Correlation between induction of DNA fragmentation and micronuclei formation in kidney cells from rats and humans and tissue-specific carcinogenic activity

Five chemicals, known to induce kidney tumors in rats, were assayed for their ability to induce DNA damage and formation of micronuclei in primary cultures of rat and human kidney cells and in the kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, and of micronuclei frequency were obtained in primary kidney cells from both rats and humans with the following concentrations of the five test compounds: lead acetate (not tested for micronuclei induction) and potassium bromate from 0.56 to 1.8 mM, phenacetin from 1 to 3.2 mM, and 1, 4-dichlorobenzene and nitrilotriacetic acid from 1.8 to 5.6 mM. In terms of DNA-damaging potency all the five chemicals were more active in rat than in human kidney cells, whereas the potencies in inducing micronuclei formation were similar in the two species with the exception of 1,4-dichlorobenzene, which was slightly more potent in human than in rat cells. Consistently with the results observed in vitro, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were detected in the kidney of rats given po a single (12 LD50) or three successive daily doses (13 LD50) of the five test compounds. 4, 4'-Methylenedianiline, a carcinogen which does not induce kidney tumors in rats, gave negative responses in both in vitro and in vivo assays. These findings give evidence that kidney carcinogens may be identified by short-term genotoxicity assays using as target kidney cells and show that the five chemicals tested produce in primary cultures of kidney cells from human donors effects similar to those observed in rats.     

DNA damage induced by 3,3'-dimethoxybenzidine in liver and urinary bladder cells of rats and humans.

3,3'-Dimethoxybenzidine (DMB), a congener of benzidine used in the dye industry and previously found to be carcinogenic in rats, was evaluated for its genotoxic activity in primary cultures of rat and human hepatocytes and of cells from human urinary bladder mucosa, as well as in liver and bladder mucosa of intact rats. A similar modest dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic DMB concentrations ranging from 56 to 180 microM. Replicating rat hepatocytes displayed a modest increase in the frequency of micronucleated cells after a 48-h exposure to 100 and 180 microM concentrations. In primary cultures of human urinary bladder mucosa cells exposed for 20 h to 100 and 180 microM DMB, the Comet assay revealed a clear-cut increase of DNA fragmentation. In rats given one-half LD50 of DMB as a single oral dose, the GSH level was reduced in both the liver and urinary bladder mucosa, whereas DNA fragmentation was detected only in the bladder mucosa. Taken as a whole, these results suggest that DMB should be considered a potentially genotoxic chemical in both rats and humans; the selective effect on the rat urinary bladder might be the consequence of pharmacokinetic behavior.     

DNA damage induced by 4,4'-methylenedianiline in primary cultures of hepatocytes and thyreocytes from rats and humans

4,4'-Methylenedianiline (MDA), an aromatic amine used in various industrial processes and previously found to induce tumor development in liver and thyroid of mice and rats, was evaluated for its DNA-damaging activity in primary cultures of hepatocytes and thyreocytes from rat and human donors. After exposure for 4 and 20 h to MDA concentrations ranging from 10 to 180 microM, a statistically significant increase in the frequency of DNA lesions was revealed by the Comet assay in primary hepatocytes and thyreocytes from donors of both species, the response being dose dependent up to 56-100 microM MDA. DNA fragmentation was more marked after 4 than after 20 h exposure in all four cell types. DNA was damaged to a lesser extent in human hepatocytes and thyreocytes than in corresponding rat cells and in both species in hepatocytes than in thyreocytes. In both rat and human hepatocytes a 20-h exposure to the same MDA concentrations elicited a modest amount of DNA repair synthesis, as evaluated by autoradiography. Evidence of a partial reduction of DNA damage, and therefore of only partial DNA repair, was observed in rat hepatocytes and in rat and human thyreocytes incubated for 16 h in MDA-free medium after a 4-h MDA treatment. A 4-h exposure to 56, 100, and 180 microM MDA did not induce DNA lesions in primary cultures of cells from three rat organs, kidney, urinary bladder mucosa, and brain, which are resistant to MDA carcinogenic activity. Under the same experimental conditions any evidence of DNA damage was absent in primary kidney and urinary bladder cells from human donors. Taken as a whole the results of this work indicate that MDA is specifically activated to DNA-damaging reactive species by hepatocytes and thyreocytes in both rats and humans and thus suggest that liver and thyroid might be the targets of the carcinogenic activity of MDA also in humans.     

Higher DNA adduct levels in urinary bladder and prostate of slow acetylator inbred rats administered 3,2'-dimethyl-4-aminobiphenyl

Human epidemiological studies suggest associations between acetylator phenotype and aromatic amine-induced tumors. The aromatic amine carcinogen 3,2'-dimethyl-4-aminobiphenyl (DMABP) induces colon, prostate, and urinary bladder tumors in the rat, and a rapid and slow acetylator rat model has been characterized. The formation of DNA adducts has been used as a valuable biomarker in tumorigenesis. In order to examine the relationship between the acetylation polymorphism and aromatic amine-induced cancer, DNA adducts were measured in three target organs (colon, prostate, and urinary bladder) and two nontarget organs (liver and heart) of male rapid (F344) and slow (WKY) acetylator inbred rats administered DMABP. Two DMABP-DNA adducts, N-(deoxyguanosin-8-yl)-DMABP (C8-DMABP) and 5-(deoxyguanosin-N2-yl)-DMABP (N2-DMABP), were identified in each target and nontarget organ examined. C8-DMABP-DNA adduct levels were highest in liver and were dose related in liver, colon, urinary bladder, and prostate. DMABP-DNA adduct levels were significantly higher in the prostate and urinary bladder of slow acetylator vs rapid acetylator rats. These studies suggest that DMABP-induced DNA damage is acetylator phenotype-dependent in urinary bladder and prostate, two target organs for DMABP-induced tumorigenesis in the rat.     

4-Aminobiphenyl and DNA reactivity: case study within the context of the 2006 IPCS Human Relevance Framework for Analysis of a cancer mode of action for humans

The IPCS Human Relevance Framework was evaluated for a DNA-reactive (genotoxic) carcinogen, 4-aminobiphenyl, based on a wealth of data in animals and humans. The mode of action involves metabolic activation by N-hydroxylation, followed by N-esterification leading to the formation of a reactive electrophile, which binds covalently to DNA, principally to deoxyguanosine, leading to an increased rate of DNA mutations and ultimately to the development of cancer. In humans and dogs, the urinary bladder urothelium is the target organ, whereas in mice it is the bladder and liver; in other species, other tissues can be involved. Differences in organ specificity are thought to be due to differences in metabolic activation versus inactivation. Based on qualitative and quantitative considerations, the mode of action is possible in humans. Other biological processes, such as toxicity and regenerative proliferation, can significantly influence the dose response of 4-aminobiphenyl-induced tumors. Based on the IPCS Human Relevance Framework, 4-aminobiphenyl would be predicted to be a carcinogen in humans, and this is corroborated by extensive epidemiologic evidence. The IPCA Human Relevance Framework is useful in evaluating DNA-reactive carcinogens.     

DNA damage and micronuclei induced in rat and human kidney cells by six chemicals carcinogenic to the rat kidney

Six chemicals, known to induce kidney tumors in rats, were examined for their ability to induce DNA fragmentation and formation of micronuclei in primary cultures of rat and human kidney cells, and in the kidney of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the Comet assay, and in micronuclei frequency, were obtained in primary kidney cells from both male rats and humans of both genders with the following subtoxic concentrations of five of the six test compounds: bromodichlorometane (BDCM) from 0.5 to 4 mM, captafol (CF) from 0.5 to 2 microM, nitrobenzene (NB) from 0.062 to 0.5 mM, ochratoxin A (OTA) from 0.015 to 1.215 microM, and trichloroethylene (TCE) from 1 to 4 mM. Benzofuran (BF), consistent with its carcinogenic activity for the kidney of female, but not of male rats, at concentrations from 0.125 to 0.5 mM gave positive responses in cells from females but did not induce DNA damage and increased the frequency of micronuclei in cells from males to a lower extent; in contrast, it was active in cells from humans of both genders. DNA-damaging and micronuclei-inducing potencies were similar in the two species. In agreement with these findings, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were obtained in the kidney of rats, given p.o. a single dose (1/2 LD50) of the six compounds, BF in this assay being more genotoxic in female than in male rats. Taken as a whole, these findings give further evidence that kidney carcinogens may be identified by short-term genotoxicity assays, using as target kidney cells, and show that the six chemicals tested produce, in primary cultures of kidney cells from human donors, effects similar to those observed in rats.     

A pharmacokinetic model to predict exposure of the bladder epithelium to urinary N-hydroxyarylamine carcinogens as a function of urine pH, voiding interval, and resorption

Estimation of urinary levels of N-hydroxyarylamines, which are believed to be ultimate carcinogens for the bladder epithelium, has been complicated by the rapid resorption of these metabolites across the bladder wall and by their formation in the urine upon hydrolysis of N-hydroxyarylamine N-glucuronide conjugates. Therefore, a three-compartment pharmacokinetic model was developed with an analog-digital hybrid computer to separate absorption, metabolism, and distribution in the whole animal from events in the bladder lumen (deposition, hydrolysis, and resorption). The total bladder exposure to the carcinogen, N-hydroxy-2-naphthylamine, was then simulated from experimental data; and urine pH and voiding interval, both of which differ widely among species, were tested as variables. For humans and dogs, urine pH was found to be considerably more acidic than monkeys or rats; while voiding intervals in rats were considerably shorter than in monkeys, dogs, or humans. The relative species susceptibilities, indicated by the model as total bladder exposure to the N-hydroxyarylamine metabolite under conditions of normal urine pH and frequency of micturition for each species, were in the order: human greater than or equal to dog greater than monkey greater than rat. This is consistent with the reported carcinogenic potency of 2-naphthylamine in these species and suggests that urine pH and voiding interval are important determinants in aromatic amine-induced bladder cancer.     

4-Aminobiphenyl and DNA Reactivity: Case Study Within the Context of the 2006 IPCS Human Relevance Framework for Analysis of a Cancer Mode of Action for Humans

The IPCS Human Relevance Framework was evaluated for a DNA-reactive (genotoxic) carcinogen, 4-aminobiphenyl, based on a wealth of data in animals and humans. The mode of action involves metabolic activation by N-hydroxylation, followed by N-esterification leading to the formation of a reactive electrophile, which binds covalently to DNA, principally to deoxyguanosine, leading to an increased rate of DNA mutations and ultimately to the development of cancer. In humans and dogs, the urinary bladder urothelium is the target organ, whereas in mice it is the bladder and liver; in other species, other tissues can be involved. Differences in organ specificity are thought to be due to differences in metabolic activation versus inactivation. Based on qualitative and quantitative considerations, the mode of action is possible in humans. Other biological processes, such as toxicity and regenerative proliferation, can significantly influence the dose response of 4-aminobiphenyl-induced tumors. Based on the IPCS Human Relevance Framework, 4-aminobiphenyl would be predicted to be a carcinogen in humans, and this is corroborated by extensive epidemiologic evidence. The IPCA Human Relevance Framework is useful in evaluating DNA-reactive carcinogens.     

N-glucuronide formation of carcinogenic aromatic amines in rat and human liver microsomes

(1) Sensitive fluorimetric assays were developed for the determination of microsomal UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine and 4-aminobiphenyl. (2) In rat liver microsomes, enzyme activity towards 1-naphthylamine was orders of magnitude higher than the activities towards 2-naphthylamine, 4-aminobiphenyl or aniline. The differences were less marked with human liver microsomes. (3) Glucuronidation of aniline and 4-aminobiphenyl was not appreciably altered in rat liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats. UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine were selectively increased (about 2-fold) by 3-methylcholanthrene-treatment. However the increases were less marked than those observed with representative substrates of the 3-methylcholanthrene-inducible enzyme form. The results suggest that the arylamines investigated are predominantly conjugated by constitutive enzyme forms in rat liver. (4) Arylamine N-glucuronides were found to be susceptible to hydrolysis by E. coli beta-glucuronidase suggesting the release of carcinogenic arylamines in the gut and their enterohepatic circulation.     

DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by four rat thyroid carcinogens

Four chemicals that are known to induce in rats thyroid follicular-cell adenomas and carcinomas were assayed for their ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measures by the Comet assay, were obtained after a 20-h exposure to the following subtoxic concentrations of the four test compounds: 2,4-diaminoanisole (DAA) from 0.10 to 1.0 mM, 4,4'-methylene-bis(N,N-dimethyl)benzenamine (MDB) from 0.32 to 1.8 mM, propylthiouracil (PTU) from 1.8 to 5.6 mM, and 4,4'-thiodianiline (THA) from 0.032 to 0.18 mM. Under the same experimental conditions, DNA repair synthesis, as evaluated by quantitative autoradiography, was present in thyreocytes exposed to DAA but absent after treatment with MDB, PTU, and THA. Consistent with their thyroid-specific carcinogenic activity, all the four chemicals, administered p.o. in rats in a single dose corresponding to 1/2 LD50, induced a statistically significant degree of DNA fragmentation in the thyroid, whereas any substantial evidence of DNA lesions was absent in liver, kidney, and lung, which, with the exception of liver tumors caused by THA, are not targets of the carcinogenic activity of the four test compounds. These findings indicate that the DNA damage observed in thyroid cells was consistent with the carcinogenicity of the four test compounds, and suggest that DAA, MDB, PTU, and THA might be carcinogenic to thyroid in humans.     

A comparative study of chemically induced DNA damage in isolated nasal mucosa cells of humans and rats assessed by the alkaline comet assay

Single-cell microgel electrophoresis (comet) assay was used to study genotoxic effects in human nasal mucosa cells and rat nasal and ethmoidal mucosa cells in vitro. Human cells were obtained from tissue samples of 10 patients (3 females/7 males), who underwent surgery (conchotomy) for treatment of nasal airway obstruction. Rat nasal mucosa cells were derived from male Sprague-Dawley rats. Cells were exposed for 1 h to either N-nitrosodiethanolamine (NDELA), epichlorohydrin (EPI), 1,2-epoxybutane (EPB), ethylene dibromide (EDB), or 1,2-dibromo-3-chloropropane (DBCP). Dimethyl sulfoxide (DMSO) was used as negative control. Alkaline comet assay was performed according to a standard protocol and DNA damage was quantified as Olive tail moment using image analysis system. All test substances induced an increase in DNA damage in human and rat cells. The absolute amount of DNA damage in rat nasal mucosa cells was usually higher than in ethmoidal mucosa cells. Human nasal mucosa cells were found to be less sensitive than rat mucosa cells to the genotoxic activities of DBCP (lowest effective concentration in human cells [LEC(human)]: 1.5, in rat cells [LEC(rat)]: 0.01 mM) and NDELA (LEC(human): 25, LEC(rat): 12.5 mM), whereas EPB-treated cells were almost equal (LEC(human) and LEC(rat) 0.78 mM). NDELA induced a marked concomitant cytotoxicity. For EPI (LEC(human) and LEC(rat): 0.097 mM) and EDB (LEC(human): 0.195, LEC(rat): 0.048 mM), pronounced interindividual differences were observed in human samples.     

The role of N-oxidation products of aromatic amines in the induction of bladder cancer in the dog

Highly sensitive gas chromatographic methods for the determination of the N-hydroxy and nitroso derivatives of 1- and 2-naphthylamine, and 4-amino-biphenyl in urine have been developed. Dogs given a single oral 70 mg/kg dose of 1-naphthylamine were found to excrete almost as much of these N-oxidation products as dogs given the same dose of 2-naphthylamine. However, when 5 mg/kg of 2-naphthylamine was given, a total of approximately 0.2% of the administered dose was found in the urine as N-oxidation products, while the same dose of 1-naphthylamine produced only faint traces. The N-oxidation products present were 2-naphthylhydroxylamine and 2-nitrosonaphthalene. In addition, blood methemoglobin production, a measure of the level of N-hydroxylation occurring, observed with 2-naphthylamine was much greater than with 1-naphthylamine at the 70 mg/kg dose. 4-Aminobiphenyl, the most potent carcinogen, produced even higher levels of N-oxidation products in the urine and blood methemoglobin than 2-naphthylamine. These results indicate that N-oxidation may be a key process in the production of bladder cancer by 2-naphthylamine and 4-aminobiphenyl.This investigation was supported by U. S. Public Health Service Grant No. CA 05449 from the National Cancer Institute.     

Dose dependency of γ-H2AX formation in the rat urinary bladder treated with genotoxic and nongenotoxic bladder carcinogens

We previously reported that immunostaining for γ-H2AX, a biomarker of DNA damage, in the rat urinary bladder is useful for early detection of bladder carcinogens in 28-day toxicity studies. Here, we aimed to examine the dose dependency of γ-H2AX formation in the urinary bladder of rats. Male F344 rats (aged 6 weeks) were orally administered N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN; 0%, 0.0001%, 0.001%, 0.01%, 0.02%, or 0.05% in drinking water), a genotoxic bladder carcinogen, and melamine (0%, 0.3%, 1.0%, or 3.0% in the diet), a nongenotoxic bladder carcinogen, for 2 days or 4 weeks. Immunohistochemical analysis showed that γ-H2AX- and Ki67-positive epithelial cells in the bladder urothelium were significantly increased, with a clear dose dependency, in both BBN- and melamine-treated groups. Additionally, γ-H2AX formation was detected from the lower-dose group, without increased Ki67 expression or histopathologic findings. The ratios of γ-H2AX-positive cells at week 4 in both BBN- and melamine-treated groups were higher than those on day 2, indicating the time-dependent increase in γ-H2AX formation. Immunofluorescence double-staining revealed that γ-H2AX single-positive cells without Ki67 expression were often found in the urothelium of BBN-treated rats, whereas most γ-H2AX-positive cells were Ki67-positive in the melamine group. Our results demonstrated that γ-H2AX formation in the urinary bladder increased in a clear dose-dependent manner and that γ-H2AX immunostaining has the potential to detect bladder carcinogens after a 2-day administration. Furthermore, the association of genotoxic mechanisms in bladder carcinogenesis could be determined by analyzing the colocalization of γ-H2AX and Ki67 in the urothelium.     

2-Naphthol levels and genotoxicity in rubber workers

Urinary bladder cancer is a historical disease of rubber workers often been associated with exposure to aromatic amines such as 2-naphthylamine. While exposure to these compounds has decreased markedly over time, the bladder cancer risk has not decreased in direct proportion. Polycyclic aromatic compounds (PAC) are candidates for urinary bladder cancer causation. We determined pre- and post-exposure urinary levels of 2-napthol (2NAP), the major metabolite of a model volatile PAC, in a group of non-smoking rubber workers. Pre- and post-exposure urine samples were collected from 43 non-smoking workers. Overall mean post-shift 2-naphthol levels were increased (13.95 ± 28.4 μg/l), but non-significantly compared to samples collected pre-exposure (7.97 ± 22.1 μg/l; p=0.29). The greatest difference was observed in the curing department where post-exposure samples were 4.5 fold higher, post shift samples were significantly higher in production workers as compared to non-production workers (p=0.02). Levels of 2NAP were not correlated with levels of carcinogen-DNA adducts in exfoliated urothelial cells nor with other estimates of exposure or effect. These data suggest that post-shift urinary 2NAP levels are increased, particularly in the curing department. However, the differences were not significantly different overall and urinary 2NAP levels did not predict the level of carcinogen DNA adducts in exfoliated urothelial cells.     

Possible Contribution of Indomethacin to the Carcinogenicity of Nongenotoxic Bladder Carcinogens That Cause Bladder Calculi

Nongenotoxic bladder carcinogens that form bladder calculi have been concluded to be of low carcinogenic risk to humans because bladder stones would be expelled or surgically removed before they had a chance to exert their carcinogenic effect. It is the aim of this report to examine the possible contribution of indomethacin to the carcinogenic risk posed by nongenotoxic bladder carcinogens that cause bladder stones. Indomethacin may act as a tumor promoter in the bladder by interfering with the synthesis of prostaglandins. Prostaglandins have a cytoprotective function in the gastric mucosa and possibly also in the urinary bladder. Diminished cytoprotection may be implicated in bladder carcinogenesis as β-naphthylamine, a human bladder carcinogen, also inhibits prostaglandin synthesis in vitro. The presence of other tumor promoters in the bladder may further ensure that tumors would be formed even if bladder stones were expelled. People who are exposed to nongenotoxic bladder carcinogens that are present in the environment and that form bladder stones, therefore, may be at an increased risk for developing bladder cancer if they are also exposed to tumor promoters, such as indomethacin.     

A concise review of the toxicity and carcinogenicity of dimethylarsinic acid

Dimethylarsinic acid (DMA) has been used as a herbicide (cacodylic acid) and is the major metabolite formed after exposure to tri- (arsenite) or pentavalent (arsenate) inorganic arsenic (iAs) via ingestion or inhalation in both humans and rodents. Once viewed simply as a detoxification product of iAs, evidence has accumulated in recent years indicating that DMA itself has unique toxic properties. DMA induces an organ-specific lesion — single strand breaks in DNA — in the lungs of both mice and rats and in human lung cells in vitro. Mechanistic studies have suggested that this damage is due mainly to the peroxyl radical of DMA and production of active oxygen species by pulmonary tissues. Multi-organ initiation-promotion studies have demonstrated that DMA acts as a promotor of urinary bladder, kidney, liver and thyroid gland cancers in rats and as a promotor of lung tumors in mice. Lifetime exposure to DMA in diet or drinking water also causes a dose-dependent increase in urinary bladder tumors in rats, indicating that DMA is a complete carcinogen. These data collectively suggest that DMA plays a role in the carcinogenesis of inorganic arsenic.     

N-Glucuronidation of N-hydroxy aromatic amines: A mechanism for their transport and bladder-specific carcinogenicity

Glucuronide conjugates of carcinogenic N-hydroxy metabolites of the primary aromatic amines, 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 1-naphthylamine (1-NA) were isolated from the urine of dogs administered the respective primary amine and from the in vitro incubation of N-hydroxy metabolites with uridine-5′-disphosphoglucuronic acid-fortified dog liver microsomes. The urinary and microsomal conjugates were purified by several sequential chromatographic procedures, including Sephadex G-15, Amberlite XAD-2, and cellulose CF-11 chromatography for microsomal conjugates and Sephadex G-10, DEAE, and Amberlite XAD-2 chromatography for urinary conjugates. The infrared spectra of purified urinary and microsomal conjugates of these three N-hydroxy aromatic amines were identical to spectra of authentic N---C glucuronides prepared by two different synthetic procedures. The urinary and microsomal conjugates comigrated with synthetic N---C glucuronides in two solvent systems. These observations in conjunction with previous studies provide evidence that N---C glucuronidation represents a general metabolic reaction of carcinogenic N-hydroxy aromatic amines which provides the means of transport of these compounds to their site of action in the bladder.     

Biochemical studies of six nitrogen-containing heterocycles in rat tissues

Female rats were dosed orally with one-fifth the LD50 of either 1-nitrosopiperidine (a carcinogen), cyclohexylamine, piperidine, 4-carboxy-1-nitrosopiperidine, 4-cyclohexyl-1-nitrosopiperidine or 2,6-dimethyl-1-nitrosopiperidine at 21 and 4 hr before they were killed. The five noncarcinogenic compounds had no effects on any experimental variables examined [hepatic DNA damage, ornithine decarboxylase (ODC) activity, serum alanine aminotransferase (SGPT) activity, cytochrome P-450 and glutathione content]. After administration of 40 mg/kg of 1-nitrosopiperidine, marked hepatic DNA damage and a 3- to 7-fold increase in the activity of hepatic ODC were observed. 1-Nitrosopiperidine (120 mg/kg, 3/5 LD50) caused DNA damage in rat liver and esophagus but not in leukocytes. This higher dose of 1-nitrosopiperidine also increased hepatic ornithine decarboxylase activity by 9-fold. Thus, this hepatic biochemical assay system correctly identified the one carcinogen and the five noncarcinogens in this series of six nitrogen-containing heterocycles.     

Immunohistochemistry of γ‐H2AX as a method of early detection of urinary bladder carcinogenicity in mice

Phosphorylated histone H2AX (γ‐H2AX) has been demonstrated as a DNA damage marker both in vitro and in vivo. We previously reported the effects of genotoxic carcinogens in the urinary bladder of rats by immunohistochemical analysis of γ‐H2AX using samples from 28‐day repeated‐dose tests. To evaluate the application of γ‐H2AX as a biomarker of carcinogenicity in the bladder, we examined species differences in γ‐H2AX formation in the urinary bladder of mice. Six‐week‐old male B6C3F₁ mice were treated orally with 12 chemicals for 4 weeks. Immunohistochemical analysis demonstrated that N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine, p‐cresidine and 2‐acetylaminofluorene (2‐AAF), classified as genotoxic bladder carcinogens, induced significant increases in γ‐H2AX levels in the bladder urothelium. In contrast, genotoxic (2‐nitroanisole, glycidol, N‐nitrosodiethylamine and acrylamide) and non‐genotoxic (dimethylarsinic acid and melamine) non‐bladder carcinogens did not upregulate γ‐H2AX. Importantly, 2‐nitroanisole, a potent genotoxic bladder carcinogen in rats, significantly increased the proportion of γ‐H2AX‐positive cells in rats only, reflecting differences in carcinogenicity in the urinary bladder between rats and mice. Significant upregulation of γ‐H2AX was also induced by uracil, a non‐genotoxic bladder carcinogen that may be associated with cell proliferation, as demonstrated by increased Ki67 expression. 2‐AAF caused γ‐H2AX formation mainly in the superficial layer, together with reduced and disorganized expression of uroplakin III, unlike in rats, suggesting the mouse‐specific cytotoxicity of 2‐AAF in umbrella cells. These results suggest γ‐H2AX is a useful biomarker reflecting species differences in carcinogenicity in the urinary bladder.     

The modulation by arylamines of the in vitro formation of superoxide anion radicals and hydrogen peroxide by rat liver microsomes

A concentration-dependent increase in the generation of the superoxide anion radical (O-2), was observed during the incubation of benzidine (0.1-5 mM), but not of the structurally related compounds 4-aminobiphenyl, 2-aminobiphenyl or 4-fluoro-4'-aminobiphenyl, with NADPH-supplemented rat liver microsomes. This increase was partially inhibited by carbon monoxide and catalase but unaffected by 4-aminobiphenyl, a substrate of the cytochrome P-450 system. Microsomes from benzo(a)pyrene-treated, but not microsomes from phenobarbitone-pretreated rats, were responsible for a larger benzidine-dependent effect compared to microsomes from control animals. In contrast to the above observations, benzidine decreased the formation of hydrogen peroxide by NADPH-supplemented microsomal preparations from untreated rats. These results indicate that free radicals of oxygen are generated during the metabolism of some arylamines.