Assessment of the genotoxic potential of indirect chemical mutagens in HepaRG cells by the comet and the cytokinesis-block micronucleus assays.

Many chemical carcinogens require metabolic activation to form genotoxic compounds in human. Standard in vitro genotoxicity assays performed with activation systems, such as rat liver S9, are recognised to lead to a high number of false positives. The aim of this study was to evaluate the suitability of differentiated human hepatoma HepaRG cells as an in vitro model system for the detection of DNA damage induced by promutagens using the comet and the cytokinesis-block micronucleus assays. Several promutagens were tested, including aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), acrylamide, N-nitrosodimethylamine (NDMA), cyclophosphamide (CPA), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Cytotoxicity of these compounds was assessed by measuring lactate dehydrogenase leakage. A 24 h exposure was generally needed to obtain an obvious positive response in differentiated HepaRG cells in the comet and in the cytokinesis-block micronucleus assays. Comet formation was observed with all compounds except IQ. B[a]P, CPA and AFB1 showed a dose-dependent increase in micronucleated cells, whereas no increase was observed with PhIP, IQ and acrylamide. These preliminary data on genotoxicity in differentiated HepaRG cells are promising but more chemicals must be tested to determine the ability of HepaRG cells to assess genotoxicity of chemicals in humans.

Lack of genotoxic effect of food dyes amaranth, sunset yellow and tartrazine and their metabolites in the gut micronucleus assay in mice.

The food dyes amaranth, sunset yellow and tartrazine were administered twice, at 24h intervals, by oral gavage to mice and assessed in the in vivo gut micronucleus test for genotoxic effects (frequency of micronucleated cells) and toxicity (apoptotic and mitotic cells). The concentrations of each compound and their main metabolites (sulfanilic acid and naphthionic acid) were measured in faeces during a 24-h period after single oral administrations of the food dyes to mice. Parent dye compounds and their main aromatic amine metabolites were detected in significant amounts in the environment of colonic cells. Acute oral exposure to food dye additives amaranth, sunset yellow and tartrazine did not induce genotoxic effect in the micronucleus gut assay in mice at doses up to 2000 mg/kg b.w. Food dyes administration increased the mitotic cells at all dose levels when compared to controls. These results suggest that the transient DNA damages previously observed in the colon of mice treated by amaranth and tartrazine by the in vivo comet assay [Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., Taniguchi, K., Tsuda, S., 2002. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat. Res. 519, 103-119] are unable to be fixed in stable genotoxic lesions and might be partly explained by local cytotoxicity of the dyes.

Long-term functional stability of human HepaRG hepatocytes and use for chronic toxicity and genotoxicity studies.

The human hepatoma HepaRG cells are able to differentiate in vitro into hepatocyte-like cells and to express various liver-specific functions, including the major cytochromes P450. This study was aimed to determine whether differentiated HepaRG cells retained their specific functional capacities for a long time period at confluence. We show that expression of transcripts encoding CYP1A2, 2B6, 3A4, and 2E1, several phase II and antioxidant enzymes, membrane transporters, including organic cation transporter 1 and bile salt export pump, the nuclear receptors constitutive androstane receptor and pregnane X receptor, and aldolase B remained relatively stable for at least the 4-week confluence period tested. Similarly, activities of CYP3A4 and CYP1A2 and their responsiveness to prototypical inducers were well preserved. Aflatoxin B(1), a potent hepatotoxicant and carcinogen, induced a dose-dependent and cumulative cytotoxicity. Furthermore, at a concentration as low as 0.1 microM, this mycotoxin caused a decrease in both CYP3A4 activity and intracellular ATP associated with morphological alterations, after 14 days following every 2-day exposure. Moreover, using the comet assay, a dose-dependent DNA damage was observed after a 3-h treatment of differentiated HepaRG cells with 1 to 5 microM aflatoxin B(1) in the absence of any cell damage, and this DNA damaging effect was strongly reduced in the presence of ketoconazole, a CYP3A4 inhibitor. These results bring the first demonstration of long-term stable expression of liver-specific markers in HepaRG hepatocyte cultures maintained at confluence and show that these cells represent a suitable in vitro liver cell model for analysis of acute and chronic toxicity as well as genotoxicity of chemicals in human liver.

New considerations regarding the risk assessment on Tartrazine An update toxicological assessment, intolerance reactions and maximum theoretical daily intake in France.

Tartrazine is an artificial azo dye commonly used in human food and pharmaceutical products. Since the last assessment carried out by the JECFA in 1964, many new studies have been conducted, some of which have incriminated tartrazine in food intolerance reactions. The aims of this work are to update the hazard characterization and to revaluate the safety of tartrazine. Our bibliographical review of animal studies confirms the initial hazard assessment conducted by the JECFA, and accordingly the ADI established at 7.5mg/kg bw. From our data, in France, the estimated maximum theoretical intake of tartrazine in children is 37.2% of the ADI at the 97.5th percentile. It may therefore be concluded that from a toxicological point of view, tartrazine does not represent a risk for the consumer. It appears more difficult to show a clear relationship between ingestion of tartrazine and the development of intolerance reactions in patients. These reactions primarily occur in patients who also suffer from recurrent urticaria or asthma. The link between tartrazine consumption and these reactions is often overestimated, and the pathogenic mechanisms remain poorly understood. The prevalence of tartrazine intolerance is estimated to be less than 0.12% in the general population. Generally, the population at risk is aware of the importance of food labelling, with the view of avoiding consumption of tartrazine. However, it has to be mentioned that products such as ice creams, desserts, cakes and fine bakery are often sold loose without any labelling.

Impact of ciprofloxacin in the human-flora-associated (HFA) rat model: comparison with the HFA mouse model.

The ecological impact of different doses of ciprofloxacin was investigated in an experimental germ-free rat model into which human fecal flora was inoculated. Animals received oral doses (gavage) of 0, 0.25, 2.5, and 25 mg/kg body weight (bw) of ciprofloxacin once daily for 5 weeks. All doses of ciprofloxacin significantly reduced aerobic populations. Elimination of Enterobacteriaceae and reduction of bifodibacteria were noticed in the group treated with 25 mg/kg of the antibiotic. The rest of the intestinal flora was not affected. These effects were reversible after the treatment ended. The percentage of resistant enterococci increased in rats treated with 2.5 and 25 mg/kg; however, this increase was not statistically significant. There was a significant (P < 0.05) emergence of ciprofloxacin-resistant Bacteroides fragilis group with 25 mg/kg bw, which is equivalent to a human therapeutic dosage of the antibiotic. The MIC values and the percentage of resistance remained elevated 2 weeks after the end of treatment in this anaerobic population. Although sub-populations of enterococci and Enterobacteriaceae showed decreased susceptibility after ciprofloxacin administration, resistance was not evident. The ability of an exogenous strain of Salmonella to colonize the intestine of animals treated with 25 mg/kg of ciprofloxacin confirmed that the drug disrupted the colonization barrier effect of the indigenous flora at the high dose level tested. No changes in the metabolic parameters occurred during the antibiotic treatment. The results obtained in the HFA rat model were similar to those obtained in our previous study using the HFA mice model where ciprofloxacin at 0.125, 1.25, and 12.5 mg/kg bw induced a decrease of enterococci and Enterobacteriaceae populations. The high dose of ciprofloxacin also induced a decrease in bifidobacteria counts, an increase in levels of resistant B. fragilis group and a significant (P < 0.05) disruption of the colonization resistance of the barrier flora in HFA mice.

Impact of residual and therapeutic doses of ciprofloxacin in the human-flora-associated mice model.

A study was conducted to evaluate the effects of therapeutic and residual doses of ciprofloxacin on the human intestinal flora implanted into germ-free mice. Ciprofloxacin was administered daily via drinking water at concentrations to provide doses of 0, 0.125, 1.25, and 12.5mg/kg b.w. Changes in the intestinal flora composition, alteration in bacterial enzyme activities, fecal short chain fatty acid concentration and bacterial cellular fatty acid profiles, overgrowth of resistant bacteria, and disruption of the colonization barrier were the endpoints evaluated in the feces of human-flora-associated (HFA) mice. Ciprofloxacin at all tested doses decreased significantly the aerobic populations and particularly the population of Enterobacteriaceae. Selection of resistant Bacteroides fragilis group was noticed in HFA mice receiving 12.5mg/kg b.w. In mice challenged with a Salmonella strain, exogenous Salmonella persisted in the feces of all treated mice indicating that the flora responsible for the colonization barrier effect was disturbed by the antibiotic treatment. None of the studied metabolic parameters of the flora were affected by ciprofloxacin at any dose level. Under the experimental conditions of the study, the no-observed-effect level of ciprofloxacin was found to be less than 0.125 mg/kg b.w.

DNA damage and DNA adduct formation in rat tissues following oral administration of acrylamide.

Acrylamide is present as a contaminant in the human diet in heated food products. It has been found to be carcinogenic in laboratory rats and has been classified as probably carcinogenic in humans. In order to clarify the possible involvement of a primary genotoxic mechanism in acrylamide-induced carcinogenicity, both the presence of DNA damage, measured by the comet assay, and the formation of N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade), derived from reaction of the active metabolite glycidamide (GA) with the DNA, analyzed by LC/MS/MS, were assessed in selected rat tissues. Rats were administered with single oral doses of acrylamide (18, 36 or 54 mg/kg body weight (b.w.) and the organs (blood leukocytes, brain, bone marrow, liver, testes and adrenals) were sampled at different times after treatment. Results from GA-induced DNA adduct measurements indicated a relatively even organ distribution of the adducts in brain, testes and liver. Organ-specificity in acrylamide carcinogenesis can therefore not be explained by a selective accumulation of GA-DNA adducts in the target organs, at least not after a single dose exposure. The DNA adduct profiles and half-lives were similar in the different organs; except that the N3-GA-Ade adduct was more rapidly removed from tissues than the N7-GA-Gua adduct. Increased extent of DNA migration, as measured by the in vivo rat comet assay, was found in brain and testes, and these specific results seem to be in accordance with the known organ-specificity in acrylamide carcinogenesis in rat. Only weak and transient DNA damage was recorded in the liver, bone marrow and adrenals. The DNA-damaging effect of the compound observed in the blood leukocytes could be a simple biomarker of acrylamide exposure and genotoxicity.

Marine toxin okadaic acid induces aneuploidy in CHO-K1 cells in presence of rat liver postmitochondrial fraction, revealed by cytokinesis-block micronucleus assay coupled to FISH.

Okadaic acid (OA), a major polyether toxin involved in diarrhetic shellfish poisoning (DSP), is a potent tumor promoter in rodent skin and glandular stomach and a specific inhibitor of the serine/threonine protein phosphatases PP1 and PP2A. A previous study, which used the cytokinesis-block micronucleus (CBMN) assay in CHO-K1 cells, showed that OA induced chromosome damage in the presence of a rat liver metabolic activation system (S9). To support OA biotransformation by S9, the same test system was performed, and DNA damage induced by OA was measured with and without metabolic activation as well as in the presence of heat-inactivated S9 fraction. The results showed that only in the presence of active S9 did OA significantly increased the frequency of micronucleated binucleated (MNBN) cells. After a 4-h treatment a 2- to 5-fold increase of MNBN cells was observed at 30 nM and at 50 nM of OA. However, without S9 or in the presence of heat-inactivated S9, OA did not induce any chromosome damage. We concluded that OA can be metabolically activated in vitro into metabolites that are more genotoxic. The CBMN assay coupled with fluorescence in situ hybridization (FISH) using a DNA probe for centromere detection was performed to discriminate between clastogenic (chromosome breakage) and aneugenic (chromosome loss) effects. FISH analysis showed that OA metabolites increased in a dose-dependent manner in centromere positive micronuclei (CEN+): 60% of CEN+ at 30 nM and 75% of CEN+ at 50 nM of OA. The uptake of OA into CHO-K1 cells and the biotransformation of the toxin are discussed.

Aneugenic potential of okadaic acid revealed by the micronucleus assay combined with the FISH technique in CHO-K1 cells.

Okadaic acid (OA) is a major toxin involved in diarrhetic shellfish poisoning in humans and has been shown to be both a potent tumor promoter in rodent skin and stomach and an inhibitor of serine/threonine protein phosphatases, specifically PP1 and PP2A. The research on the genotoxic potential of OA amounts to only a few studies, which give conflicting results. In order to evaluate the ability of OA to induce DNA damage, the cytokinesis-block micronucleus assay was performed in the CHO-K1 cell line. A statistically significant induction of micronuclei without strong cytotoxicity was obtained after a 24 h treatment with 20 (approximately 5-fold) and 30 nM (approximately 10-fold) OA. Then, in order to discriminate between a clastogenic or aneugenic effect of OA, the micronucleus assay was carried out in combination with fluorescence in situ hybridization (FISH) using a (TTAGGG)(n) DNA probe for centromere detection. FISH analysis showed that OA mainly induced centromere-positive micronuclei (68.9% induction with 20 nM OA and 77.0% with 30 nM). Therefore, OA can be considered aneugenic. Using the same assay, biotransformation of OA was studied after a 4 h treatment with and without metabolic activation. The results show that reactive metabolites of OA were generated with a significant increase in genotoxic potential. The relationship between the different components involved in the mitotic process and OA inhibition of protein phosphatase is also discussed.

Application of pharmacokinetic/pharmacodynamic and stochastic modelling to 6-mercaptopurine micronucleus induction in mouse bone marrow erythrocytes.

This study investigates the kinetics of bone marrow micronucleated polychromatic erythrocytes and some mechanistic aspects of micronuclei induction using mathematical models. Female mice were administered a single intraperitoneal injection of the purine antagonist 6-mercaptopurine at 50 mg kg(-1). The time course evolution of the drug concentrations in the plasma and the micronucleated polychromatic erythrocyte kinetic rate in bone marrow were observed. Two mathematical models were developed for this study. The first model was built from a simultaneous pharmacokinetic/pharmacodynamic approach, but was invalidated after comparing its predictions to experimental data. The second model was a stochastic model based on some biological hypotheses involved in micronuclei induction. This model predicted a wavy kinetic rate of micronucleated polychromatic erythrocytes that was confirmed by a second data set obtained from a specifically built experimental design. The biological hypotheses were then discussed. It turned out from this work that mathematical modelling could be used as a tool to explore the cellular mechanisms of toxicity of the compound: for instance, the assumptions that 6-mercaptopurine induced micronuclei mainly in cells entering the S phase, and not only during the last cell cycle but during one of the earlier cycles preceding the extrusion of the main nucleus, were confirmed. Moreover, the use of the stochastic model would help to schedule more accurately the bone marrow or blood harvesting times in the in vivo rodent micronucleus test.

Detection of topoisomerase inhibitor-induced DNA strand breaks and apoptosis by the alkaline comet assay.

The alkaline comet assay is able to identify in individual cells DNA strand breaks associated with different processes. Topoisomerase inhibitors, some of which are used as chemotherapeutic agents, stabilise topoisomerase-DNA cleavable complexes by stimulating DNA strand cleavage and inhibiting religation. This can result in the activation of stress-associated signalling pathways, inducing cell cycle arrest and activation of the biochemical cascade of apoptosis. The aim of our study was to assess the ability of the comet assay to detect stabilisation of cleavable complexes and induction of apoptosis by two topoisomerase II inhibitors, etoposide and ellipticine, and two topoisomerase I inhibitors, camptothecin and topotecan. The study was carried out on Chinese hamster ovary (CHO) cells, DC3F cells and DC3F/C-10, its camptothecin-resistant counterpart. The comet assay was able to identify stabilised cleavable complexes through the presence of DNA strand breaks after 1h treatment that disappeared within 24h after drug removal. Kinetics studies allowed to discriminate between these early DNA damages and DNA fragmentation related to apoptosis characterised by reappearance of DNA strand breaks 48h after treatment.