Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

Various recent studies have suggested that regular intake of some non-steroidal anti-inflammatory drugs (NSAIDs) have a preventative effect against several types of tumours including breast cancer. The term nanotechnology refers to technology in which one-billionth of a meter is used as a scale for chemical particle size. This work aims to study the effect of both ibuprofen and aspirin on DNA damage using peripheral blood lymphocytes from breast cancer patients and comparing the results with those from healthy females as a control using the Comet and micronucleus assays. Western blot analysis (WBA) was used to investigate the effect of these drugs on XRCC3 and p53 proteins, whereas QPCR was to evaluate this effect on p53, cox1 and cox2 genes. Two hundred fifty ng/ml of ibuprofen (NP and bulk) and 500 ng/ml of aspirin (NP and bulk) were used to treat the lymphocytes. Both aspirin and ibuprofen caused a reduction in DNA damage and micronucleus formation. Aspirin, both forms, showed a reduction in DNA damage in the Comet and micronucleus assays. Ibuprofen both forms, by contrast, showed a statistically significant reduction in micronucleus frequency in the micronucleus assay, while its preventative effect with the Comet assay was weak or insignificant. NPs of both agents were more effective than bulk sizes. Using the Comet repair assay, aspirin and ibuprofen nano form catalysed DNA repair to a greater extent than their bulk forms. Also, both sizes showed better repair with NSAIDs compared to samples repaired without NSAIDs. In WBA aspirin increased the expression of XRCC3 protein in healthy cells. However, both NSAIDs decreased that expression in cells from BC patients. Furthermore, aspirin increased p53 expression in BC patients lymphocytes. With the QPCR method, results of both aspirin forms increased the expression of the p53 gene in BC patient cells statistically significantly. Both drugs reduced cox1 expression in healthy volunteers and cancer patients lymphocytes. Moreover, cox2 reduction was only in lymphocytes from BC patients. The results of this work are consistent with the view that NSAIDs, particularly aspirin and ibuprofen, could have a promising role in cancer treatment including breast cancer.

O presente estudo teve como objetivo o estabelecimento de um método citogenotoxico, o ensaio cometa, adaptado às células meristemáticas provenientes de raízes de propágulos de mangue da espécie Rhizophora mangle para utilização em estudos sobre genotoxicidade em ambientes marinhos. Foram testados dois tipos de germinação de raízes, quatro diferentes soluções de extração de núcleos, duas soluções de lise e o não uso da lise, dois períodos de exposição à lise, dois períodos de relaxamento, dois períodos de eletroforese e a interação de duas condições de lise com dois diferentes tempos de relaxamento. A validação de método foi realizada através da exposição de núcleos a quatro diferentes concentrações de peróxido de hidrogênio. Os resultados mostraram que é possível a obtenção de cometas com núcleos extraídos de propágulos de Rhizophora mangle e que a validação de método apresenta uma relação concentração dependente entre o índice de dano de núcleos e a concentração do agente genotoxico testado. Os melhores parâmetros utilizados para obtenção de cometas através do método por nós adaptado são: PBS ou solução salina 12‰ como solução de extração, exposição à lise alcalina iônica por 60 minutos, 5 minutos de relaxamento e eletroforese em tampão pH>13, 0,8V/cm, 230mA por 20 minutos a 4ºC. / This study aimed to establish a citogenotoxic method, the comet assay, adapted to the meristematic cells from propagule roots of red mangrove, Rhizophora mangle, for use in studies of genotoxicity in marine environments. Experiments were carried out to test two ways of root germination, four different nuclei extraction solutions, two lysis solutions and without lysis, two periods of exposure to lysis, two periods of unwinding, two periods of electrophoresis and the interaction of two lysis conditions with two different times of unwinding. Experiments on validation of the method were performed by exposing the nuclei to four different concentrations of hydrogen peroxide. The results showed that it is possible to obtain comets with nuclei extracted from the root of propagules of Rhizophora mangle and the validation data showed a dose-dependent relationship between the damage index and the concentration of the genotoxic agent tested. The best parameters to obtain comets using the method adapted by us are: PBS or saline 12‰ as extraction solution, exposure to alkaline lysis for 60 minutes, 5 minutes of unwinding and electrophoresis in buffer pH> 13, 0,8V/cm, 230mA for 20 min at 4ºC.

No description available.

Nodularia spumigena is an estuarine cyanobacteria that produces the toxin nodularin. This toxic cyanobacteria is known to have caused death to domestic and wild animals and is recognised as dangerous to human health. N. spumigena causes harmful algal blooms in many parts of the world including Australia. The toxic solutes of N. spumigena are potentially dangerous when contact is made to contaminated water bodies or is ingested by primary consumers. In Australia blooms of N. spumigena are common in the Gippsland Lakes in South-eastern Victoria and cause socio - economic hardships to the local communities. This PhD investigates the toxic effects of N. spumigena and its solutes to a range of aquatic life. A method known as SPME - HPLC showed promise in environmental monitoring of N. spumigena toxins by measuring nodularin from water samples. Other research presented study into the lethal and sublethal effects of on an extract from N. spumigena to aquatic fauna. Resu lts showed the N. spumigena extract was not lethal to many aquatic fauna although zooplankton from the Gippsland Lakes showed mortality at environmental relevant levels. Biochemical studies focusing on animal detoxification and antioxidation enzymes and DNA integrity showed sublethal effects to the N. spumigena extract. Results presented in this thesis show that an extract of N. spumigena elicited detoxification and antioxidation responses in animals tested. Furthermore, the use of the COMET assay showed increased damage to DNA of animals tested. Results also showed that different organs in animals tested responded differently to the aqueous extract, suggesting mode of uptake maybe important in toxicosis. Further, feeding studies with N. spumigena help elucidate mode of uptake using enzyme response biomarkers. The overall results of this research provided an assessment of the toxic affects of N. spumigena on aquatic fauna with special reference to the Gippsland Lakes, Victoria, Australia.

Petrol attendants are exposed to petrol volatile organic compounds (VOCs) which may have genotoxic and carcinogenic effects. The single cell gel electrophoresis assay (comet assay) is a method highly sensitive to DNA damage induced by environmental and occupational exposure to carcinogenic and mutagenic agents. The aim of this study was to evaluate the level of exposure of petrol attendants to petrol VOCs and also to determine their effect on DNA damage and repair in lymphocytes of African petrol attendants. The exposed group consisted of 20 subjects, randomly selected from three petrol stations. A control group of 20 unexposed subjects was also chosen and matched for age and smoking habits with the exposed group. Sorbent tubes were used to assess personal exposure of petrol attendants. The comet assay was used to investigate the basal DNA damage and repair capacity in isolated lymphocytes of petrol attendants and control subjects. Blood samples were taken from the petrol attendants at the end of their 8 hour working shift and also from the control subjects. The petrol attendants were found to be exposed to levels of petrol VOCs lower than the occupational exposure limit (OEL) for constituent chemicals. A significant relationship was found between the volume of petrol sold during the shift and the average concentrations of benzene, toluene and the total VOCs measured. However, relative humidity had a negative correlation with the average concentrations of benzene, toluene, xylene and the total VOCs. Significantly higher basal DNA damage was observed with the exposed group compared to the control group. The period of exposure influenced the level of DNA damage and the calculated repair capacity. Smoking and age had a significant influence on the level of DNA damage. DNA repair capacity was delayed in smokers of both exposed and non-exposed group. / Thesis (M.Sc. (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2008.

Thesis (M.Sc. (Human Movement Science))--North-West University, Potchefstroom Campus, 2007.

When food is heated, a vast number of compounds are formed. Some of these are known to be toxic. Among these are furan, HMF, PhIP, IQ, and MeIQx, the subjects of this thesis. All these compounds are known or suspected carcinogens but the detailed mechanisms behind their carcinogenicity have not yet been fully examined. The aim of this thesis was to study genotoxic properties of these compounds using both in vitro and in vivo methods. Clastogenic effects of all five compounds were assessed with the flow cytometer-based micronucleus assay in vivo and for furan also with the micronucleus assay in vitro. DNA-damaging effects of HMF were studied using the comet assay. No induction of micronuclei was obtained after exposure to IQ, MeIQx or furan. Hence, it can be argued that non-genotoxic mechanisms are partly responsible for the carcinogenic properties of these compounds. PhIP, on the other hand, generated a clear response in the in vivo test. Comparing these result with previous results on acrylamide indicates that PhIP is much more potent. However, acrylamide probably poses a higher risk for humans as the intake is considerably higher. For HMF no effects were seen using the in vivo setup. To further investigate the influence of bioactivation of HMF by sulfotransferases (SULTs) the comet assay was performed in cell lines expressing different levels of SULT. However, no correlation between SULT-expression and DNA-damage was observed. Thus, the DNA-damaging effects found in our experimental setup is probably due to other factors than SULT mediated effects. Furthermore, in this thesis the effects of folic acid on chromosomal stability in healthy people were studied. A negative correlation was found between micronucleus frequency and folate status. The results gained within this thesis will hopefully contribute to the risk assessment of compounds present in our diet.

Agriculture is a valuable economic resource in western Canada, but for decades farmers have focused on intensive production practices while ignoring the long-term health and maintenance of the land. In recent years, the use of conservation agricultural techniques has been encouraged in an effort to conserve prairie landscape while sustaining cropland productivity. Sustainable agricultural practices that promote soil and water conservation and benefit wildlife and prairie biodiversity include conservation tillage and planting of winter cereal crops. Many species of wild birds nest in the ground cover provided by minimum tillage and fall seeded cropland in the spring. Although habitat quality in conservation areas is superior for birds, there is potential for eggs of ground nesting birds to be exposed to herbicides during spring weed control operations. Herbicides commonly used on the prairies to control weed growth in conservational systems include 2,4-D and Buctril-M®. Since the subtlethal effects of exposure to these herbicides may include DNA damage and immunomodulation, the overall goal of this study was to assess whether <i>in ovo</i> exposure to the herbicides 2,4-D and Buctril-M® adversely affects genetic material and/or immune system function in newly hatched domestic chickens (<i>Gallus gallus</i>) and ducks (<i>Anas platyrhynchos</i>), as surrogates for wild bird species.<p>Study design attempted to reproduce actual field exposures by use of an agricultural field spray simulator to apply formulated herbicides (as opposed to pure active ingredients) at recommended crop application rates. In three separate experiments, fertile chicken eggs were sprayed with 2,4-D ester formulation or with Buctril-M® formulation, and fertile duck eggs were sprayed with 2,4-D ester formulation, during either an early (embryonic day 6) or late (embryonic day 15 for chickens or embryonic day 21 for ducks) stage of incubation. Genotoxicity and immune system function were evaluated in the hatchlings as the main toxicological endpoints to assess potential subtle effects from herbicide exposure, but additional measures of general health and development were also evaluated. Two endpoints were used to assess subtle changes to genetic integrity. The comet assay was used to detect structural damage (strand breaks) in avian lymphocyte DNA, as an index of acute genotoxic effects. Flow cytometry was used to examine potential clastogenic effects of the herbicides, by determining if chromosomal changes resulted in variability in the DNA content of avian erythrocytes. Several endpoints were examined to evaluate potential exposure-induced effects on the immune system. Immunopathological assessment of chicks and ducklings included differential lymphocyte counts, as well as immune organ weights and histopathology. The cell-mediated and humoral immune responses in hatchlings were assessed using the delayed-type hypersensitivity test and measurement of systemic antibody production in response to immunization, respectively. Exposure of fertile chicken and duck eggs to Buctril-M® or 2,4-D had no effects on the biomarkers of genetic integrity in this study. Differences in herbicide treatment (high and low concentrations) and times of exposure (early and late incubation stages) did not translate into noticeable factor effects in final model analyses for any of the genotoxicity assay variables evaluated in newly hatched chickens exposed in ovo to 2,4-D. Similarly, comet assay outcomes in chicks exposed to Buctril-M® were not significantly associated with either herbicide treatment or time of exposure as fixed effect factors. Results of the comet assay using peripheral lymphocytes from ducklings provided evidence of potential primary genetic damage associated with the time of spray exposure in ovo. Comet tail DNA content was significantly associated (P = 0.0269) with exposure times, suggesting that ducks may be increasingly sensitive to spray exposure conditions at an early stage of embryological development. Effects of exposure timing were not attributable to herbicide treatment. Although 2,4-D exposure time was associated with DNA strand breakage in ducklings, there was no evidence of chromosomal damage. However, an association between the HPCV values (a measure of DNA content variability) and time of spray exposure was observed in the experiment where 21-day-old chickens were treated in ovo with Buctril-M®. The mean HPCV value for the early exposure group (E6) was significantly greater (P = 0.0210) than that of the group treated later in incubation (E15). However, Buctril-M® the concentration of herbicide did not have any influence on this outcome, and the reason for the difference between exposure times is uncertain, but may be attributed to stress associated with manipulations during spraying. An increase in HPCV, reflecting greater intercellular DNA variability, is indicative of increased incidence of chromosomal damage, which may be an effect of disturbance during early periods of incubation as a result of exposure conditions.<p>Among the panel of immunotoxicity tests conducted to evaluate the effects of <i>in ovo</i> exposure to 2,4-D and Buctril-M® on the developing avian immune system, only heterophil/ lymphocyte (H/L) ratios and relative immune organ weights were significantly associated with either herbicide treatment or time of spray exposure in all three experiments. In 21-day-old chicks exposed in ovo to 2,4-D, relative bursa weight was associated with the different herbicide treatments (P = 0.0006). Relative bursa weights were significantly lower in chicks in the low dose group, while the opposite effect was observed in the high dose chicks, compared with the controls. It is unlikely that the observed decrease in bursa weight in the low dose group is causally related to herbicide exposure because a consistent dose-response effect was not observed, but this outcome may be explained by a compensatory immune response. The relative spleen weights of newly hatched chickens exposed in ovo to Buctril-M® exhibited a significant association with herbicide treatment (P = 0.0137). Relative spleen weights for birds in the low dose treatment groups were significantly different than both the control (P = 0.0179) and high dose groups (P = 0.0125). However, there was no significant difference between high dose and control groups, and this outcome reduces the likelihood of a causal relationship between spleen weight and herbicide exposure. In the parallel experiment involving in ovo exposure to 2,4-D to ducklings, relative bursa weight was associated with time of spray exposure (P = 0.0434). Ducklings that hatched from eggs exposed to spray on day 6 of incubation exhibited greater mean relative bursa weights than the birds exposed to spray at a later incubation stage (E21). This result implies that spray exposure during earlier stages of development may result in conditions which affect the humoral immune response, if increased bursal weight is associated with increased B lymphocyte and antibody production. In the same experiment, mean H/L ratios in peripheral blood samples from 21-day-old ducklings were significantly different between the groups treated with the high concentration of 2,4-D and water (control) (P = 0.0395). Although ratios from the birds in the low dose groups were not significantly different from the control groups, changes in H/L ratio values demonstrate a dose dependent relationship with increasing herbicide exposure.<p>Residue analysis of chicken and duck eggs in this study measured transfer of herbicide through the shell and into the embryo 24 hours and up to 5 days (chickens only) after spraying. Mean 2,4-D residue concentrations were higher in both chicken and duck eggs from the high dose (10X) groups than in eggs exposed to the recommended field rate of herbicide application (1X). Embryo residue concentrations in the chicken eggs increased from the day following exposure to 5 days after spraying, in both low and high dose groups. This observation indicates that the risk of contaminant-induced adverse effects may continue to increase for at least several days after exposure, thereby influencing the concentration of herbicide to which the developing embryo is exposed.<p>On the Canadian prairies, wild bird eggs are potentially to be exposed to 2,4-D and Buctril-M® during various stages of embryonic development. The present study examined effects of herbicide exposure at two distinct times during incubation, and demonstrated the potential for subtle impacts on genetic integrity and the immune system. Results indicate that spray exposure during earlier stages of organogenesis may cause more significant adverse effects. Given the possible harmful consequences of the observed changes on the long-term health of wild birds, further research is needed in order to better characterize the risks of in ovo agrochemical exposure in prairie ecosystems.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.