Measurement of vitellogenin-mRNA expression in primary cultures of rainbow trout hepatocytes in a non-radioactive dot blotrRNAse protection-assay

The induction of vitellogenin synthesis both in vivo and in vitro has proven to be a reliable biomarker for assessing the estrogenic activity of individual substances and the more complex effluents of sewage treatment plants. However, due to the requirement of radioactively labelled nucleotides, the measurement of vitellogenin-mRNA has not been widely used in routine testing } even though this technique promises elevated sensitivity. In order to develop a practicable, reliable and cost-effective bioassay suitable for routine testing, a combined dot-blotrRNAse protection assay, utilising digoxigenin-labelled cRNA transcripts of plasmid psg5Vg1.1 was used for the quantification of vitellogenin-mRNA in isolated rainbow trout Oncorhynchus mykiss. hepatocytes. By re-cloning the Vg1.1 insert into a pGemZf7 y.-vector, the sense-transcript of Vg1.1 was utilized as a standard for the quantification of vitellogeninmRNA concentrations. Male rainbow trout hepatocytes were cultured as monolayers in pure M199 medium. The addition of serum supplements did not result in increased expression of vitellogenin-mRNA following 17b-estradiol administration. This indicates that for this assay no supplementation of the culture medium is necessary. After addition of 17b-estradiol, hepatocytes exhibited an exponential time-dependent expression of vitellogenin-mRNA over a period of 144 h. The dot blot system was sufficiently sensitive to detect vitellogenin-mRNA following addition of 1 mM 17b-estradiol after 6 h of incubation. However, the amount of vitellogenin-mRNA expressed was found to be a function of both incubation time and inducer concentration. Prolonged incubation times were therefore required to enhance the sensitivity of the system. After a 96-h incubation, detection limits for 17b-estradiol were between 100 pM and 1 nM. Vitellogenin-mRNA could not be detected in untreated hepatocytes. The vitellogeninmRNA dot blotrRNAse protection assay was further used as a tool for assessing the estrogenic potential of the xenoestrogens nonylphenol and bisphenol A, which exhibited estrogenic activities approximately 2000-fold less than the natural inducer 17b-estradiol. The vitellogenin-mRNA response to 17a-ethinylestradiol reached maximum efficacy down to the lowest tested concentration of 10y9 M. The assay also successfully identified estrogenic activity in selected waste water samples.