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.     

Methods for the spiral Salmonella mutagenicity assay including specialized applications

An automated approach to bacterial mutagenicity testing - the spiral Salmonella assay - was developed to simplify testing and to reduce the labor and materials required to generate dose-responsive mutagenicity information. This document provides the reader with an overview of the spiral assay and a discussion of its application for examining the mutagenic potential of pure compounds, complex environmental mixtures, and interactive effects. Guidelines for performing a routine spiral assay are presented, and alternative test methods intended to overcome a variety of technical difficulties (such as restricted sample availability, sample viscosity or volatility, etc.) are recommended. Methods for the computerized analysis of data and the interpretation of results are discussed.     

Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one (NTO)

The in vivo genotoxic activity of two inorganic lead compounds was studied in Drosophila melanogaster by measurement of two different genetic endpoints. We used the wing-spot test and the comet assay. The comet assay was conducted with larval haemocytes. The results from the wing-spot test showed that neither lead chloride, PbCl(2), nor lead nitrate, Pb(NO(3))(2), were able to induce significant increases in the frequency of mutant spots. In addition, the combined treatments with gamma-radiation and PbCl(2) or Pb(NO(3))(2) did not show significant variations in the frequency of the three categories of mutant spots recorded, compared with the frequency induced by gamma-radiation alone. This seems to indicate that the lead compounds tested do not interact with the repair of the genetic damage induced by ionizing radiation. When the lead compounds were evaluated in the in vivo comet assay with haemocytes, Pb(NO(3))(2) was effective in inducing significant increases of DNA damage with a direct dose-response pattern. These results confirm the usefulness of the comet assay with haemocytes as an in vivo model and support the assumption that there is a genotoxic risk associated with lead exposure.     

The comet assay as an indicator test for germ cell genotoxicity

The in vivo comet assay is a well-established genotoxicity test. It is currently mainly performed with somatic cells from different organs to detect a genotoxic activity of potential carcinogens. It is regarded as a useful test for follow-up testing of positive or equivocal in vitro test results and for the evaluation of local genotoxicity. However, the comet assay also has the potential to detect germ cell genotoxicity and may be used for demonstrating the ability of a substance or its metabolite(s) to directly interact with the genetic material of gonadal and/or germ cells. Such results are important for the classification of germ cell mutagens, e.g. in the context of the "Globally Harmonized System of Classification and Labelling of Chemicals" (GHS). This review summarizes and discusses available information on the use of the comet assay with germ cells and cells from the gonads in genetic toxicology. The literature contains results from in vitro studies, ex vivo studies and in vivo studies. With regard to the assessment of germ cell genotoxicity, only in vivo studies are relevant but the other kind of studies provided important information on various aspects of the methodology. Many comet assay studies with human sperm have been performed in the context of male infertility and assisted fertilization. The results of these studies are not reviewed in detail here but various aspects of the assay modifications used are discussed. Measuring DNA effects by the comet assay in sperm requires additional steps for chromatin decondensation. Many different modifications of the alkaline and the neutral comet assay are in use but a standard protocol has not been established yet. High and variable background levels of DNA effects were reported and there is still need for standardization and validation of the comet assay with sperm. Some human biomonitoring studies with human sperm were published, but it seems to be premature to use these data for hazard identification and classification of chemicals. In contrast, the standard alkaline in vivo comet assay can easily be adapted to investigations with cells from reproductive organs. Tests with cells from the gonads (testis and ovary) seem to be most appropriate and a promising tool for demonstrating that a test compound reaches the gonads and is able to interact with the genetic material of germ cells. However, studies to standardize and validate these methods are necessary before the comet assay can be usefully applied in risk assessment of germ cell mutagens.     

Use of the alkaline comet assay for the detection of transplacental genotoxins in newborn mice

Several lines of evidence show that in utero exposure to different toxicants has greater consequences than their exposure during adult life. This may be due to involvement of critical developmental stages, physiological immaturity and the long later-life span over which disease may initiate, develop and progress. The in vivo alkaline comet (single-cell gel electrophoresis) assay has been favoured by the scientific community for the evaluation of genotoxins. The objective of this study was to demonstrate the suitability of alkaline comet assay in detecting transplacental genotoxins using newborn mice. Here, we report the successful use of the comet assay in detecting multi-organ genotoxicity of known transplacental genotoxins in newborn mice. Three well known transplacental genotoxic agents, cyclophosphamide (CP), mitomycin-C (MMC) and zidovudine (AZT) were tested in pregnant Swiss mice. These compounds were administered in the late gestational period (16-20th days of pregnancy) and the comet assay was performed with lymphocytes, bone marrow, liver and kidney cells of newborn mice. Significant DNA damage was observed in all the tissues with tested transplacental genotoxins. The results of the comet assay were confirmed by the micronucleus (MN) assay of the peripheral blood of newborn mice. The results of this study provide sufficient evidence that the comet assay can be applied successfully for the detection of transplacental genotoxins in newborn mice.     

Establishment and Assessment of a New Human Embryonic Stem Cell‐Based Biomarker Assay for Developmental Toxicity Screening

A metabolic biomarker‐based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery‐phase detection of potential human developmental toxicants. In this study, metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure‐based biomarker assay using these metabolites, along with a cytotoxicity endpoint, was then developed using a 9‐point dose–response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity, an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy, but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity, 100% specificity). The assay had a high concordance (≥75%) with existing in vivo models, demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests.     

Considerations in the U.S. Environmental Protection Agency's testing approach for mutagenicity.

OPP: This paper provides the rationale and support for the decisions the OPP will make in requiring and reviewing mutagenicity information. The regulatory requirement for mutagenicity testing to support a pesticide registration is found in the 40 CFR Part 158. The guidance as to the specific mutagenicity testing to be performed is found in the OPP's Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals (referred to as the Subdivision F guideline). A revised Subdivision F guideline has been presented that becomes the current guidance for submitters of mutagenicity data to the OPP. The decision to revise the guideline was the result of close examination of the version published in 1982 and the desire to update the guidance based on developments since then and current state-of-the-science. After undergoing Agency and public scrutiny, the revised guideline is to be published in 1991. The revised guideline consists of an initial battery of tests (the Salmonella assay, an in vitro mammalian gene mutation assay and an in vivo cytogenetics assay which may be either a bone marrow assay for chromosomal aberrations or for micronuclei formation) that should provide an adequate initial assessment of the potential mutagenicity of a chemical. Follow-up testing to clarify results from the initial testing may be necessary. After this information as well as all other relevant information is obtained, a weight-of-evidence decision will be made about the possible mutagenicity concern a chemical may present. Testing to pursue qualitative and/or quantitative evidence for assessing heritable risk in relation to human beings will then be considered if a mutagenicity concern exists. This testing may range from tests for evidence of gonadal exposure to dominant lethal testing to quantitative tests such as the specific locus and heritable translocation assays. The mutagenicity assessment will be performed in accordance with the Agency's Mutagenicity Risk Assessment Guidelines. The mutagenicity data would also be used in the weight-of-evidence consideration for the potential carcinogenicity of a chemical in accordance with the Agency's Carcinogen Risk Assessment Guidelines. In instances where there are triggers for carcinogenicity testing, mutagenicity data may be used as one of the triggers after a consideration of available information. It is felt that the revised Subdivision F guideline will provide appropriate, and more specific, guidance concerning the OPP approach to mutagenicity testing for the registration of a pesticide. It also provides a clearer understanding of how the OPP will proceed with its evaluation and decision making concerning the potential heritable effects of a test chemical.(ABSTRACT TRUNCATED AT 400 WORDS)     

New approaches to estimating the mutagenic potential of chemicals

New developments in mutagenic risk assessment have appeared in the past few years. New methods have been developed such asin vitro micronucleus assay for chromosomal alterations, comet assay for primary DNA damage, use of transgenic animals to detectin vivo gene mutations, and fluorescent in situ hybridization method to detect aneuploidy. Other new methods will be developed in the few next years, including the use of DNA chips and the use of molecular biological methods. Several micromethods have been developed to test a great number of chemical compounds. New concepts have appeared concerning interpretation of data, and particularly of thresholds especially in the case of aneugens; in some cases metabolic or mechanistic thresholds were demonstrated. Genotoxic studies are best integrated into toxicological testing: for example, some genotoxicity tests can be integrated into subacute toxicology; interpretation of data includes metabolism; and toxicokinetic data relate to other toxicological studies. Conversely, genotoxicity data can be used to interpret toxicology studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

The comet assay in male reproductive toxicology

Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.     

Preclinical safety testing of biotechnology-derived pharmaceuticals

The unique and complex nature of biotechnology-derived pharmaceuticals has meant that it is often not possible to follow the conventional safety testing programs used for chemicals, and hence they are evaluated on a case-by-case basis. Nonclinical safety testing programs must be rationally designed with a strong scientific understanding of the product, including its method of manufacture, purity, sequence, structure, species specificity, pharmacological and immunological effects, and intended clinical use. This knowledge, coupled with a firm understanding of the regulatory requirements for particular product types, will ensure that the most sensitive and regulatory-compliant test systems are used to optimize the chances of gaining regulatory approval for clinical testing or marketing authorization in the shortest possible time frame.     

Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis.

Differentiating apoptosis from necrosis is a challenge in single cells and in parenchymal tissues. The techniques available, including in situ TUNEL (Terminal deoxyribonucleotide transferase-mediated dUTP-X Nick End-Labeling) staining, DNA ladder assay, and flow cytometry, suffer from low sensitivity or from a high false-positive rate. This study, using a Jurkat cell model, initially evaluated the specificity of the neutral comet assay and flow cytometry compared to the gold standard, electron microscopy, for detection of apoptosis and necrosis. Neutral comet assay distinguished apoptosis from necrosis in Jurkat cells, as evidenced by the increased comet score in apoptotic cells and the almost zero comet score in necrotic cells. These findings were consistent with those of electron microscopy and flow cytometry. Furthermore, using rats with burn or ischemia/reperfusion injury, well-established models of skeletal and cardiac muscle tissue apoptosis, respectively, we applied the comet assay to detect apoptosis in these muscles. Neutral comet assay was able to detect apoptotic changes in both models. In the muscle samples from rats with burn or ischemia-reperfusion injury, the comet score was higher than that of muscle samples from their respective controls. These studies confirm the consistency of the comet assay for detection of apoptosis in single cells and provide evidence for its applicability as an additional method to detect apoptosis in parenchymal cells.     

Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay

The present study investigated the use of whole blood from humans and rats directly for single-cell gel electrophoresis (comet) assay. As little as 20 microl of whole blood was sufficient for comet assay, and the comet images obtained from whole blood were not different from those obtained from isolated lymphocytes. The DNA remained intact up to 4 h at 4 degrees C after isolation and had no observable strand breakage, when whole blood was cryopreserved (at -80 degrees C) in 10% pre-cooled DMSO up to 60 days. To demonstrate that the whole-blood technique could be applied to in vivo studies, we injected rats with a known carcinogen Fe/NTA and measured DNA strand breaks in whole blood in comparison with isolated lymphocytes. We showed that Fe/NTA injection resulted in similar extent of DNA strand breakage in both whole blood and lymphocytes, indicating that whole-blood method can be used for in vivo genotoxic studies. One disadvantage of the whole-blood technique is that whole blood cannot be used for in vitro studies because of the interferences from red blood cell (RBC) components. However, this problem can be overcome by prior hemolysis of RBCs and a brief centrifugation to obtain white blood cells (WBCs), which can then be used for in vitro incubation with genotoxic compounds before comet assay. Overall, this whole-blood technique for comet assay is expected to provide a simple, rapid, and cost-effective alternative for the existing comet assay using isolated lymphocytes in situations such as when time and cost are limiting factors.     

Investigations on the effect of cigarette smoking in the comet assay

The comet assay (single-cell gel electrophoresis, SCG) is widely accepted as an in vitro and in vivo genotoxicity test. Because of its demonstrated ability to detect various kinds of DNA damage and its ease of application, the technique is being increasingly used in human biomonitoring. However, the assessment of small genotoxic effects as typically obtained in biomonitoring may be limited by the different sources of assay variability and the lack of an optimal protocol with high sensitivity. To better characterize the suitability of the comet assay for biomonitoring, we are performing a comprehensive investigation on blood samples from smokers and non-smokers. Because tobacco smoke is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds, smokers should be a suitable study group with relevant mutagen exposure. Here, we report our results for the first sample of 20 healthy male smokers and 20 healthy male non-smokers. Baseline and benzo[a]pyrene diolepoxide (BPDE)-induced effects were analysed by two investigators using two image analysis systems. The study was repeated within 4 months. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline and BPDE-induced DNA damage was comparatively analysed. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. None of these approaches revealed significant differences between smokers and non-smokers. Although more data is needed for a final conclusion, this study indicates some limitations of the comet assay with regard to the detection of DNA damage induced by environmental mutagens in peripheral blood cells.     

Investigations on the effect of cigarette smoking in the comet assay

The comet assay (single-cell gel electrophoresis, SCG) is widely accepted as an in vitro and in vivo genotoxicity test. Because of its demonstrated ability to detect various kinds of DNA damage and its ease of application, the technique is being increasingly used in human biomonitoring. However, the assessment of small genotoxic effects as typically obtained in biomonitoring may be limited by the different sources of assay variability and the lack of an optimal protocol with high sensitivity. To better characterize the suitability of the comet assay for biomonitoring, we are performing a comprehensive investigation on blood samples from smokers and non-smokers. Because tobacco smoke is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds, smokers should be a suitable study group with relevant mutagen exposure. Here, we report our results for the first sample of 20 healthy male smokers and 20 healthy male non-smokers. Baseline and benzo[a]pyrene diolepoxide (BPDE)-induced effects were analysed by two investigators using two image analysis systems. The study was repeated within 4 months. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline and BPDE-induced DNA damage was comparatively analysed. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. None of these approaches revealed significant differences between smokers and non-smokers. Although more data is needed for a final conclusion, this study indicates some limitations of the comet assay with regard to the detection of DNA damage induced by environmental mutagens in peripheral blood cells.     

Genotoxicity evaluation of sesamin and episesamin

Sesamin is a major lignan that is present in sesame seeds and oil. Sesamin is partially converted to its stereoisomer, episesamin, during the refining process of non-roasted sesame seed oil. We evaluated the genotoxicity of these substances through the following tests: a bacterial reverse mutation assay (Ames test), a chromosomal aberration test in cultured Chinese hamster lung cells (CHL/IU), a bone marrow micronucleus (MN) test in Crlj:CD1 (ICR) mice, and a comet assay using the liver of Sprague-Dawley (SD) rats. Episesamin showed negative results in the Ames test with and without S9 mix, in the in vitro chromosomal aberration test with and without S9 mix, and in the in vivo comet assay. Sesamin showed negative results in the Ames test with and without S9 mix. In the in vitro chromosomal aberration test, sesamin did not induce chromosomal aberrations in the absence of S9 mix, but induced structural abnormalities at cytotoxic concentrations in the presence of S9 mix. Oral administration of sesamin at doses up to 2.0g/kg did not cause a significant increase in either the percentage of micronucleated polychromatic erythrocytes in the in vivo bone marrow MN test or in the % DNA in the comet tails in the in vivo comet assay of liver cells. These findings indicate that sesamin does not damage DNA in vivo and that sesamin and episesamin have no genotoxic activity.     

In vitro bone marrow granulocyte-macrophage progenitor cultures in the assessment of hematotoxic potential of the new drugs

In pharmaceutical research, in vitro toxicity tests, for assessing the potential toxicity of new chemical entities are necessary in the early stages of the developmental process, when no information is available about the metabolism or even the target organ toxicity of the compounds to be tested. In vitro specific organ toxicity tests, such as the granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay, are useful tools for predicting the adverse effects of new compounds on the blood-forming system, provided that some reference points are available, e.g., toxicological information about compounds belonging to the same chemical class and structure-activity relationship data. Furthermore, when no information is available about metabolism, the in vitro system should cover as many possibilities as possible, to avoid false positive or false negative results. In fact, while many compounds are metabolized to a variety of inactive chemical species, some undergo bioactivation to form more active metabolites. The addition of a metabolic activation system to the CFU-GM assay enables assessment of direct and metabolism-mediated toxicity. The regulatory agencies and industry value the concept of assays performed with and without metabolic activation, since they often have to take decisions about compounds with unknown mechanisms of action. CFU-GM assay, designed in this way, is an example of such a mechanism-naive assay. It has been suggested that, for new compounds, metabolites should be generated and tested both in the presence and in the absence of the parent compound itself, to identify the possible contribution of metabolites to the hematotoxicity observed, and to determine whether there is any synergistic or antagonistic effect between metabolites and the parent compound that might affect hematotoxicity in vivo. Various approaches can be used to obtain such information.     

Risk of genetic transformation of multipotent mesenchymal stromal cells in vitro

Proof of the efficacy of cell therapy by numerous studies and clinical trials inevitably has raised the question of improving the regulatory framework that governs its use. Particular attention should be paid to the genetic safety of cell preparations. The immune, genetic, and pharmacological modification and expansion of cells in vitro can lead to an undesired effect, which not only has reduced the healing, recovery, and regulatory potential of cell therapy, but also increased the risk of accumulating genetically aberrant cells and the oncogenic transformation of cell preparations. The article has presented the estimation of the parameters of the genetic stability of cultured multipotent mesenchymal stromal cells (MSCs) derived from bone marrow and adipose tissue. The study was conducted using classic methods of genotoxicology, i.e., the individual cells gel-electrophoresis (comet assay) and the micronucleus test. We described a basic level of DNA damage and the frequency of micronucleus, identified genetically instable cultures, and conducted the comparison of genetic variability of MSCs isolated from different tissues.     

Genotoxicity of three pesticides used in Costa Rican banana plantations

The in vitro genotoxicity of imazalil and thiabendazole fungicides and the insecticide chlorpyrifos, compounds used in Costa Rican banana plantations, was evaluated with the single-cell gel electrophoresis technique (comet assay). The comet assay is a simple, rapid and low cost technique for quantification of DNA damage. This assay detects DNA single-strand breaks and alkali-labile sites in individual cells. The effects were analyzed by using human lymphocytes exposed to doses of 0, 25, 50, 75 and 100 microg/ml of each pesticide for 30 min at 37 degrees C. The cells were embedded in agarose, lysed, subjected to alkaline electrophoresis (pH >13) for 20 min at 25V, neutralized and dehydrated to be stained with a fluorescent dye and later comets visualization with the epifluorescence microscope. Chlorpyrifos and imazalil induced significant DNA damage in a dose-dependent manner. Chlorpyrifos was the major inductor of DNA breaks. These results indicate that both are genotoxic compounds in vitro. Thiabendazole fungicide did not induced DNA damage using the comet assay for all concentrations tested.     

The photo comet assay--a fast screening assay for the determination of photogenotoxicity in vitro

Different classes of chemicals can induce a phototoxic effect by absorbing light energy within the wavelength range of sunlight. The assessment of photo-safety is therefore an obligatory part of the development of new drugs. Ten UV-vis (280-800nm)-absorbing compounds (ketoprofen, promazine, chlorpromazine, dacarbazine, acridine, lomefloxacin, 8-methoxypsoralen, chlorhexidine, titanium dioxide, octylmethoxycinnamate) were tested for their photogenotoxic potential in the alkaline comet assay in the presence and absence of UV-vis. In order to establish an easy and timesaving protocol for a photo comet assay screening test, the application of 96-well plates was essential. The use of mouse lymphoma L5178Y cells, a cell line growing in suspension, allowed the determination of photocytotoxicity with the Alamar Blue assay and of photogenotoxicity with the alkaline comet assay in parallel. L5178Y cells were incubated with the test compounds for 20min and irradiated with simulated sunlight in the wavelength range from 280 to 800nm. The applied UV dose was 600mJ/cm(2) UV-A and 30mJ/cm(2) UV-B. After a post-incubation of 10min, the Alamar Blue assay and the alkaline comet assay were performed. All of the compounds which are known to be photogenotoxic (8-methoxypsoralen, acridine, chlorpromazine, dacarbazine, ketoprofen, lomefloxacin) showed a positive effect under our assay conditions. Furthermore, four UV-vis absorbing chemicals which are known to be not photogenotoxic (promazine, chlorhexidine, titanium dioxide, octylmethoxycinnamate) were analysed. For none of them an increase of the DNA damage following irradiation was observed in this study. In conclusion, all of the chemical compounds tested were classified in agreement with published data. From the data presented it is concluded that the photo comet assay with L5178Y mouse lymphoma cells is a reliable model to assess photochemical genotoxicity in vitro.     

Mutagenicity and genotoxicity of isatin in mammalian cells in vivo

Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate used for the synthesis of heterocyclic compounds and as a raw material for drug synthesis. Isatin and its derivatives demonstrate anticonvulsant, antibacterial, antifungal, antiviral, and anticancer properties. We evaluated the genotoxic and mutagenic effects of acute (24h) and repeated (14d) exposure to isatin in vivo, using the comet assay and the micronucleus test. Three doses (50, 100, and 150mg/kgb.w.) were administered to mice via gavage. Doses were selected according to the LD(50) of isatin, estimated in a preliminary test to be 1g/kgb.w. To evaluate the results, parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution. At all doses (50, 100 and 150mg/kgb.w.), after acute treatment with isatin, alterations in DNA migration (comet assay) were not observed and mutagenic effects were not seen (micronucleus test on peripheral blood cells). After repeated doses, only the highest dose of isatin (150mg/kgb.w.) induced alterations in the DNA that gave rise to micronuclei in the bone marrow and peripheral blood cells of the mice. Our results show that the mutagenic and genotoxic effects of isatin depend on dose and on period of exposure.