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 α,β-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 μM) 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 μM sulfasalazine (SS), a GST inhibitor, potentiated AA and AC toxicity in both VSMC-AA and VSMC, indicating a protective role of GST. Catalytic efficiency (Kcat/Km) 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, Kcat/Km 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 α,β-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 μM SS. Our results demonstrate that GSTs are an important defense against electrophilic atherogens and that isozymes with high activity toward α,β-unsaturated carbonyls are particularly important in the vascular wall.