Role of glutathione S-transferases A1-1, M1-1, and P1-1 in the detoxification of 2-phenylpropenal, a reactive felbamate metabolite

Felbamate has proven to be an effective therapy for treating refractory epilepsy. However, felbamate therapy has been limited due to the associated reports of hepatotoxicity and aplastic anemia. Previous research from our laboratory has proposed 2-phenylpropenal as the reactive metabolite in felbamate bioactivation and identified its mercapturates in the urine of rats and patients undergoing felbamate therapy. While the reaction between 2-phenylpropenal and GSH has been shown to occur spontaneously under physiological conditions, the potential catalysis by glutathione transferases (GST) has remained unknown. The work presented here demonstrates a role for GST in the detoxification of 2-phenylpropenal. The kinetic data show that 2-phenylpropenal is a substrate for all three isoforms tested, with a k(cat)/K-m of 0.275 + 0.035 muM(-1) s(-1) for GSTM1-1, 0.164 +/- 0.005 muM(-1) s(-1) for GSTP1-1, and 0.042 +/- 0.005 muM(-1) s(-1) for GSTA1-1. Given that electrophilic substrates such as 2-propenal have been shown to inhibit GSTs, we also examined the inhibition of GSTM1-1, GSTP1-1 and GSTA1-1 by 2-phenylpropenal. The enzyme inhibition studies demonstrate that 2-phenylpropenal inhibits GSTP1-1 and GSTM1-1. The inhibition of GSTP1-1 was completely reversible upon filtration and reconstitution in buffer containing 10 mM GSH. However, 2-phenylpropenal inhibition of GSTM1-1 was irreversible under the same conditions. The irreversible inhibition of GSTM1-1 may be important in understanding the toxicities associated with felbamate. Given that 2-phenylpropenal is both a substrate and irreversible inhibitor for GSTM1-1, GSTM1-1 represents a potential target for 2-phenylpropenal haptenization in vivo, which may in turn mediate the observed idiosyncratic reactions.