Is fluoroacetate-specific defluorinase a glutathione S-transferase?

Fluoroacetate-specific defluorinase (FSD) is a critical enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metabolism. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was determined in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our experiments showed that GSTZ1C catalysed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.

Characterization of the fluoroacetate detoxication enzymes of rat liver cytosol.

Two forms of fluoroacetate-specific defluorinase (FSD) were purified from rat hepatic cytosol. The first form, FSD1 (molecular weight 38 kDa), contained 81% of the total cytosolic fluoroacetate defluorination activity and did not bind to the glutathione-affinity, orange A or mono P columns used in the purification procedures. The second form, FSD2 (molecular weight 27 kDa), contained only 13% of the fluoroacetate defluorination activity, had a pI = 7.8, and exhibited a high glutathione S-transferase (GST)-like activity towards dichloroacetic acid. The FSD1 proteins were identified from peptide mass data and best matched with rat sorbitol dehydrogenase (SDH) (short form), although pure sheep liver SDH enzyme did not possess defluorination activity when subsequently investigated. The FSD2 protein was identified from peptide mass data and best matched with the amino acid sequence of mouse and human Zeta 1 of glutathione S-transferase (GSTZ1) and showed a high GSTZ1 specific activity. This study suggests that the major FSD component (FSD1) represents a new and unique dehalogenating or dehydrogenating enzyme present in rat liver cytosol. The minor FSD component (FSD2) is due to the GSTZ1 present in rat liver cytosol. However, it is not yet clear that FSD1 is indeed SDH and FSD2 is indeed GSTZ1, due to sequence homology being less than 60 and 45%, respectively.

Passage of silver ions through membrane-mimetic materials, and its relevance to treatment of burn wounds with silver sulfadiazine cream.

Treatment of acute burn wounds with silver sulfadiazine has raised concern of potential silver toxicity. As the wound heals, a barrier forms between the silver sulfadiazine and the blood, but this membrane is not impenetrable, and so silver absorption is still possible. In this work, we have modeled chemical systems to investigate the transport of silver sulfadiazine and silver chloride through cellulose, chitosan, collagen, and polyethylene membranes into the following media: synthetic serum electrolyte solution (SSES), SSES plus glutathione, and human serum, to simulate some of the chemical processes occurring at a burn wound during healing. Our results clearly indicate that membranes can retard the movement of silver ions, especially those that have silver-binding properties. This suggests that silver absorption at a healing wound will be minimized by entrapment of silver in the growing membrane network, and thus the likelihood of silver toxicity will be reduced.

Solubility of silver sulfadiazine in physiological media and relevance to treatment of thermal burns with silver sulfadiazine cream.

Silver sulfadiazine cream has been a standard treatment for burns over the past two decades. Although many studies have described the phenomenon of silver absorption from burn wounds treated with silver sulfadiazine, they failed to examine the chemistry underlying the absorption process: Silver chloride was assumed to form at the burn wound and absorption of silver was believed to be negligible. Here we have developed chemical model systems to investigate the interactions of silver sulfadiazine and silver chloride in direct contact with synthetic serum electrolyte solution (SSES), with SSES plus endogenous ligands or beef blood plasma, and with human serum. The results indicate that silver absorption from an acute burn site can be significant, because human serum is capable of solubilizing silver. This finding is of concern, given the potential for silver toxicity as a direct consequence of applying silver sulfadiazine to extensive burn wounds.