Endothelial Cell Injury of Carmustine (BCNU) in Hepatic Sinusoids: Contribution of Oxidation and Immune Reaction.

Abstract

Background: Carmustine (BCNU) is used for the treatment of brain tumors and is included in conditioning regimens of stem cell transplantation. It has been long believed that BCNU causes hepatotoxicity by inducing pericholangitis and intrahepatic cholestasis leading to biliary cirrhosis. Either hepatocytes or biliary ductal cells have been thought to be the target in BCNU-induced liver toxicity. However, the mechanisms operational at cell level have not been well elucidated. Possible explanations include glutathione depletion in cells, DNA damage accompanied by cellular oxidative stress and/or acceleration of apoptosis. It has been shown that BCNU treatment inhibits hepatic glutathione reductase activity in rats. Recently, a two-step process involving free radical formation and immune reaction has been proposed as a mechanism of BCNU-induced alveolar epithelial cell damage by our group (Savasan S, Ozdemir O, Buck S, et al., 95th annual meeting of the AACR, Orlando, March 27–31, 2004. Abstract#: 1133, 2004). In this study, we have investigated BCNU effect on human liver sinusoid endothelial cells (HLS-EC) in vitro.Material and Methods: Cytotoxicity in HLS-EC was measured by flow cytometric detection of annexin V/PI-positive cells, DiOC6(3) /PI staining, further quantification of fluorosphere-adjusted events and trypan blue exclusion test in repeated experiments. The generation of reactive oxygen species (ROS) was measured by flow cytometric DCF staining. Lymphokine activated killer (LAK) cell-mediated cytotoxicity against BCNU-treated and untreated HLS-EC was measured by flow cytometric cell mediated cytotoxicity assay. The influence ROS-scavenging compound butylated hydroxyanisole (BHA) and anti-inflammatory dexamethasone (DXM) on BCNU treatment was also studied.Results: Severe HLS-EC damage occurred following BCNU treatment shown by four different cytotoxicity methods. This effect was associated with oxidative stress demonstrated by significant increase in DCF staining. Although pre-treatment of cells with DXM did not affect BCNU cytotoxicity, BHA suppressed BCNU cytotoxicity by 25% (p < 0.05). BCNU treatment also resulted in increased LAK cell-mediated elimination of HLS-EC (65%) compared to untreated cells (16%). Similar to its effect on direct BCNU cytotoxicity, BHA prevented BCNU treated HLS-EC killing by LAK cells by 25%. However, DXM did not influence LAK cell-mediated killing of BCNU-treated HLS-EC cells. These results suggest that both ROS generation and immune reaction against perturbed endothelial cells play a central role in BCNU-associated HLS-EC injury. In contrary to our findings on human alveolar epithelial cells, BHA ameliorated BCNU-induced direct and immune-mediated cytotoxicity on HLS-EC, but not DXM.Conclusion: Reactive oxygen species scavenging may be useful in prevention of liver toxicity of BCNU.