Zmiany metylacji i oksydacji w próbkach DNA bydła bytującego w środowisku zanieczyszczonym metalami ciężkimi

Abstract

Background. A continuous exposure to pollutants weakens the organism and may lead to permanent changes in its cell structures. It also contributes to an increased incidence of a variety of specific health problems, notably in farm animals bred in polluted areas. The studies described in this dissertation were initiated due to diseases observed in farm animals kept in the Skidniów farm situated in the neighbourhood of Głogów Copper Smelters. The most severe problems were premature pregnancy terminations and deaths of calves during the first days of life, accounting for 22% pregnancy complications altogether. Phenotypic changes in mature animals were difficult to detect. One of the most troublesome problems was the occurrence of skin diseases which aff ected economic results of the farm. However, although these problems were observed continually and have been confi rmed by scientifi c experiments, the specific influence of contaminants present in the environment on living organisms has not yet been elucidated and is a topic of many studies all over the world. Objectives. The objective of my studies was to compare methylation levels and oxidative changes in DNA extracted from leukocytes of cattle living in the environment polluted with heavy metals as compared to a control group bred in a pollution-free region. Methods. The animals under study constituted a representation of the meat type cattle which was upgraded with different proportions of the Limousin breed blood. The progeny of bull Jaśmin was present in both groups (20 half-sisters in the experimental group and 15 among controls) to ensure a similar genetic background between the two groups. Additionally, in the experimental group were also present 16 daughters of other bull – Jasny. Animals from both groups were fed similar diets. Therefore, it can be assumed that the only factor discriminating the two groups was living in extremely different environmental conditions, with the experimental group bred in the environment polluted with heavy metals. In order to determine a possible increase of genetic instability due to environmental pollution, fingerprinting of Alu sequences was carried out. The Alu profiles were analysed by an electrophoretic analysis of PCR products generated using an Alu-specifi c primer. Moreover, methylation levels of 5-methylcytosine were measured by thin layer chromatography and direct sequencing of promoters of the Apex1 and Nudt1 genes. The levels of 8-hydroxydeoxyguanosine as ratio of 8-hydroxydeoxyguanosine (8-oxo2dG) to non-oxidased form of 2-deoxyguanosine (2dG) were determined using an HPLC system with a Gynkotek autosampler and the Hypersil BDSTMC18 column with a pre-column (Thermo Hypersil). 8-oxo2dG and 2dG were separated on a 4.6 × 150 mm column. Results. The average age of animals was 382.22 days in the control group and 556.38 days in the experimental group, and this difference was statistically signifi cant. Similarly, Jaśmin progeny was signifi cantly older than that of other animals. The methylation status of the Apex1 gene promoter was analysed for 68 animals (38 controls and 30 from the experimental group) and that of the Nudt1 gene for 42 animals (26 controls and 16 from the experimental group), both genes have been analysed for 33 of these individuals (21 from the control group and 12 from the study group). I found that changes of Alu fragment patterns in experimental animals occurred more frequent than in the control group, and this difference was highly signifi cant (P-value = 0.0001). A comparison of global methylation levels between the experimental and control groups showed a statistically signifi cant difference (Z = 5.76; P-value = 0.0000). The control group had higher levels of global DNA methylation, while the experimental group showed higher values of global DNA oxidation. Then, a comparison of promoter methylation of the Apex1 and Nudt1 genes in both groups was performed. Methylation of the Apex1 gene promoter was slightly higher in the experimental group than among controls, however this difference was not statistically signifi cant (P-value = 0.2544). Similarly, for the frequency of the Nudt1 gene promoter methylation, no signifi cant differences between the experimental and control group were observed (P-value = 0.8186). Age was negatively correlated with global methylation in all animals (R = –0.23; P-value = 0.0201), while global oxidation levels positively correlated with age, with a similarly low association strength (R = 0.25 and P-value = 0.0135). In control animals alone, a similar correlation was observed only for global oxidation, however the strength of the association was slightly higher (R = 0.44; P-value = 0.0014). In the experimental group, global oxidation was negatively associated with age of animals (R = –0.35; P-value = 0.0189). Global methylation did not correlate with age neither in the control (R = 0.14; p = 0.2734), nor in the experimental group (R = –0.22; P-value = 0.1237). Global methylation levels for the 16 daughters of the Jasny bull from the experimental group were lower than for other animals, however this difference was borderline significant (U = 478.5; P-value = 0.0691). The off spring of bull Jasny had higher global oxidation levels than other cattle (U = 253.5; P-value = 0.0002). Among the progeny of bull Jasny, the Apex1 gene promoter methylation levels were significantly lower than among other animals from the experimental group (P-value = 0.0028). Higher oxidation levels were observed in cattle from the experimental group with unmethylated Apex1 gene promoter (U = 47.5; P-value = 0.0436). In the experimental group, the cattle with methylated Apex1 gene promoter turned out to be older than cattle with unmethylated Apex1 (U = 52, P-value = 0.0350). Global methylation levels in Jaśmin progeny from the experimental group were signifi cantly lower than in those from the control group (U = 49.5; P-value = 0.0008). A comparison of global oxidation in Jaśmin progeny between the experimental and control group showed statistically significant differences (U = 22; P-value = 0.0001), with higher oxidation levels for the experimental group. There was no correlation between global methylation and global oxidation for Jaśmin progeny but the results were borderline signifi cant (R = –0.32; P-value = 0.0815). Discussion and conclusions. The experimental and control group studied here consist of unique groups of animals to test the environmental infl uence on the phenotype and the genotype due to the fact that the animals have lived in dramatically different environmental conditions. The presence of related individuals in both groups further increases the experimental value of the related biological material. I chose to analyse global methylation and global oxidation levels as markers of epigenetic changes resulting from the influence of the environment on animal organisms. It was interesting to analyse two genes involved in DNA repair, however, due to the complexity of the regulatory mechanisms of epigenetic changes, a direct association of the observed changes with the methylation or oxidation status of selected fragments of the Apex1 and Nudt1 gene promoters was not possible. A decrease of global methylation and increase in global oxidation levels with age observed for all animals was expected as a result of naturally occurring processes. However, the accumulation of oxidative damage in younger animals from the experimental group was atypical and is likely due to environmental pollution. Significantly lower global methylation levels observed in animals from the polluted area as compared to a control group confirm the influence of environment near copper smelters on genetic stability in farm animals. The 8-oxo2dG content in DNA of animals from the experimental group was significantly higher than in controls, which may be a result of the experimental group’s exposure to pollutants present in their environment. The results of a correlation analysis between global methylation and oxidation in Jaśmin progeny may suggest that the regulation of epigenetic changes may involve interactions between DNA methylation and oxidation levels. Living in a polluted environment was signifi cantly associated with the appearance of one or more changes in the Alu-PCR profiles, as was observed in the experimental group in contrast to controls. This observation is consistent with an increase of DNA genetic instability due to environmental pollution, which is widely recognised. It was not possible to determine which of the elements (like heavy metals or other pollutants) coming from the nearby Głogów I and II Copper Smelters was directly responsible for phenotypic, genetic and epigenetic changes but it is consistent with data from other studies indicating such association. It is of utmost importance to realise that the genetic and epigenetic alterations observed in animals may also occur in humans, especially that, despite a signifi cant reduction of pollutant emissions from copper smelters, we still observe the presence of heavy metal contamination in soil, water and plants. Attention should also be paid to the problem of environmental pollution caused by heavy metals resulting from their release during increasingly frequent in Poland landfi ll fires and smog.