Measurement of vitellogenin-mRNA expression in primary cultures of rainbowtrout hepatocytes in a non-radioactive dot blot/RNAse protection-assay

The induction of vitellogenin synthesis both in vivo and in vitro has prove n to be a reliable biomarker for assessing the estrogenic activity of indiv idual 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 testi ng - even though this technique promises elevated sensitivity. In order to develop a practicable, reliable and cost-effective bioassay suitable for ro utine testing, a combined dot-blot/RNAse protection assay, utilising digoxi genin-labelled cRNA transcripts of plasmid psg5Vg1.1 was used for the quant ification of vitellogenin-mRNA in isolated rainbow trout (Oncorhynchus myki ss) hepatocytes. By re-cloning the Vg1.1 insert into a pGemZf7(-)-vector, t he sense-transcript of Vg1.1 was utilized as a standard for the quantificat ion of vitellogenin-mRNA concentrations. Male rainbow trout hepatocytes wer e cultured as monolayers in pure M199 medium. The addition of serum supplem ents did not result in increased expression of vitellogenin-mRNA following 17 beta-estradiol administration. This indicates that for this assay no sup plementation of the culture medium is necessary. After addition of 17 beta- estradiol, hepatocytes exhibited an exponential time-dependent expression o f vitellogenin-mRNA over a period of 144 h. The dot blot system was suffici ently sensitive to detect vitellogenin-mRNA following addition of 1 mu M 17 beta-estradiol after 6 h of incubation. However, the amount of vitellogeni n-mRNA expressed was found to be a function of both incubation time and ind ucer concentration. Prolonged incubation times were therefore required to e nhance the sensitivity of the system. After a 96-h incubation, detection li mits for 17 beta-estradiol were between 100 pM and 1 nM. Vitellogenin-mRNA could not be detected in untreated hepatocytes. The vitellogenin-mRNA dot b lot/RNAse protection assay was further used as a tool for assessing the est rogenic potential of the xenoestrogens nonylphenol and bisphenol A, which e xhibited estrogenic activities approximately 2000-fold less than the natura l inducer 17 beta-estradiol. The vitellogenin-mRNA response to 17 alpha-eth inylestradiol reached maximum efficacy down to the lowest tested concentrat ion of 10(-9) M. The assay also successfully identified estrogenic activity in selected waste water samples. (C) 1999 Elsevier Science B.V. All rights reserved.