뱀장어 배양 간세포에서의 Cytochrome P4501A (CYP1A) 유전자 발현에 대한 중금속들의 억제효과
Inhibitory effects of heavy metals on CYP1A expression in eel hepatocyte cultures
0
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
뱀장어 간세포 배양을 이용하여 cytochrome P4501A (CYP1A) 유전자 발현에 대한 중금속들의 영향에 대해조사하였다. 첫째, CYP1A mRNA 발현에 대한 benzo [α]pyrene (B[a]P)의 농도별 조사에서, B[a]P 10-8 ∼10-5 M의 농도로 배양액에 첨가하여 배양한 후 세포를 수거하여 RT-PCR 방법으로 CYP1A mRNA 발현량을 조사하였다. CYP1A mRNA 발현은 B[a]P 농도의존적으로 유도되었으며, 10-7 M 농도부터 통계적으로 유의차를 나타냈다 (p<0.05). 둘째로 뱀장어 간세포에 B[a]P (10⁻⁵ M)와 카드뮴 (10⁻⁶, 10⁻⁵ M) 을각각 또는 함께 첨가하여 CYP1A mRNA 발현을 조사하였다. 카드뮴을 첨가한 그룹은 vehicle에 비해CYP1A 발현의 억제가 관찰되었으며, 고농도(10⁻⁵M)의 카드뮴에서 더 많이 억제되었다. 또한 B[a]P와카드뮴을 함께 처리한 그룹에서도 CYP1A 유전자 발현은 B[a]P 단독 처리에 비해 현저한 억제가 관찰되었다. 셋째로, in vivo에서 B[a]P를 주사한 뱀장어의배양 간세포를 이용하여 CYP1A 유전자 발현에 대한중금속들의 영향을 조사하였는데, 뱀장어에 10mg/kg 의 B[a]P를 주사한 후, 48시간 후에 간을 채취하여간세포 배양을 하였다. 카드뮴 ,구리, 납 및 아연 (10⁻⁶,10⁻⁵ M) 등을 각각 배양액에 첨가하여 2일간 배양한후 CYP1A mRNA 발현량을 조사하였다. 이미 B[a]P 처리에 의해 CYP1A 발현이 유도된 대조구 (vehicle)에 비해 중금속들이 첨가된 모든 그룹에서 CYP1A 유전자 발현 억제가 관찰되었다. 본 연구는 여러 어종에서 CYP1A 유전자 발현에 대한 중금속들의 영향및 중금속 독성을 연구하는데 매우 중요한 기초 자료로 활용되어질 것이다.
Abstract
Effects of heavy metal ions on the gene expression of cytochrome P4501A (CYP1A) were examined in cultured eel hepatocytes. When the expression of CYP1A mRNA was measured by RT-PCR after incubation of eel hepatocytes with benzo[α]pyrene (B[α]P) at concentrations of 10-8∼10-5 M, the CYP1A expression increased with B[α]P treatment in a dose dependent manner, showing significant increase at concentrations more than 10-7 M. When the eel hepatocyte was treated with cadmium (10-6 and 10-5 M), the expression of CYP1A was inhibited and especially at higher concentration (10-5 M). The inhibition of CYP1A expression by cadmium was also observed in cells treated with B[α]P. In another study, effects of heavy metal ions on the expression of CYP1A were examined in cultured hepatocytes isolated from eel which was treated previously with B[α]P in vivo. Hepatocytes isolated from the liver of eel taken at 48 hours after injection of B[α]P (10 mg/kg) were cultured for 2 days with cadmium, copper, lead or zinc (10-6 and 10-5 M). The expression of CYP1A was found to be suppressed by the metal ions compared with the control in which CYP1A was induced with previous treatment of B[α]P in vivo. The present results may provide an important basic information for studying the effects of heavy metal ions on CYP1A expression in other species of fish and studying toxicological mechanisms of heavy metal ions in aquatic livings.
참고문헌 (29)
- Arukwe, A., "Differential biomarker gene and protein expressions in nonylphenol and estradiol-17β treated juvenile rainbow trout (Oncorhynchus mykiss)", Comp. Biochem and Physiol., 129, pp.1~10 (2001)
- pyrene and β-naphthoflavone, "Environ. Int.", 2003, 467~473
- Canepa, E.T., "Effect of glucose on induction of delta aminllevulinic acid synthase, ferrochelatase and cytochrome P450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead", Biochim. Biophys. Acta., 841, pp.186~194 (1985)
- Degawa, M., "Preperential inhibitions of hepatic P4501A2 expression and induction by lead nitrate in the rat", Carcinogenesis, 14, pp.1091~1094 (1993)
- Falkner, K.C., "Effects of acute sodium arsenite administration on the pulmonary chemical metabolizing enzymes, cytochrome P450 monooxigenase, NAD(P)H:quinone acceptor exidoreductase and glutathione S-transferase in guinea pig: comparison with effects in liver and kidney", Chem. Biol. Interact., 86, pp.51~68 (1993)
- Ghosh, M.C., "Impact of copper on biomonitoring enzyme ethoxyresorufin-O-deethylase in cultured catfish hepatocytes", Environmental Research Section A, 86, pp.167~171 (2001)
- Greim, H., "Toxicology and risk assessment: A comprehensive introduction", pp.534~564 (2007), John Wiley and Sons
- Henczová, M., "Effects of Cu²+ and Pb²+ on different fish species: Liver cytochrome P450-dependent monooxygenase activities and FTIR spectra", Comp. Biochem and Physiol, Part C, 148, pp.53~60 (2008)
- pyrene cytotoxicity in a human lung adenocarcinoma cell line by decreasing cyclooxygenase-2 levels, "J. Toxicol. Environ. Health A", 2002, 245~263
- Iscan, M., "Combined effect of cadmium and nickel on rat hepatic monooxygenases possible stimulation of certain cytochrome P450 isozymes", Toxicol. Lett., 62, pp.191~199 (1992)
- Iscan, M., "The responses of hepatic monooxygenases of guinea pig to cadmium and nickel", Biol. Trace Elem. Res., 38, pp.129~137 (1993)
- Iscan, M., "Differential responses of hepatic monooxygenases and glutathione S-transferases of mice to a combination of cadmium and nickel", Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol., 111, pp.61~68 (1995)
- Jacobs, J.M., "Effect of arsenite on induction of CYP1A, CYP2B, and in primary cultures of rat hepatocytes", Toxicol. Appl. Pharmacol., 157, pp.51~59 (1999)
- Jönsson, M.E., "Effects of copper on CYP1A activity and epithelial barrier properties in the rainbow trout gill", Aquat. Toxicol., 79, pp.78~86 (2006)
- Korashy, H.M., "Differential effects of mercury, lead and copper on the constitutive and inducible expressin of aryl hydrocarbon receptor (AHR) regulated genes in cultured hepatoma hepa 1c1c7 cells", Toxicology, 211, pp.153~172 (2004)
- Kwon, H.C., "Involvement of growth hormone and prolactin in the induction of vitellogenin synthesis in primary hepatocyte culture in the eel, Anguilla japonica", Gen. Comp. Endocrinol., 93, pp.51~60 (1994)
- Levine, S.L., "CYP1A expression in liver and gill of rainbow trout following waterborne exposure: implications for biomarker determination", Aquat. Toxicol, 46, pp.279~287 (1999)
- Maier, A., "Disruption of dioxin-inducible phaseⅠ and phaseⅡ gene expression patterns by cadmium, chromium and arsenic", Mol. Carcinog., 28, pp.225~235 (2000)
- pyrene genotoxicity, "Mutation Research", 2002, 101~111
- Navas, J. M., "Antiestrogenicity of β -naphthoflavone and PAHs in cultured rainbow trout hepatocytes: evidence for a role of the aryl hydrocarbon receptor", Aquat, Toxicol., 51, pp.79~92 (2000)
- Risso-de Favernay, C., "The nitroxide stable radical tempo prevents metal-induced inhibition of CYP1A1 expression and induction", Toxicol. Lett., 111, pp.219~227 (2000)
- Risso-de Favernay, C., "Inhibitory effects of heavy metals on CYP1A induction in black seabream (Spondyliosoma cantharus) hepatocyte cultures" (2001), Elservier Science
- pyrene, 2,3,3’,4,4’,5-hexachlorobiphenyl (PCB-156) and cadmium on biomarker responses in flounder (Platichthys flesus L.), "Biomarkers", 1997, 153~160
- Seubert, J.M., "Acute sodium arsenite admisistration induces pulmonary CYP1A mRNA, protein, and activity in rat", J. Biochem. Mol. Toxicol., 16, pp.84~95 (2002)
- Sorrentino, C., "Co-exposure to metals modulates CYP1A mRNA inducibility in Atlantic tomcod Microgadus tomcod from two populations", Aquat. Toxicol., 75, pp.238~252 (2005)
- Tully, D.B., "Effects of arsenic, cadmium, chromium and lead on gene expression regulated by a battery of 13 different promoters in recombinant HepG2 cells", Toxicol. Appl. Phamacol., 168, pp.79~90 (2000)
- Vakharia, D.D., "Effects of metals on polycyclic aromatic hydrocarbon induction of CYP1A1 and CYP1A2 in human hepatocyte cultures", Toxicol and Pharmacol., 170, pp.93~103 (2001)
- Zhou, B., "Primary cultured cells as sensitive in vitro model for assessment of toxicants-comparison to hepatocytes and gill epithelia", Aquat. Toxicol., 80, pp.109~118 (2006)