• Proceedings of the Korea Society of Environmental Toocicology Conference
• /
• 2002.10a
• /
• pp.170-170
• /
• 2002

College of Pharmacy, Ewha womans University, Seoul, 120-750, Korea 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent halogenated aromatic hydrocarbon congener that induces expression of several genes including CYP1A1. Exposure to TCDD results in many toxic actions such as carcinogenesis, hepatotoxicity, immune suppression, and reproductive and developmental toxicity. Dramatic differences in dioxin toxicity have been observed between the sexes of some animal species, suggesting hormonal modulation of dioxin action. Many studies have been reported and propose several mechanisms of anti-estrogenic effects of TCDD. In contrast, the effect of estrogen on the regulation of CYP1A1 are not clear at present. There are several reports showing conflicting results. It seems that induction/inhibition of CYP1A1 may be dependent on cell-type and concentration. The purpose of this study was to investigate the regulation of TCDD-induced CYP1A1 gene expression by estradiol and its metabolites. We examined whether estradiol and its metabolites altered TCDD-mediated induction of CYP1A1 enzyme activity. 17 ${\beta}$ estradiol and 16 ${\alpha}$ estriol at non cytotoxic concentrations caused a significant concentration dependent decline of TCDD-induced EROD activity To determine whether reduced EROD activity reflected altered CYP1A1 mRNA expression, we measured CYP1A1 mRNA level by RT-PCR. And to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level, we also peformed transient transfection with an AhR responsive reporter plasmid containing the 5' flanking region of the human CYP1A1 gene to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level.

• Archives of Pharmacal Research
• /
• v.26 no.1
• /
• pp.47-52
• /
• 2003

The aim of this study was to investigate the effects of trauma on alterations in cytochrome P450 (CYP 450)-dependent drug metabolizing function and to determine the role of Kupffer cells in hepatocellular dysfunction. Rats underwent closed femur fracture (FFx) with associated soft-tissue injury under anesthesia, while control animals received only anesthesia. To deplete Kupffer cells in vivo, gadolinium chloride (GdCl$_3$) was injected intravenously via the tail vein at 7.5 mg/kg body wt., 1 and 2 days prior to FFx surgery. At 72 h after FFx, serum alanine aminotransferase (ALT) activity was increased, and this increase was attenuated by GdCl$_3$ pretreatment. Serum aspartate aminotransferase (AST) and lipid peroxidation levels were not changed by FFx. Hepatic microsomal CYP 450 content and aniline p-hydroxylase (CYP 2E1) activity were significantly decreased; decreases that were not prevented by GdC1$_3$. The level of CYP 2B1 activity was decreased by Kupffer cell inactivation, but not by FFx. There were no significant differences in the activities of CYP 1A1, CYP 1A2 and NADPH-CYP 450 reductase among any of the experimental groups. Our findings suggest that FFx trauma causes mild alterations of hepatic CYP 450-dependent drug metabolism, and that Kupffer cells are not essential for the initiation of such injury.

• Toxicological Research
• /
• v.13 no.1_2
• /
• pp.117-125
• /
• 1997

In order to assess the possibility whether CYP2D is involved in caffeine metabolism, we have purified and characterized the rat liver microsomal cytochrome P4502D1 (CYP2D1), equivalent to CYP2D6 in human liver, and have utilized the reconstituted CYP2D1 in the metabolism of 4 primary caffeine (1, 3, 7-trimethylxanthine) metabolites such as paraxanthine (1, 7-dimethylxanthine), 1, 3, 7-trimethylurate, theophylline (1, 3-dimethylxanthine) and theobromine (3, 7-dimethylxanthine). Rat liver CYP 2D1 has been purified to a specific content of 8.98 nmole/mg protein (13.4fold purification, 1.5% yield) using $\omega$-aminooctylagarose, hydroxlapatite, and DE52 columns in a sequential manner. As judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified CYP2D1 was apparently homogeneous. Molecular weight of the purified CYP2D1 was found to be 51, 000 Da. Catalytic activity of the purified and then reconstituted CYP2D1 was confirmed by using bufuralol, a known subsFate of CYP2D1. The reconstituted CYP2D1 was found to produce to 1-hydroxylbufuralol at a rate of 1.43$\pm$0.13 nmol/min/nmol P450. The kinetic analysis of bufuralol hydroxylation indicated that Km and Vmax values were 7.32$\mu M$ and 1.64 nmol/min/nmol P450, respectively. The reconstituted CYP2D1 could catalyze the 7-demethylation of PX to 1-methylxanthine at a rate of 12.5 pmol/min/pmol, and also the 7- and 3- demethylations of 1, 3, 7-trimethylurate to 1, 3-dimethylurate and 1, 7-dimethylurate at 6.5 and 12.8 pmol/min/pmol CYP2D1, respectively. The reconstituted CYP2D1 could also 3-demethylate theophylline to 1-methylxanthine at 5 pmol/min/pmol and hydroxylate the theophylline to 1, 3-dimethylurate at 21.8 pmol/min/pmol CYP2D1. The reconstituted CYP2D1, however, did not metabolize TB at all (detection limits were 0.03 pmol/min/pmol). This study indicated that CYP2D1 is involved in 3-and 7-demethylations of paraxanthine and theophylline and suggested that CYP2D6 (equivalent to CYP2D1 in rat liver) present in human liver may be involved in the secondary metabolism of the primary metabolites of caffeine.

• Advances in Traditional Medicine
• /
• v.7 no.4
• /
• pp.357-362
• /
• 2007

Cytochrome P450 2C19 (CYP2C19) is a clinically important enzyme involved in the metabolism of therapeutic drugs, including (S)-mephenytoin, omeprazole, proguanil, and diazepam. Individuals are characterized as either extensive metabolizers (EM) or poor metabolizers (PM) on the basis of CYP2C19 enzyme activity. The PM phenotype occurs in 2-5% of Caucasians, but in 18-23% of Asians. To clarify the association between CYP2C19 polymorphisms and gastric cancer in Koreans, we investigated CYP2C19 genotypes ($CYP2C19^*1,\;{^*2},\;and\;^*3$) in 109 patients with gastric cancer and 211 controls. Normal ($CYP2C19^*1$) and defective alleles were detected with polymerase chain reaction/restriction enzyme analysis. CYP2C19 has three hereditary genotypes: homozygous EM, with high enzymatic activity; heterozygous EM, with moderate enzymatic activity; and PM, with no enzyme activity. We found that CYP2C19 heterozygous EM is more closely associated with gastric cancer than is homozygous EM. Because the CYP2C19 genotype varies in Koreans, a genotyping test is desirable to prevent gastropathy recurrence in patients before their doses of omeprazole are reduced during maintenance therapy.

• Environmental Mutagens and Carcinogens
• /
• v.23 no.1
• /
• pp.30-34
• /
• 2003

Recent industrial society has human widely exposed to PAHs (polynuclear aromatic hydrocarbons) that are comming from the incomplete combustion of organic material as wider spread environmental contaminants. Biological activities of PAHs are not known although PAHs are considered as carcinogens. Our laboratory have been studied the effect of PAHs in the mouse liver hepa 1 cells. In this study, we examined the mouse liver hepa-l cells as a new bioassay system to evaluate bioactivity of PAHs. We have selected 13 PAHs to examine bioassay using cyp1a1-luciferase reporter gene expression system where cyp1a1 1.6 Kb 5flanking region DNA was cloned in front of luciferase reporter gene and this plasmid was transfected into hepa 1 cells transiently. This cells then used for the study to observe the effect of PAHs. We demonstrated that PAHs induced the CYP1A1 promoter and 7-ethoxyresolufin O-deethylase (EROD) activities in a concentration-dependant manner. Some of PAHs showed stronger stimulatory effect on CYP1 gene expression than TCDD. Acenaphthene, anthracene, fluorine, naphthalene, pyrene, phenanthrene, carbazole were weak responders to cyp1a1 promoter activity stimulation and EROD induction in hepa 1 cells and these chemicals seemed to respond less to EROD than cyp1a1 promoter activity. Benz(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, chrysene, and dibenzo(a,h)anthracene showed strong response to cyp1a1 promoter activity stimulation and also EROD induction in hepa 1cells. Results of dose response study suggested that four strong responding PAHs, such as benzo(a)anthracene benzo(k)fluoranthene, chrysene, and dibenzo(a, h)anthracene might be mediated through arylhydrocarbon receptor system in hepa1 cells.

• Preventive Nutrition and Food Science
• /
• v.19 no.4
• /
• pp.268-273
• /
• 2014

The induction of detoxification enzymes by benzyl isothiocyanate (BITC) and its synthetic N-acetyl-L-cysteine (NAC) conjugate (NAC-BITC) was examined in Hepa1c1c7 murine hepatoma cells. BITC and NAC-BITC inhibited Hepa1c1c7 cell growth in a dose-dependent manner. Cell growth was 4.5~57.2% lower in Hepa1c1c7 cells treated with $0.1{\sim}1.0{\mu}M$ BITC than in control-treated Hepa1c1c7 cells. The NAC-BITC treatment had a similar inhibitory pattern on Hepa1c1c7 cell growth; $0.5{\mu}M$ and $10{\mu}M$ NAC-BITC decreased cell growth by 13.6% and 47.4%, respectively. Treatment of Hepa1c1c7 cells with $0.1{\sim}2.0{\mu}M$ BITC also elicited a dose-response effect on the induction of quinone reductase quinone reductase (QR) activity and QR mRNA expression. Treatment with $1{\mu}M$ and $2{\mu}M$ BITC caused 1.8- and 2.8-fold inductions of QR mRNA, respectively. By comparison, treatment with $1{\mu}M$ and $2{\mu}M$ NAC-BITC caused 1.6-and 1.9-fold inductions of QR mRNA, respectively. Cytochrome P450 (CYP) 1A1 and CYP2E1 induction were lower in $0.1{\sim}2{\mu}M$ BITC-treated cells than in control-treated cells. CYP2E1 activity was 1.2-fold greater in $0.1{\mu}M$ NAC-BITC-treated cells than in control-treated cells. However, the CYP2E1 activity of cells treated with higher concentrations (i.e., $1{\sim}2{\mu}M$) of NAC-BITC was similar to the activity of control-treated cells. Considering the potential of isothiocyanatesto prevent cancer, these results provide support for the use of BITC and NAC-BITC conjugates as chemopreventive agents.

• Toxicological Research
• /
• v.15 no.1
• /
• pp.89-93
• /
• 1999

2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) is known to induce cytochrome p450 1A1 and to activate c-Src kinase and p21 Ras. This study examined the molecular interactions of adaptor proteins including Shc, Grb2, and Sos in rat primary hepatocytes and their relationship to the induction of CYP1A1 by TCDD. TCDD induced CYP1A1 level and EROD activity in a dose-dependent mode. Sos/Grb2 association isincreased by TCDDㅑㅜ a dose dependent mode. Tyrosine phosphorylated Shc, mainly p152, onloads to Grb2/Sos complex upon TCDD stimulation. The electrophoretic mobility shift of Sos is showed by TCDD. These results indicate that TCDD modulated the molecular interaction features of adaptor compoes proteins including Shc, Grb2, and Cnl in early signaling pathway of TCDD-mediated CYP 1A1 induction of rat primary hepatocyte.

• Archives of Pharmacal Research
• /
• v.29 no.7
• /
• pp.570-576
• /
• 2006

Phytoestrogen biochanin A is an isoflavone derivative isolated from red clover Trifolium pratense with anticarcinogenic properties. This study examined the action of biochanin A with the carcinogen activation pathway that is mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 breast carcinoma cells. Treating the cells with biochanin A alone caused the accumulation of CYP1A1 mRNA and an increase in CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity in a dose dependent manner. A concomitant treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and biochanin A markedly reduced the DMBA-inducible EROD activity and CYP1A1 mRNA level. In addition, the biochanin A treatment alone activated the DNA-binding capacity of the AhR for the dioxin-response element (DRE) of CYP1A1, as measured by the electrophoretic-mobility shift assay (EMSA). EMSA revealed that biochanin A reduced the level of the DMBA-inducible AhR-DRE binding complex. Furthermore, biochanin A competed with the prototypical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), for binding to the AhR in an isolated rat cytosol. The biochanin A competitively inhibited the metabolic activation of DMBA, as measured by the formation of the DMBA-DNA adducts. These results suggest that biochanin A may thus be a natural ligand to bind on AhR. Therefore, biochanin A may be due to act an antagonist/agonist of the AhR pathway.

• Proceedings of the Korean Society of Toxicology Conference
• /
• 2003.10b
• /
• pp.169-169
• /
• 2003

CYP1 enzymes, CYP1A1, CYP1A2 and CYP1B1, are known to bioactivate procarcinogens particularly polyaromatic compounds. Flavonoids are a class of natural compounds that are present in edible plants. Structurally, these compounds are polyphenols with two aromatic rings (A, B) and a heterocycyclic ring (C).(omitted)

• Korean Journal of Environmental Biology
• /
• v.22 no.1
• /
• pp.177-183
• /
• 2004

Cytochrome P45O (CYP) contents and 7-ethoxyresorufin-O-deethylase (EROD) activity were determined in hepatic microsome of olive flounder (Paralichthys olivaceus) exposed to tributyltin chloride (TBTC), tributyltin oxide (TBTO), and triphenyltin chloride (TPTC). In addition, effects of in vivo (intraperitoneal injection of 7.5 mg $kg^{-1}$ BW) exposure of flounder to TPTC on CYP, NADPH cytochrome c reductase, NADH cytochrome b5 yeductase and EROD levels were measured. In in vitro exposure of hepatic microsome to organotins, TBTC, TBTO and TPTC reduced CYP contents and inhibited EROD activity. The TPTC was the strongest inhibitor, which is followed by TBTO and TBTC. The degree of inhibition, especially EROD acitivity, depended on the exposure duration. In addition, all the target enzymes in flounder were inhibited by TPTC with the in vivo exposure to TPTC. As EROD activity was the most sensitive to the inhibitions and demonstrated good reproducibility of the results, it could be used as a helpful tool toy monitor effects of organotin compounds on mixed funciton oxygenase system in marine fish.