Presence Of S9 Research Articles

In Vitro Mutagenic and Genotoxic Assessment of Anatoxin-a Alone and in Combination with Cylindrospermopsin.

Anatoxin-a (ATX-a) is a cyanobacterial toxin whose occurrence has been reported worldwide and has attracted increasing scientific interest due to its toxicity. Moreover, in nature, ATX-a usually appears together with other cyanotoxins, such as cylindrospermopsin (CYN), so possible interaction phenomena could happen and should be considered for risk assessment purposes. For this reason, the aim of this work was to explore the potential mutagenicity and genotoxicity of pure ATX-a and an ATX-a/CYN mixture using a battery of in vitro assays, including the bacterial reverse-mutation assay in Salmonella typhimurium (OECD 471) and the micronucleus test (MN) (OECD 487) on L5178Y Tk+/- cells. The results showed that ATX-a was not mutagenic either alone or in combination with CYN under the conditions tested. Nevertheless, genotoxic effects were observed for both ATX-a and its mixture with CYN following the in vitro MN assay. The genotoxicity exhibited by ATX-a was only observed in the absence of S9 mix, whereas in the cyanotoxin mixture the concentration-dependent genotoxicity of ATX-a/CYN in vitro was observed only in the presence of S9. Thus, the toxicity induced by cyanotoxin mixtures may vary from that produced by toxins alone, and consequently more studies are necessary in order to perform more realistic risk assessments.

Toxicity and mutagenicity studies of 6PPD-quinone in a marine invertebrate species and bacteria.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone), an oxidation product of the tire additive, 6PPD, has been associated with high mortality of salmonids (0.1 μg/L). The objective of this study was to determine the acute toxicity using neonates and mutagenicity (micronuclei in hemolymph of exposed adults) of 6PPD-quinone in the marine amphipod Parhyale hawaiensis. Also, we studied its mutagenicity in the Salmonella/microsome assay using five strains of Salmonella with and without metabolic system (rat liver S9, 5%). 6PPD-quinone did not present acute toxicity to P. hawaiensis from 31.25 to 500 μg/L. Micronuclei frequency increased after 96 h-exposure to 6PPD-quinone (250 and 500 μg/L) when compared to the negative control. 6PPD-quinone also showed a weak mutagenic effect for TA100 only in the presence of S9. We conclude that 6PPD-quinone is mutagenic to P. hawaiensis and weakly mutagenic to bacteria. Our work provides information for future risk assessment of the presence of 6PPD-quinone in the aquatic environment.

Evaluation of the genotoxic effect of tartrazine using a metabolic activation system in human lymphocyte culture under cytokinetic block conditions

The expansion of the spectrum of use of food additives and, in particular, food dyes (FD), increases the risk of increasing human exposure to genotoxicants. Since in real life, not pure substances with proven genetic safety are in contact with a person, but complex mixtures of unknown composition, even minor impurities in which can become an additional source or modifier of genome instability effects. Of particular concern in this aspect are synthetic FD azo and diazo compounds which can be transformed by human intestinal microflora to some forms of genotoxicants. The purpose of the work is to evaluate the genotoxic effects of 02 mg/mL of Tartrazine FD (E102) purchased in a retail network in a micronucleus test on human blood cells cultured under cytokinetic block conditions in parallel in presence and without rat S9 hepatocyte metabolic activation system.
 Genotoxic effects were found in cultures without metabolic activation at 0.00002560,00064 mg/mL and 0.4 mg/mL of tartrazine, and in the presence of S9 at 0.0000256 mg/mL, 0,000128 mg/mL and 0.16 mg/mL of tartrazine. For the first time, a dose-dependent suppression of mitotic and proliferative activity of lymphocytes induced by the tested tartrazine sample was revealed, as well as a dose-dependent U-shaped curve in the frequency of apoptosis. The data obtained indicate the presence of genotoxic activity of the studied sample.
 We discuss the necessity to create the system for evaluation the genotoxic safety of FD real mixtures from a retail network.

Evaluation of the Genotoxic Potential of the Selective COX-2 Inhibitor Enflicoxib in a Battery of in vitro and in vivo Genotoxicity Assays.

Aim Enflicoxib, a selective COX-2 inhibitor approved for the treatment of pain and inflammation associated with osteoarthritis in dogs (Daxocox ® [Ecuphar/Animalcare Group]) was assessed for its genotoxic potential in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse mutation assay (Ames test), an in vitro human lymphocyte chromosome aberration assay and an in vivo mouse bone marrow micronucleus assay. Methods Relevant vehicle and positive control cultures and animals were included in all assays. In the Ames test, enflicoxib was tested at concentrations of up to 5000 μg/plate. Signs of cytotoxicity were observed at the highest tested concentrations for several of the bacterial strains, both in absence and presence of S9. In human lymphocytes, enflicoxib was assessed for the induction of chromosomal aberrations when exposed at concentrations of up to 62.5 (3 hours) and 29.6 µg/mL (20 hours) in the absence of S9, and up to 66.7 µg/mL (3 hours) in presence of S9. Signs of cell toxicity, evidenced as a decrease in the mitotic index, were observed at these concentrations. In the mouse micronucleus assay, enflicoxib dose levels of up to 2000 mg/kg were administered (single dose) to male and female animals, and bone marrow samples were taken 24 and 48 hours (high-dose animals only) after administration. Results Enflicoxib was shown to lack genotoxic activity in the conducted assays. Conclusions The administration of enflicoxib as a therapeutic analgesic agent would not pose a genotoxic risk to animals or humans.

Micronucleus Formation Induced by Glyphosate and Glyphosate-Based Herbicides in Human Peripheral White Blood Cells.

Glyphosate is the most commonly used herbicide around the world, which led to its accumulation in the environment and consequent ubiquitous human exposure. Glyphosate is marketed in numerous glyphosate-based herbicide formulations (GBHs) that include co-formulants to enhance herbicidal effect of the active ingredient, but are declared as inert substances. However, these other ingredients can have biologic activity on their own and may interact with the glyphosate in synergistic toxicity. In this study, we focused to compare the cytogenetic effect of the active ingredient glyphosate and three marketed GBHs (Roundup Mega, Fozat 480, and Glyfos) by investigating cytotoxicity with fluorescent co-labeling and WST-1 cell viability assay as well as genotoxicity with cytokinesis block micronucleus assay in isolated human mononuclear white blood cells. Glyphosate had no notable cytotoxic activity over the tested concentration range (0–10,000 μM), whereas all the selected GBHs induced significant cell death from 1,000 μM regardless of metabolic activation (S9). Micronucleus (MN) formation induced by glyphosate and its formulations at sub-cytotoxic concentrations (0–100 μM) exhibited a diverse pattern. Glyphosate caused statistically significant increase of MN frequency at the highest concentration (100 μM) after 20-h exposure. Contrarily, Roundup Mega exerted a significant genotoxic effect at 100 μM both after 4- and 20-h exposures; moreover, Glyfos and Fozat 480 also resulted in a statistically significant increase of MN frequency from the concentration of 10 μM after 4-h and 20-h treatment, respectively. The presence of S9 had no effect on MN formation induced by either glyphosate or GBHs. The differences observed in the cytotoxic and genotoxic pattern between the active principle and formulations confirm the previous concept that the presence of co-formulants in the formulations or the interaction of them with the active ingredient is responsible for the increased toxicity of herbicide products, and draw attention to the fact that GBHs are still currently in use, the toxicity of which rivals that of POEA-containing formulations (e.g., Glyfos) already banned in Europe. Hence, it is advisable to subject them to further comprehensive toxicological screening to assess the true health risks of exposed individuals, and to reconsider their free availability to any users.

Assessment of the mutagenicity of the technical product
 of N-(1-ethylpropyl)-2,6-dinitro -3,4-xylidinen

Introduction. Evaluation of genotoxicity of the pesticide technical products is one of the mandatory requirements for their toxicological and hygienic assessment. The data about mutagenic property is ambiguous for some pesticides. This may be due to the use of various active ingredients of technical products of the pesticide for testing, as they may have different profiles of relevant impurities, some of which may be potentially genotoxic. Material and methods. A technical product of N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine was tested using the bacterial reverse mutation method with Salmonella typhimurium (Ames test) and the in vivo mammalian micronucleus analysis in mouse bone marrow erythrocytes. Results. Statistically significant dose-dependent mutagenic effects of the technical product of N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine were revealed for TA97 (+S9 / -S9); TA100 (+S9 / -S9); TA102 (+S9 / -S9) and TA98 (+S9 / -S9) strains. In all cases, the fold increase of the revertant numbers mediated by the tested substance compared with the concurrent negative control was > 2 except TA98 in the presence of S9. In the micronucleus test, the technical product did not induce a statistically significant increase in the frequency of the micronucleated polychromatophilic erythrocytes in CD-1 mouse bone marrow up to 2000 mg/kg bw. Conclusion. The data suggest all technical products of pesticides entering the market should be tested for the potential genotoxicity. In such a case it is necessary to use at least two methods on different test systems for obtaining reliable results.

Assessment of the mutagenicity of the technical product
 of N-(1-ethylpropyl)-2,6-dinitro -3,4-xylidinen

Introduction. Evaluation of genotoxicity of the pesticide technical products is one of the mandatory requirements for their toxicological and hygienic assessment. The data about mutagenic property is ambiguous for some pesticides. This may be due to the use of various active ingredients of technical products of the pesticide for testing, as they may have different profiles of relevant impurities, some of which may be potentially genotoxic. Material and methods. A technical product of N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine was tested using the bacterial reverse mutation method with Salmonella typhimurium (Ames test) and the in vivo mammalian micronucleus analysis in mouse bone marrow erythrocytes. Results. Statistically significant dose-dependent mutagenic effects of the technical product of N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine were revealed for TA97 (+S9 / -S9); TA100 (+S9 / -S9); TA102 (+S9 / -S9) and TA98 (+S9 / -S9) strains. In all cases, the fold increase of the revertant numbers mediated by the tested substance compared with the concurrent negative control was > 2 except TA98 in the presence of S9. In the micronucleus test, the technical product did not induce a statistically significant increase in the frequency of the micronucleated polychromatophilic erythrocytes in CD-1 mouse bone marrow up to 2000 mg/kg bw. Conclusion. The data suggest all technical products of pesticides entering the market should be tested for the potential genotoxicity. In such a case it is necessary to use at least two methods on different test systems for obtaining reliable results.

3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing.

MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/β-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.

Evaluation of Potential DNA-Damaging Effects of Nitenpyram and Imidacloprid in Human U937-Cells Using a New Statistical Approach to Analyse Comet Data

Even if the two neonicotinoids nitenpyram and imidacloprid have been considered safe for humans, their potential genotoxicity still remains a matter of discussion. The DNA-damaging effects of these two compounds were therefore evaluated in a lymphoma cell line of human origin (U-937) using the comet assay after 3-h exposure to up to 50 μM, with or without metabolic activation using S9 from human liver. The comet data were analysed using a traditional one-way ANOVA after pooling the data on cellular level, and a new alternative approach we have called Uppsala Comet Data Analysis Strategy (UCDAS). UCDAS is a proportional odds model tailored to continuous outcomes, taking the number of pooled cultures, slides and cells into consideration in the same analysis. To the best of our knowledge, the UCDAS approach when analysing comet data has never been presented before. Without metabolic activation, no increase in DNA damage was observed in the neonicotinoide-exposed cells. Nitenpyram was also without DNA-damaging effects when S9 was added. However, in the presence of S9, imidacloprid was found to increase the level of DNA damage. Whereas the ANOVA showed an increase (P < 0.001) both at 5 and 50 μM, UCDAS showed an increase only at the lowest concentration (P < 0.001). Based on these findings, the two neonicotinoids seem to be of little concern when it comes to their potential genotoxicity. However, since the U-937 cells were rather resistant to our positive controls, they may not be the best cells to use when evaluating potential genotoxicity of chemicals.

Mutagenicity, genotoxicity and gene expression of Rad51C, Xiap, P53 and Nrf2 induced by antimalarial extracts of plants collected from the middle Vaupés region, Colombia

Due to Plasmodium resistance to antimalarial drugs, it is important to find new therapeutic alternatives for malaria treatment and control. Based on the knowledge of Colombian indigenous communities, we collected extracts of plants with potential antimalarial effects from the middle Vaupés region. To evaluate the mutagenic and genotoxic effects, as well as the gene expression of Rad51C, Xiap, P53 and Nrf2 induced by four ethanolic extracts with antimalarial activity (R001, T002, T015 and T028). We evaluated four ethanolic extracts with antimalarial activity using the Ames test to assess mutagenicity, and the comet assay on HepG2 cells to determine the genotoxicicity. We also evaluated the expression of Rad51C, Xiap, P53 and Nrf2 from HepG2 cells stimulated with the four extracts. None of the four extracts was mutagenic in Salmonella typhimurium TA98 strain in the presence and absence of S9 metabolic activity. Extracts R001, T015 and T028 were weakly mutagenic on the TA100 strain in the presence of S9, with mutagenic indexes (MI) of 1.58, 1.53 and 1.61, respectively. The T015 strain showed the same behavior without S9 with an MI of 1.36. The results of the comet assay showed that the four extracts produced category 1 or 2 damage, with comets between 36.7 and 51.48 μm in length. However, the genetic damage index suggested that most of the cells were affected by the treatments. Regarding gene expression, extracts R001 and T028 induced an overexpression of genes Xiap and P53 with an 1.84 to 3.99 fold-change compared with untreated cells. These results revealed that the T002 extract was the safest as it had antimalarial activity and was not cytotoxic on HepG2 cells. Moreover, it was not mutagenic and it only produced category 1 damage on the DNA. Also, the extract did not induce a change in the expression of the tested genes.

The influence of exogenous metabolism on the specificity of in vitro mammalian genotoxicity tests.

A two-part study was designed to determine whether the inclusion of the rodent liver 'S9' exogenous metabolic activating system contributes to the generation of misleading positive results by the regulator-required in vitro mammalian genotoxicity tests. The mono-oxygenase enzymes in S9 produce direct-acting DNA-reactive electrophiles, and are included in in vitro genotoxicity tests to enhance the detection of substances which only become genotoxic following metabolism. However, as the S9 system lacks 'detoxifying' phase 2 factors it was hypothesised that increased chemical metabolism per se may lead to an increase in irrelevant S9 test outcomes in safety assessment. To test this, 89 compounds with positive or negative carcinogenicity data were identified, which produced negative Ames test data (+/- S9), and only produced positive in vitro mammalian test data in the presence of S9. This allowed a determination of whether or not misleading predictions of carcinogenicity by the in vitro mammalian tests were more or less prevalent in the presence of S9. A subset of these compounds was then tested with and without S9 in the GADD45a-GFP genotoxicity test, in order to determine whether misleading in vitro mammalian positive results were generally more prevalent with S9, or reflected particular tests' liabilities. This study suggests that the use of S9 metabolic activation in in vitro genotoxicity tests does not increase the prevalence of misleading positive results in in vitro mammalian genotoxicity assays, at least amongst Ames negative compounds.

Abstract 4079: Pre-clinical toxicology and safety of SH7139: The first of a new class of targeted therapeutics for non-Hodgkin's lymphoma and other cancers

Abstract Selective High Affinity Ligands (SHALs) are small molecules that can be designed to bind selectively and with high affinity to any protein target by linking together several ligands that recognize and bind to different sites on the protein’s surface. SH7139, our first SHAL therapeutic for treating advanced non-Hodgkin’s lymphoma, has shown remarkable efficacy in B-cell lymphoma xenograft models by providing permanent cures for up to two-thirds of the animals at a human equivalent dose as low as 0.4 μg/kg. Functioning similar to an antibody-drug conjugate and a pro-drug, SH7139 targets and binds to a unique structural epitope within the antigen-binding pocket of HLA-DR10. HLA-DRs containing this epitope are expressed in approximately 85% of B-cell lymphomas. Upon binding to its HLA-DR target, SH7139 is carried into the cytoplasm where it is concentrated and subsequently metabolized, releasing toxic metabolites (derived from each of the linked ligands) that inhibit a series of cellular activities required for tumor cell function. In preparation for an IND application submission and advancing SH7139 into clinical trials, acute dose range finding (i.v. bolus) and multiple dose (i.v. bolus on days 1, 8, and 15) toxicology and safety studies have been conducted with SH7139 in rats and beagle dogs. In the rat studies, doses up to 30 mg/kg (NOAEL = 30 mg/kg, 11,854 fold higher than the efficacy dose), were well tolerated with no findings associated with the drug. Beagle dogs were found to be the most sensitive species (NOAEL = 0.3mg/kg, 395 fold higher than the efficacy dose), with doses at 1 and 10mg/kg producing a reversible allergic-type reaction. A maximum tolerated dose (MTD) was reached in rats (100 mg/kg). No attempt was made to identify the MDT in dogs. In vitro assays conducted to evaluate potential cardiac safety showed SH7139 had no effect on potassium (hERG) and calcium (hCaV1.2) ion channel function up to the highest concentration tested (25 μM). A detectable effect above background was only observed with the sodium channel hNaV1.5 at 25 μM, a plasma concentration that would only be achieved in patients administered a dose ~2,000 fold higher than the efficacy dose. SH7139 was found to be negative in inducing gene mutation in five tester strains of Salmonella and E. coli (AMES assay). In a CHO-K1 micronucleus assay, no genotoxicity was observed in the presence of S9 over the entire concentration range tested. In the absence of S9, a positive response (3.3 fold) just above the triggering threshold (3 fold) of the assay was observed at the highest SH7139 concentration (500 μM). Based on these results and others derived from mouse xenograft efficacy studies, a maximum recommended Phase I trial starting dose has been tentatively set at 0.5 μg/kg with a safety factor of 324. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843. Citation Format: Rod Balhorn, Monique Cosman Balhorn. Pre-clinical toxicology and safety of SH7139: The first of a new class of targeted therapeutics for non-Hodgkin's lymphoma and other cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2017-4079

Genotoxic risk of ethyl-paraben could be related to telomere shortening.

The ability of parabens to promote the appearance of multiple cancer hallmarks in breast epithelium cells provides grounds for regulatory review of the implication of the presence of parabens in human breast tissue. It is well documented that telomere dysfunction plays a significant role in the initiation of genomic instability during carcinogenesis in human breast cancer. In the present study, we evaluated the genotoxic effect of ethyl 4-hydroxybenzoate (ethyl-paraben), with and without metabolic activation (S9), in studies following OECD guidelines. We observed a significant increase in genotoxic damage using the Mouse Lymphoma Assay and in vitro micronucleus (MN) tests in the L5178Y cell line in the presence of S9 only after a short exposure. A high frequency of MN was observed in the TK6 cells after a short exposure (3 h) in the presence of S9 and a long exposure (26 h) without S9. We found significant increases in the MN frequency and induced chromosomal aberrations in the lymphocytes of only one donor after ethyl-paraben exposure in the presence of S9 after a short exposure. Cytogenetic characterization of the paraben-treated cells demonstrated telomere shortening associated with telomere loss and telomere deletions in L5178Y and TK6 cells and lymphocytes of the paraben sensitive-donor. In a control cohort of 68 human lymphocytes, telomere length and telomere aberrations were age-dependent and showed high inter-individual variation. This study is the first to link telomere shortening and the genotoxic effect of ethyl paraben in the presence of S9 and raises the possibility that telomere shortening may be a proxy for underlying inter-individual sensitivity to ethyl-paraben. Copyright © 2016 John Wiley & Sons, Ltd.

Sister chromatid exchange test in river buffalo lymphocytes treated in vitro with furocoumarin extracts.

Furocoumarin extracts from Psoralea morisiana, the endemic Sardinian legume species, were tested for their mutagenic potential on river buffalo blood cells. The results obtained performing the sister chromatid exchange (SCE) test in blood cultures of five river buffalo calves (exposure to furocoumarins for 72h) and five cows (exposure to furocoumarins for 3h, in the absence and presence of S9 metabolic activator) are reported. Significant differences in mean values of SCEs were observed in cells of calves compared to control cells (unexposed), but no differences in SCE mean values were found between treated and untreated cells of cows in the presence or absence of S9. SCE mean values were much higher in cells of cows (exposed and control) than in cells of calves. Indeed, in calf cells, SCE mean values/cell (±SD) were 6.66±2.45 in the control and 7.63±3.01, 9.03±3.90, 9.53±3.60 and 9.99±3.41 in treated cells at 50, 100, 200 and 400 µg/ml of furocoumarin extracts, respectively. In cow cells, grown in presence of S9, SCE mean values/cell were 11.49±4.78 and 11.65±5.19 in treated cells at 100 and 200 µg/ml of furocoumarins and 11.66±5.45 in the control. In cow cells grown in absence of S9, SCE mean values were 11.81±6.14 in the control and 12.35±7.09 and 12.01±5.43, respectively, in the presence of 100 and 200 µg/ml of furocoumarins. Despite their higher SCE values in the absence of S9, no statistically significant differences were found when these values were compared with those shown in presence of S9, suggesting no mutagenic action of furocoumarins in cows, at the doses used in this study.

In vitro evaluation of the cyto-genotoxic potential of Ruthenium(II) SCAR complexes: a promising class of antituberculosis agents

Tuberculosis is a top infectious disease killer worldwide, caused by the bacteria Mycobacterium tuberculosis. Increasing incidences of multiple drug-resistance (MDR) strains are emerging as one of the major public health threats. However, the drugs in use are still incapable of controlling the appalling upsurge of MDR. In recent years a marked number of research groups have devoted their attention toward the development of specific and cost-effective antimicrobial agents against targeted MDR-Tuberculosis. In previous studies, ruthenium(II) complexes (SCAR) have shown a promising activity against MDR-Tuberculosis although few studies have indeed considered ruthenium toxicity. Therefore, within the preclinical requirements, we have sought to determine the cyto-genotoxicity of three SCAR complexes in this present study. The treatment with the SCARs induced a concentration-dependent decrease in cell viability in CHO-K1 and HepG2 cells. Based on the clonogenic survival, SCAR 5 was found to be more cytotoxic while SCAR 6 exhibited selectivity action on tumor cells. Although SCAR 4 and 5 did not indicate any mutagenic activity as evidenced by the Ames and Cytokinesis block micronucleus cytome assays, the complex SCAR 6 was found to engender a frameshift mutation detected by Salmonella typhimurium in the presence of S9. Similarly, we observed a chromosomal damage in HepG2 cells with significant increases of micronuclei and nucleoplasmic bridges. These data indicate that SCAR 4 and 5 complexes did not show genotoxicity in our models while SCAR 6 was considered mutagenic. This study presented a comprehensive genotoxic evaluation of SCAR complexes were shown to be genotoxic in vitro. All in all, further studies are required to fully elucidate how the properties can affect human health.

Eco- and Genotoxicological Assessments of Two Reactive Textile Dyes

Contamination of natural waters has been one of the major problems of modern society and the textile industry is rated as an important polluting source, due to the generation of large amounts of wastewaters. The aim of this study was to assess textile dyes Reactive Blue 19 (RB19, anthraquinone dye) and Reactive Red 120 (RR120, azo dye) in terms of the potential to induce adverse effects on aquatic organisms and humans. Thus, these dyes were tested using the following assays: Microtox assay (Vibrio fischeri); brine shrimp (Artemia salina); Daphnia similis; and Comet with normal human dermal fibroblasts as well as Ames test (TA98, TA100, YG1041, YG1042—with and without S9). RB19 was relatively nontoxic to all aquatic bioindicators analyzed with an EC50 of more than 100 mg/L, whereas RR120 was only moderately toxic to A. salina with a EC50-48h of 81.89 mg/L. Mutagenicity through base pair substitution was observed with RB19 in the presence of S9 (Ames-positive). The comet assay did not demonstrate any apparent genotoxic effects for any tested dye. Although mutagenicity was detected with RB19, the mutagenic effect observed may be considered weak compared to the ability to induce DNA damage by other classes of dyes such as disperse dyes. Therefore, these dyes may be classified as nonmutagens (RR120) or weak mutagens (RB19) and relatively nontoxic for aquatic organisms. However, it is noteworthy that the weak acute toxicity to A. salina induced by RR120 is sufficient to suggest potential damage to the aquatic ecosystem and emphasizes the need for biomonitoring dye levels in wastewater systems.

Development of mutagenicity during degradation of N-nitrosamines by advanced oxidation processes

Development of mutagenicity of five N-nitrosamines (N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-propylamine (NDPA), N-nitrosopyrrolidine (NPYR) and N-nitrosodiphenylamine (NDPhA)) was investigated during oxidative processes involving UV-photolysis, ozone and OH radicals. The mutagenicity was detected by the Ames test with 3 different strains, TA98, TAMix and YG7108, a strain which is sensitive for N-nitrosamines, in presence and absence of metabolic activation (S9). UV photolysis of mutagenic N-nitrosamines (NDMA, NDEA, NDPA and NPYR) leads to the removal of their specific mutagenic activity as detected in YG7108 in the presence of S9. A formation of mutagens during UV photolysis was detected only in case of NDPhA in the strain TA98. Oxidation products of NDMA, NDEA and NDPhA did not show any significant mutagenicity in the strains used, whereas oxidation of NDPA and NPYR by hydroxyl radicals seems to lead to the formation of direct mutagens (mutagenic in the absence of S9) in YG7108 and TAMix. Oxidation by hydroxyl radicals of N-nitrosamines with chains longer than ethyl can mimic metabolic activation of N-nitrosamines in vivo.

Evidence for different mechanisms of action behind the mutagenic effects of 4-NOPD and OPD: the role of DNA damage, oxidative stress and an imbalanced nucleotide pool

The mutagenicity of 4-nitro-o-phenylenediamine (4-NOPD) and o-phenylenediamine (OPD) was compared using the Mouse Lymphoma Assay (MLA) with or without metabolic activation (S9). As expected, OPD was found to be a more potent mutagen than 4-NOPD. To evaluate possible mechanisms behind their mutagenic effects, the following end points were also monitored in cells that had been exposed to similar concentrations of the compounds as in the MLA: general DNA damage (using a standard protocol for the Comet assay); oxidative DNA damage (using a modified procedure for the Comet assay in combination with the enzyme hOGG1); reactive oxygen species (ROS; using the CM-H2DCFDA assay); and the balance of the nucleotide pool (measured after conversion to the corresponding nucleosides dC, dT, dG and dA using high-performance liquid chromatography). Both compounds increased the level of general DNA damage. Again, OPD was found to be more potent than 4-NOPD (which only increased the level of general DNA damage in the presence of S9). Although less obvious for OPD, both compounds increased the level of oxidative DNA damage. However, an increase in intracellular ROS was only observed in cells exposed to 4-NOPD, both with and without S9 (which in itself induced oxidative stress). Both compounds decreased the concentrations of dA, dT and dC. A striking effect of OPD was the sharp reduction of dA observed already at very low concentration, both with and without S9 (which in itself affected the precursor pool). Taken together, our results indicate that indirect effects on DNA, possibly related to an unbalanced nucleotide pool, mediate the mutagenicity and DNA-damaging effects of 4-NOPD and OPD to a large extent. Although induction of intracellular oxidative stress seems to be a possible mechanism behind the genotoxicity of 4-NOPD, this pathway seems to be of less importance for the more potent mutagen OPD.

Utility of Rat Liver S9 Fractions to Study Skin-Sensitizing Prohaptens in a Modified KeratinoSens Assay

Prohaptens are chemicals, which may cause skin sensitization after being converted into electrophilic molecules by skin enzymes. Aroclor-induced rat liver S9 fractions represent the metabolic activation system most commonly used in in vitro toxicology. This system contains much higher enzyme activities compared with those reported in skin, but it may still serve as a surrogate system to study the potential of chemicals to act as prohaptens. To test this concept, the luciferase induction in KeratinoSens reporter cells treated with chemicals in presence and absence of S9 fractions was measured. Suspected prohaptens such as methyl isoeugenol, eugenol, or trans-anethole gave no, or only weak, ge ne induction in absence of S9 fractions, and a significantly enhanced luciferase induction in presence of S9, proving their prohapten status. Direct-acting haptens like 2,4-dinitrochlorobenzene or cinnamic aldehyde gave a reduced response in presence of S9. We evaluated whether this metabolic activation assay might be implemented in a tiered screening strategy to counter-screen negatives in the KeratinoSens assay to enhance sensitivity. To this aim, all chemicals classified negative were retested with this activation step. Among the 77 chemicals found as correct-negatives, 73 were also negative in presence of metabolic activation, thus this counterscreen would reduce specificity only slightly. However, this comprehensive screening showed that only a small fraction of the known skin sensitizers need activation by the S9 system. Therefore, the KeratinoSens-S9 assay appears useful for the in vitro evaluation of specific classes of potential prohaptens and to mechanistically rationalize their prohapten status.

The position of imidazopyridine and metabolic activation are pivotal factors in the antimutagenic activity of novel imidazo[1,2-a]pyridine derivatives

The antimutagenic activity of eight novel imidazo[1,2-a]pyridine derivatives (I-VIII) against sodium azide (NaN3) and benzo[a]pyrene (B[a]P) was evaluated using the Salmonella reverse mutation assay. At non-toxic concentrations (12.5–50µM), imidazopyridines I, II, III, and V with a terminal imidazopyridine group were mutagenic, while derivatives VII and VIII with a central imidazopyridine group were not mutagenic. Compounds IV, VII, and VIII exerted a moderate antimutagenic activity against NaN3 under pre-exposure conditions, and a strong activity (>40%) against B[a]P in the presence of S9 under both pre- and co-exposure conditions and mostly independent on the dose. Imidazopyridines possibly inhibited the microsomal-dependent activation of B[a]P. The demethylated derivative VII was the most active antimutagen. All imidazopyridines had a low to moderate antioxidant activity. The antibacterial activity of imidazopyridines was sporadic and moderate probably due to the failure of bacteria to convert imidazopyridines into active metabolites. The position of imidazopyridine was a pivotal factor in the mutagenic/antimutagenic activity. The strong antimutagenic compounds were dicationic planar compounds with a centered imidazo[1,2-a]pyridine spacer. With LD50 of 60mg/kg in mice for both derivatives VII and VIII, it is safe to investigate the anticancer activity of these derivatives in animal models.