Loss of CUL4A expression is underlying cisplatin hypersensitivity in colorectal carcinoma cells with acquired trabectedin resistance.

BACKGROUND: Colorectal carcinoma (CRC) is the third most common cancer worldwide. Platinum-based anticancer compounds still constitute one mainstay of systemic CRC treatment despite limitations due to adverse effects and resistance development. Trabectedin has shown promising antitumor effects in CRC, however, again resistance development may occur. In this study, we aimed to develop strategies to circumvent or even exploit acquired trabectedin resistance in novel CRC treatment regimens. METHODS: Human HCT116 CRC cells were selected for acquired trabectedin resistance in vitro and characterised by cell biological as well as bioinformatic approaches. In vivo xenograft experiments were conducted. RESULTS: Selection of HCT116 cells for trabectedin resistance resulted in p53-independent hypersensitivity of the selected subline against cisplatin. Bioinformatic analyses of mRNA microarray data suggested deregulation of nucleotide excision repair and particularly loss of the ubiquitin ligase CUL4A in trabectedin-selected cells. Indeed, transient knockdown of CUL4A sensitised parental HCT116 cells towards cisplatin. Trabectedin selected but not parental HCT116 xenografts were significantly responsive towards cisplatin treatment. CONCLUSIONS: Trabectedin selection-mediated CUL4A loss generates an Achilles heel in CRC cancer cells enabling effective cisplatin treatment. Hence, inclusion of trabectedin in cisplatin-containing cancer treatment regimens might cause profound synergism based on reciprocal resistance prevention.

Effects of unconjugated bilirubin on chromosomal damage in individuals with Gilbert's syndrome measured with the micronucleus cytome assay.

Circulating unconjugated bilirubin (UCB) has been reported to protect against lung and colorectal cancer. The present study aimed to explore, for the first time, whether mildly elevated circulating UCB, as found in Gilbert`s syndrome (GS), is associated with changes of DNA damage. A random 76 individuals, matched for age and gender, were recruited from the general population and allocated into the GS group (UCB ≥ 17.1 µM; n = 38) or control group (UCB <17.1 µM; n = 38). Chromosomal and cytological changes were determined in lymphocytes and buccal cells using the cytokinesis-block micronucleus cytome assay (CBMN) and buccal micronucleus cytome assay (BMcyt). No significant differences were found between GS subjects and the control group in the CBMN and BMcyt determined endpoints. Subsequently, when age dependency of effects were analysed, lower formation of buccal micronucleated cells (by 73.3%) and buccal nuclear buds (by 70.9%) in the GS subgroup ≥ 30 years were found, compared to the GS subgroup <30 years. These findings suggest DNA protection in epithelial tissue of older individuals with GS.

Intake of a resveratrol-containing dietary supplement has no impact on DNA stability in healthy subjects.

It has been postulated that the beneficial health effects of dietary supplements and of red wines which contain resveratrol (RES) are due to the anti-oxidative properties of this phenolic compound, but evidence for protection against reactive oxygen species is mainly based on results of in vitro experiments and high-dose animal experiments. Aim of this study was to find out if intake of a RES-containing supplement protects healthy humans against oxidative DNA-damage and alters their redox status. Therefore, an intervention trial was conducted in which the participants (n=12) consumed a RES-containing supplement over a period of five days. At the start, after one day and after five days of consumption, and after a washout period DNA stability was measured in single cell gel electrophoresis (SCGE) assays with peripheral blood lymphocytes. These tests were conducted (a) under standard conditions, which reflect single- and double-strand DNA breaks, (b) after treatment of the cells with hydrogen peroxide, which enables detection of alterations of the ROS sensitivity, and (c) by use of formamidopyrimidine DNA-glycosylase (FPG), which provides information on formation of oxidatively damaged bases (pyrimidines). Furthermore, the biochemical parameters TAC (total antioxidant capacity) and oxLDL (oxidized low-density lipoprotein), which reflect the redox status, and C-reactive protein (CRP), a marker of inflammation, were monitored. The intake of the supplement had no significant impact on the DNA stability parameters and on the different biomarkers of the redox status. Our results indicate that intake of 6mg RES per day via the supplement does not cause DNA-protective or antioxidant effects. This amount is equivalent to or lower than that reached after intake of many (ca. 50%) of the RES-containing preparations which are currently on the market in Middle Europe, and is contained in 0.3-2L red wine.

Detoxification of patulin and ochratoxin A, two abundant mycotoxins, by lactic acid bacteria.

Aim of the present study was to investigate the detoxification of two abundant mycotoxins, namely ochratoxin A (OTA) and patulin (PAT) which are frequently found in human foods, by lactic acid bacteria. The removal of the two mycotoxins from liquid medium by thirty different LAB strains was analyzed in a screening trial by the use of HPLC coupled with UV- or fluorescence detection. Two highly effective strains were identified; Lactobacillus acidophilus VM 20 caused a decrease of OTA by > or = 95% and Bifidobacterium animalis VM 12 reduced PAT levels by 80%. Subsequently experiments showed that the binding of these compounds depends on different parameters, i.e. the concentration of toxins, the cell density, the pH-value and on the viability of the bacteria. To proof that the decrease of the toxins by LAB from liquid medium results in a reduction of their toxic properties, micronucleus (MCN) assays were conducted with a human derived hepatoma cell line (HepG2). Indeed, a substantial decrease (39-59%) of OTA and PAT induced MCN formation was observed with the most effective strains detected in the chemical analyses. Furthermore, also the inhibition of the cell division rates by the toxins was significantly reduced. These findings indicate that certain LAB strains are able to detoxify the two toxins and may be useful to protect humans and/or animals against the adverse health effects of these compounds.

Genotoxic, mutagenic and cytotoxic effects of the commercial dye CI Disperse Blue 291 in the human hepatic cell line HepG2.

Textile dyes are discarded into the aquatic ecosystem via industrial effluents and potentially expose humans and local biota to adverse effects. The commercial dye CI Disperse Blue 291 which contains the aminoazobenzene 2-[(2-bromo-4,6-dinitrophenyl)azo]-5(diethylamino)-4-methoxyacetanilide (CAS registry no. 56548-64-2), was tested for genotoxicity and cytotoxicity in the human hepatoma cell line HepG2, using the comet assay, micronucleus (MN) test and a cell viability test. Five different concentrations of the test compound were examined: 200 microg/ml, 400 microg/ml, 600 microg/ml, 800 microg/ml and 1000 microg/ml. An increase in comet tail length and in the frequency of MN was detected with exposure of cells to concentrations of the commercial dye from 400 microg/ml. Furthermore, the dye was found to decrease cell viability. The results of this study demonstrate for the first time the genotoxic and mutagenic effects of the dye CI Disperse Blue 291 in mammalian cells, thus stressing the need to develop non-mutagenic dyes and to invest in improving the treatment of effluents. These measures will help to prevent harmful effects that these compounds can have on humans and aquatic organisms that come in contact with them.

Coffee consumption protects human lymphocytes against oxidative and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) induced DNA-damage: results of an experimental study with human volunteers.

Aim of the study was to investigate the impact of coffee on DNA-stability in humans. DNA-damage was monitored in lymphocytes of eight individuals with single cell gel electrophoresis assays before and after consumption of 600 ml coffee (400 ml paper filtered and 200 ml metal filtered/d) for five days. Under standard conditions, no alteration of DNA-migration was seen, but a strong reduction of DNA-migration attributable to endogenous formation of oxidised purines and pyrimidines was detected with restriction enzymes; furthermore DNA-damage caused by reactive oxygen radicals (H2O2 treatment) and by the heterocyclic aromatic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole-acetate was significantly reduced after coffee consumption by 17% and 35%, respectively. Also in in vitro experiments, inhibition of H2O2 induced DNA-damage was observed with coffee at low concentrations (

Monitoring, removal and risk assessment of cytostatic drugs in hospital wastewater.

Cytostatic agents are applied in cancer therapy and subsequently excreted into hospital wastewater. As these substances are known to be carcinogenic, mutagenic and toxic for reproduction, they should be removed from wastewater at their source of origin. In this study the fate and effects of the cancerostatic platinum compounds (CPC) cisplatin, carboplatin, oxaliplatin, 5-fluorouracil (5-FU) and the anthracyclines doxorubicin, daunorubicin and epirubicin were investigated in hospital wastewater. Wastewater from the in-patient treatment ward of a hospital in Vienna was collected and monitored for the occurrence of the selected drugs. A calculation model was established to spot the correlation between administered dosage and measured concentrations. To investigate the fate of the selected substances during wastewater treatment, the oncologic wastewater was treated in a pilot membrane bioreactor system (MBR) and in downstream advanced wastewater treatment processes (adsorption to activated carbon and UV-treatment). Genotoxic effects of the oncologic wastewater were assessed before and after wastewater treatment followed by a risk assessment. Monitoring concentrations of the selected cytostatics in the oncologic wastewater were in line with calculated concentrations. Due to different mechanisms (adsorption, biodegradation) in the MBR-system 5 - FU and the anthracyclines were removed < LOD, whereas CPC were removed by 60%. In parallel, genotoxic effects could be reduced significantly by the MBR-system. The risk for humans, the aquatic and terrestrial environment by hospital wastewater containing cytostatic drugs was classified as small in a preliminary risk assessment.

Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside.

A new synthetic drug, benzamide riboside (BR) exhibited strong oncolytic activity against leukemic cells in the 5-10 microM range. Higher BR-concentrations (20 microM) predominantly induced necrosis which correlated with DNA strand breaks and subsequent depletion of ATP- and dATP levels. Replenishment of the ATP pool by addition of adenosine prevented necrosis and favoured apoptosis. This effect was not a pecularity of BR-treatment, but was reproduced with high concentrations of all trans-retinoic acid (120 microM) and cyanide (20 mM). Glucose was also capable to suppress necrosis and to favour apoptosis of HL-60 cells, which had been treated with necrotic doses of BR and cyanide. Apoptosis eliminates unwanted cells without affecting the microenvironment, whereas necrosis causes severe inflammation of surrounding tissues due to spillage of cell fluids into the peri-cellular space. Thus, the monitoring and maintenance of cellular energy pools during therapeutic drug treatment may help to minimize nonspecific side effects and to improve attempted drug effects.

Coffee diterpenes prevent the genotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and N-nitrosodimethylamine in a human derived liver cell line (HepG2).

Aim of the present experiments was to study the genotoxic effects of coffee diterpenoids, namely cafestol palmitate and a mix of cafestol and kahweol (C+K) in human derived hepatoma (HepG2) cells. Furthermore, we investigated the potential protective properties of these substances towards carcinogens contained in the human diet, namely N-nitrosodimethylamine (NDMA) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). C+K and cafestol palmitate were tested over a broad dose range in micronucleus (MN) assays and no indication for genotoxic effects was seen. In combination experiments with PhIP (300 microM), pronounced inhibition (approximately 1.7-fold) of MN formation was observed with C+K and cafestol palmitate at dose levels > or = 0.9 and 1.7 microg/ml, respectively. Enzyme measurements indicate that the protection is due to inhibition of sulfotransferase, an enzyme involved in the activation of the amine, and/or to induction of UDP-glucuronosyltransferase which detoxifies the DNA-reactive metabolites of PhIP. Furthermore, a significant increase of glutathione-S-transferase was seen, whereas the activities of cytochrome P-450 1A1 and N-acetyltransferase 1 were not significantly altered. Also in combination experiments with C+K and NDMA, strong protective effects (50% reduction of genotoxicity) were seen at low dose levels (> or = 0.3 microg/ml). Since inhibition of MN was also observed when C+K were added after incubation with NDMA, it is likely that the chemoprotective effects are due to induction of DNA repair enzymes. Comparison of data on the effects of C+K on the cholesterol metabolism, which was investigated in earlier in vivo studies, with the present findings suggests that DNA-protective effects take place at exposure levels which are substantially lower than those which cause hypercholesterolemia.

Studies on the metabolic activation of diethanolnitrosamine in animal-mediated and in vitro assays using Escherichia coli K-12 343/113 as an indicator.

The mutagenic activity of diethanolnitrosamine (NDELA), a carcinogenic compound which leads to inconsistent results in standard in vitro procedures was tested in vitro and in animal-mediated assays with the indicator strain Escherichia coli (E. coli) K-12 343/113. This strain allows the simultaneous detection of forward and back mutations arising in several genes of the E. coli chromosome. In animal-mediated assays in which mice were used as hosts for i.v. injected E. coli indicator cells, s.c. application of NDELA induced a dose dependent increase of galactose fermenting mutants in cells recovered from the livers of animals exposed for 3 h to the mutagen. Comparison with results obtained with diethylnitrosamine (DENA) in the same test system revealed that the two compounds apparently cause different types of mutagenic lesions. Induction of arg+ mutations by DENA and several other aliphatic nitrosamines is mainly due to base pair substitutions, whereas NDELA is rather mutagenic in the galRs system. This latter system is, in addition, sensitive to frameshifts and deletions. These differences in mutagenic specificity suggest that NDELA and DENA, although structurally closely related, are activated via different molecular mechanisms. In fact, evidence is accumulating that alcohol dehydrogenase (ADH) could be involved in the activation of NDELA. On the other hand, the effective mutagenesis of NDELA obtained in vitro with E. coli upon addition of rat liver microsomal fraction would not be expected if ADH is involved in the activation since the S-9 Mix used in the present experiments was devoid of cofactors (NAD, NADP), necessary to accomplish oxidation by ADH. Therefore, further in vivo studies were performed, in which pyrazole, a potent blocker of ADH, was administered prior (1 and 24 h) to the injection of the mutagen. The observation that a dose dependent increase of mutants in the liver (and to a lower extent in the spleens) of treated animals takes place under conditions in which ADH activity is blocked, whereas several microsomal enzymes are stimulated, indicated that besides oxidation of NDELA by ADH other metabolic activation pathways are involved. Apparently enzymes contained in the liver homogenate, possibly NADPH dependent enzymes of the microsomal ethanol oxidizing system, play an important role in the formation of mutagenic metabolites of NDELA.

Mutation spectra of Glu-P-1 in Salmonella: induction of hotspot frameshifts and site-specific base substitutions.

We used colony probe hybridization and PCR/DNA sequence analysis to determine the mutations in approximately 1,640 revertants of the -1 frameshift allele hisD3052 and approximately 260 revertants of the base substitution allele hisG46 of Salmonella typhimurium induced by the heterocyclic amine cooked food mutagen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). All of the mutations were at sites containing guanine, which is the base at which Glu-P-1 forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100% of the Glu-P-1-induced mutations at the frameshift allele in strains TA1978 (uvr+) and TA1538 (delta uvrB) and 99% in TA98 (delta uvrB, pKM101). To explain the induction of these hotspot mutations by Glu-P-1, we describe here a more detailed version of our recently proposed correct incorporation/slippage model [Genetics:136:731, 1994]. We propose that after cytosine is incorporated correctly opposite a Glu-P-1-adducted guanine, various slipped intermediates may form (a total of 18), depending on which guanine is adducted and whether it remains within the helix or becomes extrahelical. This variety of mutational pathways may account for the high mutability of the hotspot sequence by Glu-P-1. Although the pKM101 plasmid does not influence the mutagenic potency or mutational spectrum of Glu-P-1 at the frameshift allele, it is required by Glu-P-1 to revert the base substitution allele, where Glu-P-1 induces G-C --> T-A transversions (75%) and G-C --> tA-T transitions (25%) exclusively at a single site (the second position of the CCC codon of the hisG46 allele). The limited (20-30 times less) base substitution mutagenic potency of Glu-P-1 relative to its frameshift mutagenic potency as well as the extreme site specificity exhibited by Glu-P-1 for base substitutions may have bearing on the lack of base substitutions identified in ras genes in Glu-P-1-induced rat colon tumors.

Studies on the antimutagenic activities of garlic extract.

Experiments with Salmonella tester strains indicated that aqueous garlic extract possesses antimutagenic properties toward ionizing radiation, peroxides, adriamycin, and N-methyl-N'-nitro-nitrosoguanidine. The assumption that radical scavenging garlic constituents, i.e., molecules with sulfur moieties, might be responsible for the inhibitory effect of aqueous extract toward mutagenesis induced by radiation and radiomimetic compounds was confirmed by the results of subsequent experiments; 1) garlic extract attenuated the lethal effects of gamma-rays on repair-deficient E. coli strains; 2) the garlic constituent allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is partly responsible for the reduced radiation-induced mutagenesis in Salmonella typhimurium TA 102. No such inhibitory effects were detected with alliin (S-allyl-L-cysteine sulfoxide) or cysteine; 3) aqueous garlic extract inhibited hydrogen-peroxide-induced lipid peroxidation. Results obtained in preliminary experiments with Chinese hamster ovary cells suggest that the antimutagenic properties of garlic extract are not restricted to procaryotic cells.

Induction of genotoxic effects by chlorohydroxyfuranones, byproducts of water disinfection, in E. coli K-12 cells recovered from various organs of mice.

The genotoxic effects of three chlorohydroxyfuranones (CHFs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), 3-chloro-4-(chloromethyl)-5-hydroxy-2[5H]furanone (CMCF) and 3,4,-dichloro-5-hydroxy-2[5H]furanone (MCA), which are formed as byproducts of water disinfection with chlorine, were investigated in bacterial differential DNA repair assays in vitro and in animal-mediated assays in vivo. As indicators of DNA damage, E. coli K-12 strains were used that differ in their repair capacity (uvrB/recA vs. uvr+/rec+). Liquid incubation of the compounds without metabolic activation caused a pronounced reduction of the viability of the repair-deficient strain relative to the repair-proficient wild-type strain. The order of potency of genotoxic activity in vitro (dose range 0.004-10 micrograms/ml) was MX > CMCF > MCA. Addition of mouse S-9 mix or bovine serum albumin to the incubation mixtures resulted in an almost complete loss of the activity of all three test compounds. In the animal-mediated assays, mixtures of the indicator bacteria were injected intravenously into mice which were subsequently treated with the test compounds (200 mg/kg b.w.). Two hours later, the cells were recovered from various organs and the relative survival frequencies determined. Under these conditions, all three compounds caused pronounced genotoxic effects, MX and CMCF being stronger genotoxins than MCA. The strongest effects were consistently found in the gastrointestinal tract, but statistically significant DNA damage was also observed in indicator cells recovered from lungs, liver, spleen and kidneys. In a further experiment, the effects of lower doses of MX (4.3, 13 and 40 mg/kg) were investigated. In these experiments dose-dependent effects were measured in all organs. CMCF and MA caused only marginal effects at 40 mg/kg except in the stomach where approximately a 50% reduction of relative survival frequency was observed with CMCF. The results of these animal-mediated assays indicate that (i) all three CHFs cause genotoxic effects in the living animal, and (ii) the potencies of the three compounds observed under in vivo conditions are not commensurate with their extremely high activities measured in vitro. One possible explanation for the weaker responses observed in the animal-mediated assays might be that CHFs are inactivated by non-specific protein binding.

Genotoxic effects of heavy metals: comparative investigation with plant bioassays.

The potential use of micronucleus assays in plants for the detection of genotoxic effects of heavy-metal ions was investigated. Three different plant systems were comparatively investigated in micronucleus tests with Tradescantia pollen mother cells (Trad MCN) and micronucleus tests with meristematic root tip cells of Allium cepa and Vicia faba (Allium/ Vicia MCN). As3+, Pb2+, Cd2+, Zn2+ caused a dose-dependent increase of MCN frequencies in all three test systems. Cu2+ gave consistently negative responses in all three tests; Zn2+ caused only a moderate, statistically not significant increase of MCN frequencies in Vicia. The ranking of genotoxic potencies in all three tests was in the descending order: As3+ > Pb2+ > Cd2+ > Zn2+ Cu2+. In experiments with Tradescantia, induction of MCN was observed in a concentration range between 1 and 10 mM, whereas in tests with root tip cells, higher concentrations (10-1,000 mM) were required to show significant effects. Further increase of the exposure levels caused toxic effects (reduction of root growth), cell division delays, and a decrease of MCN frequencies. Comparisons by linear regression analyses indicated that the sensitivity of the three bioassays for heavy metals decreases in the order: Trad MCN > Vicia root MCN > Allium root MCN. In further experimental series, a soil sample which contained high concentrations of the five metals and a control soil were investigated. Aqueous soil extracts induced only weak effects in Trad MCN tests and no effects in the root tip assays, whereas cultivation of the plants in the soils resulted in a pronounced induction of MCN in the Tradescantia system and moderate effects in Vicia and Allium. In conclusion, the results of the study indicate that the Trad MCN assay detects the genotoxic effects of heavy metals and can be used for biomonitoring metal-contaminated soils.

Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants; current state of knowledge.

This article gives an overview of the results of genotoxicity tests, which have been conducted within the last 5 years with the human liver cell line HepG2. It is an update of an earlier review from 1998 (by Knasmüller et al.). In addition, a number of publications are discussed which are relevant for the use of human derived liver cell lines in genetic toxicology. They concern the establishment of new endpoints, the development of new cell lines and possible pitfalls and problems. HepG2 cells have been used to test a wide variety of compounds over the last years. The most interesting observations are that the cells are highly sensitive toward polycyclic aromatic hydrocarbons and that genotoxic effects are seen with a number of carcinogenic mycotoxins, that give negative results in other in vitro assays. Carcinogenic metals such as As and Cd caused positive results as well, whereas only marginal or negative results were seen with nitrosamines. The low sensitivity toward these latter carcinogens is probably due to a lack of cytochrome P4502E1 which catalyses their activation. Also, a number of structurally different synthetic pesticides as well as bioactive plant constituents ("natural pesticides") have been tested and with some of them genotoxic effects were found. In most experiments, the formation of micronuclei was used as an endpoint; however also the single cell gel electrophoresis assay is increasingly used. Several transfectant lines of HepG2 have been constructed which express increased levels of phase I enzymes (such as CYP1A1, CYP1A2, CYP2E1 etc.); furthermore, cell lines became available which express human glutathione-S-transferases. These new clones might be particularly useful for the investigation of specific classes of genotoxicants and also for mechanistic studies. Apart from HepG2 cells, a number of other human derived liver cell lines have been isolated, but so far no data from genotoxicity experiments are available, except for Hep3B cells, which were compared with HepG2 and found to be less sensitive in general. Studies with HepG2 clones of a different origin indicate that the cells differ in regard to their sensitivity toward genotoxicants; also medium effects and the cultivation time might affect the outcome of genotoxicity studies. Overall, the results support the assumption that HepG2 cells are a suitable tool for genotoxicity testing.

Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection.

This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AalphaC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm x 2 mm, 4 microm and LiChrospher 60 RP-select B, 250 mm x 4 mm, 5 microm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and--after isolation of the analytes by several clean-up steps--for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.

Investigation of the genotoxic effects of 2-amino-9H-pyrido[2,3-b]indole in different organs of rodents and in human derived cells.

Aim of the present study was the investigation of the genotoxicity of amino-alpha-carboline (AalphaC) in human derived cells and of its organ-specific effects in laboratory rodents. This heterocyclic amine (HA) is contained in fried meat and fish in higher concentrations than most other cooked food mutagens. In the present experiments, AalphaC caused dose-dependent induction of micronuclei in the human derived hepatoma cell line HepG2 at concentrations > or =50 microM. In contrast, no significant effects were seen in Hep3B, another human hepatoma cell line, which may be explained by the concurrent lower activity of sulfotransferase (SULT), an enzyme playing a key role in the activation of AalphaC. A positive result was also obtained in the single cell gel electrophoresis (SCGE) assay in peripheral human lymphocytes, but the effect was only significant at the highest concentration (1000 microM). In Fischer F344 rats and ICR mice, the liver was the main target organ for the formation of DNA adducts (at > or =50 mg/kg bw), and in lungs and colon substantially lower levels were detected. Identical organ specificity as in the DNA adduct measurements was seen in SCGE assays with rats, whereas in mice the most pronounced induction of DNA migration was observed in the colon. Comparison of our results with data from earlier experiments indicate that the genotoxic potency of AalphaC is equal to that of other HAs, which are contained in human foods in much smaller amounts. Therefore, our findings can be taken as an indication that the human health risk caused by exposure to AalphaC is higher than that of other HAs that are formed during the cooking of meat and fish.

Effect of intestinal microfloras from vegetarians and meat eaters on the genotoxicity of 2-amino-3-methylimidazo[4,5-f]quinoline, a carcinogenic heterocyclic amine.

Aim of this study was to investigate the impact of intestinal microfloras from vegetarians and non-vegetarians on the DNA-damaging activity of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), a carcinogenic heterocyclic amine that is found in fried meats. Floras from four vegetarians (Seventh Day Adventists) and from four individuals who consumed high amounts of meats were collected and inoculated into germfree F344 rats. The rats were kept on isocaloric diets that either contained animal derived protein and fat (meat consumers group) or proteins and fat of plant origin (vegetarian groups). IQ (90 mg/kg bw) was administered orally, after 4 h the extent of DNA-damage in colon and liver cells was determined in single cell gel electrophoresis assays. In all groups, the IQ induced DNA-migration was in the liver substantially higher than in the colon. In animals harbouring floras of vegetarians, the extent of damage was in both organs significantly (69.2% in the liver, P<0.016 and 64.7%, P<0.042 in the colon, respectively) lower than in the meat consumer groups. Our findings show that diet related differences in the microfloras have a strong impact on the genotoxic effects of IQ and suggest that heterocyclic amines are less genotoxic and carcinogenic in individuals that consume mainly plant derived foods.

Genotoxic effects of allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC).

Two isothiocyanates (ITCs) commonly found in human diet, allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC), were tested for genotoxic effects in a battery of assays: Salmonella/microsome assay with TA 98 and TA 100, differential DNA repair assay with E. coli and micronucleus (MN) induction assay with human derived Hep G2 cells. Albeit to a different degree, both ITCs induced genotoxic effects in all test systems. AITC was more genotoxic in bacterial test systems than in Hep G2 cells; in contrast, the effect of PEITC was stronger in Hep G2 cells. In in vivo assays with E. coli indicators in which mice were exposed to relatively high doses of the compounds (90 and 270 mg/kg), AITC induced moderate but significant effects; PEITC failed to induce significant effects in any of the organs. To find out the reason for the weak genotoxicity of AITC and PEITC under in vivo test conditions, we exposed E. coli indicator cells to the test substances in the absence or presence of rat liver homogenate (with and without cofactors), bovine serum albumin (BSA) and human saliva. All of them markedly attenuated the genotoxicity of AITC and PEITC, implying that the test substances are detoxified by direct non-enzymatic binding to proteins. Additional experiments carried out on the mechanistic aspects of AITC and PEITC-induced genotoxicity showed that the compounds induce the formation of thiobarbituric acid reactive substances (TBARS) in Hep G2 cells. Furthermore, in in vitro assays with E. coli, radical scavengers reduced the differential DNA damage induced by AITC and PEITC. The latter two findings give a clue that reactive oxygen species might be involved in the genotoxic effect of the ITCs. Although ITCs have been repeatedly advocated as very promising anticancer agents, the data presented here indicate that the compounds are genotoxic, and probably carcinogenic, in their own right.

On the distribution of genotoxic factors in various organs of mice treated with cycasin.

The distribution of genotoxic factors in various organs of mice treated orally with methylazoxymethanol-beta-D-glycoside (cycasin) was investigated using the DNA-repair host mediated assay. Indicator of genotoxic activity was a pair of streptomycin dependent Escherichia coli strains differing vastly in DNA repair capacity; uvrB/recA vs. uvr+/rec+. The animal-mediated assays were performed by injecting mixtures of the two strains i.v. and orally into mice, which were subsequently treated with the test chemical and from which the differential survival of the indicator bacteria present in several organs was determined. The same strains and selection procedures were also used for assessing the DNA-damaging activity in vitro. In the animal-mediated assays in which cycasin was applied orally, significant effects were observed at doses of 100 and 500 mg/kg body weight. The organ distribution of genotoxic factors in the host animal was as follows: the highest genotoxic activity was observed in the liver, followed by intestine and stomach; a clear effect was also observed in the kidneys and, to a lower extent, in the blood stream and in the lungs at the highest dose administered (500 mg/kg body weight). Under in vitro conditions a marginal genotoxic effect was observed even in the absence of liver homogenate, indicating that the test compound is possible activated (hydrolysed) by the E. coli cells. Therefore the genotoxic activity of cycasin observed in the gastrointestinal tract was not unexpected, since the substance was applied orally, thereby exposing the indicator bacteria in these organs to high levels of unmetabolised compound, especially in the stomach. In the intestine members of the microbial flora probably contribute to the metabolic activation of the test compound. The occurrence of genotoxic factors remote from the gastrointestinal tract shows that the present compound or active metabolites thereof penetrate through the intestinal barrier. The extraordinarily high genotoxic activity observed in the liver suggests that the compound is additionally activated in this organ. In compliance with previous in vitro findings this second activation step might lead to the formation of the highly reactive aldehydic form of methylazoxymethanol (MAMAL) mediated by dehydrogenases. Comparison with carcinogenicity studies indicates a good correlation between the distribution of genotoxic effects as determined in the present studies and the localisation of tumors in various organs of rodents treated with cycasin.