Mechanisms of in vitro immunosuppression by hepatocyte-generated cyclophosphamide metabolites and 4-hydroperoxycyclophosphamide

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.