Ribrag V, Massaad L, Janot F, Bissery M C, Parise O, Gouyette A, Chabot G G
Département de Pharmacotoxicologie et de Pharmacogénétique, CNRS URA 147, Institut Gustave-Roussy, Villejuif, France.
Leuk Res. 1994 Nov;18(11):829-35. doi: 10.1016/0145-2126(94)90163-5.
1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance has been mostly studied in vitro. In an attempt to better understand BCNU resistance in the in vivo situation, we compared the principal drug-metabolizing enzyme systems in two L1210 leukemia lines, one sensitive and one resistant to BCNU (L1210/BCNU), passaged in vivo in mice. The following enzymes were assayed by immunoblotting: cytochromes P-450 (1A1/1A2, 2B1/2B2, 2C8-10, 2E1, 3A), epoxide hydrolase (EH) and glutathione S-transferase (GST-alpha, -mu and -pi). The following enzymes and cofactors were assayed fluorometrically or spectrophotometrically: 1-chloro-2-4 dinitrobenzene-GST (CDNB-GST), total glutathione (GSH), UDP-glucuronosyltransferase, beta-glucuronidase, sulfatase and sulfotransferase. Results showed that cytochrome P-450 1A1/1A2 was the only isoenzyme detected in both L1210 and L1210/BCNU. CDNB-GST activity was significantly higher in L1210/BCNU compared with L1210. The isoenzyme GST-alpha was more abundant in L1210/BCNU compared with L1210, whereas GST-pi was expressed less in the BCNU-resistant leukemia line. GST-mu was not detected in either L1210 leukemia lines. GSH levels were similar in the two L1210 lines. No significant difference was observed between the two leukemia lines for the conjugative enzymes UDP-glucuronosyltransferase and sulfotransferase, whereas their corresponding hydrolytic enzymes beta-glucuronidase and sulfatase were about two-fold lower in the BCNU-resistant leukemia line. Epoxide hydrolase was 1.3-fold higher in L1210/BCNU compared with L1210 and this level was about three-fold higher than in mouse liver. In conclusion, these studies showed the presence of cytochrome P-450 1A1/1A2 in the two L1210 leukemia lines studied, and indicated noteworthy differences between the two leukemia lines for many enzyme systems such as GST, beta-glucuronidase, sulfatase and epoxide hydrolase. These data are of importance to better understand the mechanisms of drug resistance to nitrosoureas in vivo.
1,3-双(2-氯乙基)-1-亚硝基脲（卡莫司汀，BCNU）耐药性大多是在体外进行研究的。为了更好地了解体内情况下的BCNU耐药性，我们比较了两种L1210白血病细胞系中的主要药物代谢酶系统，一种对BCNU敏感，另一种对BCNU耐药（L1210/BCNU），它们在小鼠体内传代。通过免疫印迹法检测了以下几种酶：细胞色素P-450（1A1/1A2、2B1/2B2、2C8 - 10、2E1、3A）、环氧化物水解酶（EH）和谷胱甘肽S-转移酶（GST-α、-μ和-π）。通过荧光法或分光光度法检测了以下几种酶和辅助因子：1-氯-2,4-二硝基苯-GST（CDNB-GST）、总谷胱甘肽（GSH）、UDP-葡萄糖醛酸基转移酶、β-葡萄糖醛酸酶、硫酸酯酶和磺基转移酶。结果显示，细胞色素P-450 1A1/1A2是在L1210和L1210/BCNU中均检测到的唯一同工酶。与L1210相比，L1210/BCNU中的CDNB-GST活性显著更高。与L1210相比，GST-α同工酶在L1210/BCNU中更为丰富，而GST-π在BCNU耐药白血病细胞系中的表达较少。在两种L1210白血病细胞系中均未检测到GST-μ。两种L1210细胞系中的GSH水平相似。在两种白血病细胞系之间，结合酶UDP-葡萄糖醛酸基转移酶和磺基转移酶未观察到显著差异，而它们相应的水解酶β-葡萄糖醛酸酶和硫酸酯酶在BCNU耐药白血病细胞系中约低两倍。与L1210相比，L1210/BCNU中的环氧化物水解酶高1.3倍，且该水平比小鼠肝脏中的高约三倍。总之，这些研究表明在所研究的两种L1210白血病细胞系中存在细胞色素P-450 1A1/1A2，并表明在许多酶系统如GST、β-葡萄糖醛酸酶、硫酸酯酶和环氧化物水解酶方面，两种白血病细胞系之间存在显著差异。这些数据对于更好地理解体内亚硝基脲类药物耐药机制具有重要意义。
