The conventional battery for genotoxicity testing is not well-suited to assessing the large number of chemicals needing evaluation. Traditional in vitro tests lack throughput capacity, provide little mechanistic information, and have poor specificity in predicting in vivo genotoxicity. The Health Canada GeneTox21 research program is developing a multi-endpoint platform for modernized in vitro genotoxicity assessment. The GeneTox21 assays include the TGx-DDI transcriptomic biomarker (i.e., 64-gene expression signature to identify DNA damage-inducing (DDI) substances), the MicroFlow® assay (i.e., a flow cytometry-based micronucleus (MN) test), and the MultiFlow® assay (i.e., a multiplexed flow cytometry-based reporter assay that yields mechanism-of-action (MoA) information). As part of GeneTox21 development, the objective of this study was to investigate the utility of the TGx-DDI transcriptomic biomarker, multiplexed with the MicroFlow® and MultiFlow® assays, as an integrated testing strategy for screening data-poor substances prioritized by Health Canada’s New Substances Assessment and Control Bureau. Human lymphoblastoid TK6 cells were exposed to 3 control and 10 data-poor substances, using a 6-point concentration range. Cells were exposed for 4 hours with or without exogenous metabolic activation. Gene expression profiling was conducted using the targeted TempO-SeqTM assay, and the TGx-DDI classifier was applied to the dataset. Classifications were compared with those based on the MicroFlow® and MultiFlow® assays. Benchmark Concentration (BMC) modeling was used for potency ranking. The results of the integrated hazard calls indicate that five data-poor compounds are genotoxic in vitro, causing DNA damage via a clastogenic MoA, and one is positive via a pan-genotoxic MoA. Two compounds are likely irrelevant positives in the MN test; two are considered possibly genotoxic causing DNA damage via an ambiguous MoA. From quantitative analyses of concentration-response data, we observed nearly identical potency rankings for each assay with two main potency groups being observed. This ranking was maintained when all endpoint BMCs were converted into a single score using the Toxicological Prioritization (ToxPi) approach. Overall, this study contributes to the establishment of a modernized approach for effective genotoxicity assessment and chemical prioritization for further regulatory scrutiny. We conclude that integration of the TGx-DDI biomarker with other GeneTox21 assays is an effective NAM-based strategy for genotoxicity assessment of data-poor compounds.

A exposição a tintas, as quais contêm solventes orgânicos e metais, pode levar a danos no DNA e formação de espécies reativas (ER), que podem lesar diversas classes de moléculas. Em vista disso, este trabalho teve como objetivo avaliar possíveis danos oxidativos, genotóxicos e mutagênicos em 33 indivíduos, do sexo masculino, ocupacionalmente expostos a tintas há, no mínimo, 6 meses. Para o grupo controle, foram selecionados 29 indivíduos saudáveis, não expostos a tintas, pareados em idade com o grupo exposto. A fim de verificar a influência do descanso do fim de semana, foram realizadas coletas na segunda-feira pela manhã e na sexta-feira ao final da jornada de trabalho. Os danos oxidativos foram avaliados pelos produtos de reação com o ácido tiobarbitúrico (TBARS), proteínas carboniladas (PC) e atividade das enzimas antioxidantes superóxido dismutase (Sod) e catalase (Cat). Foram medidos, ainda, o ácido hipúrico (AH) e o ácido deltaaminolevulínico (ALA), marcadores urinários de exposição ao tolueno e ao chumbo, respectivamente. A genotoxicidade foi avaliada pelo ensaio cometa (em sangue periférico) e pelo teste de micronúcleos (MN) (em linfócitos e células da mucosa bucal). Não foi observado aumento significativo nos níveis de TBARS no grupo exposto quando comparado ao grupo controle. Entretanto, verificou-se, neste grupo, um maior índice de danos aos lipídeos nas amostras coletadas na sexta-feira comparado com as amostras coletadas na segunda-feira (p = 0,008; z = -2,637). Ao final da semana (amostras coletadas na sexta-feira), os indivíduos expostos a tintas apresentaram mais danos às proteínas em comparação com o grupo controle (p = 0,032; z = -2,14). Observou-se também, que os trabalhadores expostos a tintas tiveram uma diminuição nas atividades de Sod (p = 0,003; z = 2,935) e Cat (p = 0,025; z = -2,247) nas amostras de segunda-feira, bem como valores mais elevados de AH (p = 0,010; z = - 2,591) e de ALA (p = 0,000; z = -4,487). A exposição a tintas induziu um aumento significativo dos danos ao DNA (principalmente classes um e dois), tanto nas amostras coletadas na segunda (p = 0,000; z = - 5,356) quanto nas de sexta-feira (p = 0,000; z = -6,456). Apesar de não ter sido encontrado um aumento na frequência de MN em linfócitos ou em células da mucosa bucal no grupo exposto, observou-se um aumento de nuclear buds (NBUDs) (segunda-feira, p = 0,004, z = - 2,894), uma diminuição do índice de divisão nuclear (IDN) (sexta-feira, p = 0,000, z = -4,78) nos linfócitos e um aumento na frequência de células com cromatina condensada nas células da mucosa bucal (segunda-feira, p = 0,000, z = -4,503; sexta-feira, p = 0,000, z = -5,203), indicativo de amplificação gênica e indução de mecanismos apoptóticos nestas células. Observou-se uma correlação positiva entre o índice de danos no DNA (ensaio cometa) e o tempo de exposição a tintas (r = 0,376; p = 0,031), assim como entre o tempo diário de exposição a tintas e a frequência de micronúcleos (segunda-feira, r = 0,450; p = 0,018) e de NBUDs (sexta-feira, (r = 0,402; p = 0,038) nos indivíduos expostos. Embora outros estudos sejam necessários, esses resultados mostram que a exposição ocupacional a tintas pode induzir um aumento de danos no DNA, os quais parecem estar sendo reparados durante o descanso do final de semana. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-05-29T19:53:19Z No. of bitstreams: 1 Dissertacao Carina Cassini.pdf: 626115 bytes, checksum: 4477a810de80e2c9702e22c8302fba89 (MD5) / Made available in DSpace on 2014-05-29T19:53:19Z (GMT). No. of bitstreams: 1 Dissertacao Carina Cassini.pdf: 626115 bytes, checksum: 4477a810de80e2c9702e22c8302fba89 (MD5) / Organic solvents and metals, widely used in paints, can lead to DNA damages and reactives species (RS) generation. The aim of this study was to evaluate possibles oxidative, genotoxic and mutagenic damages in 33 male workers exposed for at least six months to paint. To constitute the control group 29 healthy individuals were choosen, without paint exposure, which matched in age with exposed group. Two sampling were performed to verify a possible DNA repair during the weekend: in the beginning and at the end of work week. The oxidative damages were evaluated by thiobarbituric acid reaction products (TBARS), carbonylated proteins (CP), superoxide dismutase (Sod) and catalase (Cat) activities. Hippuric acid (HA) and delta-aminolevulinic acid (ALA) were used as toluene and lead markers exposure, respectively. The genotoxicity was evaluated by comet assay (in peripherical blood) and by micronucleus (MN) test (in limphocytes and exfoliated buccal cells). The results showed no significant increase in TBARS levels in exposed group in relation to the control group. However, the lipidic damages was higher in Friday samples comparing to Monday samples (p = 0.008; z = -2.637). The proteins damage was higher in exposed group in comparison to control group exclusively in Friday samples (p = 0.032; z = -2.14). It was also observed that the workers exposed to paints showed lower Sod (p = 0.003; z = 2.935) and Cat (p = 0.025; z = -2.247) activities in Monday samples. The exposed group presented HA levels (p = 0.010; z = - 2.591) and ALA levels (p = 0.000; z = -4.487) higher than the control group. The workers exposed to paints presented a significant increase in DNA damage in both Monday (p = 0.000; z = -5.356) and Friday (p = 0.000; z = -6.456) samples. No increase was observed in MN frequency in limphocytes and buccal cells. However, the individuals exposed to paints showed an increase in nuclear buds (NBUDS) (Monday samples, p = 0.004, z = -2.894), a reduction in nuclear division index (NDI) (Friday samples, p = 0.000, z = -4.78) in lymphocytes and an increase in condensed chromatin frequency in buccal cells (Monday samples, p = 0.000, z = -4.503; Friday samples, p = 0.000, z = -5.203), indicating genic amplification and apoptosis induction. The DNA damage index (comet assay) correlated positively with average working time (r = 0.376; p = 0.031). It was also observed a positive correlation between time daily exposure and MN (Monday samples, r = 0.450; p = 0.018) and NBUDs (Friday samples: r = 0.402; p = 0.038) frequency. These results showed that paint exposure is able to generate DNA damages and these damages are being repaired during the weekend.

A exposição a tintas, as quais contêm solventes orgânicos e metais, pode levar a danos no DNA e formação de espécies reativas (ER), que podem lesar diversas classes de moléculas. Em vista disso, este trabalho teve como objetivo avaliar possíveis danos oxidativos, genotóxicos e mutagênicos em 33 indivíduos, do sexo masculino, ocupacionalmente expostos a tintas há, no mínimo, 6 meses. Para o grupo controle, foram selecionados 29 indivíduos saudáveis, não expostos a tintas, pareados em idade com o grupo exposto. A fim de verificar a influência do descanso do fim de semana, foram realizadas coletas na segunda-feira pela manhã e na sexta-feira ao final da jornada de trabalho. Os danos oxidativos foram avaliados pelos produtos de reação com o ácido tiobarbitúrico (TBARS), proteínas carboniladas (PC) e atividade das enzimas antioxidantes superóxido dismutase (Sod) e catalase (Cat). Foram medidos, ainda, o ácido hipúrico (AH) e o ácido deltaaminolevulínico (ALA), marcadores urinários de exposição ao tolueno e ao chumbo, respectivamente. A genotoxicidade foi avaliada pelo ensaio cometa (em sangue periférico) e pelo teste de micronúcleos (MN) (em linfócitos e células da mucosa bucal). Não foi observado aumento significativo nos níveis de TBARS no grupo exposto quando comparado ao grupo controle. Entretanto, verificou-se, neste grupo, um maior índice de danos aos lipídeos nas amostras coletadas na sexta-feira comparado com as amostras coletadas na segunda-feira (p = 0,008; z = -2,637). Ao final da semana (amostras coletadas na sexta-feira), os indivíduos expostos a tintas apresentaram mais danos às proteínas em comparação com o grupo controle (p = 0,032; z = -2,14). Observou-se também, que os trabalhadores expostos a tintas tiveram uma diminuição nas atividades de Sod (p = 0,003; z = 2,935) e Cat (p = 0,025; z = -2,247) nas amostras de segunda-feira, bem como valores mais elevados de AH (p = 0,010; z = - 2,591) e de ALA (p = 0,000; z = -4,487). A exposição a tintas induziu um aumento significativo dos danos ao DNA (principalmente classes um e dois), tanto nas amostras coletadas na segunda (p = 0,000; z = - 5,356) quanto nas de sexta-feira (p = 0,000; z = -6,456). Apesar de não ter sido encontrado um aumento na frequência de MN em linfócitos ou em células da mucosa bucal no grupo exposto, observou-se um aumento de nuclear buds (NBUDs) (segunda-feira, p = 0,004, z = - 2,894), uma diminuição do índice de divisão nuclear (IDN) (sexta-feira, p = 0,000, z = -4,78) nos linfócitos e um aumento na frequência de células com cromatina condensada nas células da mucosa bucal (segunda-feira, p = 0,000, z = -4,503; sexta-feira, p = 0,000, z = -5,203), indicativo de amplificação gênica e indução de mecanismos apoptóticos nestas células. Observou-se uma correlação positiva entre o índice de danos no DNA (ensaio cometa) e o tempo de exposição a tintas (r = 0,376; p = 0,031), assim como entre o tempo diário de exposição a tintas e a frequência de micronúcleos (segunda-feira, r = 0,450; p = 0,018) e de NBUDs (sexta-feira, (r = 0,402; p = 0,038) nos indivíduos expostos. Embora outros estudos sejam necessários, esses resultados mostram que a exposição ocupacional a tintas pode induzir um aumento de danos no DNA, os quais parecem estar sendo reparados durante o descanso do final de semana. / Organic solvents and metals, widely used in paints, can lead to DNA damages and reactives species (RS) generation. The aim of this study was to evaluate possibles oxidative, genotoxic and mutagenic damages in 33 male workers exposed for at least six months to paint. To constitute the control group 29 healthy individuals were choosen, without paint exposure, which matched in age with exposed group. Two sampling were performed to verify a possible DNA repair during the weekend: in the beginning and at the end of work week. The oxidative damages were evaluated by thiobarbituric acid reaction products (TBARS), carbonylated proteins (CP), superoxide dismutase (Sod) and catalase (Cat) activities. Hippuric acid (HA) and delta-aminolevulinic acid (ALA) were used as toluene and lead markers exposure, respectively. The genotoxicity was evaluated by comet assay (in peripherical blood) and by micronucleus (MN) test (in limphocytes and exfoliated buccal cells). The results showed no significant increase in TBARS levels in exposed group in relation to the control group. However, the lipidic damages was higher in Friday samples comparing to Monday samples (p = 0.008; z = -2.637). The proteins damage was higher in exposed group in comparison to control group exclusively in Friday samples (p = 0.032; z = -2.14). It was also observed that the workers exposed to paints showed lower Sod (p = 0.003; z = 2.935) and Cat (p = 0.025; z = -2.247) activities in Monday samples. The exposed group presented HA levels (p = 0.010; z = - 2.591) and ALA levels (p = 0.000; z = -4.487) higher than the control group. The workers exposed to paints presented a significant increase in DNA damage in both Monday (p = 0.000; z = -5.356) and Friday (p = 0.000; z = -6.456) samples. No increase was observed in MN frequency in limphocytes and buccal cells. However, the individuals exposed to paints showed an increase in nuclear buds (NBUDS) (Monday samples, p = 0.004, z = -2.894), a reduction in nuclear division index (NDI) (Friday samples, p = 0.000, z = -4.78) in lymphocytes and an increase in condensed chromatin frequency in buccal cells (Monday samples, p = 0.000, z = -4.503; Friday samples, p = 0.000, z = -5.203), indicating genic amplification and apoptosis induction. The DNA damage index (comet assay) correlated positively with average working time (r = 0.376; p = 0.031). It was also observed a positive correlation between time daily exposure and MN (Monday samples, r = 0.450; p = 0.018) and NBUDs (Friday samples: r = 0.402; p = 0.038) frequency. These results showed that paint exposure is able to generate DNA damages and these damages are being repaired during the weekend.

Glyphosate (N-phosphonomethylglycine) is an organophosphate compound which was developed by the Monsanto Company in 1971 and is the active ingredient found in several herbicide formulations. The use of glyphosate-based herbicides in South Africa for the control of alien invasive plants and weeds is well established, extensive and currently unregulated, which vastly increases the likelihood of glyphosate contamination in environmental water systems. Although the use of glyphosate-based herbicides is required for economic enhancement in industries such as agriculture, the presence of this compound in natural water systems presents a potential risk to human health. Glyphosate and glyphosate formulations were previously considered safe, however their toxicity has become a major focal point of research over recent years. The lack of monitoring protocols for pesticides in South Africa is primarily due to limited financial capacity and the lack of analytical techniques.

Chan Kwan-ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 91-105). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Contents --- p.vi / List of figures and tables --- p.ix / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Literature review --- p.1 / Chapter 1.1.1 --- The effect of pollutants on the genetic materials of aquatic organisms --- p.1 / Chapter 1.1.1.1 --- Response of individual to genotoxicants --- p.1 / Chapter 1.1.1.2 --- Effects of genotoxicants on population structure --- p.3 / Chapter 1.1.2 --- Application of genetic markers in monitoring water pollution --- p.3 / Chapter 1.1.2.1 --- DNA adduct --- p.4 / Chapter 1.1.2.2 --- Sister chromatid exchange (SCE) test --- p.5 / Chapter 1.1.2.3 --- Micronucleus --- p.6 / Chapter 1.1.2.4 --- DNA strand breaks --- p.7 / Chapter 1.1.3 --- Single-cell gel electrophoresis (comet) assay --- p.9 / Chapter 1.1.4 --- Test organisms for comet assay --- p.12 / Chapter 1.2 --- Objective of the present study --- p.13 / Chapter Chapter 2 --- Genotoxicity of pollutants on Hyale crassicornis / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.2 --- Materials and methods --- p.24 / Chapter 2.2.1 --- Sampling of amphipods --- p.24 / Chapter 2.2.2 --- Acclimation --- p.24 / Chapter 2.2.3 --- Acute toxicity test --- p.26 / Chapter 2.2.4 --- The effect of test duration on DNA damage --- p.27 / Chapter 2.2.5 --- Effect of toxicants on DNA damage --- p.28 / Chapter 2.2.6 --- Comet assay --- p.29 / Chapter 2.2.7 --- Chemicals --- p.34 / Chapter 2.2.8 --- Data analysis --- p.34 / Chapter 2.3 --- Results --- p.34 / Chapter 2.4 --- Discussion --- p.47 / Chapter Chapter 3 --- Genotoxicity of hydrogen peroxide on different tissue types of Metapenaeus ensis / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and Methods --- p.57 / Chapter 3.2.1 --- Collection and acclimation of shrimps --- p.57 / Chapter 3.2.2 --- Incubation --- p.59 / Chapter 3.2.3 --- Comet Assay --- p.60 / Chapter 3.2.4 --- Chemicals --- p.61 / Chapter 3.2.5 --- Data analysis --- p.61 / Chapter 3.3 --- Results --- p.61 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4 --- Genotoxicity of wastewater on Hyale crassicornis / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Materials and Methods --- p.72 / Chapter 4.2.1 --- Collection of wastewater samples --- p.72 / Chapter 4.2.2 --- Metal content analysis --- p.73 / Chapter 4.2.3 --- Genotoxic effect of wastewater samples on Hyale crassicornis --- p.74 / Chapter 4.2.4 --- Chemicals --- p.76 / Chapter 4.2.5 --- Statistical analysis --- p.77 / Chapter 4.3 --- Results --- p.77 / Chapter 4.3.1 --- Metals content in water samples --- p.77 / Chapter 4.3.2 --- DNA damage --- p.79 / Chapter 4.4 --- Discussion --- p.79 / Chapter Chapter 5 --- Conclusions --- p.89 / References --- p.91

PiRNAs and their Piwi family protein partners are part of a germline specific epigenetic regulatory mechanism essential for proper spermatogenesis, silencing of transposable elements, and maintaining germline genome integrity, yet their role in the response of the male germline to genotoxic stress is unknown. Ionizing radiation (IR) is known to cause transgenerational genome instability that is linked to carcinogenesis. Although the molecular etiology of IR-induced transgenerational genomic instability is not fully understood, it is believed to be an epigenetically mediated phenomenon. IR-induced alterations in the expression pattern of key regulatory proteins involved in the piRNA pathway essential for paternal germline genome stability may be directly involved in producing epigenetic alterations that can impact future generations. Here we show whole body and localized X-irradiation leads to significant altered expression of proteins that are necessary for, and intimately involved in, the proper functioning of the germline specific piRNA pathway in mice and rats. In addition we found that IR-induced alterations to piRNA pathway protein levels were time and dose dependent. / ix, 123 leaves : ill. (some col.) ; 29 cm

Thesis (Ph.D.)--Uppsala Universitet, 2008. / This website links to the complete document in PDF format. Title from title screen (viewed on November 22, 2009). Includes bibliographical references.