| dc.description.abstract | Disease in the male reproductive system seems to increase in industrialised countries. Examples are increasing rates of poor semen quality, and more cases of testicular cancer, - with Denmark and Norway having the World’s highest rates. Male reproductive health is crucial for the ability to reproduce, and these changes gives rise to concern. This project studies whether - and how - environmental factors contribute to this development. In particular, we have investigated genotoxic effects of one specific compound, benzo(a)pyrene (BaP), using several experimental approaches.
BaP, which is a carcinogen present in cigarette smoke, is among the agents proposed to mediate negative effects on reproduction. Smoking causes DNA damage in sperm such as BaP DNA adducts, known to be transmitted to the early embryo. BaP can also lead to oxidised base damage through generation of reactive oxygen species (ROS).
One would expect the cells of the male germ line to be able to preserve the integrity of their DNA by exhibiting active DNA repair. This is however not the case with respect to excision repair. Human male germ cells exhibit low or non-functional nucleotide transition repair (NER) as well as limited repair of oxidised DNA such as 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) by base excision repair (BER). The consequences and importance of these characteristics in the testes are not known. In particular, it is essential to understand whether human male germ cells are particularly sensitive to genotoxic agents in the environment.
We have previously proposed a transgenic mouse line to be used as a model for human male reproductive toxicity. The enzyme 8-oxoguanine-DNA glycosylase (Ogg1; the mammalian homologue of E. coli Fpg) removes oxidised base lesions such as 8-oxoG. The functional homologue has been disrupted in the mouse line, giving rise to repair deficient (Ogg1-/-) animals mimicking the normal human male with respect to repair of oxidative DNA lesions in the testis.
In this work we have examined whether exposure to BaP (150 mg/kg i.p.) leads to oxidative DNA lesions in the testes in vivo, in both Ogg1-/- and Ogg1+/+ mice. A mechanical method for isolating nuclei was established, allowing analysis of DNA damage by the comet assay. To better understand the response in the testis, the expression of selected genes involved in the metabolism of BaP was measured with real-time RT PCR. The genes included Akr1a4 (required for induction of oxidised DNA) as well as Cyp1a1, -1a2 and -1b1 (required for induction of bulky DNA adducts). We found that BaP did indeed induce statistically increased levels of oxidative DNA damage in the testis of Ogg1-/- mice at day 17 after the exposure. DNA damage was also induced in somatic tissues such as the lung, and there were indications of damage in the liver (not significant). Such damage was not observed in wild type (Ogg1+/+) mice. Gene expression analysis of Akr1a4 and Cyp1a1 showed that these gene products were induced in the testis and liver of both genotypes, to variable degrees, implying that BaP may be metabolised to induce DNA adducts as well as oxidised base damage in these tissues. Hence, the data corroborate the fact that increased levels of oxidised base damage were found in the testis. Both Cyp1a2 and -1b1 were induced in the liver of both genotypes. In the testis, a different pattern was observed; Cyp1a2 was not clearly induced in the testis of Ogg1-/- mice, and was down-regulated in the testis of Ogg1+/+ mice. Cyp1b1 was induced neither in the testis of Ogg1-/- nor Ogg1+/+ mice.
We also studied the in vivo repair of Fpg-sensitive DNA lesions in Ogg1-/- and Ogg1+/+ mice exposed to X-rays (10 Gy), as part of evaluating the Ogg1-/- mouse model. Results from the in vivo repair experiment showed that repair of Fpg-sensitive sites was not detectable in the testis, liver or lung of Ogg1-/- mice following X-rays, indicating no significant back-up repair by alternative repair pathways in these tissues. As expected, Ogg1+/+ mice did repair Fpg-sensitive sites.
Taken together, we have established experimental protocols for DNA damage analysis after in vivo exposure, that are highly suitable for large-scale animal experiments and inter-laboratory collaborations. Our results show that BaP gives rise to oxidative DNA lesions in testicular cells of Ogg1-/- mice; this could explain the elevated levels of such lesions observed in sperm of smoking men. The Ogg1-/- mouse model obviously requires further evaluation, but our results give strength to the belief that it could serve as a model for human male germ cells in the study of possible effects of genotoxic agents. | eng |