| Summary: | 碩士 === 國立臺灣大學 === 毒理學研究所 === 92 === 第一章
Territrem is a tremogenic mycotoxin isolated from the chloroform extracts of rice culture of Aspergillus terreus 23-1. The previous study showed that three metabolites MA1, MAX and MA2 of territrem A (TRA) were formed in male rats, and gender difference in TRA metabolism of Wistar rats from that only MA1 formation was formed in female rats. Further study of cytochrome P450 involvement TRA metabolism suggested CYP3A1 is mainly responsible for MA1 formation and CYP3A2 is responsible for MA1, MAX, and MA2 formation. It has been reported that four metabolites of territrem B (TRB), designated as MB1, MB2, MB3, and MB4 and one metabolite of territrm C (TRC), designated as MB1, were formed from liver microsomes of both sexs Wistar rats. The metabolism of TRB and TRC related to gender difference and CYP450 family involved were not investigated.
To investigate which cytochrome P450 isoforms were involved in TRB and TRC metabolism, four CYP450 isoform-specific inhibitors (
furafylline, orphenadrine, cimetidine, and troleandomycin) and antibodies against CYP1A2, CYP2B1, CYP2C11, or CYP3A2 were used. The gender difference in TRB and TRC metabolism in liver microsomes of 14-week-old male and female Wistar rats were also investigated. The following results indicated that (1) Metabolism of TRB to MB2, and metabolism of TRC to MB1 were observed in both sexes. The amounts of MB2, MB4, and MB1 formed were higher in male rats.(2) Formation of MB2, MB4, and MB1 was markedly inhibited by cimetidine and troleandomycin, but less by furafylline and orphenadrine. (3) Anti-CYP3A2 antibody markedly inhibited MB2, MB4, and MB1 formation, while antibodies against CYP1A1, CYP2B1, or CYP2C11 had little effect, and (4) Of the seven tested supersomes from baculovirus-transformed insect cells expressing rat CYP450 isoforms ( 1A1, 1A2, 2B1, 2C11, 2C12, 3A1, and 3A2), only those expressing CYP3A1 and CYP3A2 metabolized TRB and TRC. (5) The amounts of MB2, MB4, and MB1 were related to the 6��-testosterone hydroxylase activity and CYP3A1/2 protein content. (6) Immunoblotting showed that CYP3A1 was expressed in both sexes, but at different levels, while CYP3A2 was only expressed in male. These results suggest that the formation of MB2, MB4, and MB1 in liver microsomes from 14-week-old rats of either sex is mediated by CYP3A1 and CYP3A2.
第二章
Cytochrome P450s constitute a multigene family of hemoproteins responsible for the metabolism of numerous xenobiotics, including therapeutic drugs, environmental chemicals, and dietary constituents, as well as such endogenous compounds as steroid and bile acids. Among them, CYP3A enzymes are the most abundant P450 enzymes and involved two thirds of xenobiotics. In the rat exists as two isoenzymes, CYP3A1 and CYP3A2. The protein level of CYP3A2 was expressed in 20-day-old rat of both genders, constitutively expressed in male rats, but decreased after puberty in female rats and extinguished in the adult female. Previous studies showed that (1) the sex difference in TRA metabolism was observed. (2) CYP3A1 and CYP3A2 are mainly responsible for TRA metabolism. (3) Treatment of Wistar rats with either dexamethasone (DEX) or Phenobarbital (PB) markedly increased in CTP3A protein level . Whereas, a similar rise in the metabolism of TRA. (4) In the gonadectomized male rats, MAX and MA2 formation were reversed when the animals were further treated by testosterone. Whereas, CYP3A2 protein levels was reversed by further testosterone treatment with gonadectomized male rats.
Therefore it suggested that testosterone may regulate CYP3A2 gene expression. For this purpose, two cultured cell lines, called McA-RH8994 and McA-RH7777, were established from rat hepatoma cells by in vitro culture with cytochrome P450 inducer, such as DEX and PB, and sex hormone, such as dihydrotestosterone (DHT) and ���{estradiol investigated protein levels, mRNA, and transcription factors responsible for CYP3A1 and CYP3A2. Whereas, it was also investigated 6��-testosterone hydroxylase activity and the metabolism of TRA in these two cell lines..
In the present study, the following results were obtained : (1) CYP3A1 mRNA and protein level are increased by DEX and PB 72 hr treatment. (2) Only MA1 formed from TRA by DEX treatmentthe. (3) 6β-hydroxytestosterone formation was higher in DEX and PB treatment. (4) For nuclear transcription factor of CYP3A, PXR/RXR�� mRNA and protein level are increased by DEX treatment, the protein level of glucocorticoid receptor (GR) was markedly decreased, and the protein levels of androgen receptor (AR) was not changed.
These results suggest that (1) In vitro cell culture, CYP3A1 was markedly induced by xenobiotics, while endogenous, such as sex hormone, may be indirectly regulated CYP3A2 gene expression. (2) DEX induce CYP3A1 level through PXR/RXR?rather than protypical GR pathway.
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