Takahashi Eizo, Fujita Ken-ichi, Kamataki Tetsuya, Arimoto-Kobayashi Sakae, Okamoto Keinosuke, Negishi Tomoe
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.
Mutat Res. 2002 Oct 31;508(1-2):147-56. doi: 10.1016/s0027-5107(02)00212-9.
Recently we have shown that anthraquinone food pigments such as purpurin and alizarin suppress the genotoxic activities of several mutagens including heterocyclic amines and polycyclic aromatic hydrocarbons in the Drosophila DNA repair test and in the Ames test. To investigate the mechanism of this inhibition, we have now examined the effects of these anthraquinone pigments on enzymes that metabolize xenobiotics. The activities of eight human recombinant cytochrome P450 (CYP) isozymes were measured in the presence of purpurin, alizarin or carminic acid. Purpurin and alizarin strongly inhibited the activities of CYP1A1, CYP1A2 and CYP1B1, and weakly suppressed those of CYP2A6 and CYP2E1 in a dose-dependent manner, but did not inhibit those of CYP2C19, CYP3A4 and CYP3A5. Carminic acid did not affect the activities of any CYPs tested. CYP1B1 was the most strongly affected CYP molecule by purpurin and alizarin among CYPs examined in this study. From kinetic analysis, it was shown that the inhibition by purpurin on CYP1B1 was both competitive and non-competitive, and that by alizarin was competitive. The values of slopes obtained from Lineweaver-Burk plots are proportional to the square of purpurin concentration. This observation suggests that two molecules of purpurin are interacting with one molecule of CYP1B1. The K(m) value of CYP1B1 was 11 microM, and the K(i) value of purpurin and alizarin against CYP1B1 was 0.7 microM(2) and 0.5 microM, respectively. We also examined the effects of these pigments on the mutagenicities of MeIQx and B[a]P in the Ames test, using Salmonella typhimurium TA1538 co-expressing each form of human CYP and NADPH-cytochrome P450 reductase (OR). The mutagenicity of MeIQx in TA1538 1A2/OR or 1B1/OR was suppressed by purpurin and alizarin but not by carminic acid. Purpurin also reduced the mutagenicity of B[a]P in TA1538 1A1/OR or 1B1/OR. These results suggest that the antigenotoxic activities of purpurin and alizarin can be explained by inhibition of CYP activities responsible for activating the mutagens.
最近我们发现，诸如紫红素和茜素之类的蒽醌类食用色素在果蝇DNA修复试验和艾姆斯试验中可抑制包括杂环胺和多环芳烃在内的多种诱变剂的基因毒性活性。为了研究这种抑制作用的机制，我们现在检测了这些蒽醌类色素对代谢外源性物质的酶的影响。在存在紫红素、茜素或胭脂红的情况下，测定了八种人重组细胞色素P450（CYP）同工酶的活性。紫红素和茜素以剂量依赖的方式强烈抑制CYP1A1、CYP1A2和CYP1B1的活性，并微弱抑制CYP2A6和CYP2E1的活性，但不抑制CYP2C19、CYP3A4和CYP3A5的活性。胭脂红对所检测的任何CYP的活性均无影响。在本研究检测的CYP分子中，CYP1B1是受紫红素和茜素影响最强烈的CYP分子。通过动力学分析表明，紫红素对CYP1B1的抑制作用既有竞争性又有非竞争性，而茜素的抑制作用是竞争性的。从Lineweaver - Burk图得到的斜率值与紫红素浓度的平方成正比。这一观察结果表明，两个紫红素分子与一个CYP1B1分子相互作用。CYP1B1的K(m)值为11 microM，紫红素和茜素对CYP1B1的K(i)值分别为0.7 microM(2)和0.5 microM。我们还使用共表达每种形式的人CYP和NADPH - 细胞色素P450还原酶（OR）的鼠伤寒沙门氏菌TA1�38在艾姆斯试验中检测了这些色素对MeIQx和B[a]P诱变性的影响。紫红素和茜素可抑制TA1538 1A2/OR或1B1/OR中MeIQx的诱变性，但胭脂红无此作用。紫红素还降低了TA1538 1A1/OR或1B1/OR中B[a]P 的诱变性。这些结果表明，紫红素和茜素的抗基因毒性活性可通过抑制负责激活诱变剂的CYP活性来解释。
