Conjugation of isoprene monoepoxides with glutathione, catalyzed by alpha, mu, pi and theta-class glutathione S-transferases of rat and man.
In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.
Bogaards
J J
JJ
Toxicology Division, TNO Nutrition and Food Research Institute, Zeist, The Netherlands. bogaards@voeding.tno.nl
Venekamp
J C
JC
Salmon
F G
FG
van Bladeren
P J
PJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology; D051379:Mice; D051381:Rats; D017208:Rats, Wistar; D013045:Species Specificity; D013347:Subcellular Fractions / Q000378:metabolism
D004852:Epoxy Compounds; C103970:3,4-epoxy-3-methyl-1-butene; C103971:3,4-epoxy-2-methyl-1-butene; D005982:Glutathione Transferase; D005978:Glutathione