Quantifying fish metallothionein transcript by real time PCR for its utilization as an environmental biomarker

The study is aimed at introducing the hepatic level of metallothionein transcript in the fish Lithognathus mormyrus as environmental biomarker, including: (a) establishing real time PCR procedure for the evaluation of metallothionein and 18S ribosomal RNA transcript levels, (b) examining the suitability of two alternate normalization factors, 18S- and total RNA, (c) partially characterizing hepatic metallothionein transcript expression––(1) in two samples of feral fish, aimed at determining within-sample variability, (2) during a 318 days depuration experiment aimed at determining basal transcript level, (3) after cadmium injection aimed at determining maximal induced level. Hepatic transcript levels of cytochrome P4501A measured in the same individuals and published elsewhere [Environ. Toxicol. Chem. 22 (2003)], were re-analysed concurrent with the metallothionein ones. 18S rRNA was chosen as normalizing agent of choice, compensating for demonstrated partial RNA degradation in part of the preparations. Minimal and maximal metallothionein transcript levels were determined, 61 ± 47 and 2159 ± 905 atomol/pmol 18S rRNA, respectively. Within-sample variability of the two feral fish samples, or of similarly treated experimental fish groups, expressed as percentage of the standard deviation from the average transcript level, ranged between 41% and 80%, and as low as 3.8-fold difference between pairs of feral or experimental groups was statistically significant.