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4-Aminobiphenyl (4-ABP) may cause DNA damage in human liver cells (HepG2 and L-02). Propolis exhibits antioxidant properties through reactive oxygen species (ROS) scavenging. We determined the effects of propolis in alleviating 4-ABP -induced DNA damage using the comet assay. Results revealed that propolis could significantly alleviated oxidative damaged DNA by 4-ABP. Furthermore, we proved that inhibition of cytochrome P450 2E1 (CYP2E1) expression by propolis could contribute to the decreased oxidative DNA damage in the treated cells, as the conversion of 4-ABP into its metabolite, N-hydroxy-ABP (HOABP), was blocked; after all, HOABP showed more genotoxic than its parent chemical, 4-ABP. With the homologous recombination assay, propolis failed to induce DNA repair enzymes. Furthermore, the expression of RAD51, Ku70/Ku80, and OGG1 in treated cells were determined with the western blot, revealing that the expression of these protein were unchanged in comparison with those in nontreated cells. However, propolis could protect the treated cells from DNA damage. In conclusion, propolis could antagonize 4-ABP-induced oxidative DNA damage though the removal of ROS and inhibition of CYP2E1 expression in the treated cells.

Purpose: The present study evaluated biochemical as well as biophysical mechanisms behind the synergistic effects of curcumin and resveratrol during prostate carcinogenesis.Methods: The rats were s...

Rationale The small molecule Kartogenin (KGN) promotes cartilage regeneration in osteoarthritis (OA) by activating stem cells differentiation, but its pharmacological mode-of-action remains unclear. KGN can be cleaved into 4-aminobiphenyl (4-ABP) and phthalic acid (PA) following enzymolysis of an amide bond. Therefore, this study investigated whether 4-ABP or PA exerted the same action as KGN. Methods KGN, 4-ABP and PA were analyzed in cartilage of mice after oral, intravenous or intra-articular administration of KGN by liquid chromatography-mass spectrometry method. Their effect on proliferation and chondrogenic differentiation of mesenchymal stem cells (MSC) was evaluated in vitro. Furthermore, their effect on cartilage preservation was tested in mice OA model induced by destabilization of medial meniscus. OA severity was quantified using OARSI histological scoring. Transcriptional analysis was used to find the possible targets of the chemicals, which were further validated. Results We demonstrated that while oral or intra-articular KGN delivery effectively ameliorated OA phenotypes in mice, only 4-ABP was detectable in cartilage. 4-ABP could induce chondrogenic differentiation and proliferation of MSC in vitro and promote cartilage repair in OA mouse models mainly by increasing the number of CD44+/CD105+ stem-cell and prevention of matrix loss. These effect of 4-ABP was stronger than that of KGN. Transcriptional profiling of 4-ABP-stimulated MSC suggested that RPS6KA2 and the PI3K-Akt pathway were 4-ABP targets; 4-ABP could activate the PI3K-Akt pathway to promote MSC proliferation and repair OA injury, which was blocked in RPS6KA2-knockdown MSC or RPS6KA2-deficient mice. Conclusion 4-ABP bio-distribution in cartilage promotes proliferation and chondrogenic differentiation of MSC, and repairs osteoarthritic lesions via PI3K-Akt pathway activation.

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.

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.

Epidemiological studies have linked aromatic amines (AAs) from tobacco smoke and some occupational exposures with bladder cancer risk. Several epidemiological studies have also reported a plausible role for structurally related heterocyclic aromatic amines present in tobacco smoke or formed in cooked meats with bladder cancer risk. DNA adduct formation is an initial biochemical event in bladder carcinogenesis. We examined paired fresh-frozen (FR) and formalin-fixed paraffin-embedded (FFPE) non-tumor bladder tissues from 41 bladder cancer patients for DNA adducts of 4-aminobiphenyl (4-ABP), a bladder carcinogen present in tobacco smoke, and 2-amino-9H-pyrido[2,3-b]indole, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, possible human carcinogens, which occur in tobacco smoke and cooked meats. These chemicals are present in urine of tobacco smokers or omnivores. Targeted DNA adduct measurements were done by ultra-performance liquid chromatography-electrospray ionization multi-stage hybrid Orbitrap MS. N-(2′-Deoxyguanosin-8-yl)-4-ABP (N-(dG-C8)-4-ABP) was the sole adduct detected in FR and FFPE bladder tissues. Twelve subjects (29%) had N-(dG-C8)-4-ABP levels above the limit of quantification, ranging from 1.4 to 33.8 adducts per 10(9) nucleotides (nt). DNA adducts of other human AA bladder carcinogens, including 2-naphthylamine (2-NA), 2-methylaniline (2-MA), 2,6-dimethylaniline (2,6-DMA), and lipid peroxidation (LPO) adducts were screened for in bladder tissue, by our untargeted data-independent adductomics method, termed wide-selected ion monitoring (wide-SIM)/MS(2). Wide-SIM/MS(2) successfully detected N-(dG-C8)-4-ABP, N-(2′-deoxyadenosine-8-yl)-4-ABP and the presumed hydrazo linked adduct, N-(2′-deoxyguanosin-N(2)-yl)-4-ABP, and several LPO adducts in bladder DNA. Wide-SIM/MS(2) detected multiple DNA adducts of 2-NA, 2-MA and, 2,6-DMA, when calf thymus DNA was modified with reactive intermediates of these carcinogens. However, these AA-adducts were below the limit of detection in unspiked human bladder DNA (< 1 adduct per 10(8) nt). Wide-SIM/MS(2) can screen for many types of DNA adducts formed with exogenous and endogenous electrophiles and will be employed to identify DNA adducts of other chemicals that may contribute to the etiology of bladder cancer.

4-Aminobiphenyl (ABP) and its structure analog 2-aminofluorene (AF) are well-known carcinogens. In the present work, an unusual sequence effect in the 5′-CTTCTG1G2TCCTCATTC-3′ DNA duplex is reported for ABP- and AF-modified G. Specifically, the ABP modification at G1 resulted in a mixture of 67% major groove B-type (B) and 33% stacked (S) conformers, while at the ABP modification at G2 exclusively resulted in the B-conformer. The AF modification at G1 and G2 lead to 25%:75% and 83%:17% B:S population ratios, respectively. These differences in preferred conformation are due to an interplay between stabilizing (hydrogen bonding and stacking that is enhanced by lesion planarity) and destabilizing (solvent exposure) forces at the lesion site. Furthermore, while the B-conformer is a thermodynamic stabilizer and the S-conformer is a destabilizer in duplex settings, the situation is reversed at the single strands/double strands (ss/ds) junction. Specifically, the twisted biphenyl is a better stacker at the ss/ds junction than the coplanar AF. Therefore, the ABP modification leads to a stronger strand binding affinity of the ss/ds junction than the AF modification. Overall, the current work provides conformational insights into the role of sequence and lesion effects in modulating DNA replication.

Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotypes. NAT2 gene dose response (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A) was observed towards the N-acetylation of the NAT2-specific drug sulfamethazine by human hepatocytes both in vitro and in situ. N-acetylation of 4-aminobiphenyl, an arylamine carcinogen substrate for both N-acetyltransferase 1 and NAT2, showed the same trend both in vitro and in situ although the differences were not significant (p > 0.05). The N-acetylation of the N-acetyltransferase 1-specific substrate p-aminobenzoic acid did not follow this trend. In comparisons of NAT2 intermediate acetylator genotypes, differences in N-acetylation between NAT2*4/*5B and NAT2*4/*6B hepatocytes were not observed in vitro or in situ towards any of these substrates. These results further support phenotypic heterogeneity among NAT2 slow acetylator genotypes, consistent with differential risks of drug failure or toxicity and cancer associated with carcinogen exposure.

Rationale: Cartilage stem/progenitor cells (CSPC) are a promising cellular source to promote endogenous cartilage regeneration in osteoarthritis (OA). Our previous work indicates that ribosomal s6 kinase 3 (RSK-3) is a target of 4-aminobiphenyl, a chemical enhancing CSPC-mediated cartilage repair in OA. However, the primary function and mechanism of RSK-3 in CSPC-mediated cartilage pathobiology remain undefined. Methods: We systematically assessed the association of RSK-3 with OA in three mouse strains with varying susceptibility to OA (MRL/MpJ>CBA>STR/Ort), and also RSK-3-/- mice. Bioinformatic analysis was used to identify the possible mechanism of RSK-3 affecting CSPC, which was further verified in OA mice and CSPC with varying RSK-3 expression induced by chemicals or gene modification. Results: We demonstrated that the level of RSK-3 in cartilage was positively correlated with cartilage repair capacities in three mouse strains (MRL/MpJ>CBA>STR/Ort). Enhanced RSK-3 expression by 4-aminobiphenyl markedly attenuated cartilage injury in OA mice and inhibition or deficiency of RSK-3 expression, on the other hand, significantly aggravated cartilage damage. Transcriptional profiling of CSPC from mice suggested the potential role of RSK-3 in modulating cell proliferation. It was further shown that the in vivo and in vitro manipulation of the RSK-3 expression indeed affected the CSPC proliferation. Mechanistically, ribosomal protein S6 (rpS6) was activated by RSK-3 to accelerate CSPC growth. Conclusion: RSK-3 is identified as a key regulator to enhance cartilage repair, at least partly by regulating the functionality of the cartilage-resident stem/progenitor cells.

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.

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.

Cathepsin B plays key roles in tumor progression with its overexpression being associated with invasive and metastatic phenotypes and is a primary target of protease activated antibody-directed prodrug therapy. It therefore represents a potential therapeutic and diagnostic target and effort has been made to develop fluorescent probes to report on Cathepsin B activity in cells and animal models of cancer. We have designed, synthesized, and thoroughly evaluated four novel “turn on” probes that employ a lysosomotropic dansylcadaverine dye to report on Cathepsin B activity. Enzyme activity assays using a recombinant human enzyme and cancer cell lysates coupled with confocal microscopy experiments demonstrated that one of the probes, derivatized with the self-immolative prodrug linker p-aminobenzyl alcohol, can selectively report on Cathepsin B in biological samples including live cells.

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.

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.

Tobacco smoking contributes to about 50% of the bladder-cancer (BC) cases in the United States. Some aromatic amines in tobacco smoke are bladder carcinogens; however, other causal agents of BC are uncertain. Exfoliated urinary cells (EUCs) are a promising noninvasive biospecimen to screen for DNA adducts of chemicals that damage the bladder genome, although the analysis of DNA adducts in EUCs is technically challenging because of the low number of EUCs and limiting quantity of cellular DNA. Moreover, EUCs and their DNA adducts must remain viable during the time of collection and storage of urine to develop robust screening methods. We employed RT4 cells, a well-differentiated transitional epithelial bladder cell line, as a cell-model system in urine to investigate cell viability and the chemical stability of DNA adducts of two prototypical bladder carcinogens: 4-aminobiphenyl (4-ABP), an aromatic amine found in tobacco smoke, and aristolochic acid I (AA-I), a nitrophenanthrene found in Aristolochia herbaceous plants used for medicinal purposes worldwide. The cell viability of RT4 cells pretreated with 4-ABP or AA-I in urine exceeded 80%, and the major DNA adducts of 4-ABP and AA-I, quantified by liquid chromatography-mass spectrometry, were stable for 24 h. Thereafter, we successfully screened EUCs of mice treated with AA-I to measure DNA adducts of AA-I, which were still detected 25 days following treatment with the carcinogen. EUCs are promising biospecimens that can be employed for the screening of DNA adducts of environmental and dietary genotoxicants that may contribute to the development of BC.

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 CdCl(2)), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f] quinoline (MelQ) 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 4 h. Only CdCl(2) 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 (BMD(10), BMD(50), BMD(100) and BMD(200)), 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 CdCl(2)(-S9) > BaP(+S9) > CdCl(2)(+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 (BMD(10)) 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.

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.

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.

The urinary metabolites 2-cyanoethylmercapturic acid and 4-aminobiphenyl have been correlated with tobacco smoke exposure. Similarly, 2-cyanoethylvaline and 4-aminobiphenyl haemoglobin adducts have been used as biomarkers of effective dose for the exposure to acrylonitrile and 4-aminobiphenyl, respectively. Each pair of biomarkers is derived from the same parent chemical; however, the correlation between the urinary and the haemoglobin biomarkers has not been investigated. Using clinical study samples, we report a weak correlation between urinary and haemoglobin biomarkers due to different accumulation and elimination rates. Time course analysis showed that a reduction in exposure was paralleled by a delayed reduction in haemoglobin adducts.

The aromatic amine 4-aminobiphenyl (ABP) is a liver procarcinogen in mice, requiring enzymatic bioactivation to exert its tumorigenic effect. To assess the role of arylamine N-acetyltransferase (NAT)-dependent acetylation capacity in the risk for ABP-induced liver tumors, we compared 1-year liver tumor incidence following the postnatal exposure of wild-type and NAT-deficient Nat1/2(−/−) mice to ABP. At an ABP exposure of 1200 nmol, male Nat1/2(−/−) mice had a liver tumor incidence of 36% compared to 69% in wild-type males, and at 600 nmol there was a complete absence of tumors compared to 60% in wild-type mice. Only one female wild-type mouse had a tumor using this exposure protocol. However, levels of N-deoxyguanosin-8-yl-ABP-DNA adducts did not correlate with either the strain or sex differences in tumor incidence. These results suggest that female sex and NAT deficiency reduce risk for ABP-induced liver tumors, but by mechanisms unrelated to differences in DNA-damaging events.

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.

Overexposure to biphenyl amine compounds, which are found in smoke and azo-dyes, is linked to the occurrence of bladder cancer. However, the molecular mechanisms of biphenyl amine compound-induced bladder cancer are still unclear. Many studies have demonstrated that overexpression of cyclooxygenase-2 (COX-2) in neoplastic lesions is associated with carcinogenesis. In this study, we have demonstrated that 2-aminobiphenyl (2-ABP) up-regulated the expression of COX-2 in a dose- and time-dependent manner in TSGH-8301 bladder cancer cells. This 2-ABP-induced COX-2 expression was attenuated by ROS scavenger NAC and NADPH oxidase inhibitors apocynin and DPI. The p22phox subunit of NADPH oxidase, but not p67, and Nox2 was up-regulated by 2-ABP. Knocking down p22phox by siRNA significantly reduced 2-ABP-induced COX-2 expression. Furthermore, 2-ABP also activated the ERK/JNK-AP1 pathways, and this effect was also abolished by NADPH oxidase inhibitors. Blocking the ERK/JNK-AP1 signaling pathways by pharmacological inhibitors attenuated 2-ABP-induced COX-2 expression. Overexpression of the upstream ERK activator MEK1 significantly and consistently increased 2-ABP-mediated COX-2 expression. Transfection of a dominant negative c-Jun mutant, TAM-67, blocked 2-ABP-mediated COX-2 expression, demonstrating that c-Jun was responsible for the transcriptional activation. Taken together, these results demonstrate that 2-ABP induces the carcinogenic factor COX-2 and that this induction is mediated through NADPH oxidase-derived ROS-dependent JNK/ERK-AP-1 pathways.

Background: This multicenter, observational study was conducted in three European countries (Germany, Switzerland, and the United Kingdom) to determine the exposure of adult cigarette smokers and nonsmokers to selected cigarette smoke constituents: 1,3-butadiene, 2-naphthylamine, 4-aminobiphenyl, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acrolein, benzene, carbon monoxide, nicotine, pyrene, and o-toluidine. Methods: Smokers were grouped by tar category (TC) according to the tar yield of their regular cigarette brand: TC1: ≤4 mg tar, TC2: 5–7 mg tar, and TC3: ≥8 mg tar [to the legal tar yield ceiling in the respective countries (10 or 12 mg tar)]. Levels of biomarkers of exposure to the aforementioned cigarette smoke constituents were compared between smokers and nonsmokers, and within smokers across tar categories. Results: The full population consisted of 1,631 subjects (1,223 smokers and 408 nonsmokers). Biomarkers of exposure were analyzed for 1,558 subjects (valid case population) as follows: 1,159 smokers (TC1: n = 402, TC2: n = 379, TC3: n = 378), and 399 nonsmokers. Exposure levels were higher in smokers than nonsmokers and increased with increasing tar yield and cigarette consumption. An association of tar category and exposure level was observed for all smoke constituents, except pyrene, 4-aminobiphenyl, and o-toluidine, whereas only NNK exposure was different in all three tar categories. Conclusions: Smoking status and, among smokers, daily cigarette consumption and tar yield were observed to affect biomarker of exposure levels. Impact: This research provides a comprehensive evaluation of smoke constituent exposure of adult cigarette smokers and nonsmokers in three European countries. Cancer Epidemiol Biomarkers Prev; 20(7); 1524–36. ©2011 AACR.

DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 10(7) DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation.

Stavudine is a hepatotoxic antiretroviral nucleoside analogue that also inhibits the replication of mitochondrial DNA (mtDNA). To elucidate the mechanism and consequences of mtDNA depletion, we treated HepG2 cells with stavudine and either redoxal, an inhibitor of de novo pyrimidine synthesis, or uridine, from which pyrimidine pools are salvaged. Compared with treatment with stavudine alone, co-treatment with redoxal accelerated mtDNA depletion, impaired cell division, and activated caspase 3. These adverse effects were completely abrogated by uridine. Intracellular ATP levels were unaffected. Transcriptosome profiling demonstrated that redoxal and stavudine acted synergistically to induce CDKN2A and p21, indicating cell cycle arrest in G1, as well as genes involved in intrinsic and extrinsic apoptosis. Moreover, redoxal and stavudine showed synergistic interaction in the up-regulation of transcripts encoded by mtDNA and the induction of nuclear transcripts participating in energy metabolism, mitochondrial biogenesis, oxidative stress, and DNA repair. Genes involved in nucleotide metabolism were also synergistically up-regulated by both agents; this effect was completely antagonized by uridine. Thus, pyrimidine depletion sensitizes cells to stavudine-mediated mtDNA depletion and enhances secondary cell toxicity. Our results indicate that drugs that diminish pyrimidine pools should be avoided in stavudine-treated human immunodeficiency virus patients. Uridine supplementation reverses this toxicity and, because of its good tolerability, has potential clinical value for the treatment of side effects associated with pyrimidine depletion.

Colon cancer is second leading cause of cancer-related deaths in Western countries. Diet and smoking, which contain aromatic and heterocyclic amines, are major risk factors for colon cancer. Colorectal cancers have a natural history of long latency and therefore provide ample opportunities for effective chemoprevention. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is an experimental aromatic amine that causes cancer in rat colon and serves as an experimental model for arylamine and heterocyclic amine mutagens derived from diet and smoking. In this study, we investigated the effects of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor on DMABP-induced DNA adduct formation in rat liver and colon. Male F-344 rats (5-week old) were provided free access to modified AIN-76A rat chow containing 0 (control), 500, 1000, or 1500 ppm celecoxib. Two weeks later, the rats received a subcutaneous injection of 100mg/kg DMABP in peanut oil. Two days after DMABP treatment, the rats were killed and DMABP-derived adducts were analyzed in colon and liver DNA by butanol extraction-mediated (32)P-postlabeling. Two major DNA adducts, identified as dG-C8-DMABP and dG-N(2)-DMABP, were detected in liver and colon of rats treated with DMABP. These DNA adducts were diminished approximately 35-40% with 500 ppm and 65-70% with 1,000 ppm celecoxib. In the colon, no further decline in DNA adducts was observed at 1500 ppm. The same DMABP-DNA adducts also were detected in the liver and were also diminished by celecoxib treatment. The reduction in DMABP-DNA adduct levels in celecoxib-treated animals provides further support for celecoxib as a chemopreventive agent for colorectal cancer.

Since recombinant human cyclooxygenase (COX) enzymes have been shown to activate environmental and dietary carcinogens implicated in human colorectal cancer etiology, we hypothesized that COX-2 inhibitors reduce arylamine-induced aberrant crypts (AC) and foci (ACF), preneoplastic lesions of colorectal cancer. Male weanling F344 inbred rats were fed modified AIN-76A control diet or the same diets supplemented with 320 ppm sulindac or 500, 1000, or 1500 ppm celecoxib. At 7 weeks of age, rats received a subcutaneous injection of 3,2′-dimethyl-4-aminobiphenyl (DMABP), an aryl-amine colon carcinogen, once weekly for two weeks. Ten weeks after the initial DMABP or vehicle treatment (at 17 weeks of age), rats were euthanized with CO2, and the entire colorectum was removed and scored for ACF and AC. ACF possessing one to five AC were identified in the colorectum of rats administered DMABP, whereas no AC/ACF were identified in vehicle-treated controls. Significant reductions (p < 0.001) in ACF and AC frequencies were observed in DMABP-treated rats supplemented with sulindac or celecoxib. Celecoxib reduced AC and ACF more than sulindac, but this difference was not significant (p > 0.05). Reductions in both AC and ACF were highest following treatment with 1000 ppm celecoxib. These results provide additional experimental support for the chemopreventive effects of COX inhibitors in arylamine-induced colorectal cancer.

Pseudomonas aeruginosa is an important opportunistic pathogen of the human urinary bladder. Similar to rat liver S9, the cell-free extract from P. aeruginosa caused significant increase of histidine reversion numbers with the Salmonella typhimurium tester strain TA98 in the Ames Salmonella mutagenicity assay in the presence of either 2-aminofluorene, 4-aminobiphenyl, or benzidine procarcinogens. The presence of cytochrome P-450 protein in the cell-free extract was demonstrated by the carbon monoxide difference spectrum. We employed gene knockout technology to inactivate one of the three known putative cytochrome P-450 genes of P. aeruginosa , namely CYP107S1, which we postulated to be the most likely to induce activation. The ampicillin resistant gene from PUC19 DNA confers carbenicillin resistance to P. aeruginosa . We inserted a synthetic ampicillin gene flanked by 40 base-pairs of the 5′ and 3′ untranslated region of the CYP gene by electroporating the synthetic gene into electrocompetent P. aeruginosa cells. CYP107S1 knockout strains were selected on 1000 μg/ml carbenicillin plates. A single cloned carbenicillin resistant colony was isolated and used to determine its mutagenic capacity using Ames Salmonella mutagenicity assay. The results showed that Salmonella TA98 tester strain returned the number of revertants to its baselines level indicating the lack of metabolic activation of procarcinogens in the P. aeruginosa CYP107S1 knockout cell-free extract. In addition, the characteristic cytochrome P-450 peak determined by the carbon monoxide difference spectrum was completely absent in the cell-free extract from this CYP107S1 knockout strain bacterium. Homologous recombination of the synthetic ampicillin gene on the CYP 107S1 P-450 locus was confirmed by PCR on purified genomic DNA extracted from the knockout bacterium. The metabolic activation of tested procarcinogens is, therefore, carried out by CYP107S1 in P. aeruginosa .

The possible mechanisms of antimutagenicity against 4-nitroquinoline-N-oxide (4-NQO; a direct mutagen) and 3,2'-dimethyl-4-amino-biphenyl (DMAB; an indirect mutagen) were examined in fermented soymilk prepared with a coculture of Streptococcus thermophilus and Bifidobacterium infantis. The antimutagenicity in the fermented soymilk was not due to the bioantimutagenic effect of modulation of DNA repair processes. The mutagenicity of DMAB decreased with increased preincubation of fermented soymilk and the DMAB metabolite but not with intact DMAB or an S9 mixture. Mutagenicity of 4-NQO was not affected by preincubation of fermented soymilk with this mutagen. Mutagenicity of both 4-NQO and DMAB was reduced when Salmonella Typhimurium TA 100 was pretreated with fermented soymilk, indicating that fermented soymilk affected the function of the bacterial cell, which might also lead to reduced mutagenicity of the tested mutagens. Desmutagenic and blocking effects were the main mechanisms of antimutagenicity in the fermented soymilk against DMBA. In contrast, the antimutagenic effect of the fermented soymilk on 4-NQO was primarily due to a blocking effect.

Prilocaine, a widely used local anesthetic, is metabolized to o-toluidine which is classified as human carcinogen. We aimed to assess the impact of prilocaine-treatment on hemoglobin adducts from o-toluidine. Blood samples were obtained before and 24h after receiving prilocaine local anesthesia (Xylonest, 100mg) from 20 head and neck surgery patients and 6 healthy volunteers. Hemoglobin adducts of o-toluidine and 4-aminobiphenyl were determined by gas chromatography/mass spectrometry. Hemoglobin adducts of o-toluidine were significantly increased 24h after 100mg prilocaine-treatment by 21.6+/-12.8ng/g hemoglobin (mean+/-S.D., N=26; P

A series of biphenylaminocyclopropane carboxamide based bradykinin B1 receptor antagonists has been developed that possesses good pharmacokinetic properties and is CNS penetrant. Discovery that the replacement of the trifluoropropionamide in the lead structure with polyhaloacetamides, particularly a trifluoroacetamide, significantly reduced P-glycoprotein mediated efflux for the series proved essential. One of these novel bradykinin B1 antagonists (13b) also exhibited suitable pharmacokinetic properties and efficient ex vivo receptor occupancy for further development as a novel approach for the treatment of pain and inflammation.

We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10 9 bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d 9 ) dG-C8-ABP (MW = 443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H + ] +1 , at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444 → 328. The method was validated by analysis of DNA (100 μg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 μg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10 9 bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.

DNA mismatch repair (MMR) deficiencies result in increased frequencies of spontaneous mutation and tumor formation. In the present study, we tested the hypothesis that a chemically-induced mutational response would be greater in a mouse with an MMR-deficiency than in the MMR-proficient mouse models commonly used to assay for chemical carcinogenicity. To accomplish this, the induction of H-ras codon 61 CAA-->AAA mutation was examined in Pms2 knockout mice (Pms2-/-, C57BL/6 background) and sibling wild-type mice (Pms2+/+). Groups of five or six neonatal male mice were treated with 0.3 micromol 4-aminobiphenyl (4-ABP) or the vehicle control, dimethylsulfoxide. Eight months after treatment, liver DNAs were isolated and analysed for levels of H-ras codon 61 CAA-->AAA mutation using allele-specific competitive blocker-PCR. In Pms2-proficient and Pms2-deficient mice, 4-ABP treatment caused an increase in mutant fraction (MF) from 1.65x10(-5) to 2.91x10(-5) and from 3.40x10(-5) to 4.70x10(-5), respectively. Pooling data from 4-ABP-treated and control mice, the approximately 2-fold increase in MF observed in Pms2-deficient as compared with Pms2-proficient mice was statistically significant (P=0.0207) and consistent with what has been reported previously in terms of induction of G:C-->T:A mutation in a Pms2-deficient background. Pooling data from both genotypes, the increase in H-ras MF in 4-ABP-treated mice, as compared with control mice, did not reach the 95% confidence level of statistical significance (P=0.0606). The 4-ABP treatment caused a 1.76-fold and 1.38-fold increase in average H-ras MF in Pms2-proficient and Pms2-deficient mice, respectively. Furthermore, the levels of induced mutation in Pms2-proficient and Pms2-deficient mice were nearly identical (1.26x10(-5) and 1.30x10(-5), respectively). We conclude that Pms2-deficiency does not result in an amplification of the H-ras codon 61 CAA-->AAA mutational response induced by 4-ABP.

The mechanisms underlying the susceptibility of neonatal mice to genotoxic carcinogens were investigated by analyzing the DNA adducts and mutations induced in the livers of neonatal and adult Big Blue transgenic mice by 4-aminobiphenyl (4-ABP), a potent human and rodent carcinogen. Neonatal and adult mice were treated with a regimen of 4-ABP known to induce tumors in neonatal mice. Animals were sacrificed 1 day after the last treatment for DNA adduct analysis and 8 weeks after the last treatment for analysis of lacI and cII mutant frequency (MF). N-(Deoxyguanosin-8-yl)-4-ABP was the major DNA adduct identified in the livers of the 4-ABP-treated mice and levels of this adduct were significantly higher in treated animals than in the controls for both the neonates and adults. Adduct levels for adult females (44.0 ± 4.8 adducts/106 nucleotides) were higher than in neonatal females (25.9 ± 2.2 adducts/106 nucleotides), while adduct levels in adult males (13.5 ± 2.0 adducts/106 nucleotides) were lower than in neonatal males (33.8 ± 4.1 adducts/106 nucleotides). 4-ABP treatment significantly increased the liver cII MFs in both sexes of neonatal mice but not in adult mice. Sequence analysis of cII mutant DNA revealed that 4-ABP induced a unique spectrum of mutations in neonatal mice, characterized by a high frequency of G:CT:A transversion, while the mutation spectrum in 4-ABP-treated adults was similar to that of control mice. Our results indicate that DNA adduct formation by 4-ABP depends as much on sex as it does on age, whereas the conversion of DNA adducts into mutations differed with animal age. These observations suggest that neonates are more sensitive than adults to genotoxic carcinogens because the relatively high levels of cell division in the developing animal facilitate the conversion of DNA damage into mutation. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html © 2005 Wiley-Liss, Inc.

The utility of liver H-ras codon 61 CAA to AAA mutant fraction as a biomarker of liver tumor development was investigated using neonatal male mice treated with 4-aminobiphenyl (4-ABP). Treatment with 0.1, 0.3, or 1.0 mumol 4-ABP produced dose-dependent increases in liver DNA adducts in B6C3F(1) and C57BL/6N mice. Eight months after treatment with 0.3 mumol 4-ABP or the DMSO vehicle, H-ras codon 61 CAA to AAA mutant fraction was measured in liver DNA samples (n = 12) by allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). A significant increase in average mutant fraction was found in DNA of 4-ABP-treated mice, with an increase from 1.3 x 10(-5) (control) to 44.9 x 10(-5) (treated) in B6C3F(1) mice and from 1.4 x 10(-5) to 7.0 x 10(-5) in C57BL/6N mice. Compared with C57BL/6N mutant fractions, B6C3F(1) mutant fractions were more variable and included some particularly high mutant fractions, consistent with the more rapid development of liver foci expected in B6C3F(1) mouse liver. Twelve months after treatment, liver tumors developed in 79.2% of 4-ABP-treated and 22.2% of control B6C3F(1) mice; thus measurement of H-ras mutant fraction correlated with subsequent tumor development. This study demonstrates that ACB-PCR can directly measure background levels of somatic oncogene mutation and detect a carcinogen-induced increase in such mutation.

Background: Some members of the arylamine family of compounds, specifically 4-aminobiphenyl (ABP), 2-naphthylamine, and benzidine, are established human bladder carcinogens. Cigarette smoking and use of permanent hair dye contribute substantially to current arylamine exposure. Low levels of 4-ABP exposure have been associated with non‐smokingrelated bladder cancer. Other arylamine compounds coming from as yet unidentified environmental sources may also be human bladder carcinogens. Methods: We conducted a population-based case‐control study in Los Angeles County, California, involving 298 case subjects with bladder cancer and 308 control subjects, who were matched on age, sex, race/ethnicity, and neighborhood of residence. In-person interviews provided information on tobacco smoking and other potential risk factors for bladder cancer. To assess arylamine exposure, levels of arylamine‐hemoglobin adducts of nine selected alkylanilines (2,3-dimethylaniline [2,3DMA], 2,4-DMA, 2,5-DMA, 2,6-DMA, 3,4-DMA, 3,5-DMA, 2-ethylaniline [2-EA], 3-EA, 4-EA) were measured in peripheral blood collected from study subjects. Analysis of covariance and conditional logistic regression methods were used to analyze the relationship between arylamine‐hemoglobin adducts and bladder cancer risk. All statistical tests were two-sided. Results: Levels of all arylamine‐hemoglobin adducts, with the exception of 2,6-DMA, were higher in smokers than in nonsmokers, and levels of all arylamine‐hemoglobin adducts were higher in case subjects than in control subjects. Arylamine‐hemoglobin adducts of 2,6-DMA, 3,5DMA, and 3-EA were all independently, statistically significantly (all P

The modifying effects of late neonatal administration of p-nonylphenol (NP), a suspected xenoestrogen, on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced prostatic carcinogenesis were investigated in male F344 rats. Three-week-old rats received 25, 250 or 2000 ppm of NP in the diet for 3 weeks prior to DMAB treatment and were sacrificed at 67 weeks of age for histopathological assessment of lesions and Ki-67 immunohistochemical analysis of cell cycle kinetics. Dietary administration of NP during the sexually immature period had no effects on maturation of male sex organs. Incidence, multiplicity and areas of neoplastic lesions in the prostate and seminal vesicles, and Ki-67 labeling indices in normal-looking epithelium were not significantly different among the experimental groups. These results indicate that late neonatal treatment with NP has no modulating effects on DMAB-induced rat prostatic carcinogenesis.

Background: Metabolites of the potent human carcinogen 4-aminobiphenyl (ABP) induce oxidative stress and form DNA adducts that are associated with hepatic and urinary bladder toxicity and bladder tumorigenesis. Results of in vitro and cell culture studies have suggested that cytochrome P450 1A2 (CYP1A2) is the major metabolic activator of ABP. We used Cyp1a2(–/–) knockout mice to examine the role of CYP1A2 in ABP–DNA adduct formation in the liver and the bladder. Methods: Cyp1a2(+/+) wild-type and Cyp1a2(–/–) mice (total of four mice per group) were treated topically with 10 mg/kg ABP for various times, with or without pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an inducer of CYP1A2 activity. We evaluated ABP-induced toxicity by carrying out quantitative histology (of the liver, skin, and bladder), oxidative stress by measuring hepatic thiol levels, and liver and bladder DNA adduct formation by using 32 P-postlabeling. Data were analyzed by general linear models and analysis of variance. All statistical tests were two-sided. Results: At the experimental times selected, we observed no histologic evidence of toxicity in the liver, skin, or bladder. Overall, Cyp1a2(+/+) mice had fewer DNA adducts 24 hours after ABP treatment than similarly treated Cyp1a2(–/–) mice. Compared with male mice, female mice had more DNA adducts in the liver but fewer adducts in the bladder, regardless of Cyp1a2 genotype. TCDD pretreatment was associated with a decrease in ABP–DNA adduct levels overall. After 2 hours of ABP treatment, hepatic thiol levels underwent statistically significant declines of severalfold in Cyp1a2(+/+) and Cyp1a2(–/–) males and in Cyp1a2(–/–) females. Conclusions: Contrary to our expectations, CYP1A2 expression was not associated with ABP-induced hepatic oxidative stress or with ABP–DNA adduct formation. Either CYP1A2 is not the major metabolic activator of ABP or other enzymes metabolically activate ABP in mice in the absence of CYP1A2. [J Natl Cancer Inst 2003;95:1227–37]

Inbred, congenic and transgenic strains of mice were characterized for acetylation of p -aminobenzoic (PABA) and the carcinogen 4-aminobiphenyl (4ABP). C57Bl/6 mice have the NAT2*8 allele, A/J mice have NAT2*9 and congenic B6.A mice have NAT2*9 on the C57Bl/6 background. The first transgenic strain with human NAT1 , the functional equivalent of murine NAT2 , was also tested. The murine NAT2*9 allele correlated with a slow phenotype measured with the murine NAT2 selective substrate PABA. The two strains having this allele also had a lower capacity to acetylate 4ABP. A line with five copies of the human NAT1 transgene was bred for at least five generations with either C57Bl/6 or A/J mice. There was no significant change in PABA NAT activity on the C57Bl/6 background but a 2.5-fold increase was seen in hNAT1:A/J compared with A/J. The effect of variation in NATs on 4ABP genotoxicity was assessed in these strains. Twenty-four hours after exposure to a single oral dose of 120 mg 4ABP/kg, hepatic 4ABP-DNA adducts were detected by immunofluoresence in all strains. Nuclear fluorescence intensities (mean±S.D.) were 41.1±3.6 for C57Bl/6, 37.9±1.11 for A/J and 36.3±2.44 for B6.A. There was no correlation between murine NAT2 alleles and 4ABP-DNA adduct levels. Similar results were seen with the transgenic strains. The data indicate that the range of variation present in these strains of mice was insufficient to alter susceptibility to 4ABP genotoxicity. The impact of these relatively modest differences in the acetylation of the activation of 4ABP may be masked by other competing biotransformation reactions since 4ABP is a substrate for both NAT1 and NAT2. Mouse models with variation in both isoforms are needed to adequately assess the role of variation in NATs in susceptibility to 4ABP genotoxicity.

Age-related changes in the expression of xenobiotic biotransformation enzymes can result in differences in the rates of chemical activation and detoxification, affecting responses to the therapeutic and/or toxic effects of chemicals. Despite recognition that children and adults may exhibit differences in susceptibility to chemicals, information about when in development specific biotransformation enzymes are expressed is incomplete. N-acetyltransferases (NATs) are phase II enzymes that catalyze the acetylation of arylamine and hydrazine carcinogens and therapeutic drugs. The postnatal expression of NAT1 and NAT2 was investigated in C57Bl/6 mice. Hepatic NAT1 and NAT2 messenger RNAs (mRNAs) increased with age from neonatal day (ND) 4 to adult in a nonlinear fashion. The presence of functional proteins was confirmed by measuring NAT activities with the isoform selective substrates p-aminobenzoic acid and isoniazid, as well as the carcinogens 2-aminofluorene and 4-aminobiphenyl (4ABP). Neonatal liver was able to acetylate all of the substrates, with activities increasing with age. Protein expression of CYP1A2, another enzyme involved in the biotransformation of arylamines, showed a similar pattern. The genotoxicity of 4ABP was assessed by determining hepatic 4ABP-DNA adducts. There was an age-dependent increase in 4ABP-DNA adducts during the neonatal period. Thus, developmental increases in expression of NAT1 and NAT2 genes in neonates are associated with less 4ABP genotoxicity. The age-related pattern of expression of biotransformation enzymes in mice is consistent with human data for NATs and suggests that this may play a role in developmental differences in arylamine toxicity.

Breast cancer is one of the major cancers around the world but its etiology is still not well understood. Only approximately 50% of the disease is associated with known risk factors including highly penetrant genes and lifestyle factors. Thus, environmental carcinogens may play an important role in the etiology of breast cancer. The arylamine 4-aminobiphenyl (4-ABP) is a tobacco smoke constituent, an environmental contaminant, and a well-established bladder carcinogen in rodents and humans. In this study, we investigated the role of 4-ABP in the etiology of human breast cancer by measuring 4-ABP-DNA adducts using a monoclonal antibody based immunoperoxidase method that had been validated by comparison with gas chromatography/mass spectroscopy analysis of liver tissues from 4-ABP-treated mice. Adducts were analyzed in 150 paraffin-embedded breast tumors and in 55 adjacent normal tissues collected from cases in the Long Island Breast Cancer Study Project. The role of polymorphisms in genes involved in the metabolism of 4-ABP including N-acetyl transferase 2 (NAT2), cytochrome P4501A2 (CYP1A2) and glutathione S-transferase M1 (GSTM1) and the nucleotide excision repair gene XPD was also explored in the same patients. The mean log-transformed relative staining intensity for 4-ABP-DNA adducts was higher in normal (5.93 +/- 0.54) than in the corresponding tumor (5.44 +/- 0.62, P < 0.0001) tissues. However, a highly significant positive correlation was observed between the levels of 4-ABP-DNA in both tissues (r = 0.72, P < 0.0001). Smoking status was correlated with the levels of 4-ABP-DNA in tumor adjacent normal tissues with a significant linear trend (P = 0.04) for current, former and never smokers; adducts were not related to smoking status in tumor tissues. No correlation was observed between the levels of 4-ABP-DNA and polymorphisms in the genes analyzed even when subjects were stratified by smoking status. These results demonstrate that smoking is associated with increased levels of 4-ABP-DNA adducts in human mammary tissue. In this study, genetic polymorphisms did not significantly affect the formation of 4-ABP-DNA adducts in breast cancer cases, perhaps due to the small number of samples.

The results of quantitative structure-activity relationship (QSAR) studies on six series of compounds exhibiting indirect mutagenic activity are reported. These findings demonstrate the importance of frontier orbital energies and, in some cases, frontier orbital electronic populations to overall mutagenicity in diverse polyaromatic hydrocarbons, benzidines and aminobiphenyls, benzonitrofurans, nitrogenous cooked-food mutagens, benzanthracenes, and chrysenes. The correlations between structural parameters and mutagenic potency vary from R=0.81 to R=0.97, and these findings are discussed in the context of possible molecular mechanisms of mutagenicity. In particular, it is generally regarded that cytochrome P450-mediated activation of polyaromatic hydrocarbons and their amino derivatives plays an important role in mutagenic activity. In this respect, it is apparent that enzymes of the cytochrome P4501 (CYP1) family are closely associated with the metabolic activation of polyaromatic mutagens and carcinogens via the generation of reactive intermediates (usually electrophilic in nature) that attack DNA. The findings presented in this study indicate that QSAR analyses on several series of compounds are consistent with the known evidence of procarcinogen activation mechanisms, particularly for polyaromatic hydrocarbons and their heterocyclic/amino derivatives, pointing to the importance of frontier orbital energy values in particular. Teratogenesis Carcinog. Mutagen. Suppl. 1:187–193, 2003. © 2003 Wiley-Liss, Inc.

Mutant spectra analysis was conducted with spontaneous hisG46 revertants of Salmonella typhimurium strain YG1029 and revertants induced by the plant- and mammalian S9-activation of benzidine and 4-aminobiphenyl (4-ABP). Under preincubation conditions, YG1029 cells were exposed to benizidine or 4-ABP with mammalian S9 activation or to a high molecular weight fraction that contained the plant-activated products. The induced revertants were isolated at mutagen concentrations that caused an increased mutant frequency of approximately 4- to 10-fold above background. Genomic DNA from each revertant was isolated and the hisG region was amplified using polymerase chain reaction (PCR). Using a series of specific probes and a modified version of the ECL3's-oligolabelling and detection system, each of the six possible base-pair substitution mutations at hisG46 that leads to a reversion event was determined. Of the YG1029 spontaneous revertants, transition mutations were 31.8% and transversion mutations were 68.2%. The YG1029 spontaneous mutant spectrum differed significantly from the spontaneous spectrum of TA1535 but did not significantly differ from the spontaneous TA100 mutant spectrum. The differences of the spontaneous mutant spectra among these highly related strains illustrate that the introduction of the plasmid pKM101 into S. typhimurium increased the frequency of transversions (CCC→ACC; CCC→CAC) and reduced site 2 (CCC→CTC) transitions. With plant-activated benzidine, 21.1% of recovered revertants resulted from transitions and 78.9% from transversions while S9 activated-benzidine induced revertants were recovered as 14.2% from transition and 85.8% from transversion mutations. Plant-activated 4-ABP recovered 20.0% transitions and 80.0% transversions. S9-activated 4-ABP-induced 21.4% transitions and 78.6% transversions. Chi-square analysis of mutant spectra indicated that the DNA lesions that resulted in reversion at the hisG46 allele induced by plant-activated benzidine or 4-ABP were different from those generated after mammalian S9 activation of these promutagens. The plant-activated benzidine and 4-ABP induced statistically identical mutant spectra. Also, the mammalian-activated benzidine and 4-ABP induced statistically similar mutant spectra. These data show that the plant-activated and mammalian-activated aromatic amine products inflicted different types or distributions of DNA lesions that were reflected in the resulting induced mutant spectra. Teratogenesis Carcinog. Mutagen. Suppl. 1:47–60, 2003. © 2003 Wiley-Liss, Inc.

4-Aminobiphenyl (ABP) is a trace component of cigarette smoke and hair dyes, a suspected human carcinogen and a potent rodent liver carcinogen. Postnatal exposure of mice to ABP results in a higher incidence of liver tumors in males than in females, paralleling the sex difference in human liver cancer incidence. A traditional model of ABP tumorigenesis involves initial CYP1A2-mediated N-hydroxylation, which eventually leads to production of mutagenic ABP-DNA adducts that initiate tumor growth. However, several studies have found no correlation between sex or CYP1A2 function and the DNA-damaging, mutagenic, or tumorigenic effects of ABP. Oxidative stress may be an important etiological factor for liver cancer, and it has also been linked to ABP exposure. The goals of this study were to identify novel enzyme(s) that contribute to ABP N-oxidation, and to investigate a potential role for oxidative stress in ABP liver tumorigenicity. Isozyme-selective inhibition experiments using liver microsomes from wild-type and genetically modified mice identified CYP2E1 as a major ABP N-hydroxylating enzyme. The N-hydroxylation of ABP by transiently expressed CYP2E1 produced oxidative stress in cultured mouse hepatoma cells. In vivo postnatal exposure of mice to a tumorigenic dose of ABP also produced oxidative stress in male wild-type mice, but not in male Cyp2e1(-/-) mice or in female mice. However, a stronger NRF2-associated antioxidant response was observed in females. Our results identify CYP2E1 as a novel ABP-N-oxidizing enzyme, and suggest that sex differences in CYP2E1-dependent oxidative stress and antioxidant responses to ABP may contribute to the observed sex difference in tumor incidence.

Carcinogenic aromatic amines such as 4-aminobiphenyl, which is contained in tobacco smoke, are one of the causal factors of urothelial epithelial cancers. 4-Aminobiphenyl has been shown to be bioactivated through N-hydroxylation by hepatic cytochrome (CYP) 1A2 and subsequently through O-sulfation and O-acetylation by phenol sulfating sulfotransferase, ST1A3 (SULT1A1), and arylamine N-acetyltransferase, NAT2, respectively. In a case-control study for urothelial epithelial cancers, low activity alleles of NAT2 are overall high-risk alleles (OR 2.11; 95% CI 1.08-4.26). Wild-type ST1A3*1 ((213)Arg) alleles were slightly overrepresented in nonsmoking urothelial cancer patients (82.6% vs. 69.7%) and in smoking cancer patients (76.7% and 74.3%) compared to a variant ST1A3*2 ((213)His) allele. In combination of ST1A3 and NAT2 genotypes for analyses of urothelial cancer risk, the highest OR of 2.45 (95% CI 1.04-5.98) was obtained with ST1A3*1 and NAT2 slow genotype among the 4 combinations. Recombinant ST1A3*1 enzyme showed a tendency of catalyzing higher in vitro 3'-phosphoadenosine 5'-phosphosulfate-dependent DNA adduct formation than ST1A3*2 (2.84 +/- 0.49 and 2.22 +/- 0.11 adducts/10(8) nucleotides). Combined analyses of different alleles of carcinogenic aromatic amine-activating phase II enzymes were applied to urothelial cancer risk for the first time and showed the highest risk combination of ST1A3 and NAT2 alleles.

Methemoglobin formation, as well as hemoglobin or DNA adducts, are useful biomarkers of occupational exposure to certain arylamines. It has been suggested that, in liver from animals not treated with a cytochrome P450 (CYP) inducer, hepatic CYP1A2 is the major P450 involved in N-hydroxylation. This is the first step in the metabolic activation of many arylamines, such as the human urinary bladder carcinogen 4-aminobiphenyl (ABP). The product of this catalytic step, N-hydroxy-4-ABP, reacts in the blood with oxyhemoglobin to form methemoglobin and nitrosobiphenyl. We therefore examined the role of CYP1A2 in causing methemoglobinemia in ABP-treated Cyp1a2(-/-) knockout mice. Application of ABP (100 micromol/kg body wt) to the skin resulted in a marked depletion in the levels of the hepatic thiols (reduced glutathione and cysteine) after 2 h, which rebounded to basal levels 24 h later, and we found no differences between the Cyp1a2(-/-) and wild-type Cyp1a2(+/+) animals. Unexpectedly, the methemoglobin levels were significantly (p < 0.05) higher in Cyp1a2(-/-) than Cyp1a2(+/+) mice at 2, 7, and 24 h following topical ABP. Treatment with dioxin, 24 h prior to ABP, decreased methemoglobin levels by about half at each of the time points in both the Cyp1a2(-/-) and Cyp1a2(+/+) mice. These data suggest that CYP1A2 does not play a positive role in methemoglobin formation via the activation of ABP; rather, the absence of CYP1A2 enhances ABP-induced methemoglobinemia. Because liver CYP1A2 levels are known to vary more than 60-fold between humans, our findings may be relevant to patients who are exposed to arylamines in the workplace.

4-Aminobiphenyl (4-ABP) is a major etiological agent for human bladder cancer. Metabolically activated 4-ABP is able to interact with DNA to form adducts that may induce mutations and initiate carcinogenesis. Thirty to sixty percent of bladder cancer has a mutation in the tumor suppressor p53 gene, and the mutational spectrum bears unique features. To date the DNA binding spectrum of 4-ABP in the p53 gene is not known due to the lack of methodology to detect 4-ABP-DNA adducts at nucleotide sequence level. We have found that UvrABC nuclease, a nucleotide excision repair complex isolated from Escherichia coli, is able to incise specifically and quantitatively DNA fragments modified with N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), an activated intermediate of 4-ABP. Using the UvrABC nuclease incision method, we mapped the binding spectrum of N-OH-4-ABP in DNA fragments containing exons 5, 7, and 8 of the human p53 gene and also determined the effect of C5 cytosine methylation on N-OH-4-ABP-DNA binding. We found that codon 285, a mutational hotspot at a non-CpG site in bladder cancer, is the preferential binding site for N-OH-4-ABP. We also found that C5 cytosine methylation greatly enhanced N-OH-4-ABP binding at CpG sites, and that two mutational hotspots at CpG sites, codons 175 and 248, became preferential binding sites for N-OH-4-ABP only after being methylated. These results suggest that both the unique DNA binding specificity of 4-ABP and cytosine methylation contribute to the mutational spectrum of the p53 gene in human bladder cancer.

The modifying effects of dietary feeding of an estrogenic compound 4- n -octylphenol on 3,2′-dimethyl-4-aminobiphenyl (DMAB) induced prostatic carcinogenesis were investigated in male F344 rats. The authors also assessed the effects of test compound on proliferating cell nuclear antigen (PCNA) index in induced neoplasms, hyperplastic lesions, and nonlesional glands in the prostrate. Rats were given intraperitoneal injections of DMAB (25 mg/kg body wt) every other week, 10 times, to induce prostatic neoplasms. They also received the experimental diet containing 10 or 100 ppm 4- n -octylphenol for 20 weeks, starting one week after the last dosing of DMAB. DMAB exposure produced prostatic adenocarcinoma with an incidence of 21% at the end of the study (Week 39). Dietary administration of 4- n -octylphenol did not affect the incidence of prostatic adenocarcinoma: 17% in DMAB→10 ppm 4- n -octylphenol group; and 12% in DMAB→100 ppm 4- n -octylphenol group. The PCNA indices in adenocarcinomas, hyperplastic lesions, and nonlesional glands in rats treated with DMAB and 4- n -octylphenol were slightly lower than that of the DMAB alone group, but the differences were not statistically significant. These results might suggest that dietary feeding of the weak estrogenic compound 4- n -octylphenol did not have modulating effects on DMAB-induced rat prostatic carcinogenesis.

A previous investigation demonstrated the anticarcinogenicity of acetaminophen (APAP) against colon carcinogenesis in rats induced by 3,2'-dimethyl-4-aminobiphenyl (DMAB). DMAB was selected as a structurally related surrogate for heterocyclic amines, formed during cooking of protein, which are believed to be involved in human colon cancer. The objective of the present study was to ascertain whether the early initiating effects of this colon carcinogen are inhibited by APAP. Six groups of male F344 rats were treated over a 6-week period as follows: (1) vehicle (corn oil) for 6 weeks; (2) APAP in the diet at 1000 ppm daily for 6 weeks; (3) 50 mg/kg DMAB by gavage once a week for the last 4 weeks; (4) 5 mg/kg DMAB as for (3); (5) 1000 ppm APAP for 6 weeks and 50 mg/kg DMAB for the last 4 weeks; and (6) 1000 ppm APAP and 5 mg/kg DMAB as for (5). Colonic tissue was within normal limits in the control and APAP groups. In the APAP only group, apical enterocytic hypertrophy and hyperaemia over the entire surface epithelium was present. In the high-dose DMAB group, in the lower third of the crypts, foci of enlarged glands with hypertrophic cells exhibiting karyomegaly and anisokaryosis (FHE) of 3+ degree of severity were evident in 100% of the animals. Also, there were increases in periglandular fibrocytes, matrix and mononuclear cells (PF). In the low-dose DMAB group both FHE and PF changes with the same degree of severity were reduced. In rats given the low dose of DMAB plus APAP, FHE and PF with the same degree of severity (3+) was absent. Both DMAB exposures increased significantly the replicating fraction of colonic enterocytes in an exposure-related fashion and the replicating fractions were significantly reduced by APAP. In 32P-postlabelling of colon, liver and urinary bladder DNA, high-dose DMAB produced 2-6 distinct dose-related spots reflecting DNA adducts. These spots were reduced or were no longer detectable in all three tissues when APAP was given 2 weeks before and during DMAB exposure. Using immunohistochemical detection of DMAB adducts in the colon, a dose-related colour intensity was present for both doses of DMAB. APAP reduced this by 94-fold. Thus, APAP produced a marked protective effect in colonic enterocytes against several parameters of neoplastic development by the carcinogen.