Kawabata T T, Chapman M Y, Kim D H, Stevens W D, Holsapple M P
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
Biochem Pharmacol. 1990 Sep 1;40(5):927-35. doi: 10.1016/0006-2952(90)90476-2.
Cyclophosphamide (CY) is metabolized to 4-hydroxy-CY which spontaneously breaks down to the reactive intermediates, phosphoramide mustard (PAM) and acrolein. The alkylating property of PAM is thought to mediate the anti-proliferative and cytotoxic actions of CY. Acrolein is known to bind sulfhydryl groups of cellular molecules and may contribute to the action of CY. However, the role of acrolein in the CY-induced immunosuppression remains unclear. The results of studies in which a hepatocyte co-culture system was used suggest that acrolein may play an important role in the cytotoxic action of CY, whereas those investigations using activated derivatives of CY indicate that acrolein is not an important factor. Thus, both approaches of CY exposure were utilized in the present study. Splenocytes of B6C3F1 mice were incubated with syngeneic isolated hepatocytes and CY or with 4-hydroperoxycyclophosphamide (4-HC) (which spontaneously decomposes to 4-hydroxy-CY). The in vitro antibody forming cell (AFC) response was found to be suppressed with both methods of exposure to CY metabolites. The addition of DNA to bind extracellular PAM was ineffective in preventing the suppression produced by hepatocyte-activated CY. However, it was also observed that DNA was able to attenuate the PAM-induced suppression. The sulfhydryl compounds 2-mercaptoethanesulfonate (MESNA) (15 microM) or reduced glutathione (GSH) (1 mM) inhibited the suppression of the AFC response of splenocytes incubated with CY and mouse hepatocytes. The suppression produced by 4-HC, however, was not affected by MESNA and only slightly inhibited by GSH. Similarly, the PAM-induced suppression was not affected by MESNA and slightly attenuated with GSH. In contrast, both MESNA and GSH were very effective in abrogating the acrolein-induced suppression, whereas DNA was ineffective. The findings of this study suggest that in the hepatocyte co-culture system, the immunosuppressive actions of CY are mediated by acrolein generated outside of the splenocyte, whereas the 4-HC induced suppression is not mediated by extracellular acrolein. Thus, this difference may explain the contradictory findings of previous studies that used different means of exposing cells to activated CY.
环磷酰胺（CY）代谢生成4-羟基环磷酰胺，其会自发分解为活性中间体磷酰胺氮芥（PAM）和丙烯醛。PAM的烷基化特性被认为介导了CY的抗增殖和细胞毒性作用。已知丙烯醛会与细胞分子的巯基结合，可能对CY的作用有影响。然而，丙烯醛在CY诱导的免疫抑制中的作用仍不清楚。使用肝细胞共培养系统的研究结果表明，丙烯醛可能在CY的细胞毒性作用中起重要作用，而那些使用CY活化衍生物的研究表明丙烯醛不是一个重要因素。因此，本研究采用了两种CY暴露方法。将B6C3F1小鼠的脾细胞与同基因分离的肝细胞以及CY或4-氢过氧环磷酰胺（4-HC，其会自发分解为4-羟基环磷酰胺）一起孵育。发现两种CY代谢物暴露方法均能抑制体外抗体形成细胞（AFC）反应。添加DNA以结合细胞外PAM并不能有效防止肝细胞活化的CY产生的抑制作用。然而，也观察到DNA能够减弱PAM诱导的抑制作用。巯基化合物2-巯基乙烷磺酸钠（MESNA）（15微摩尔）或还原型谷胱甘肽（GSH）（1毫摩尔）可抑制与CY和小鼠肝细胞一起孵育的脾细胞AFC反应的抑制作用。然而，4-HC产生的抑制作用不受MESNA影响，仅被GSH轻微抑制。同样，PAM诱导的抑制作用不受MESNA影响，被GSH轻微减弱。相反，MESNA和GSH在消除丙烯醛诱导的抑制作用方面都非常有效，而DNA则无效。本研究结果表明，在肝细胞共培养系统中，CY的免疫抑制作用是由脾细胞外产生的丙烯醛介导的，而4-HC诱导的抑制作用不是由细胞外丙烯醛介导的。因此，这种差异可能解释了先前使用不同方法使细胞暴露于活化CY的研究中相互矛盾的结果。
