TY - JOUR
T1 - Role of glutathione S-transferase 8-8 in allylamine resistance of vascular smooth muscle cells in vitro
AU - He, Nonggao
AU - Singhal, Sharad S.
AU - Awasthi, Sanjay
AU - Zhao, Tiejun
AU - Boor, Paul J.
N1 - Funding Information:
This work was supported by Grant HL-26189 from the National Institutes of Health.
PY - 1999/7/15
Y1 - 1999/7/15
N2 - 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 (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 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.
AB - 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 (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 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.
KW - Acrolein
KW - Allylamine
KW - Allylamine-resistant cell line
KW - Glutathione S-transferases
KW - Rat GST8-8
KW - Semicarbazide-sensitive amine oxidase
KW - Vascular smooth muscle cells
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U2 - 10.1006/taap.1999.8700
DO - 10.1006/taap.1999.8700
M3 - Article
C2 - 10406932
AN - SCOPUS:0033566203
SN - 0041-008X
VL - 158
SP - 177
EP - 185
JO - Toxicology and Applied Pharmacology
JF - Toxicology and Applied Pharmacology
IS - 2
ER -