Genotoxicity Biomarkers Research Articles

Multiple biomarker responses in Prochilodus lineatus subjected to short-term in situ exposure to streams from agricultural areas in Southern Brazil

In order to assess the quality of streams susceptible to contamination by pesticides we apply biochemical and genotoxic biomarkers in the Neotropical fish Prochilodus lineatus submitted to in situ tests. Fish were caged, for 96h, in two streams located in areas with intensive use of pesticides, the Apertados (AP) and the Jacutinga (JC), and in a small stream (Godoy stream — GD) found inside a forest fragment adjacent to a State Park. Biochemical parameters, such as biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST), non-protein thiols (NPSH), lipoperoxidation (LPO), protein carbonylation (PCO) and acetylcholinesterase (AChE) were evaluated in various fish organs, as well as genotoxic biomarkers (damage to DNA and occurrence of micronuclei and erythrocyte nuclear abnormalities). Samples of water and sediment were collected for analysis of metals (Cu, Cr, Pb, Ni, Mn, Cd and Zn), organochloride pesticides, and triazine and glyphosate herbicides. We observed an increase in liver GST activity in fish at AP and gill GST activity in fish at JC. An increase in liver LPO was also observed in fish exposed to AP and JC. The same animals also exhibited increased DNA damage and erythrocyte nuclear abnormalities (ENAs) compared to the fish kept in GD. A number of compounds showed concentrations higher than the permitted levels, in particular, dichlorodiphenyltrichloroethane (DDT), its metabolites dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD), hexachlorocyclohexanes (HCH), heptachloride, diclofluanid and aldrins. These pesticides were detected at higher concentrations in water and sediment samples from AP, followed by JC and GD. The Integrated Biomarker Response Index (IBR) indicated that AP and JC (AP: 21.7>JC: 18.5>GD: 12.6) have the worst environmental quality. Integrated biomarker analysis revealed that the alterations observed related well with the levels of environmental contaminants, demonstrating the effectiveness of this biomonitoring approach.

Early genotoxic response and accumulation induced by waterborne copper, lead, and arsenic in European seabass, Dicentrarchus labrax

Cu, Pb, and As, which are among the most abundant metals in the aquatic environment, are also among the most health-threatened by causing diverse cellular injuries. The aim of this study was to assess and compare the potential early induction of genotoxic effects after waterborne Cu, Pb, and As exposure in European seabass, Dicentrarchus labrax, a commercial widely cultured fish, using the micronucleus (MN) assay in peripheral blood erythrocytes. Fish were exposed under laboratory conditions to nominal solutions ranging 0-10 mg/L for 24 and 96 h. Furthermore, actual metal ion concentrations were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) or differential pulse anodic stripping voltammetry (DPASV) in water and four fish tissues differentially related to environmental exposition and metal accumulation, i.e. the gills, liver, muscle, and brain. Dose-dependent increases of micronuclei (MNi) frequency were observed after these very short exposures; based on measured metal concentrations in water, the genotoxic effect ordered as Cu > As > Pb. Significant genotoxic effect at 0.009 mg/L Cu, 0.57 mg/L Pb, and 0.01 mg/L As was seen. For Cu and Pb these are only slightly higher, but for As it is notably lower than the USEPA criteria of maximum concentration to prevent acute toxicity in aquatic organisms. Furthermore, genotoxicity was differentially related to metal accumulation. MNi frequency correlated positively with the content of Pb in all the organs, with the content of As in liver and gills and only with the content of Cu in the brain. In conclusion, our findings raised environmental concerns because these depicted a genotoxic potential of Cu, Pb, and As after a very short exposure to low but environmentally relevant concentrations, too close to regulatory thresholds. In addition, the MN test in D. labrax could be considered an early biomarker of genotoxicity induced by these metals in fish.

Usefulness of biomarkers as intermediate endpoints in health risks posed by occupational lead exposure.

The article concerns potential harmful effects of exposure to lead. Although the occurrence of severe lead poisoning has receded in several countries, occupational exposure resulting in moderate and clinically symptomatic toxicity is still common. An earlier and precise characterization of an individual response is obligatory in order to assess the possible risks for human health. Biomarkers may fill important gaps in the path from exposure to a disease. Specifically speaking, emerging (DNA double strand breaks and telomeric DNA erosion) and validated (micronuclei induction and chromosomal aberrations) biomarkers of genotoxicity seem to provide evidence for the assessment of molecular and cellular damage. Moreover, identification of genetic variability with a key role in modulating genotoxic damage may help minimize risks for susceptible subjects. Further investigations are naturally needed to properly define their diagnostic and/or prognostic value as "early warning" signs of a long-term risk for a subsequent clinically overt disease.

Abstract CN08-03: How does air pollution cause cancer?

Abstract In October 2013, the International Agency for Research on Cancer (IARC) evaluated outdoor air pollution and associated particulate matter (PM) as group 1 (known) human carcinogens for the lung and probable carcinogens for the bladder. Although the IARC monograph on outdoor air (Vol. 109) will not be published until late 2014, this talk reviews some of the main mechanistic data that supported this evaluation. The IARC working group consisted of four sub-groups: epidemiology, animal cancer, exposure, and mechanistic data. Six people comprised the mechanistic sub-group, and this talk is an overview of their review of the literature, which covered more than 30 years and 800 publications on the genetic and related effects of air pollution. More than 2,500 measurements of the mutagenicity in Salmonella (Ames assay) of outdoor air samples from around the world showed that the mutagenicity of outdoor air ranges over 6 orders of magnitude in potency (revertants/m3); however, the mutagenic potency of the extractable organics from the PM ranges only over 1 order of magnitude. This indicates that the general chemical composition and mutagenic potency of the PM is similar around the world; what varies is the amount of this PM per cubic meter of air, i.e., its concentration. Worldwide, air is generally more mutagenic during working hours (due to traffic) and during winter (due to heating emissions). More than 90 measurements compared genotoxicity biomarkers between people whose daily work involved chronic exposure to outdoor air/traffic (traffic police, toll takers, gate keepers, street vendors, etc.) versus people without such exposures (typically office workers). Approximately 90% of these studies found that chronic exposure to traffic exhaust enhanced a range of such biomarkers, including cytogenetic damage, DNA damage (DNA adducts and strand breaks), altered telomere length, altered gene expression, and epigenetic changes (DNA methylation). Some of these biomarkers are associated with increased risk for cancer and provide mechanistic support for how air pollution causes cancer—i.e., by causing DNA damage, mutation, and/or altered gene expression. A large number of studies showed that organic extracts of outdoor PM induced DNA adducts in mammalian cells in culture as well as in rodents, and oxidative DNA damage and DNA strand breaks are also induced not only in people exposed chronically to air pollution but to cells in culture and rodents exposed to outdoor PM. Efforts are underway to associate through global positioning coordinates the air mutagenicity data in Salmonella relative to PM concentrations and the lung cancer risk values relative to PM concentrations from epidemiology studies. Such data will provide additional evidence for the linkage between air mutagenicity and lung cancer risk. A growing literature is indicating the important role of epigenetic changes (DNA methylation) associated with air pollution, resulting in increased risk for cancer and other diseases associated with air pollution due to altered gene expression. A substantial literature documents the genotoxic effects of air pollution in plants, including DNA damage, chromosome aberration, and gene mutation. Collectively, the large body of data reviewed by the mechanistic sub-group provided strong mechanistic support for the ability of various components of outdoor air pollution to cause DNA damage, which if not repaired, could be processed into mutations (primarily G to T base substitutions) in key cancer-associated genes (TP53 and KRAS), leading to lung and possibly bladder cancer. The nitroarenes, primarily from diesel exhaust, are the likely cause of the bladder cancer due to their conversion to aromatic amines, which are known bladder carcinogens. Polycyclic aromatic hydrocarbons (PAHs) clearly play a large role in both the lung and bladder carcinogenesis associated with outdoor air pollution. Citation Format: David M. DeMarini. How does air pollution cause cancer? [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr CN08-03.

Environmental and biological monitoring of occupational formaldehyde exposure resulting from the use of products for hair straightening

The evaluation of formaldehyde (FD) exposure in beauty salons, due to the use of hair straightening products, and its relation with genotoxicity biomarkers was performed in this study. Regardless of official recommendations, the inappropriate use of homemade hair creams has became a popular practice in Brazil, and high formaldehyde content in the "progressive straightening" creams can contain mutagens that could increase the incidence of neoplasia in those people who use them. Damage to DNA was assessed by conducting a micronuclei test (MNT) on buccal cells and the comet assay on heparinized venous blood samples. A total of 50 volunteers were recruited at six different beauty salons (labeled A to F). At two salons that used products that did not contain FD (salons D and E), environmental FD concentrations were 0.04 and 0.02 ppm. In contrast, the products used at salons A, B, C, and F contained 5.7, 2.61, 5.9, and 5.79% of FD, and these salons had environmental FD concentrations of 0.07, 0.14, 0.16, and 0.14 ppm, respectively. Comparison of the beauty salon workers from each of the six beauty salons revealed significant differences in urinary formic acid (FA) concentration before exposure (p = 0.016), urinary FA after exposure (p = 0.004), variation in FA concentration before and after exposure (p = 0.018), environmental FD concentration (p < 0.001), cytogenetic damage detected by the comet assay according to both damage index (p < 0.001) and frequency of damage (p < 0.001), and for karyorrhexis only according to the MNT (p = 0.001).

Toxic Assessment of Triclosan and Triclocarban on Artemia salina.

In this study, we investigated the possible acute toxic and genotoxic effects of triclosan (TCS) and triclocarban (TCC) on Artemia salina. Genotoxicity was evaluated using single-cell gel electrophoresis and apoptotic frequency assays (Annexin V-FITC/PI assay). Acute toxicity test results showed that TCC (LC50-24h=17.8µg/L) was more toxic than TCS (LC50-24h=171.1µg/L). Significant increases in both genotoxic biomarkers were observed at 24h after initial exposure, indicating that these two chemicals are potentially dangerous for this aquatic biological model. Although further studies are required, a comparison of data both in vitro and in vivo allowed us to suggest possible mechanisms of action for TCS and TCC in this sentinel organism.

Evaluation of genotoxicity using automated detection of γH2AX in metabolically competent HepaRG cells.

The in situ detection of γH2AX was recently reported to be a promising biomarker of genotoxicity. In addition, the human HepaRG hepatoma cells appear to be relevant for investigating hepatic genotoxicity since they express most of drug metabolizing enzymes and a wild type p53. The aim of this study was to determine whether the automated in situ detection of γH2AX positive HepaRG cells could be relevant for evaluation of genotoxicity after single or long-term repeated in vitro exposure compared to micronucleus assay. Metabolically competent HepaRG cells were treated daily with environmental contaminants and genotoxicity was evaluated after 1, 7 and 14 days. Using these cells, we confirmed the genotoxicity of aflatoxin B1 and benzo(a)pyrene and demonstrated that dimethylbenzanthracene, fipronil and endosulfan previously found genotoxic with comet or micronucleus assays also induced γH2AX phosphorylation. Furthermore, we showed that fluoranthene and bisphenol A induced γH2AX while no effect had been previously reported in HepG2 cells. In addition, induction of γH2AX was observed with some compounds only after 7 days, highlighting the importance of studying long-term effects of low doses of contaminants. Together, our data demonstrate that automated γH2AX detection in metabolically competent HepaRG cells is a suitable high-through put genotoxicity screening assay.

Abstract 4580: Expression of γH2AX as a biomarker of genotoxic carcinogen in the urinary bladder of rats

Abstract Background: Bladder cancer is the seventh most common cancer in males worldwide. Recent studies have demonstrated that DNA double-strand breaks are recognized to be a serious injury leading to genome instability and cancer development. The rapid phosphorylation of histone H2AX at Serine 139, to become γH2AX, occurs in response to DNA double-strand breaks. Roles of γH2AX in carcinogenesis are still unclear, and there are few reports on the association with bladder cancer. We have previously demonstrated that γH2AX expression is induced in urinary bladders of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated rat (Toyoda et al., J Toxicol Pathol, 26: 215-221, 2013). To evaluate the value of γH2AX as a biomarker for carcinogenicity and genotoxicity to the urinary bladder, we performed an immunohistochemical analysis of γH2AX expression in chemical-treated rats. Methods: Six-week-old male F344 rats were treated with 0.05% BBN, 1.8% 2-nitroanisole (2-NA), 2% 2,2-bis (bromomethyl)-1,3-propanediol (BMP), 0.1% phenethyl isothiocyanate (PEITC), 3% melamine, or 3% uracil in either drinking water (BBN) or diet (other chemicals) for 4 weeks. Animals were sacrificed at 4 or 6 weeks of the experiment, and immunohistochemistry for γH2AX in urinary bladders was performed. Results: At week 4, γH2AX expression in the urothelium were significantly increased in BBN and 2-NA groups compared with the controls. γH2AX expression ratio (positive cells/1000 cells) in BBN, 2-NA and control groups were 76±21, 111±37 and 0.8±0.6, respectively. γH2AX was recognized as dot-like foci in nuclei of epithelial cells, and the positive cells were mainly located near the basal layer of the mucosa. In BMP and PEITC groups, γH2AX expression was mainly observed in umbrella cells and intermediate cells, respectively. In melamine and uracil groups, there were diffuse urothelial hyperplasia and focal expression of γH2AX especially in the basal layer. The expression ratios of γH2AX in BMP, PEITC, melamine and uracil groups were 8.3±6.6, 26±9.0, 16±11 and 11±11, respectively. Two weeks after withdrawal of administration, although the expression ratio of γH2AX was markedly reduced in all groups, it remained high in BBN (17±1.9) and 2-NA (19±3.4) groups compared with the controls (0.6±0.9). Discussion and conclusion: BBN and 2-NA, genotoxic bladder carcinogens, induced more extensive γH2AX expression in the urothelium than other non-genotoxic (melamine and uracil) or non-carcinogenic chemicals (BMP and PEITC), and γH2AX-positive cells remained in relatively large numbers even 2 weeks after withdrawal of its administration. In addition, ratio of basal cells, intermediate cells and umbrella cells in γH2AX-positive cells showed similar patterns in BBN/2-NA and melamine/uracil groups. These data indicate that γH2AX has potential as a biomarker for early detection of genotoxic carcinogen in the urinary bladder. Citation Format: Takeshi Toyoda, Young-Man Cho, Jun-ichi Akagi, Yasuko Mizuta, Kumiko Ogawa. Expression of γH2AX as a biomarker of genotoxic carcinogen in the urinary bladder of rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4580. doi:10.1158/1538-7445.AM2015-4580

Differential Effects of Glycyrrhiza Species on Genotoxic Estrogen Metabolism: Licochalcone A Downregulates P450 1B1, whereas Isoliquiritigenin Stimulates It.

Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG > GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as nontoxic and 4-MeOE1 as genotoxic biomarkers in the nontumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 μg/mL), and LigC (1 μM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1, 10 μM) decreased cytokine- and TCDD-induced P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50 = 12.3 μM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 μg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that, of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for women's health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds.

GENOTOXICITY OF FORMALDEHYDE IN PERIPHERAL BLOOD LYMPHOCYTES AMONG PATHOLOGY LABORATORY WORKERS

Introduction: Formaldehyde (FA) is a chemical, traditionally used in pathology and anatomy laboratories as a tissue preservative. Many studies clearly indicated that FA can induce genotoxic effects in peripheral blood lymphocytes. Aim of work: to evaluate the genotoxic effects of formaldehyde among medical and paramedical personnel in The Histopathology Laboratory. Materials and Methods: The study was conducted in histopathology laboratory including 30 workers occupationally exposed to formaldehyde and 29 persons from other lab in kasr Al Aini hospital matching the exposed group for age, sex and socioeconomic status with no history of occupational exposure to formaldehyde. A questionnaire was done including inquiries about age, sex, occupational history and special habits. All participants were subjected to the following laboratory investigations: measuring DNA protein crosslink (DPC), sister chromatid exchange (SCE) and micronucleus (Mn) frequency in peripheral blood lymphocytes. Environmental monitoring of the work place was done to measure the level of FA at different place in the pathology lab. Results: This study showed statistically significant increase of DPC with significant increase in sister chromatid exchange (SCE) and micronucleus MN in peripheral blood lymphocytes. Conclusion: The studied group was exposed to formaldehyde level above the exposure limits recommended by NIOSH, ACGIH and OSHA with adverse health effects in the form of increase in DPC, SCE and Mn as genotoxicity biomarkers. These genotoxicity biomarkers are considered to play an important role in the carcinogenesis of FA.

Dose-Response for Multiple Biomarkers of Exposure and Genotoxic Effect Following Repeated Treatment of Rats with the Alkylating Agents, MMS and MNU.

The nature of the dose-response relationship for various in vivo endpoints of exposure and effect were investigated using the alkylating agents, methyl methanesulfonate (MMS) and methylnitrosourea (MNU). Six male F344 rats/group were dosed orally with 0, 0.5, 1, 5, 25 or 50mg/kg bw/day (mkd) of MMS, or 0, 0.01, 0.1, 1, 5, 10, 25 or 50 mkd of MNU, for 4 consecutive days and sacrificed 24h after the last dose. The dose-responses for multiple biomarkers of exposure and genotoxic effect were investigated. In MMS-treated rats, the hemoglobin adduct level, a systemic exposure biomarker, increased linearly with dose (r (2) = 0.9990, P < 0.05), indicating the systemic availability of MMS; however, the N7MeG DNA adduct, a target exposure biomarker, exhibited a non-linear dose-response in blood and liver tissues. Blood reticulocyte micronuclei (MN), a genotoxic effect biomarker, exhibited a clear no-observed-genotoxic-effect-level (NOGEL) of 5 mkd as a point of departure (PoD) for MMS. Two separate dose-response models, the Lutz and Lutz model and the stepwise approach using PROC REG both supported a bilinear/threshold dose-response for MN induction. Liver gene expression, a mechanistic endpoint, also exhibited a bilinear dose-response. Similarly, in MNU-treated rats, hepatic DNA adducts, gene expression changes and MN all exhibited clear PoDs, with a NOGEL of 1 mkd for MN induction, although dose-response modeling of the MNU-induced MN data showed a better statistical fit for a linear dose-response. In summary, these results provide in vivo data that support the existence of clear non-linear dose-responses for a number of biologically significant events along the pathway for genotoxicity induced by DNA-reactive agents.

NSAID-manufacturing plant effluent induces geno- and cytotoxicity in common carp (Cyprinus carpio)

The pharmaceutical industry generates wastewater discharges of varying characteristics and contaminant concentrations depending on the nature of the production process. The main chemicals present in these effluents are solvents, detergents, disinfectants – such as sodium hypochlorite (NaClO) – and pharmaceutical products, all of which are potentially ecotoxic. Therefore, this study aimed to evaluate the geno- and cytotoxicity induced in the common carp Cyprinus carpio by the effluent emanating from a nonsteroidal anti-inflammatory drug (NSAID)-manufacturing plant. Carp were exposed to the lowest observed adverse effect level (LOAEL, 0.1173%) for 12, 24, 48, 72 and 96h, and biomarkers of genotoxicity (comet assay and micronucleus test) and cytotoxicity (caspase-3 activity and TUNEL assay) were evaluated. A significant increase with respect to the control group (p<0.05) occurred with all biomarkers from 24h on. Significant positive correlations were found between NSAID concentrations and biomarkers of geno- and cytotoxicity, as well as among geno- and cytotoxicity biomarkers. In conclusion, exposure to this industrial effluent induces geno- and cytotoxicity in blood of C. carpio.

Oxidative stress, genotoxicity and histopathology biomarker responses in Mugil cephalus and Dicentrarchus labrax gill exposed to persistent pollutants. A field study in the Bizerte Lagoon: Tunisia

The use of biomarkers has become an important tool for modern environmental assessment as they can help to predict pollutants involved in the monitoring program. Despite the importance of fish gill in several functions (gaseous exchange, osmotic and ionic regulation, acid-base balance and nitrogenous waste) its use in coastal water biomonitoring focusing on protection and damage is scarce.This field study investigates biochemical (catalase, superoxide dismutase, lipid peroxidation), molecular (DNA integrity) and morphological (histology) parameters in gill of mullet (Mugil cephalus) and sea bass (Dicentrarchus labrax) and originating from Bizerte lagoon (a coastal lagoon impacted by different anthropogenic activities) and from the Mediterranean Sea (a reference site). Remarkable alterations in the activities of oxidative stress enzymes and DNA integrity in the tissue of the two studied fish species were detected in Bizerte Lagoon. The study of histopathological alterations of gills in both two fish species from Bizerte Lagoon suggest thickening of primary lamellae, cellular hyperplasia, aneurism, curving, shortening and fusion of secondary lamellae.The adopted approach, considering simultaneously protection responses and damaging effects, revealed its usefulness on the pollution assessment.

Antigenotoxic potential of curcumin and carvacrol against malathion-induced DNA damage in cultured human peripheral blood and its relation to GSTM1 and GSTT1 polymorphism

The aerial application of malathion, a widely used organophosphate insecticide, has raised public concerns about its potential adverse health effects. We, therefore, studied the antigenotoxic potential of curcumin and carvacrol against malathion-induced DNA damage using sister chromatid exchange (SCE) as a biomarker of genotoxicity. To observe the antigenotoxic potential of curcumin and carvacrol, heparinized fresh blood from healthy individuals was treated with 30 μg/mL of malathion in the presence of curcumin and carvacrol. Curcumin at concentrations of 25 μg/mL and 50 μg/mL had significantly reduced (p < 0.05) the frequency of SCE as compared to malathion-exposed sample. Similarly, carvacrol showed significant (p < 0.05) antigenotoxic effect at concentrations of 2.5 μg/mL and 5.0 μg/mL against malathion. We also studied the effect of GSTT1 and GSTM1 on the genotoxicity of malathion and antigenotoxic potential of curcumin and carvacrol. We observed that there is a statistically significant (p < 0.05) reduction in the frequency of SCE in case of curcumin and carvacrol as compared to malathion, but we did not observe any significant relationship (p > 0.05) between GSTT1 and GSTM1 polymorphism and the genotoxicity of malathion and antigenotoxic potential of curcumin and carvacrol.

Development of a toxicogenomics signature for genotoxicity using a dose-optimization and informatics strategy in human cells.

The development of in vitro molecular biomarkers to accurately predict toxicological effects has become a priority to advance testing strategies for human health risk assessment. The application of in vitro transcriptomic biomarkers promises increased throughput as well as a reduction in animal use. However, the existing protocols for predictive transcriptional signatures do not establish appropriate guidelines for dose selection or account for the fact that toxic agents may have pleiotropic effects. Therefore, comparison of transcriptome profiles across agents and studies has been difficult. Here we present a dataset of transcriptional profiles for TK6 cells exposed to a battery of well-characterized genotoxic and nongenotoxic chemicals. The experimental conditions applied a new dose optimization protocol that was based on evaluating expression changes in several well-characterized stress-response genes using quantitative real-time PCR in preliminary dose-finding studies. The subsequent microarray-based transcriptomic analyses at the optimized dose revealed responses to the test chemicals that were typically complex, often exhibiting substantial overlap in the transcriptional responses between a variety of the agents making analysis challenging. Using the nearest shrunken centroids method we identified a panel of 65 genes that could accurately classify toxicants as genotoxic or nongenotoxic. To validate the 65-gene panel as a genomic biomarker of genotoxicity, the gene expression profiles of an additional three well-characterized model agents were analyzed and a case study demonstrating the practical application of this genomic biomarker-based approach in risk assessment was performed to demonstrate its utility in genotoxicity risk assessment.

Micronucleus as biomarker of genotoxicity in birds from Brazilian Cerrado

Birds are considered efficient bioindicators, by their behavioral characteristics, diversified diet, and use of several vegetation layers, including in contaminated environments. The accumulation of contaminants can harm the reproductive process and survival of species, in addition to causing severe metabolic disorders. Air pollution can also affect the birds' health. Micronucleus analysis, a technique able to evaluate the organisms' sensitivity to contaminant agents, has been regarded as a practical tool for evaluating and monitoring the clastogenic and aneugenic effects caused by pollutants. The purpose of this study was to evaluate the presence of micronuclei in bird species that use forest environments and their surroundings; and to verify if the frequency and amount of micronuclei varies between species, areas and populations. Birds transiting between the Brazilian Cerrado forest and open formations were analyzed, coming from four forest fragments of Triângulo Mineiro, two close to urban areas and two more distant. Birds were captured with mist-nets for collecting blood extensions, which were used for counting micronuclei. In total, 103 individuals of 21 species were captured, and the micronucleus rate for every 5000 erythrocytes analyzed was 1.30. Only six populations had sampling sufficiency. There was no difference between the number of individuals with and without micronuclei (χ²=3.18, df=1, p=0.08). In areas closer to the urban perimeter, the micronuclei averages in birds were greater compared to the most isolated areas (H=27.534, df=3, p<0,001). In São José, the individuals of Myiothlypis flaveola presented a number of micronuclei significantly greater than the Galheiro and Água Fria (H=9.601, df=2, p=0.008). M. flaveola clearly reflected the area quality. The micronuclei analysis in birds was effective for evaluating the area quality as well as the intensity with which the birds respond to impacts caused by the surrounding matrix.

Genotoxicity evaluation of environmental pollutants using analysis of nucleolar alterations.

Nucleolar alterations resulting from the action of either chemical or physical agents can serve as important genotoxicity biomarkers. In this study, the efficiency of AgNOR banding technique to identify the presence of nucleoli in micronucleus and assess nucleolar alterations in aberrant cells of Allium cepa was evaluated. Seeds of this plant were exposed to both water samples from a river that receives untreated urban effluent and to the trifluralin herbicide (0.84mg/L concentration), both analyzed in two different seasons (summer and winter seasons). Samples induced significant frequencies of chromosomal and nuclear aberrations and micronuclei, as observed in cells submitted to conventional chromosomal staining. The herbicide caused a significant increase in the number of nucleoli and micronuclei, interpreted as due to the elimination of excessive nucleolar material resulting from polyploidization. The use of the AgNOR technique enabled the identification of both the presence of the nucleolus in some micronuclei and the nucleolar organizer region (NOR) behavior of aberrant cells. The NOR-banding technique showed to be an efficient tool for studying the genotoxic effects caused by a xenobiotics and a complex environmental sample.

γH2AX assay as DNA damage biomarker for human population studies: defining experimental conditions.

H2AX histone phosphorylation represents an early event in the cellular response against DNA double-strand breaks (DSBs), and plays a central role in sensing and repairing DNA damage. Therefore, the analysis of H2AX phosphorylated (γH2AX) may be possibly used as biomarker of genotoxicity and genomic instability with a number of applications in human epidemiology. However, the lack of an experimental standard leads to a wide heterogeneity in the results obtained and their interpretation, affecting the reliability of the assay. To address the most critical issues limiting the use of the γH2AX assay in human population studies, a flow cytometry analysis was performed to establish differences in γH2AX levels between fresh or cryopreserved peripheral blood lymphocytes, and to assess the influence of phytohemagglutinin (PHA) stimulation. To this purpose, cells were treated with 4 known genotoxic chemicals with different mechanisms of DSB induction, ie, bleomycin, methyl methanesulfonate, camptothecin, and actinomycin. According to our results, both unstimulated and stimulated fresh lymphocytes can be efficiently employed to evaluate γH2AX levels, but the sensitivity of the assay is depending upon the kind of damage observed. On the other hand, cryopreserved lymphocytes require PHA stimulation since unstimulated cells showed too high basal damage. Consequently, the protocol conditions will depend on the expected mechanism of production of DSB and the characteristics of the study design (sample collection and storage conditions, type of epidemiological study). Further studies are required to standardize the protocol of γH2AX assay to be employed as biomarker of genotoxicity or genomic instability in human population studies.

Influence of Serum Levels of Vitamins A, D, and E as well as Vitamin D Receptor Polymorphisms on Micronucleus Frequencies and Other Biomarkers of Genotoxicity in Workers Exposed to Formaldehyde

Background/Aim: Formaldehyde is classified as carcinogenic to humans, making it a major concern, particularly in occupational settings. Fat-soluble vitamins, such as vitamins A, D, and E, are documented as antigenotoxic and antimutagenic and also correlate with the cell antioxidant potential. This study investigates the influence of these vitamins on genotoxicity biomarkers of formaldehyde-exposed hospital workers. Methods: The target population were hospital workers exposed to formaldehyde (n = 55). Controls were nonexposed individuals (n = 80). The most used genotoxicity biomarkers were the cytokinesis-block micronucleus assay for lymphocytes and the micronucleus test for exfoliated buccal cells. Vitamins A and E were determined by high-performance liquid chromatography with a diode array detector (HPLC-DAD) and vitamin D receptor (VDR) polymorphisms by real-time PCR. Results: Significant correlations were found between genotoxicity biomarkers and between vitamins A and E in controls. Multiple regression showed that vitamin A was significantly associated with a higher mean of nucleoplasmic bridges (p < 0.001), and vitamin E was significantly associated with a decreased frequency of nuclear buds (p = 0.045) in the exposed group. No effect of vitamin D was observed. The VDRBsmI TT genotype carriers presented higher means of all the genotoxicity biomarkers; however, we found no significant associations. Conclusions: The study suggests that vitamin levels may modulate direct signs of genotoxicity.

Genotoxic and Cytotoxic Damage by Cyclophosphamide and Adriamycin as a Response to Treatment in Breast Cancer Patients: Pilot Study

The aim of this study was to evaluate the genotoxicity induced by cyclophosphamide-adriamycin treatment in breast cancer patients through the frequencies of Sister Chromatid Exchange (SCE), Replication Index (RI), Mitotic Index (MI) and Cell Proliferation Index (CPI) and to study the possible association between biomarkers of genotoxicity and the early response to treatment. The frequencies were obtained before and immediately after therapy from 17 patients with breast cancer (p