The kinetics of aflatoxin B-1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4
The kinetics of aflatoxin B-1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4
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DOI：
10.1006/taap.1996.0326
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发表时间：
1996-12-01
影响因子：
3.8
通讯作者：
Eaton, DL
中科院分区：
文献类型：
--
作者：
Gallagher, EP;Kunze, KL;Eaton, DL
CYP1A2 和 3A4 共同存在，两者均可将黄曲霉毒素 B-1 (AFB(1)) 氧化为反应性黄曲霉毒素 B-1-8,9-环氧化物 (AFBO) 以及羟基化灭活产物黄曲霉毒素 M(1) (AFM(1)) 和黄曲霉毒素 Q(1) (AFQ(1))，这使得人肝脏中 AFB(1) 氧化的动力学分析变得非常复杂微粒体。在本研究中，我们检查了人肝微粒体（HLM，N = 3）和人 CYP3A4 和 CYP1A2 cDNA 表达的淋巴母细胞微粒体中 AFB(1) 氧化的反应动力学，目的是鉴定在接近饮食中可能遇到的低底物浓度下负责 AFB(1) 氧化的 CYP 亚型。 cDNA 表达的人 CYP1A2 形成的 AFBO 遵循 Michaelis-Menten 动力学（K-m = 41 mu M，V-max = 2.63 nmol/min/nmol P450）。此外，在 HLM 中形成的 AFBO 部分被呋拉茶碱（一种基于 CYP1A2 的特定机制的抑制剂）消除，也遵循 Michaelis-Menten 动力学（K-m = 32-47 μM，V-max = 0.36-0.69 nmol/min/nmol P450）。 cDNA 表达的人 CYP3A4 微粒体中 AFBO（激活产物）和 AFQ（解毒产物）的形成呈 S 形且与底物激活动力学一致。因此，应用相当于 Hill 方程的 s 形 V-max 模型对 cDNA 表达的 CYP3A4 数据以及来自用呋拉茶碱预处理以去除 CYP1A2 的 HLM 的数据产生了极好的拟合。 Hill 模型预测两个底物结合位点参与 CYP3A4 介导的 AFB 催化，并且 AFB(1) 对这两个位点的平均亲和力为 140-180 muM。 AFQ(1) 形成的反应比 AFBO 的反应大 10 倍，两者的总底物周转率为 67 nmol/min/nmol CYP3A4。使用 CYP1A2 和 3A4 的推导动力学参数来模拟低底物浓度下 AFB 体外激活率，预测 CYP1A2 在 0.1 μM AFB 下对人肝微粒体中 95% 以上的 AFB 激活有贡献。 DNA 结合研究支持了 CYP1A2 在低底物浓度下体外激活 AFB 中的重要作用。对照 HLM（反映 CYP1A2 和 CYP3A4 的贡献）和呋拉茶碱预处理的微粒体（仅反映 CYP3A4 的贡献）中的 AFB(1)-DNA 结合分别催化 1.71 和 0.085 pmol 当量的 AFB(1) 与 DNA 的结合，表明 CYP1AZ 负责 95% 的 AFB(1)-DNA在 0.133 mu M AFB 下形成加合物。这些结果表明，在亚微摩尔浓度下，CYP1A2 在体外人肝微粒体中主导 AFB 的激活，并支持这样的假设：在人类饮食中遇到的相对较低的饮食浓度下，CYP1A2 是体内人肝中 AFBO 激活的主要酶，即使在世界上 AFB 暴露较高的地区也是如此。然而，由于膳食暴露后肝脏中 AFB 的实际浓度不确定，因此需要对暴露人群进行更多研究。人类 AFM(1) 和 AFQ(1) 排泄（分别是 CYP1A2 和 3A4 活性的潜在生物标志物）相对速率的定量数据将有助于验证这两种酶对 AFB（体内氧化）的实际贡献。 (C) 1996 学术出版社
The combined presence of CYP1A2 and 3A4, both of which oxidize aflatoxin B-1 (AFB(1)) to the reactive aflatoxin B-1-8,9-epoxide (AFBO) and to hydroxylated inactivation products aflatoxin M(1) (AFM(1)) and aflatoxin Q(1) (AFQ(1)), substantially complicates the kinetic analysis of AFB(1) oxidation in human liver microsomes. In the present study, we examine the reaction kinetics of AFB(1) oxidation in human liver microsomes (HLMs, N = 3) and in human CYP3A4 and CYP1A2 cDNA-expressed lymphoblastoid microsomes for the purpose of identifying the CYP isoform(s) responsible for AFB(1) oxidation at low substrate concentrations approaching those potentially encountered in the diet. AFBO formation by cDNA-expressed human CYP1A2 followed Michaelis-Menten kinetics (K-m = 41 mu M, V-max = 2.63 nmol/min/nmol P450). Furthermore, the portion of AFBO formed in HLMs which was eliminated by furafylline, a specific mechanism-based inhibitor of CYP1A2, also followed Michaelis-Menten kinetics (K-m = 32-47 mu M, V-max = 0.36-0.69 nmol/min/nmol P450). The formation of AFBO (activation product) and AFQ, (detoxification product) in cDNA-expressed human CYP3A4 microsomes was sigmoidal and consistent with the kinetics of substrate activation. Accordingly, application of a sigmoid V-max model equivalent to the Hill equation produced excellent fits to the cDNA-expressed CYP3A4 data and also to the data from HLMs pretreated with furafylline to remove CYP1A2 The Hill model predicted that two substrate binding sites are involved in CYP3A4-mediated AFB, catalysis and that the average affinity of AFB(1) for the two sites was 140-180 mu M. V-max values for AFQ(1) formation were 10-fold greater than those for AFBO, and total substrate turnover to both was 67 nmol/min/nmol CYP3A4. Using the derived kinetic parameters for CYP1A2 and 3A4 to model the in vitro rates of AFB activation at low substrate concentrations, it was predicted that CYP1A2 contributes to over 95% of AFB activation in human liver microsomes at 0.1 mu M AFB. The important role of CYP1A2 in the in vitro activation of AFB at low substrate concentrations was supported by DNA binding studies. AFB(1)-DNA binding in control HLMs (reflecting the contribution of CYP1A2 and CYP3A4) and furafylline-pretreated microsomes (reflecting the contribution of CYP3A4 only) catalyzed the binding of 1.71 and 0.085 pmol equivalents of AFB(1) to DNA, respectively, indicating that CYP1AZ was responsible for 95% of AFB(1)-DNA adduct formation at 0.133 mu M AFB, These results demonstrate that CYP1A2 dominates the activation of AFB in human liver microsomes in vitro at submicromolar concentrations and support the hypothesis that CYP1A2 is the predominant enzyme responsible for AFBO activation in human liver in vivo at the relatively low dietary concentrations encountered in the human diet, even in high AFB exposure regions of the world. However, because the actual concentrations of AFB in liver in vivo following dietary exposures are uncertain, additional studies in exposed human populations are needed. Quantitative data on the relative rates of AFM(1) and AFQ(1) excretion (potential biomarkers for CYP1A2 and 3A4 activity, respectively) in humans would be useful to validate the actual contributions of these two enzymes to AFB, oxidation in vivo. (C) 1996 Academic Press, Inc.