Biological assessment of chemical DNA damage in germ cells of male rabbits

DNA damage represents a key step in mutagenesis and carcinogenesis. Excision repair has been described to be the major pathway restoring damaged DNA. Assaying DNA repair may thus illuminate the mechanism of mutagenesis and carcinogenesis and serve as an indicator of the mutagenic and carcinogenic potential of environmental chemicals and drugs. The paper demonstrates a practical application of these concepts. DNA damage induced by chemical substances in male germ cells of rabbits was assessed by the demonstration of DNA repair synthesis in meiotic and post-meiotic maturation stages. Incorporation of tritium-labeled thymidine was monitored in spermatozoa obtained by serial ejaculation. The test was validated with several standard mutagens and carcinogens, and its usefulness was demonstrated with a study on 3 suspected genotoxic drugs, i.e. hycanthone, isoniazid, and metronidazole.     

Pro-oxidant Induced DNA Damage in Human Lymphoblastoid Cells: Homeostatic Mechanisms of Genotoxic Tolerance

Oxidative stress contributes to many disease etiologies including ageing, neurodegeneration, and cancer, partly through DNA damage induction (genotoxicity). Understanding the i nteractions of free radicals with DNA is fundamental to discern mutation risks. In genetic toxicology, regulatory authorities consider that most genotoxins exhibit a linear relationship between dose and mutagenic response. Yet, homeostatic mechanisms, including DNA repair, that allow cells to tolerate low levels of genotoxic exposure exist. Acceptance of thresholds for genotoxicity has widespread consequences in terms of understanding cancer risk and regulating human exposure to chemicals/drugs. Three pro-oxidant chemicals, hydrogen peroxide (H(2)O(2)), potassium bromate (KBrO(3)), and menadione, were examined for low dose-response curves in human lymphoblastoid cells. DNA repair and antioxidant capacity were assessed as possible threshold mechanisms. H(2)O(2) and KBrO(3), but not menadione, exhibited thresholded responses, containing a range of nongenotoxic low doses. Levels of the DNA glycosylase 8-oxoguanine glycosylase were unchanged in response to pro- oxidant stress. DNA repair-focused gene expression arrays reported changes in ATM and BRCA1, involved in double-strand break repair, in response to low-dose pro-oxidant exposure; however, these alterations were not substantiated at the protein level. Determination of oxidatively induced DNA damage in H(2)O(2)-treated AHH-1 cells reported accumulation of thymine glycol above the genotoxic threshold. Further, the H(2)O(2) dose-response curve was shifted by modulating the antioxidant glutathione. Hence, observed pro- oxidant thresholds were due to protective capacities of base excision repair enzymes and antioxidants against DNA damage, highlighting the importance of homeostatic mechanisms in "genotoxic tolerance."     

Assessment of antimutagenic and genotoxic potential of senna (Cassia angustifolia Vahl.) aqueous extract using in vitro assays.

Senna (Cassia angustifolia Vahl.) is widely used as a laxative, although potential side effects, such as toxicity and genotoxicity, have been reported. This study evaluated genotoxic and mutagenic effects of senna aqueous extract (SAE) by means of four experimental assays: inactivation of Escherichia coli cultures; bacterial growth inhibition; reverse mutation test (Mutoxitest) and DNA strand break analysis in plasmid DNA. Our results demonstrated that SAE produces single and double strand breaks in plasmid DNA in a cell free system. On the other hand, SAE was not cytotoxic or mutagenic to Escherichia coli strains tested. In effect, SAE was able to avoid H(2)O(2)-induced mutagenesis and toxicity in Escherichia coli IC203 (uvrA oxyR) and IC205 (uvrA mutM) strains, pointing to a new antioxidant/antimutagenic action of SAE.     

On the mechanism of genotoxicity of ethephon on embryonic fibroblast cells

Ethephon is one of the most widely used plant growth regulator in agriculture that its application has been increased in recent years. Many reports have raised concern over the safety of this organophosphorus compound. The aim of the current study was to assess the potential genotoxic effect of ethephon on murine embryonic fibroblast (MEF) cell line, using two genotoxicity endpoints: γH2AX expression and comet assay. γH2AX served as an early and sensitive biomarker of genotoxic damage. Oxidative stress biomarkers, including reactive oxygen species (ROS), lipid peroxidation (LPO) and total antioxidant capacity were also examined. The results showed a significant increase in cell proliferation, 24?h post-treatment with 10, 40,160?μg/ml ethephon, while at the higher concentrations cytotoxic effect was observed. The γH2AX expression and γH2AX foci count per cell were significantly increased at non-cytotoxic concentrations of ethephon, accompanied with increased DNA damage as illustrated by comet assay. LPO and ROS levels were elevated only at 160?μg/ml and higher doses. The results interestingly showed that low non-cytotoxic doses of ethephon promoted DNA damage inducing cell proliferation, raising the possibility of ethephon mutagenicity. The genotoxic effect of ethephon at low doses might not relate to oxidative damage and that increased in the level of ROS and LPO generation at higher doses could account for the cytotoxic effect of ethephon. Taken together, our study provides strong in vitro evidence on potential genotoxicity of ethephon at low doses. More precise studies are needed to clarify the mutagenic effect of chronic exposure to ethephon.     

Mutagenicity and genotoxicity of ethylvinyl ketone in bacterial tests

The mutagenic and genotoxic effects of ethylvinyl ketone were investigated. This alpha, beta-unsaturated carbonyl compound is widely distributed in the environment, in particular in food. Whereas ethylvinyl ketone shows only weak genotoxicity in the SOS Chromotest with Escherichia coli PQ37, it was distinctly mutagenic per se in the Salmonella preincubation assay with TA100. Using SKF 525 (an inhibitor of microsomal monooxygenase) and trichloropropene oxide (an inhibitor of epoxide hydrolase) we found indication for additional activation via epoxidation by S9 mix. The need for further investigation of the genotoxic, mutagenic and carcinogenic effects of this compound is strongly indicated.     

Genotoxicity of phthalates

Many of the environmental, occupational and industrial chemicals are able to generate reactive oxygen species (ROS) and cause oxidative stress. ROS may lead to genotoxicity, which is suggested to contribute to the pathophysiology of many human diseases, including inflammatory diseases and cancer. Phthalates are ubiquitous environmental chemicals and are well-known peroxisome proliferators (PPs) and endocrine disruptors. Several in vivo and in vitro studies have been conducted concerning the carcinogenic and mutagenic effects of phthalates. Di(2-ethylhexyl)-phthalate (DEHP) and several other phthalates are shown to be hepatocarcinogenic in rodents. The underlying factor in the hepatocarcinogenesis is suggested to be their ability to generate ROS and cause genotoxicity. Several methods, including chromosomal aberration test, Ames test, micronucleus assay and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation test and Comet assay, have been used to determine genotoxic properties of phthalates. Comet assay has been an important tool in the measurement of the genotoxic potential of many chemicals, including phthalates. In this review, we will mainly focus on the studies, which were conducted on the DNA damage caused by different phthalate esters and protection studies against the genotoxicity of these chemicals.     

Effects of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites on DNA damage and repair under in vitro conditions

3-monochloropropane-1,2-diol (3-MCPD) is a food contaminant that occurs during industrial production processes and can be found mainly in fat and salt containing products. 3-MCPD has exhibited mutagenic activity in vitro but not in vivo, however, a genotoxic mechanism for the occurrence of kidney tumors has not so far been excluded. The main pathway of mammalian 3-MCPD metabolism is via the formation of β – chlorolactatic acid and formation of glycidol has been demonstrated in bacterial metabolism. The aim of this study was to investigate genotoxic and oxidative DNA damaging effects of 3-MCPD and its metabolites, and to provide a better understanding of their roles in DNA repair processes. DNA damage was assessed by alkaline comet assay in target rat kidney epithelial cell lines (NRK-52E) and human embryonic kidney cells (HEK-293). Purine and pyrimidine base damage, H₂O₂ sensitivity and DNA repair capacity were assessed via modified comet assay. The results revealed in vitro evidence for increased genotoxicity and H₂O₂ sensitivity. No association was found between oxidative DNA damage and DNA repair capacity with the exception of glycidol treatment at 20 μg/mL. These findings provide further insights into the mechanisms underlying the in vitro genotoxic potential of 3-MCPD and metabolites.     

Differential genotoxicity and cytotoxicity of phomoxanthone A isolated from the fungus Phomopsis longicolla in HL60 cells and peripheral blood lymphocytes

Phomoxanthone A (PhoA) is a compound isolated from the endophytic fungus Phomopsis longicolla, associated with marine algae Bostrychia radicans. Although this metabolite was previously described regarding its high biological potential, there are no reports concerning the effects of this compound on DNA integrity. This study aimed to evaluate, in lymphocytes and promyelocytic leukemia HL60 cells, the cytotoxicity of this compound through MTT and neutral red (NR) assays, as well as its genotoxicity and mutagenicity by alkaline comet assay and cytokinesis-block micronucleus cytome assay (CBMN-Cyt), respectively. Cells were treated with PhoA concentrations ranging from 0.01 to 100.0 μg/mL, and the results show that this molecule did not exhibit cytotoxicity, genotoxicity or mutagenicity in lymphocytes at any tested concentration. Furthermore, PhoA was highly cytotoxic, genotoxic and mutagenic to HL60 cells, establishing a differential response of this natural product in normal and cancer cells. PhoA was highly selective towards HL60 compared to lymphocytes, causing no damage in the latter cell line, suggesting that this compound could be a promising compound in antitumoral drug development.     

A review of the genotoxicity of triallate

Triallate is a selective herbicidal chemical used for control of wild oats in wheat. It has an extensive genotoxicity database that includes a variety of in vitro and in vivo studies. The chemical has produced mixed results in in vitro assay systems. It was genotoxic in bacterial mutation Ames assays, predominantly in Salmonella typhimurium strains TA100 and TA1535 in the presence of S9. Weaker responses have been observed in TA100 and TA1535 in the absence of S9. Mixed results have been observed in strain TA98, whereas no genotoxicity has been observed in strains TA1537 and TA1538. The presence and absence of S9 and its source seem to play a role in the bacterial response to the chemical. There have also been conflicting results in other test systems using other bacterial genera, yeast, and mammalian cells. Chromosome effects assays (sister-chromatid exchange and cytogenetics assays) have produced mixed results with S9 but no genotoxicity without S9. Triallate has not produced any genotoxicity in in vitro DNA damage or unscheduled DNA synthesis assays using EUE cells, human lymphocytes, and rat and mouse hepatocytes. In a series of in vivo genotoxicity assays (cytogenetics, micronucleus, dominant lethal, and unscheduled DNA synthesis), there has been no indication of any adverse genotoxic effect. Metabolism data indicate that the probable explanation for the differences observed between the in vitro studies with S9 and without S9 and between the in vitro and the in vivo studies is the production of a mutagenic intermediate in vitro at high doses of triallate is expected to be at most only transiently present in in vivo studies. The weight of evidence strongly suggests that triallate is not likely to exert mutagenic activity in vivo due to toxicokinetics and metabolic processes leading to detoxification.     

Evaluation of the genotoxicity of cisplatin,paclitaxel and 5-fluorouracil combined treatment in the Drosophila wing-spot test

The somatic mutation and recombination test in Drosophila melanogaster was applied to analyze the mutagenic and recombinagenic activity of the chemotherapeutic drugs cisplatin, paclitaxel, and 5-fluorouracil, comparing the effects observed in combinatory treatments with those observed in single administrations. The results obtained in two different genotypes allowed to quantitatively and qualitatively estimate the contribution of genotoxic effects. The results obtained with the individual drug treatments showed that cisplatin and 5-fluorouracil were genotoxic, being able to increase the frequency of total spots on both genotypes. While cisplatin preferentially induced DNA damage of recombinational origin, all the damages induced by 5-fluorouracil were caused by gene and/or chromosome mutations, and the aneuploidogenic compound paclitaxel was not genotoxic. The combination of these drugs does not exert a synergist genotoxic effect in both genotypes compared to the single-agent administration. Instead, it was observed a modification in the proportion of mutation and recombination to the final genotoxicity observed. The antiproliferative activity of PAC could be responsible for the non-synergic genotoxic effect observed. Based on our results it is possible to suggest that cisplatin/paclitaxel/5-fluorouracil treatment regimen cannot impose a higher risk of the development of genotoxicity-associated secondary tumors in comparison to their individual applications.     

Protective effects of Hibiscus tiliaceus L. methanolic extract to V79 cells against cytotoxicity and genotoxicity induced by hydrogen peroxide and tert-butyl-hydroperoxide

Plants of the genus Hibiscus thrives produce a diversity of molecules with bioactive properties. In a previous study of Hibiscus tiliaceus L. methanolic extract (HME) using bacteria and yeast, as test media, it has been shown that HME strongly inhibited the mutagenic action of H₂O₂ or tert-butyl-hydroperoxide (t-BHP). Here, our interest is to evaluate the genotoxicity and the antigenotoxic/antimutagenic properties of HME using oxidative challenge with H₂O₂ and t-BHP in V79 cells. We determined cytotoxicity using clonal survival assay; evaluated DNA damage using the comet assay and the micronucleus test in binucleated cells besides of the lipid peroxidation degree and the reduced glutathione content. We examined the ability of HME in quenching hydroxyl radical by means of a HPLC-based method utilizing the hypoxanthine/xanthine oxidase assay. At concentrations ranging from 0.001 to 0.1 mg/mL, HME was not cytotoxic, genotoxic or mutagenic. Treatment with non-cytotoxic concentrations of HME increased cell survival after H₂O₂ and t-BHP exposure and prevented DNA damage. The pre-treatment with HME also was able to decrease the mutagenic effect of these genotoxins, evaluated using the micronucleus test. HME prevented the increase in lipid peroxidation and decrease in GSH content in response to the oxidative challenge. Therefore, the ability in preventing against H₂O₂- and t-BHP-induced GSH depletion and lipid peroxidation was probably a major contribution to the cytoprotective effects. Moreover, HME acts as a hydroxyl radical scavenger. In summary, HME did not have a harmful or inhibitory effect on the growth of V79 cells and presented antioxidant activity, consequently, both antigenotoxic and antimutagenic effects against oxidative DNA damage.     

Mutagenicity and genotoxicity of sorbic acid-amine reaction products.

Sorbic acid as well as potassium and calcium sorbate (E202 and E203) are legally used as preservatives in numerous processed foods. Owing to its system of conjugated double bonds, sorbic acid is likely to undergo a nucleophilic attack, which may turn it into mutagenic products. The cyclic derivatives resulting from a double addition reaction between sorbic acid and various amines at two different temperatures (50 degrees C and 80 degrees C) have been analysed. A genotoxicity study has been performed with HeLa cells and plasmid DNA. A mutagenesis study has been carried out by using the Ames test. A SOS spot test and a cytotoxicity study have been realised as well. The results showed that the products involved exhibited neither mutagenic nor genotoxic activities.     

Mutagenicity of malachite green and leucomalachite green in in vitro tests.

The genotoxic potential of the fungicide malachite green (MG) and its reduced derivative leucomalachite green (LMG) was assessed in bacteria and mammalian cells using the standard Salmonella typhimurium/Ames and CHO/HGPRT tests. In vitro potential DNA damaging effects of MG and LMG were tested using the single-cell gel electrophoresis (Comet) assay on CHO cells. Malachite green was found to be extremely cytotoxic to bacteria and mammalian cells. It did not have any mutagenic activity, in any bacterial strains, in the presence or absence of metabolic activation for doses up to 10 microg per plate. In the CHO/HGPRT test, the mutagenic potential of MG could be evaluated only for very low concentrations ranging from 0.001 to 0.05 microg ml(-1) medium. When S9 fraction was added to the medium, the highest tested dose could be increased to 1 microg ml(-1). In these experimental conditions, MG did not increase the number of thioguanine-resistant mutants. Leucomalachite green was less toxic than MG to Salmonella typhimurium and did not have mutagenic activity in the Ames' test for doses up to 2000 microg per plate. It was also less cytotoxic than MG to CHO cells and was tested at doses ranging from 5 to 100 microg ml(-1). Overall results indicated that LMG was not mutagenic in the HGPRT test. In the Comet assay, MG induced DNA damage only at cytotoxic doses. Loss of cell viability was observed for doses of > or = 3 microg ml(-1), with parallel increase in DNA alterations as measured by the tail moment. After metabolic activation, however, DNA damage was observed at doses (15-20 microg ml(-1)) inducing only low cytotoxicity. In this case, the direct genotoxicity of MG metabolites could not be excluded. In the absence or presence of metabolic activation, LMG did not have any effect on cell viability or DNA damage for doses up to 500 microg ml(-1). This study indicates that LMG, which is the main residue found in fish tissues after treatment with MG, did not have any mutagenic or clastogenic effects in the experimental conditions used.     

Evaluation of the potential genotoxicity of chromium picolinate in mammalian cells in vivo and in vitro.

Chromium picolinate (CrPic) is a synthetic nutritional supplement primarily used for weight loss and muscle building. Recent studies have indicated that CrPic might be genotoxic and these findings together with the wide-spread consumer use, have increased the concern about its safety. In the present study we investigated the potential genotoxicity of CrPic in mice given a single intraperitoneal injection (up to 3 mg/kgb.wt.) by evaluating the frequency of micronucleated polychromatic erythrocytes (fMNPCE) in peripheral blood, and DNA damage in lymphocytes and hepatocytes. The fMNPCE was evaluated after 42 h and DNA damage after 16 h. Using the Comet assay DNA damage was also monitored in extended-term cultures of human lymphocytes and in L5178Y mouse lymphoma cells that had been exposed for 3h to 500 microM CrPic under different exposure conditions. A slight, but significant CrPic-induced increase in DNA damage (P<0.001) was observed in the human lymphocytes, but only when these cells were exposed in the absence of serum. In all other experiments CrPic was found to be without genotoxic effects, both in vivo and in vitro. Taken together, our results suggest that a high concentration of CrPic might be DNA damaging, but only under non-physiological conditions.     

Co-exposure of iron oxide nanoparticles and glyphosate-based herbicide induces DNA damage and mutagenic effects in the guppy (Poecilia reticulata)

Iron oxide nanoparticles (IONPs) have been tested to remediate aquatic environments polluted by chemicals, such as pesticides. However, their interactive effects on aquatic organisms remain unknown. This study aimed to investigate the genotoxicity and mutagenicity of co-exposure of IONPs (γ-Fe₂O₃ NPs) and glyphosate-based herbicide (GBH) in the fish Poecilia reticulata. Thus, fish were exposed to citrate-functionalized γ-Fe₂O₃ NPs (0.3 mg L⁻¹; 5.44 nm) alone or co-exposed to γ-Fe₂O₃ NPs (0.3 mg L⁻¹) and GBH (65 and 130 μg of glyphosate L⁻¹) during 14 and 21 days. The genotoxicity (DNA damage) was analyzed by comet assay, while the mutagenicity evaluated by micronucleus test (MN test) and erythrocyte nuclear abnormalities (ENA) frequency. The co-exposure induced clastogenic (DNA damage) and aneugenic (nuclear alterations) effects on guppies in a time-dependent pattern. Fish co-exposed to NPs and GBH (130 μg glyphosate L⁻¹) showed high DNA damage when compared to NPs alone and control group, indicating synergic effects after 21 days of exposure. However, mutagenic effects (ENA) were observed in the exposure groups after 14 and 21 days. Results showed the potential genotoxic and mutagenic effects of maghemite NPs and GBH co-exposure to freshwater fish. The transformation and interaction of iron oxide nanoparticles with other pollutants, as herbicides, in the aquatic systems are critical factors in the environmental risk assessment of metal-based NPs.     

The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology. Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organ-specific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data. Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.     

Protective role of curcumin against nicotine-induced genotoxicity on rat liver under restricted dietary protein

Nicotine, the well known addictive chemical of tobacco and active medication for several diseases, has proven to be a potential genotoxic compound. Although it is absorbed through lungs with smoking and mainly metabolized in liver, its effect on liver injuries is not clear. This study was designed to evaluate the genotoxicity of nicotine and corresponding the protective role of curcumin against nicotine on liver of female populations particularly who used tobacco but deprived of healthy diet. The effects were investigated by measurement of total DNA concentration of liver tissues and Comet assay of liver tissue DNA damage of female rats maintained under normal and restricted protein diets. Total DNA contents in the liver tissues were observed to decrease more significantly (P<0.001) by nicotine in both dietary conditions. Significant (P<0.01) increase of total DNA content in normal dietary condition and more significant (P<0.001) increase of total DNA content in protein restricted condition of the liver tissues were observed due to curcumin supplementations. Highly significant (P<0.001) DNA damages (37% in normal diet and 56% in protein restricted diet) of the liver tissues were observed due to nicotine treatment. Curcumin reduced the nicotine-induced DNA damage percentage of the liver tissues more significantly (P<0.001) in protein restricted condition. Curcumin proved its potential to function against genotoxic effect by reducing the DNA damage activity of nicotine and minimized the percentage of DNA damage (50-60%) in protein restricted dietary condition. The degree of nicotine-induced genotoxicity therefore can be effectively compensated by the protective effect of curcumin in protein stress condition.     

Chemopreventive effect and lack of genotoxicity and mutagenicity of the exopolysaccharide botryosphaeran on human lymphocytes

Carbohydrate biopolymers of fungal-origin are an important natural resource in the search for new bioagents with therapeutic and nutraceutical potential. In this study the mutagenic, genotoxic, antigenotoxic and antioxidant properties of the fungal exopolysaccharide botryosphaeran, a (1 → 3)(1 → 6)-β-D-glucan, from Botryosphaeria rhodina MAMB-05, was evaluated. The mutagenicity was assessed at five concentrations in Salmonella typhimurium by the Ames test. Normal and tumor (Jurkat cells) human T lymphocyte cultures were used to evaluate the genotoxicity and antigenotoxicity (Comet assay) of botryosphaeran alone and in combination with the mutagen methyl methanesulfonate (MMS). The ability of botryosphaeran to reduce the production of reactive oxygen and nitrogen species (RONS) generated by hydrogen peroxide was assessed using the CM-H₂DCFDA probe in lymphocyte cultures under different treatment times. None of the evaluated botryosphaeran concentrations were mutagenic in bacteria, nor induced genotoxicity in normal and tumor lymphocytes. Botryosphaeran protected lymphocyte DNA against damage caused by MMS under simultaneous treatment and post-treatment conditions. However, botryosphaeran was not able to reduce the RONS generated by H₂O₂. Besides the absence of genotoxicity, botryosphaeran exerted a protective effect on human lymphocytes against genotoxic damage caused by MMS. These results are important in the validation of botryosphaeran as a therapeutic agent targeting health promotion.     

Poly(ethylene imine) nanocarriers do not induce mutations nor oxidative DNA damage in vitro in MutaMouse FE1 cells

Genotoxicity information on polymers used for gene delivery is scant, but of great concern, especially when developing polymeric nanocarriers as nonviral vector systems for cancer treatment. The genotoxicity of some engineered nanomaterials, e.g., metal oxides like ZnO, TiO?, and CuO but also carbon based materials like carbon black or nanotubes, has commonly been related to oxidative stress, and subsequent inflammation. Recent studies of poly(ethylene imine) (PEI)-based polymers, important nonviral vector systems for pDNA and siRNA, might raise concerns because of their toxic effects dominated by cellular oxidative stress and inflammatory responses, similar to the mentioned effects of engineered nanoparticles. In this study, we employed a FE1-MutaMouse lung epithelial cell line based mutation assay to determine the genotoxicity of three PEI-based polymers and nanosized zinc oxide particles (NZO), all of which have previously been shown to trigger oxidative stress and inflammation. In addition, oxidative DNA damage (8-OH-dG) in FE1 cells was assessed by ELISA. The well-known carcinogen benzo[a]pyrene (B[a]P) was used as positive control. FE1 lung epithelial cells were exposed for eight sequential 72 h incubations, and reporter-gene mutation frequency or 8-OH-dG formation was determined to assess mutagenicity and oxidative DNA damage, respectively. No cytotoxic effects were detected at the exposure levels examined, which are representative of PEI concentrations normally used in in vitro transfection studies. In contrast to B[a]P, neither PEI-polymers nor NZO showed any significant mutagenic activity or oxidative DNA damage in the exposed cells, although PEI-based polymers have been shown to generate significant levels of cellular stress and inflammatory responses. We suggest that the lack of any detectable mutagenic/genotoxic activity of the PEI-based polymers studied here is a crucial step toward a safe use of such nanocarriers in clinical trials.     

Environmental nitration processes enhance the mutagenic potency of aromatic compounds

This work is an attempt to establish if aromatic nitration processes are always associated with an increase of genotoxicity. We determined the mutagenic and genotoxic effects of Benzene (B), Nitrobenzene (NB), Phenol (P), 2‐Nitrophenol (2‐NP), 2,4‐Dinitrophenol (2,4‐DNP), Pyrene (Py), 1‐Nitropyrene (1‐NPy), 1,3‐Dinitropyrene (1,3‐DNPy), 1,6‐Dinitropyrene (1,6‐DNPy), and 1,8‐Dinitropyrene (1,8‐DNPy). The mutagenic activities were evaluated with umuC test in presence and in absence of metabolic activation with S9 mix. Then, we used both cytokinesis‐blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan‐centromeric DNA probes on human lymphocytes in order to evaluate the genotoxic effects. Analysis of all results shows that nitro polycyclic aromatic hydrocarbons (PAHs) are definitely environmental genotoxic/mutagenic hazards and confirms that environmental aromatic nitration reactions lead to an increase in genotoxicity and mutagenicity properties. Particularly 1‐NPy and 1,8‐DNPy can be considered as human potential carcinogens. They seem to be significant markers of the genotoxicity, mutagenicity, and potential carcinogenicity of complex PAHs mixtures present in traffic emission and industrial environment. In prevention of environmental carcinogenic risk 1‐NPy and 1,8‐DNPy must therefore be systematically analyzed in environmental complex mixtures in association with combined umuC test, CBMN assay, and FISH on cultured human lymphocytes. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.