Xenoestrogens and telomerase: A potential mechanism for their teratogenic effects?

• Main Author: Pook, Christopher James
• Other Authors: School of Biological Sciences
• Format: Thesis
• Language: English
• Published: University of Plymouth 2005
• Subjects: Pacific; Crassostrea gigas; Pacific oyster
• Online Access: http://hdl.handle.net/10026.2/778

The developmental life stages of all organisms have been demonstrated to be especially vulnerable to exposure to exogenous compounds that mimic, modulate or antagonize the action of endogenous hormones. One target for xenoestrogens is the ribonucleoprotein telomerase. Transcription of the ratelimiting telomerase reverse transcriptase [TERT] subunit of telomerase is directly induced via an oestrogen responsive element in the gene's promoter region and oestrogen also modulates Telomerase activity via post-translational modulation of the TERT molecule. This investigation examined the potential for environmentally relevant concentrations of two xenoestrogens, the antioestrogenic therapeutant tamoxifen and the synthetic oestrogen 17-P-ethinyloestradiol, to modulate telomerase activity in freshly fertilised embryos of the commercially and ecologically important pacific oyster Crassostrea gigas. Telomerase activity has not previously been demonstrated in C. gigas, therefore this was the first objective of this investigation. Oysters were strip-spawned and their gametes combined under controlled conditions to yield freshly fertilised embryos that were raised under controlled conditions for 24 hours. Samples were obtained of unfertilised female gametes, 4 hour old embryos and 24 hour old embryos. A second batch of larvae were raised and exposed immediately after fertilisation to 4 different concentrations; 1000, 100, 10 and Ing 1"^ of both the antioestrogen tamoxifen and the synthetic oestrogen 17p-ethinyloestradiol, as well as seawater and solvent controls in order to determine their effect upon telomerase activity. Material was pelleted and the seawater removed before washing the pellet with TE buffer and extracting telomerase using the CHAPS lysis buffer that was supplied with the commercially available TRAPeze S7700 telomerase assay from Chemicon International. Extensive optimisation of the PCR was required in order to produce viable results from the TRAPeze assay and ultimately a reliable and consistent modified protocol was developed. The manufacturer's semiquantitative method did not produce statistically viable data, despite various measures taken to account for potentially confounding artifacts in the gel images produced by the TR^Peze assay. An alternative quantification method was developed although it could not be incontrovertibly validated using the data available. Due to the lack of a validated quantification method no analysis of telomerase activity could be performed although activity was demonstrated in the extracts studied. Such activity confirms that telomerase is conserved almost ubiquitously in eukaryotes and that the crucial role it plays in errorfree cell replication may be vulnerable to perturbation by developmental exposure to xenoestrogens, however it was not possible to demonstrate such an effect in this investigation School of Biological Sciences