Role of glutathione S-transferase 8-8 in allylamine resistance of vascular smooth muscle cells in vitro

Allylamine (AA) is a cardiovascular toxin that causes lesions resembling atherosclerosis in several mammalian species. AA's toxic effects are thought to be exerted through its conversion to acrolein (AC), a potent electrophilic alkylating agent and atherogen. Semicarbazide sensitive amine oxidase (SSAO) catalyzes the oxidation of AA to AC. Glutathione S-transferases (GST) can catalyze the first step of detoxification of AC to mercapturic acid. Our previous studies suggest that the isozyme rGST8-8 is a principal defense against electrophilic stress exerted by alpha,beta-unsaturated carbonyls such as AC. In the present studies, we use cultured rat vascular smooth muscle cells (VSMC) to examine the relative roles of SSAO and rGST8-8 in the cytotoxic effects of the atherogens, AA and AC. Exposure derived AA-resistant cells (VSMC-AA) were 3.5-fold more resistant to AA when compared to VSMC and 1.8-fold more resistant to acrolein. SSAO activity was 2-fold higher in VSMC-AA than in VSMC. Consistent with the role of SSAO in biotransformation of AA, the SSAO inhibitor semicarbazide (SC; 100 mu M) provided nearly complete protection from AA to both VSMC-AA and VSMC. As expected, SC did not affect the cytotoxicity of AC. Pretreatment with 100 mu M sulfasalazine (SS), a GST inhibitor, potentiated AA and AC toxicity in both VSMC-AA and VSMC, indicating a protective role of GST. Catalytic efficiency (K-cat/K-m) of GSTs was higher toward 4-hydroxynonenal (4-HNE) (0.65 mM(-1) s(-1)) than toward 1-chloro-2,4-dinitrobenzene (CDNB) (0.14 mM(-1) s(-1)) for VSMC. In VSMC-AA, K-cat/K-m was increased 4.1-fold toward CDNB (0.58 mM(-1) s(-1)) and 6-fold toward 4HNE (3.9 mM(-1) s(-1)) when compared to VSMC, indicating a preferential increase in VSMC-AA of GST isozymes which utilize alpha,beta-unsaturated carbonyls. Western blots confirmed induction of rGST8-8 in VSMC-AA. Expression of recombinant mGSTA4 (the mouse homolog of rGST8-8) in VSMC caused a 1.6-fold increase in resistance to AA and AC. This resistance was fully reversed by 50 mu M SS. Our results demonstrate that GSTs are an important defense against electrophilic atherogens and that isozymes with high activity toward alpha,beta-unsaturated carbonyls are particularly important in the vascular wall, (C) 1999 Academic Press.