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The potential of the hepatic transcriptome expression profile evaluated in a sentinel feral fish to serve as an environmental biomarker was examined. Expression profiles of Lithognathus mormyrus individuals were exhibited using cDNA microarray and were related to the set of exposure conditions at their sites and dates of collection. Expression profiles of individual fish were reasonably clustered according to the fish samples. In addition, several sample-specific gene clusters were determined, designated sample gene signatures. The selection procedure for future optimal reference RNA is discussed. The relationship between transcriptome expression and fish samples indicated a potential for using the former as an environmental biomarker. [ABSTRACT FROM AUTHOR]

The study was aimed at examining the effects of tert-butyl hydroperoxide (tBHP) on hepatic transcriptome expression patterns of the teleost fish Lithognathus mormyrus. tBHP is an organic hydro-peroxide, widely used as a model pro-oxidant. It generates the reactive oxygen species (ROS) tert-butoxyl and tert-butylperoxyl. Complementary DNAs of tBHP-treated vs control fish were applied onto a previously produced cDNA microarray of ∼ 1500 unique sequences. The effects of the tBHP application were demonstrated by leukocyte infiltration into the liver and by differential expression of various genes, some already known to be involved in ROS-related responses. Indicator genes of putative ROS effects were: aldehyde dehydrogenase 3A2, Heme oxygenase and the hemopexin-like protein. Putative indicators of transendothelial leukocyte migration and function were: p22phox, Rac1 and CD63-like genes. Interestingly, 7-dehydrocholesterol reductase was significantly down-regulated in response to all treatments. Several non-annotated genes revealed uniform directions of differential expression in response to all treatments. [ABSTRACT FROM AUTHOR]

Abstract: The levels of contaminant-affected gene products (transcripts and proteins) are increasingly utilized as environmental biomarkers, and their appropriate implementation as diagnostic tools is discussed. The required characteristics of a gene product biomarker are accurate evaluation using properly normalized absolute units, aiming at long-term comparability of biomarker levels over a wide geographical range and among many laboratories. Quantitative RT-PCR and competitive ELISA are suggested as preferred evaluation methods for transcript and protein, respectively. Constitutively expressed RNAs or proteins which are part of the examined homogenate are suggested as normalizing agents, compensating for variable processing efficiency. Essential characterization of expression patterns is suggested, providing reference values to be compared to the monitored levels. This comparison would enable estimation of the intensity of biological effects of contaminants. Contaminant-independent reference expression patterns should include natural fluctuations of the biomarker level. Contaminant-dependent patterns should include dose response to model contaminants chronically administered in two environmentally-realistic routes, reaching extreme sub-lethal affected levels. Recent studies using fish as environmental sentinel species, applying gene products as environmental biomarkers, and implementing at least part of the depicted methodologies are reviewed. [Copyright &y& Elsevier]

Hepatic cytochrome P4501A (CYP1A) expression was partially characterized in the striped sea bream (Lithognathus mormyrus) from the Mediterranean coast of Israel as part of the process of establishing the CYP1A gene as an environmental biomarker. Reverse transcription--competitive polymerase chain reaction, competitive enzyme-linked immunosorbent assay, and ethoxyresorufin O-deethylase (EROD) assay were used for evaluating transcript, protein, and catalytic activity levels, respectively, in absolute units. Highest elucidated transcript, protein, and catalytic activity levels were 0.264 ± SD 0.084 fmol/µg total RNA, 0.88 ± 0.52 pmol/µg total protein, and 1.11 ± 0.52 pmol resorufin/min/µg total protein, respectively, and the lower levels were 0.009 ± 0.007 fmol/µg total RNA, 0.17 ± 0.08 pmol/µg total protein, and 0.11 ± 0.06 pmol resorufin/min/µg total protein, respectively, demonstrating substantial induction potential. All alternate pairs of seven examined field samples, revealing a transcriptlevel ratio higher than 1.7, also demonstrated a significant difference between their transcript levels, indicating a potential to detect relatively small biomarker changes (1.7-fold) caused by environmental effects. Simultaneous triple measurements of transcript, protein, and catalytic activity were carried out in individuals from two field samples and during a 318-d decay experiment. Fish from the field samples revealed significant alternate bivariate correlation between transcript, protein, and enzymatic activity. Conflicting results were found when analyzing the decay experiment, in which both protein and catalytic activity levels decreased significantly to basal levels, in contrast to no significant change in transcript levels throughout the experiment. No significant difference was observed between males and females regarding the levels of CYP1A transcript, protein, and EROD. [ABSTRACT FROM AUTHOR]

Abstract: Copper transporting ATPase, ATP7A, is an ATP dependent copper pump present in all vertebrates, critical for the maintenance of intracellular and whole body copper homeostasis. Effects of copper treatment on ATP7A gene expression in fibroblast cells (SAF1) of the sea bream (Sparus aurata) were investigated by qRT-PCR and by a medium density microarray from a closely related species, striped sea bream (Lithognathus mormyrus). To discriminate between the effects of Cu and other metals, SAF1 cells were exposed to sub-toxic levels of Cu, Zn and Cd. Expression of Cu homeostasis genes copper transporter 1 (CTR1), Cu ATPase (ATP7A), Cu chaperone (ATOX1) and metallothionein (MT) together with the oxidative stress markers glutathione reductase (GR) and Cu/Zn superoxide dismutase (CuZn/SOD) were measured 0, 4 and 24 hours post-exposure by qRT-PCR. Microarray was conducted on samples from 4 hours post Cu exposure. Cu, Zn and Cd increased MT and GR mRNA levels, while only Cu increased ATP7A mRNA levels. Microarray results confirmed the effects of Cu on ATP7A and MT and in addition showed changes in the expression of genes involved in protein transport and secretion. Results suggest that ATP7A may be regulated at the transcriptional level directly by Cu and by a mechanism that is different from that exerteted by metals on MT genes. [Copyright &y& Elsevier]