Metabolism of lomustine in dogs and comparative cytotoxicity of lomustine and its major metabolites and O6-methylguanine-DNA methyltransferase expression in canine cells
The nitrosourea drug lomustine is used clinically for treating a wide variety of malignancies, most commonly brain tumors and lymphoma. Lomustine undergoes hydrolysis in vivo to form isomeric metabolites, primarily trans-4-hydroxylomustine (trans-4) and cis-4- hydroxylomustine (cis-4) in various animal species including humans. Despite its widespread usage to treat canine lymphoma, the metabolism of lomustine has not been studied in dogs. It is reported that 4’-hydroxylation products of lomustine (trans-4 and cis-4) have enhanced alkylating activity and reduced toxic effects relative to lomustine, resulting in a better therapeutic index of each of the metabolites relative to the parent compound. Our results show that the metabolic profile of lomustine in dogs is similar to that in humans with trans-4 being the major metabolite and cis-4 as the minor metabolite. Comparative cytotoxicity studies of lomustine and its trans-4 and cis-4 metabolites in canine lymphoma cell lines 17-71 and GL-1 show that there is no difference in the cytotoxicity of the three compounds. In addition, a concentration and time-dependent cell killing was seen in both of these cell lines. Also primary canine cells like peripheral blood mononuclear cells (PBMC) from lymphoma dogs and canine hepatocytes for normal dogs did not show any sensitivity towards lomustine and its metabolites. Alkylation is thought to be the reason for the efficacy of lomustine while carbamylation has been implicated as the cause for the side effects seen with lomustine treatment such as hepatotoxicity. In the alkylation study we show that lomustine and its metabolites form similar levels of the DNA adducts N7 hydroxyethylguanine and O6 hydroxyethyldeoxyguanosine. These adducts may be used as molecular dosimeters of therapeutic response thus correlating to the similar cytotoxicity of these compounds. In terms of carbamylation, lomustine showed greater extent of carbamylation in the canine hepatocytes and lymphoma cell lines. We hypothesize that if carbamylation of proteins is indeed one of the reasons for the side effects of lomustine, then using the metabolites instead may be a better option in terms of side effects. The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of tumor cells to bifunctional nitrosoureas, like lomustine. There is no data available regarding MGMT expression/activity in canine cells or tissues. Our study shows that there is low MGMT activity in the canine lymphoma cell line 17-71 while the GL-1 cells did not show any detectable enzyme activity or mRNA expression. The MGMT enzyme activity measured in canine hepatocytes is about 250-350 fmol/mg protein as compared to about 90 fmol/mg protein in 17-71 cells. We also show that MGMT mRNA expression in 17-71 cells and canine hepatocytes positively correlates with its enzyme activity in these cells.
Lomustine, metabolites, cytotoxicity, alkylation, carbamylation, O6-methylguanine-DNA methyltransferase (MGMT)
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign