rabbits challenged with 4-ABP-SNP-modified human DNA-induced high-titer immunogen-specific antibodies, which showed Cross-reaction with modified/unmodified DNA bases and ss-DNA in competitive inhibition assay. The immunogen specificity of induced antibodies against 4-ABP-SNP-modified human DNA was further confirmed in gel retardation assay. It may be concluded that induction of anti-modified DNA antibodies could be due to perturbation in the DNA structure and its subsequent recognition by immunoregulatory cells as a foreign molecule.

Genotoxic effect of N-hydroxy-4-acetylaminobiphenyl on human DNA: implications in bladder cancer.

BACKGROUND: The interaction of environmental chemicals and their metabolites with biological macromolecules can result in cytotoxic and genotoxic effects. 4-Aminobiphenyl (4-ABP) and several other related arylamines have been shown to be causally involved in the induction of human urinary bladder cancers. The genotoxic and the carcinogenic effects of 4-ABP are exhibited only when it is metabolically converted to a reactive electrophile, the aryl nitrenium ions, which subsequently binds to DNA and induce lesions. Although several studies have reported the formation of 4-ABP-DNA adducts, no extensive work has been done to investigate the immunogenicity of 4-ABP-modified DNA and its possible involvement in the generation of antibodies in bladder cancer patients. METHODOLOGY/PRINCIPAL FINDINGS: Human DNA was modified by N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), a reactive metabolite of 4-ABP. Structural perturbations in the N-OH-AABP modified DNA were assessed by ultraviolet, fluorescence, and circular dichroic spectroscopy as well as by agarose gel electrophoresis. Genotoxicity of N-OH-AABP modified DNA was ascertained by comet assay. High performance liquid chromatography (HPLC) analysis of native and modified DNA samples confirmed the formation of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4ABP) in the N-OH-AABP damaged DNA. The experimentally induced antibodies against N-OH-AABP-modified DNA exhibited much better recognition of the DNA isolated from bladder cancer patients as compared to the DNA obtained from healthy individuals in competitive binding ELISA. CONCLUSIONS/SIGNIFICANCE: This work shows epitope sharing between the DNA isolated from bladder cancer patients and the N-OH-AABP-modified DNA implicating the role of 4-ABP metabolites in the DNA damage and neo-antigenic epitope generation that could lead to the induction of antibodies in bladder cancer patients.

Influence of arylamine N-acetyltransferase, sex, and age on 4-aminobiphenyl-induced in vivo mutant frequencies and spectra in mouse liver.

One model for cancer initiation by 4-aminobiphenyl (ABP) involves N-oxidation by cytochrome P450 CYP1A2 followed by O-conjugation by N-acetyltransferase(s) NAT1 and/or NAT2 and decomposition to a DNA-binding nitrenium ion. We recently observed that neonatal ABP exposure produced liver tumors in male but not in female mice, and that NAT deficiency reduced liver tumor incidence. However, ABP-induced liver tumor incidence did not correlate with liver levels of N-(deoxyguanosin-8-yl)-ABP adducts 24 hr after exposure. In this study, we compared in vivo ABP-induced DNA mutant frequencies and spectra between male and female wild-type and NAT-deficient Muta™Mouse using both the tumor-inducing neonatal exposure protocol and a 28-day repetitive dosing adult exposure protocol. ABP produced an increase in liver DNA mutant frequencies in both neonates and adults. However, we observed no sex or strain differences in mutant frequencies in neonatally exposed mice, and higher frequencies in adult females than males. Neonatal ABP exposure of wild-type mice increased the proportion of G-T transversions in both males and females, while exposure of Nat1/2(-/-) mice produced increased G-T transversions in males and a decrease in females, even though females had higher levels of N-(deoxyguanosin-8-yl)-4-ABP adducts. There was no correlation of mutant frequencies or spectra between mice dosed as neonates or as adults. These results suggest that observed sex- and NAT-dependent differences in ABP-induced liver tumor incidence in mice are not due to differences in either mutation rates or mutational spectra, and that mechanisms independent of carcinogen bioactivation, covalent DNA binding and mutation may be responsible for these differences.

Rationally designing safer anilines: the challenging case of 4-aminobiphenyls.

We describe how we have been able to design 4-aminobiphenyls that are nonmutagenic (inactive in the Ames test). No such 4-aminobiphenyls were known to us, but insights provided by quantum mechanical calculations have permitted us to design and synthesize some examples. Importantly, the quantum mechanical calculations could be combined with predictions of other properties of the compounds that contained the 4-aminobiphenyls so that these remained druglike. Having found compounds that are not active, the calculations can provide insight into which factors (electronic and conformational in this case) are important. The calculations provided SAR-like information that was able guide the design of further examples of 4-aminobiphenyls that are not active in the Ames test.