Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutants

Abstract

The sequence of cytochrome P4501A (CYP1A) cDNA of medaka (Oryzias latipes) was determined, and its messenger ribonucleic acid (mRNA) regulation by beta-naphthoflavone (betaNF) was evaluated. The determined cDNA sequence contained 2,349 base pairs (bp), and the open reading frame contained a total of 1,563 bp encoding 521 predicted amino acids. The induction of CYP1A mRNA in medaka was evaluated using reverse transcription-polymerase chain reaction. The concentration-dependent induction of CYP1A mRNA in the liver was observed after exposure to betaNF at nominal concentrations of 20, 100, and 500 microg/L for 2 d. Time-dependent changes of CYP1A mRNA levels were also observed in the liver, gill, gut, and caudal fin tissues of medaka exposed to 100 microg/L of betaNF for 7 d. Our results showed that the degree of CYP1A mRNA induction in the gill, gut, and caudal fin after exposure to betaNF was relatively higher than that in the liver, possibly because of low basal levels of CYP1A mRNA in the gill, gut, and caudal fin of nonexposed fish. The induction of medaka CYP1A mRNA was also observed after exposure to an environmental sample, landfill leachate. The CYP1A mRNA inductions in the gill, gut, and caudal fin were also higher than that in the liver as shown in the betaNF-treated groups. These results show that CYP1A mRNA determination in the gill, gut, and caudal fin, which are in direct contact with the polluted water, may become a useful method for monitoring CYP1A-inducible chemicals.

The feasibility of using mosquitofish (Gambusia affinis) for detecting endocrine‐disrupting chemicals in the freshwater environment

“…The EROD activity of European eel (Anguilla anguilla) is significantly increased by BNF exposure through water at 0.1 mM (27.2 mg/L) for 4 h or more and at 0.3 mM (81.7 mg/L) for 6 h or more, but the levels of the EROD activity were not dose-dependent and reached peaks at 8 h of exposure [25]. These findings agree with our results that the transcript of the cyp1a gene decreased over time after 24 h with no dose-dependency, whereas gene transcription also may reach a peak sooner than 24 h. Because several studies using fish other than mosquitofish, with other PAHs or different administration routes, report the dose-dependency of EROD activity and Cyp1a protein and its mRNA levels [24,[26][27][28], the response of the Cyp1A appears to vary among species or compounds. The cyp1a mRNA in medaka, which has habitats similar to those of mosquitofish, is also significantly increased by BNF exposure, but the medaka liver responded at 100 mg/L or more for 2 d, but not at 20 mg/L [24].…”

“…These findings agree with our results that the transcript of the cyp1a gene decreased over time after 24 h with no dose-dependency, whereas gene transcription also may reach a peak sooner than 24 h. Because several studies using fish other than mosquitofish, with other PAHs or different administration routes, report the dose-dependency of EROD activity and Cyp1a protein and its mRNA levels [24,[26][27][28], the response of the Cyp1A appears to vary among species or compounds. The cyp1a mRNA in medaka, which has habitats similar to those of mosquitofish, is also significantly increased by BNF exposure, but the medaka liver responded at 100 mg/L or more for 2 d, but not at 20 mg/L [24]. Thus, the transcriptional response of mosquitofish to BNF is sufficiently sensitive when compared with that of other fish for mosquitofish to be considered a suitable test organism.…”

“…The responses were somewhat different between the sexes and in males reached their peak at 24 h of exposure to 50 mg/L. b-Naphthoflavone exerts activating effects on liver Cyp1a of other fish species as well [21][22][23][24]. The EROD activity of European eel (Anguilla anguilla) is significantly increased by BNF exposure through water at 0.1 mM (27.2 mg/L) for 4 h or more and at 0.3 mM (81.7 mg/L) for 6 h or more, but the levels of the EROD activity were not dose-dependent and reached peaks at 8 h of exposure [25].…”

Statistical analysis of cytochrome P4501A biomarker measurements in fish

“…Fifteen papers equating to 44 studies were retained for the analysis of RT‐PCR control/reference data [106–120], but none met the requirements to be included in the dose‐response analysis. No data from Western blot analysis or IHC were used in the control/reference examination, but two papers equating to two studies using Western blot analysis [33,38] and two papers equating to three studies using IHC [26,33] met the criteria for inclusion in the dose‐response examination.…”

Molecular cloning of CYP1A gene and its expression by benzo(a)pyrene from goldfish (Carassius auratus)

“…Therefore, in fish, the CYP1A gene has been used as a useful biomarker of dioxin-like compounds for assessing the level of contamination in an aquatic environment (Williams et al, 1998;Moore et al, 2003;Fent, 2003). Recently, there are reports of CYP1A molecular studies in freshwater fish, such as zebrafish , rainbow trout (Heilmann et al, 1988), killifish (Morrison et al, 1998), medaka (Ryu et al, 2004), and tomcod (Roy et al, 1995).…”