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PubMed2006

Genotoxicity and carcinogenicity studies of antihypertensive agents.

This survey is a compendium of genotoxicity and carcinogenicity information of antihypertensive drugs. Data from 164 marketed drugs were collected. Of the 164 drugs, 65 (39.6%) had no retrievable genotoxicity or carcinogenicity data; this group was comprised largely of drugs marketed in a limited number of countries. The remaining 99 (60.4%) had at least one genotoxicity or carcinogenicity test result. Of these 99, 48 (48.5%) had at least one positive finding: 32 tested positive in at least one genotoxicity assay, 26 in at least one carcinogenicity assay, and 10 gave a positive result in both at least one genotoxicity assay and at least one carcinogenicity assay. In terms of correlation between results of the various genotoxicity assays and absence of carcinogenic activity in both mice and rats 2 of 44 non-carcinogenic drugs tested positive in the in vitro bacterial mutagenesis assay, 2 of 9 tested positive in the mouse lymphoma assay, none of 14 tested positive for gene mutation at the hprt locus, 5 of 25 tested positive in in vitro cytogenetic assays, none of 31 in in vivo cytogenetic assays, and none of 14 in inducing DNA damage and/or repair in in vitro and/or in vivo assays. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, 75 drugs had both genotoxicity and carcinogenicity data; of these 37 (49.3%) were neither genotoxic nor carcinogenic, 14 (18.7%) were non-carcinogens which tested positive in at least one genotoxicity assay, 14 (18.7%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, and 10 (13.3%) were both genotoxic and carcinogenic. Only 42 of the 164 marketed antihypertensives (25.6%) had all data required by the guidelines for testing of pharmaceuticals.

Animals↗

PubMed1997

Detection of genotoxic substances in bivalve molluscs from the Saguenay Fjord (Canada), using the SOS chromotest.

Few studies have employed bioassays to investigate the accumulation of genotoxins in aquatic biota that inhabit areas contaminated with industrial and municipal wastes. This study employed the SOS Chromotest, a short-term bacterial genotoxicity assay, to investigate the presence of genotoxins in bivalve molluscs from the Saguenay Fjord (Canada). Genotoxicity analyses were performed on dichloromethane extracts of Mya arenaria and Mytilus edulis collected downstream from several aluminum refineries and forestry products industries known to produce and release genotoxic substances. The results confirmed that bivalve molluscs inhabiting downstream regions are contaminated with both direct-acting and pro-genotoxic substances. In several cases, SOS response induction factors exceeded 3.0. The results failed to reveal a clear downstream trend of decreasing genotoxicity with increasing distance from the presumed industrial sources(s). A significant relationship (r2 = 0.61, p < 0.007) between a demographic variable (population near shoreline) and lipid-corrected genotoxic potency suggest that the accumulated direct-acting genotoxins may be of municipal origin. Significant relationships between tissue extract genotoxicity (r2 = 0.75, p < 0.003) and tissue PAH contamination (r2 = 0.77, p < 0.0001) and drainage basin area suggests that the bivalves are accumulating airborne contaminants deposited on the surface of the relevant drainage basins. In spite of contamination with genotoxic PAHs, the addition of rat liver microsomal enzymes reduced the genotoxic potency of all samples investigated (31-94% decrease). The results also revealed a significant relationship between tissue extract genotoxicity and PAH concentration (r2 = 0.72, p < 0.0005). Further analyses confirmed that a variable portion (7-97%) of the S9-activated tissue extract genotoxicity can be attributed to the detected PAHs. Although the sources, identity and effects of genotoxins accumulated by bivalves of the Saguenay Fjord remain to be determined, the study has confirmed the utility of the SOS Chromotest in environmental monitoring of aquatic biota.

Animals↗

PubMed2002

Mechanisms of genotoxicity of particles and fibers.

With regard to genotoxicity testing and cancer risk assessment, particles and fibers form a rather specific group among all toxicants. First, the physicochemical behavior of fibrous and nonfibrous particles is usually very different from that of nonparticulate, chemical carcinogens. Reactive oxygen species (ROS) are believed to play a major role in primary genotoxicity of particles, which may derive from their surface properties, the presence of transition metals, intracellular iron mobilization, and lipid peroxidation. Other aspects relevant to primary genotoxicity are particle size, shape, crystallinity (e.g., silica), and solubility, and may also include particle uptake, interaction with cell division machinery (e.g., asbestos), and the presence of mutagens carried with the particle (e.g., diesel exhaust particles, DEP). Excessive and persistent formation of ROS from inflammatory cells is considered as the hallmark of the secondary genotoxicity of nonfibrous and fibrous particles. Since lung inflammation is known to occur and persist only at sufficient particle dose, this secondary pathway is considered to contain a threshold (Greim et al., 2001). Identification of (mechanisms of) particle genotoxicity has been/can be achieved via (1) acellular assays, (2) in vitro tests, (3) in vivo studies, usually in mice or rats, and finally (4) biomarker studies in humans with (occupational) exposure. The significance of acellular assays and biomarker studies for risk assessment is limited, but has provided some mechanistic insights (e.g., in oxidant generating properties of quartz and asbestos) and may also contribute to hazard identification. In vitro studies have lead to identification of primary genotoxic properties of particles, whereas recent in vivo studies provide further support for the correlation between particle-induced lung inflammation and secondary genotoxicity. Proper risk assessment of particles necessitates identification of the relative impact of primary versus secondary genotoxicity in realistic exposure conditions. However, since it is impossible to discern between primary and secondary genotoxicity with current in vivo tests, concomitant in vitro assays are required to determine primary genotoxicity. In vivo tests should ideally be designed using different doses to allow dose-effect analysis for both inflammation and genotoxicity.

Animals↗

PubMed1992

Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation and recombination in wing cells of Drosophila melanogaster.

Sixteen pyrrolizidine alkaloids (PAs) were examined for their genotoxic potency in the wing spot test of Drosophila melanogaster following oral application. This in vivo assay tests for the induction of somatic mutation and mitotic recombination in cells of the developing wing primordia. All PAs tested except the C9-monoester supinine were clearly genotoxic. Depending on their chemical structure, however, genotoxicity of the PAs varied widely in a range encompassing about three orders of magnitude. In general, macrocyclic diester-type PAs were the most and 7-hydroxy C9-monoester types the least genotoxic representatives studied, while open diesters were intermediate in this respect. Stereoisomeric PAs mostly showed similar, but sometimes also clearly unequal genotoxicity. An increasing number of hydroxy groups in the PA molecule seemed to reduce its genotoxic potency. With respect to the structure/activity relationships, there appears to be a good correlation between hepatotoxicity of PAs in experimental rodents and genotoxicity in the wing spot test of Drosophila. This suggests that PAs are bioactivated along similar pathways in the mammalian liver and in the somatic cells of Drosophila. The genotoxic potential of PAs in the Drosophila wing spot test and their carcinogenic potential in mammals also seem correlated, although the information in the literature on carcinogenicity of the non-macrocyclic PAs with moderate to low genotoxic potency is concededly limited. Comparisons with other genotoxicity tests suggest that the wing spot test is particularly suitable for genotoxins like PAs, on the one hand because of the versatile metabolic bioactivation system of Drosophila and on the other hand also because of its excellent sensitivity to the crosslinking agents among the genotoxins.

Animals↗

PubMed1986

Use of the DNA-repair host-mediated assay for determining the organ distribution of genotoxic factors in mice treated orally with nitro-aromatic compounds.

The distribution of genotoxic factors in various organs of mice treated orally with nitro-aromatic compounds of actual or potential use as chemotherapeutic (antiprotozoal and anthelminthical) agents was investigated in the DNA-repair host-mediated assay, with mice as host animals and a pair of E. coli K12 strains differing in DNA-repair capacity as indicators of genotoxicity. The test substances were derivatives of nitroimidazole (metronidazole), nitrofuran (SQ 18 506) and nitrodiphenylamine (amoscanate). Animal-mediated assays were performed by injecting mixtures of the two E. coli strains both intravenously and orally into mice, which were subsequently treated with the test chemicals, and from which the differential survival of indicator bacteria present in liver, lungs, spleen, kidneys, stomach, small intestine, colon and the blood stream was determined on selective agar medium. The same strains and selection procedures were used for assessing the genotoxic activity of the compounds in vitro. All three compounds displayed genotoxic activity in vitro, the order of potency on the basis of exposure concentration being SQ 18 506 greater than metronidazole greater than amoscanate. In the animal-mediated assays the same ranking order of genotoxic activity was observed, but the exposure levels required to produce significant genotoxic effects in vivo were (substantially) higher than in the in vitro tests: SQ 18 506 was active at 0.1 mg/kg body weight, metronidazole at 4 mg/kg, and amoscanate at dosages higher than 10 mg/kg. In host-mediated assays the highest genotoxic activity for all three chemicals was observed in organs of the gastro-intestinal tract (usually in the stomach). All three chemicals also induced genotoxic effects in organs remote from the gastro-intestinal tract although with substantially lower activity, the order of potency being again SQ 18 506 greater than metronidazole greater than amoscanate. In the case of SQ 18 506 and metronidazole, dose-dependent genotoxic activities were observed in liver, spleen, lungs, kidneys and the blood stream, with no clear indication of a preferential target or non-target organ, while the minor genotoxic effects of amoscanate were restricted to bacteria present in the blood stream. This can be taken as an indication that the substances (or active metabolites thereof) have been transported from the intestinal tract into the blood stream and distributed evenly in organ tissues, without an indication of organ specific deactivation during the time periods (less than 180 min) presently investigated.(ABSTRACT TRUNCATED AT 400 WORDS)

5-Amino-3-((5-nitro-2-furyl)vinyl)-1,2,4-oxadiazol↗

PubMed2000

Human biological relevance and the use of threshold-arguments in regulatory genotoxicity assessment: experience with pharmaceuticals.

Issues of biological relevance and thresholds for genotoxicity are discussed here based upon the background of experience with the submissions for the approval of new pharmaceuticals to the German regulatory authority over the period between 1990 and 1997. This experience shows that out of the genotoxicity test systems which are required according to existing guidelines in the European Union (EU), the in vitro tests for chromosomal aberrations (CA) and the mouse lymphoma tk assays (MLA) yield a rate of positives that is about four-fold higher than that of other genotoxicity tests. A detailed analysis of chemical and pharmacological classes of compounds and their effects in these systems reveals that in addition to direct DNA reactivity several mechanisms of indirect genotoxicity such as nucleoside analogue incorporation into DNA, interaction with microtubule assembly, topoisomerase inhibition and high levels of cytotoxicity are relevant. New pharmaceuticals, for which the latter mechanisms apply, often display threshold-like characteristics in their genotoxic effects in vitro or even in vivo in experimental animals. This casts doubt upon the relevance of positive in vitro test results for such compounds. However, the discussion of examples shows that it may not be easy to demonstrate the exact thresholded mechanism of genotoxicity in a given case. In particular, the demonstration of a coincidence of genotoxicity and high levels of cytotoxicity, which seems to be a major factor for biologically non-relevant in vitro positive new pharmaceuticals, usually requires quite extensive testing. Hence, for new pharmaceuticals it is practice to provide in addition to in vitro results that may be thresholded a wealth of information from in vivo studies on genotoxicity, carcinogenicity, metabolism, pharmacokinetics, etc. the results of which help in assessing the biological relevance of in vitro positives. The regulatory acknowledgement of biologically non-relevant, thresholded mechanisms of (in vitro) genotoxicity in addition to those that are considered relevant for human risk ensures a better understanding of test results and is needed for the credibility of genotoxicity testing practice in general.

Animals↗

PubMed1999

Genotoxicity testing of biotechnology-derived products. Report of a GUM task force. Gesellschaft für Umweltmutationsforschung.

Various aspects of genotoxicity testing of biotechnology-derived products are discussed based on information gathered from a questionnaire which was sent to about 30 predominantly European companies. Feedback was received from 13 companies on 78 compounds, mostly recombinant proteins but also on a number of nonrecombinant proteins, which had been assessed for genotoxicity in a total of 177 tests. Four of the 78 compounds appeared to elicit reproducible genotoxic effects. For one of these compounds, the activity could be related to a nonpeptidic linker molecule. No scientifically convincing rationale for the other three compounds could be established, although, at least for two compounds, their activity may be connected with the enzymatic/hormonal activity. In addition to the survey, published reports on genotoxicity testing of biotechnology products were reviewed. The data are discussed relative to whether genotoxicity testing is a valuable exercise when assessing potentially toxic liabilities of biotechnology-derived compounds. It is concluded that genotoxicity testing is generally inappropriate and unnecessary, a position which is in accordance with the available guidelines addressing this area. For the 'average' protein, electrophilic reactions are difficult to envision. Indirect reactions via DNA metabolism and growth regulation seem possible for only very specific proteins such as nucleases, growth factors, cytokines. No information on testing of different types of biotechnology-derived products (e.g., ribozymes, antisense-oligonucleotides, DNA vaccines) has been received in the questionnaires. Discussion of their potential to cause genotoxic changes was based on literature reports. Even for those products for which concerns of genotoxic/tumourigenic potential cannot be completely ruled out, e.g., because of their interaction with DNA metabolism or proliferation control, the performance of standard genotoxicity assays generally appears to be of little value. All information, including also information on the occurrence of genotoxic impurities, has been utilized to formulate a decision tree approach for the genotoxicity testing of biotechnology-derived products.

Animals↗

PubMed2006

Genotoxic risk identification of soil contamination at a major industrialized city in northeast China by a combination of in vitro and in vivo bioassays.

The present study evaluated the genotoxicity of field soils in the Tianjin area, one of the most industrialized contaminated areas in northeast China. The genotoxicity of organic extracts of 41 soils was assayed by an in vitro SOS/ umu bioassay with Salmonella typhimurium TA 1535/pSK 1002. From the 41 soil samples, 11 samples were selected to confirm the genotoxic effect by in vivo single-cell gel electrophoresis (comet assay) using earthworms (Eisenia fetida). The results obtained demonstrated that, in the in vitro assay, genotoxicity expressed as induction ratios (IR) ranged from 1.00 to 4.60, and in the in vivo assay, the genotoxicity expressed as tail moment (TM) varied from 14.6 to 57.8 microm. All samples with high genotoxicity assessed by the SOS/umu bioassay possessed significantly high genotoxic effects in the comet assay, and there was a correlation (R2 = 0.736, p < 0.05) between IR and TM in both bioassays. It is concluded that soils in the Tianjin area were seriously contaminated by organic genotoxicants and higher levels of genotoxic effects existed in soils in the urban area of Tianjin as well as in areas near the coastal towns in the northeast part of the city. It can be concluded that a combination of in vivo and in vitro bioassays as a powerful and efficient genotoxicity-assessing tool could facilitate the assessment of genotoxic risk at a regional scale.

Animals↗

PubMed2002

Mammalian cell cytotoxicity and genotoxicity analysis of drinking water disinfection by-products.

Cytotoxicity and genotoxicity assays were used to analyze drinking water disinfection by-products (DBPs) in Chinese hamster ovary (CHO) AS52 cells. The DBPs were chosen because they are common in drinking water, resulting from conventional disinfection using chlorination and chloramination. Data were also available to compare these results with cytotoxicity and mutagenicity studies in Salmonella typhimurium. The rank order in decreasing chronic cytotoxicity measured in a microplate-based assay was bromoacetic acid (BA) >> 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) > dibromoacetic acid (DBA) > chloroacetic acid (CA) > KBrO(3) > tribromoacetic acid (TBA) > EMS (ethylmethanesulfonate, positive control) > dichloroacetic acid (DCA) > trichloroacetic acid (TCA). The induction of DNA strand breaks by these agents was measured by alkaline single-cell gel electrophoresis (SCGE, comet assay) and the rank order in decreasing genotoxicity was BA >> MX > CA > DBA > TBA > EMS > KBrO(3), while DCA and TCA were refractory. BA was more cytotoxic (31x) and genotoxic (14x) than MX in CHO cells. BA was over 400x more genotoxic than potassium bromate. The brominated haloacetic acids (HAAs) were more cytotoxic and genotoxic than their chlorinated analogs. The HAAs expressed a statistically significant inverse relationship in CHO cell cytotoxicity and genotoxicity as a function of increased numbers of halogen atoms per molecule. A quantitative comparison was conducted with results from a previous study with cytotoxicity and mutagenicity in S. typhimurium. There was no correlation between chronic CHO cell and bacterial cell cytotoxicity. DBP-induced CHO cell cytotoxicity was not related to mutagenic potency in S. typhimurium. Cytotoxicity in CHO cells was statistically significant and highly correlated to CHO cell genotoxicity. Finally, we determined that the DBP genotoxic potency in CHO cells and the mutagenic potency in S. typhimurium were not related. This suggests that toxicity data in S. typhimurium did not quantitatively predict the toxic effects of DBPs in mammalian cell systems. The microplate CHO cell cytotoxicity and genotoxicity assays were well suited for the analysis of DBPs, especially when the quantity of test material is limited.

Animals↗

PubMed2004

Changes in toxicity and genotoxicity of industrial sewage sludge samples containing nitro- and amino-aromatic compounds following treatment in bioreactors with different oxygen regimes.

GOALS, SCOPE AND BACKGROUND: From 2005, deposition of organic waste will be banned in Sweden. Likewise, in Germany and Austria, similar bans are being planned, and further countries will probably follow. Thus, there is a need to develop new methods and to refine established techniques for sludge management in the whole of the European Union. For this end, there is also an urgent need for appropriate ecotoxicological approaches to elucidate and assess the hazard potential of sewage sludge. Therefore, the present study was designed to assess the capacity of various established sludge treatment methods using different oxygen regimes to degrade recalcitrant nitro-substituted organic compounds and reduce their toxicity. Sewage sludge samples from a wastewater treatment plant in Sweden (Cambrex Karlskoga AB, industrial area Björkborn) receiving wastewater from industries manufacturing pharmaceutical substances, chemical intermediates and explosives were processed with different sludge treatment methods. Among other treatment methods, bioreactors (for anaerobic and aerobic sludge treatment) were used. In the present investigation, a battery of in vitro bioassays was employed to compare the cytotoxic and genotoxic potentials of different fractions of sludge samples in order to elucidate whether the treatments were suitable to reduce the toxicity of the sludge. METHODS: In order to investigate the cytotoxicity of the extracts of treated and untreated sludge samples, the acute cytotoxicity test with the permanent cell line RTL-W1 was used. Genotoxicity was tested by means of the comet assay (single cell gel electrophoresis) with RTL-W1 cells, and mutagenicity was assessed with the Ames test using the Salmonella typhimurium strains TA98, TA98NR and TA100. Sludge toxicity was tested in different fractions of organic extracts produced by acetone and hexane extractions. The subsequent clean-up procedure (silica gel chromatography and elution with hexane and dichloromethane) resulted in two fractions, a lipophilic hexane-fraction and a semi-lipophilic dichloromethane-fraction. For the genotoxicity and mutagenicity tests, these fractions were reunited at equal ratios. RESULTS AND DISCUSSION: The acute cytotoxicity test with RTL-W1 cells revealed a high cytotoxic potential for the semi-lipophilic DM-fractions of all sludge samples with NR50 values (= effective concentration for 50% cell death in the neutral red test) from 8.9 up to 20 mg sludge d.w./ml medium. A low cytotoxic potential for the hexane fractions of the untreated sludge samples (NR50 400 to > 400 mg sludge d.w./ml medium) was observed, whereas the hexane fractions of the treated sludge samples showed elevated cytotoxicity increasing further with treatment in the bioreactors. The comet assay indicated that three out of eight of the reunited fractions had a significant genotoxic potential. Whereas the genotoxic potential of one sample treated anaerobically was very high with an induction factor of 11.6, a similar sample (taken from the same anaerobic reactor four months later) and one untreated sample showed lower potentials. The samples treated in another anaerobic bioreactor as well as the samples treated aerobically showed no genotoxic potential. Results indicate that aerobic treatment was basically adequate for reducing the genotoxicity of the sludge, whereas anaerobic treatment was only partly useful for reduction of genotoxicity. The Ames test revealed a very high mutagenic potential for the reunited fractions of the untreated sludge samples with strain TA98 (maximum induction factors (IFmax) up to 45) and a relatively high potential for one of the samples treated aerobically (S2, IFmax = 18 (TA98, S9-)), thus documenting the suitability of both anaerobic and aerobic treatments to reduce the mutagenicity of the samples, however, with the aerobic treatment being less effective. CONCLUSIONS: Overall, none of the microbiological treatments for wastewater sludge in bioreactors was found to be ideal for general toxicity reduction of the sludge samples. Whereas cytotoxicity of the sludge increased or levelled off in most cases following either treatment, genotoxicity both increased or decreased after anaerobic treatment, depending on the specific sample. However, mutagenicity could generally be reduced by anaerobic treatment and, to a lesser degree, by aerobic treatment. RECOMMENDATIONS AND PERSPECTIVES: The complex modification of the diverse damage potentials of sludge sample extracts by use of an in vitro biotest battery following treatment for toxicity reduction in bioreactors showed that considerations of different toxicological endpoints is essential for an adequate hazard assessment. Whereas in the case of cytotoxicity reduction, the reactors proved ineffective, mutagenicity could be reduced significantly at least in some cases in this case study.

Biological Assay↗

PubMed1996

Genotoxicity and DNA adduct formation of incense smoke condensates: comparison with environmental tobacco smoke condensates.

Indoor air pollution has now been recognized as a potentially important problem for public health, since people spend most of their day in closed environments. Incense burning is possibly associated with elevated risks of leukemia and brain tumor in children from the epidemiological studies. Thus, evaluation of the genotoxicity of smoke condensates from incense burning is needed. We examined the genotoxicity of incense smoke condensates (ISC) using the Ames test in S. typhimurium strains with different mutagenic specificity and level of metabolic enzyme, the SOS chromotest in E. coli PQ37, and sister chromatid exchange assay in Chinese hamster ovary cells (SCE/CHO). The genotoxicity of environmental tobacco smoke condensates (TSC) was also evaluated by the three assays to compare with the genotoxicity of ISC. ISC showed a positive response in TA98, but not in TA100. It suggested that ISC only contained frame shift mutagens. The mutagenicity of ISC in both strains of TA98NR with deficient nitroreductase and TA98/1,8-DNP6 with deficient O-acetyltransferase was markedly decreased compared to that in TA98 strain. However, the mutagenicity was enhanced in YG1024 with overexpression of O-acetyltransferase activity. Thus, nitroarenes seemed to be responsible in part for the mutagenicity of ISC. Interestingly, all of the four ISC and two TSC samples showed a dose-dependent genotoxic response in the SOS chromotest with E. coli PQ37 but a low SCE induction of those samples were observed in CHO cells. When the genotoxicity was analyzed based on the condensates per one gram of original samples, the genotoxicity of two TSC condensates in prokaryotic cells was higher than that of four ISC samples except for the genotoxicity of TSC-2 in TA98 strain. However, the genotoxicity of certain ISC in eukaryotic cells based on the SCE/CHO assay was higher than that of TSC. To compare the covalent binding of DNA reactive intermediates of ISC and TSC to S. typhimurium TA98, the DNA adducts were evaluated by the 32P-postlabeling method with butanol extraction version. Similar diagonal radioactive zone (DRZ) was observed between ISC and CSC. However, DNA adduct levels induced by TSC were much greater than that of ISC.

Acetyltransferases↗

PubMed1992

Non-genotoxic factors in the carcinogenetic process: problems of detection and hazard evaluation.

In the classical two-stage models of carcinogenesis, initiation has been usually related to a DNA-damage/gene-mutation event, while promotion has been related to the non-genotoxic effects of clonal expansion of preneoplastic cells and/or modulation of cell differentiation. It is now clear that the process of carcinogenesis is linked to more than one irreversible alteration in the genome. Likewise, we can envisage that non-genotoxic events can take place after perhaps 0, 1, 2 or more irreversible alterations in the genome. Initiating and promoting activities of a chemical can be considered clearly separated in theory, but in practice, the chemicals we work with only rarely will be purely of the genotoxic or non-genotoxic type. We will discuss an empirical approach to classify genotoxic or prevalently non-genotoxic chemical carcinogens. For prevalently non-genotoxic carcinogens we will analyze what fraction of them can be detected as promoters of in vivo rat liver carcinogenesis. We will analyze carcinogenic potency of genotoxic and non-genotoxic carcinogens.

Animals↗

PubMed2004

Genotoxicity of environmental air pollution in three European cities: Prague, Kosice and Sofia.

The genotoxic potential of extractable organic matter (EOM) associated with the respirable particulate matter (PM <10 microm) of atmospheric pollution has been determined in three European cities--Prague (Czech Republic, two monitoring sites, Libus and Smíchov), Kosice (Slovak Republic) and Sofia (Bulgaria) using the alkaline single-cell gel electrophoresis (the comet assay). The EOM samples were extracted by dichloromethane from ambient airborne particles collected daily (24 h intervals) during 3-month sampling periods in winter and summer seasons. The human metabolically competent cell line Hep G2 was used as a test system and benzo[a]pyrene (BaP), a known carcinogen, was applied as a positive control (internal standard) in each electrophoretic run. Two-hour exposure of Hep G2 cells to equivalent EOM concentrations ranging from 5 to 150 microg EOM/ml resulted in a linear dose-dependent increase of DNA migration (r > 0.9, P < 0.01). A less significant dose-response (r = 0.61) was only induced by the EOM sample from the locality Prague-Libus (PRG-LB) in the winter. Generally, a 1.5 to four-fold increase of DNA strand breaks over the background control level was determined in EOM-exposed cells. In order to compare the genotoxic potential of individual EOMs, a mathematical model was used to correct the 'real' data. No substantial location- or season-related differences were found in EOM genotoxicity (EOM microg/ml), except for the EOM sample from Sofia, collected in the summer. This EOM sample induced a nearly two-fold lower level of DNA damage in comparison with other EOMs. On the other hand, clear statistically significant location- and season-related differences (P < 0.001) in ambient air genotoxicity were determined when the EOM quantity per cubic meter of air (microg/m3) was taken into account. In that case, the genotoxicity of winter air pollution was six- to 10-fold higher in comparison with summer air. The air pollution genotoxicity in individual localities rose during the winter season in the order: PRG-LB < Kosice < Prague-Smíchov (PRG-SM) < Sofia, while during the summer season the highest ambient air genotoxicity was revealed in the locality Prague-Smíchov and approximately equal air pollution genotoxicity was determined among localities Prague-Libus, Kosice and Sofia (PRG-LB approximately Kosice approximately Sofia < PRG-SM). The greatest overall air pollution genotoxicity was determined in the locality Sofia during the winter season. In a time course study to evaluate the kinetics of DNA strand break rejoining it was shown that the level of DNA strand breaks in EOM-exposed cells has returned to near the background level within 24 h after the treatment.

Air Pollutants↗

PubMed2005

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity.

The performance of a battery of three of the most commonly used in vitro genotoxicity tests--Ames+mouse lymphoma assay (MLA)+in vitro micronucleus (MN) or chromosomal aberrations (CA) test--has been evaluated for its ability to discriminate rodent carcinogens and non-carcinogens, from a large database of over 700 chemicals compiled from the CPDB ("Gold"), NTP, IARC and other publications. We re-evaluated many (113 MLA and 30 CA) previously published genotoxicity results in order to categorise the performance of these assays using the response categories we established. The sensitivity of the three-test battery was high. Of the 553 carcinogens for which there were valid genotoxicity data, 93% of the rodent carcinogens evaluated in at least one assay gave positive results in at least one of the three tests. Combinations of two and three test systems had greater sensitivity than individual tests resulting in sensitivities of around 90% or more, depending on test combination. Only 19 carcinogens (out of 206 tested in all three tests, considering CA and MN as alternatives) gave consistently negative results in a full three-test battery. Most were either carcinogenic via a non-genotoxic mechanism (liver enzyme inducers, peroxisome proliferators, hormonal carcinogens) considered not necessarily relevant for humans, or were extremely weak (presumed) genotoxic carcinogens (e.g. N-nitrosodiphenylamine). Two carcinogens (5-chloro-o-toluidine, 1,1,2,2-tetrachloroethane) may have a genotoxic element to their carcinogenicity and may have been expected to produce positive results somewhere in the battery. We identified 183 chemicals that were non-carcinogenic after testing in both male and female rats and mice. There were genotoxicity data on 177 of these. The specificity of the Ames test was reasonable (73.9%), but all mammalian cell tests had very low specificity (i.e. below 45%), and this declined to extremely low levels in combinations of two and three test systems. When all three tests were performed, 75-95% of non-carcinogens gave positive (i.e. false positive) results in at least one test in the battery. The extremely low specificity highlights the importance of understanding the mechanism by which genotoxicity may be induced (whether it is relevant for the whole animal or human) and using weight of evidence approaches to assess the carcinogenic risk from a positive genotoxicity signal. It also highlights deficiencies in the current prediction from and understanding of such in vitro results for the in vivo situation. It may even signal the need for either a reassessment of the conditions and criteria for positive results (cytotoxicity, solubility, etc.) or the development and use of a completely new set of in vitro tests (e.g. mutation in transgenic cell lines, systems with inherent metabolic activity avoiding the use of S9, measurement of genetic changes in more cancer-relevant genes or hotspots of genes, etc.). It was very difficult to assess the performance of the in vitro MN test, particularly in combination with other assays, because the published database for this assay is relatively small at this time. The specificity values for the in vitro MN assay may improve if data from a larger proportion of the known non-carcinogens becomes available, and a larger published database of results with the MN assay is urgently needed if this test is to be appreciated for regulatory use. However, specificity levels of <50% will still be unacceptable. Despite these issues, by adopting a relative predictivity (RP) measure (ratio of real:false results), it was possible to establish that positive results in all three tests indicate the chemical is greater than three times more likely to be a rodent carcinogen than a non-carcinogen. Likewise, negative results in all three tests indicate the chemical is greater than two times more likely to be a rodent non-carcinogen than a carcinogen. This RP measure is considered a useful tool for industry to assess the likelihood of a chemical possessing carcinogenic potential from batteries of positive or negative results.

Animals↗

PubMed2006

Development of genotoxicity test procedures with Episkin, a reconstructed human skin model: towards new tools for in vitro risk assessment of dermally applied compounds?

Today reconstructed skin models that simulate human skin, such as Episkin, are widely used for safety or efficacy pre-screening. Moreover, they are of growing interest for regulatory purposes in the framework of alternatives to animal testing. In order to reduce and eventually replace results of in vivo genotoxicity testing with in vitro data, there is a need to develop new complementary biological models and methods with improved ability to predict genotoxic risk. This can be achieved if these new assays do take into account exposure conditions that are more relevant than in the current test systems. In an attempt to meet this challenge, two new applications using a human reconstructed skin model for in vitro genotoxicity assessment are proposed. The skin is the target organ for dermally exposed compounds or environmental stress. Although attempts have been made to develop genotoxicity test procedures in vivo on mouse skin, human reconstructed skin models have not been used for in vitro genotoxicity testing so far, although they present clear advantages over mouse skin for human risk prediction. This paper presents the results of the development of a specific protocol allowing to perform the comet assay, a genotoxicity test procedure, on reconstructed skin. The comet assay was conducted after treatment of Episkin with UV, Lomefloxacin and UV or 4-nitroquinoline-N-oxide (4NQO). Treatment with the sunscreen Mexoryl was able to reduce the extent of comet signal. A second approach to use reconstructed epidermis in genotoxicity assays is also proposed. Indeed, the skin is a biologically active barrier driving the response to exposure to chemical agents and their possible metabolites. A specific co-culture system (Figure 1) using Episkin to perform the regular micronucleus assay is presented. Micronucleus induction in L5178Y cells cultured underneath Episkin was assessed after treatment of the reconstructed epidermis with mitomycin C, cyclophosphamide or apigenin. This second way of using human reconstructed skin for genotoxicity testing aims at improving the relevance of exposure conditions in in vitro genotoxicity assays for dermally applied compounds.

4-Nitroquinoline-1-oxide↗

PubMed2006

Genotoxic activity of nitroarene-contaminated industrial sludge following large-scale treatment in aerated and non-aerated sacs.

An industrial sludge containing a complex mixture of nitroaromatic compounds was treated in industrial large-scale aerobic and anaerobic biodegradation processes, performed in compost sacs. The goal was to study changes in genotoxicity during the two different oxygen regimes using the umuC genotoxicity assay. The composting sac was actively aerated during 3 months and allowed to mature for another 3 months. The anaerobic sac was not aerated for 5 months and aerated during the last month in order to enhance degradation of remaining organic carbon. The sludge was obtained from the wastewater treatment plant at an industrial area in Karlskoga, Sweden. The biodegradation study was performed at a commercial waste treatment plant in Stockholm, according to the company routine procedure when treating household waste in sealed sacs. The material from the non-aerated system showed increased genotoxicity in the acetone-soluble fraction after treatment, as did the water-soluble fraction. The subsequent aeration period did not decrease the toxicity below the genotoxicity limit. The increase in the water-soluble genotoxic compounds may pose an environmental problem during secondary storage or use of sludge treated this way, since leakage of water-dissolved genotoxic compounds may occur. The composting process also generated genotoxicity, but this was restricted to acetone-soluble compounds, while the water-soluble compounds remained low in genotoxicity. The aerated process therefore seems more favorable in term of risk reduction of this industrial sludge, although it is necessary to optimize the aerated process in order to achieve non-toxic levels of potential genotoxic compounds extractable by organic solvents.

Bacteria↗

PubMed1986

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.

Animals↗

PubMed2001

Catechins are not major components responsible for anti-genotoxic effects of tea extracts against nitroarenes.

The anti-genotoxic properties of tea leaf extracts were examined in a Salmonella umu-test. Seven non-fermented teas (green tea), one semi-fermented tea (oolong tea), two fermented teas (black tea and Chinese pu er tea) and two other teas were examined for their anti-genotoxic abilities and for their catechins contents. This was to study the relationship between catechins contents and anti-genotoxic activity of various tea leaf extracts. All types of tea extracts showed more potent suppressive effects against umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK 1002 induced by four nitroarenes (1-nitropyrene, 2-nitrofluorene, 3-nitrofluoranthene and a mixture of 1,6- and 1,8-dinitropyrene) rather than 4-NQO, AF-2 and MNNG. The anti-genotoxic effect of 12 tea leaf extracts on 1-NP, 2-NF, 3-NF and DNP decreased in the order: oolong tea (semi-fermented tea)>black tea (fermented tea)>sencha (non-fermented tea, an ordinary grade green tea)>tocyucya (other tea)>Chinese pu er tea (fermented tea). The amount of catechins (EGC, C, EGCG, EC and ECG) in various teas in decreasing order was non-fermented tea>semi-fermented tea>fermented tea>other tea. A remarkable feature was the effectiveness of black tea and Chinese pu er tea in suppressing the genotoxicity induced by nitroarenes, in spite of the fact that these fermented teas do not have high catechins contents. Statistical analysis showed that no significant (P<0.01) correlation was found between the anti-genotoxicity of tea extracts against nitroarenes and the catechins contents in tea leaf extracts. In further experiment, fractionation of sencha extract by HPLC revealed that anti-genotoxicity of the peak fraction corresponding to catechins accounted for <10% of the total anti-genotoxic activity of sencha extract against for 1-nitropyrene. These results suggest that catechins are not major components responsible for the anti-genotoxic effects of tea leaf extracts against direct-acting nitroarenes.

Antimutagenic Agents↗