Quantitative evaluation of hepatic cytochrome P4501A transcript, protein, and catalytic activity in the striped sea bream (Lithognathus mormyrus).
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/microg total RNA, 0.88 +/- 0.52 pmol/microg total protein, and 1.11 +/- 0.52 pmol resorufin/min/microg total protein, respectively, and the lower levels were 0.009 +/- 0.007 fmol/microg total RNA, 0.17 +/- 0.08 pmol/microg total protein, and 0.11 +/- 0.06 pmol resorufin/min/microg 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.
Tom
Moshe
M
Israel Oceanographic and Limnological Research, P.O. Box 8030, Haifa, 31080 Israel. tom@ocean.org.il
Shmul
Merav
M
Shefer
Edna
E
Chen
Nir
N
Slor
Hanoch
H
Rinkevich
Baruch
B
Herut
Barak
B
D000818:Animals; D002384:Catalysis; D019363:Cytochrome P-450 CYP1A1 / Q000032:analysis / Q000096:biosynthesis* / Q000494:pharmacology*; D004785:Environmental Pollutants / Q000633:toxicity; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005819:Genetic Markers; D007557:Israel; D008099:Liver / Q000201:enzymology; D010473:Perciformes / Q000502:physiology*; D016133:Polymerase Chain Reaction
D004785:Environmental Pollutants; D005819:Genetic Markers; D019363:Cytochrome P-450 CYP1A1