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Quantitative evaluation of hepatic cytochrome P4501A transcript, protein, and catalytic activity in the striped sea bream ( Lithognathus mormyrus )
Author(s) -
Tom Moshe,
Shmul Merav,
Shefer Edna,
Chen Nir,
Slor Hanoch,
Rinkevich Baruch,
Herut Barak
Publication year - 2003
Publication title -
environmental toxicology and chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.1
H-Index - 171
eISSN - 1552-8618
pISSN - 0730-7268
DOI - 10.1897/02-275
Subject(s) - cytochrome p450 , biology , enzyme , gene expression , messenger rna , reverse transcription polymerase chain reaction , microbiology and biotechnology , cytochrome , chemistry , biochemistry , gene
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 transcript‐level 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.