title: Glutathione-dependent biotransformation of 1-chloro-2,4-dinitrobenzene in arterial and venous blood of the striped bass (Morone saxitilis).
abstract: Glutathione (GSH) mediated metabolism of a model electrophile, 1-chloro-2,4-dintrobenzene (CDNB), by striped bass (Morone saxitilis) whole blood and reconstituted erythrocytes equilibrated to 'arterial' and 'venous' blood gas concentrations was assessed. Reduced GSH was found to be an abundant nucleophile (2-2.4 mM) in striped bass erythrocytes. Incubation of whole blood and intact erythrocytes, reconstituted in phosphate buffer solution (PBS), with CDNB resulted in rapid depletion of >98% of cellular GSH levels and rapid, though not stoichiometric, accumulation of a glutathione S-conjugate, 2,4-dintrophenyl S-glutathione (DNP-SG) within the 60 min exposure period. The GSH depletion half-lives were 11.0+/-2.6 min and 10.3+/-2.0 min, respectively, for 'arterial' and 'venous' trials using whole blood compared to 6.4+/-1.8 min and 7.1+/-0.9 min, respectively, for erythrocytes in PBS. The conjugate was not metabolized further within erythrocytes, but was slowly transported linearly from the cells to the plasma or PBS medium. Reconstitution of erythrocytes in PBS produced significantly greater rates of CDNB biotransformation compared to whole blood, although the total amount of DNP-SG formed per unit of GSH depleted was significantly lower. Differences may be attributed to altered rates of CDNB membrane permeation and/or efflux and changes in the rate of GSH oxidation in the presence of plasma versus PBS. Whole blood and erythrocytes in PBS equilibrated to 'arterial' and 'venous' blood gas concentrations showed no significant difference in rates of GSH depletion, DNP-SG formation, or conjugate efflux. GSH concentration decreased after exposure to CDNB and was not regenerated within the 60 min incubation period. GSH reacted nonenzymatically with CDNB at a very slow and relatively insignificant rate under physiological blood gas concentrations. Enzymatic biotransformation of lipophilic electrophiles by erythrocytic GSH results in rapid depletion of the protective thiol and constitutes the first step in mercapturic acid formation. This investigation provides strong evidence for the presence of significant levels of glutathione S-transferase in striped bass erythrocytes.
authors.affiliation: Department of Ocean Sciences, University of California, 95064, Santa Cruz, CA, USA
authors.initials: MS
authors.lastname: Stoelting
authors.initials: RS
authors.lastname: Tjeerdema