ABSTRACT
Treatment for 48 h of murine Hepa 1c1c7 cells in culture with the cancer chemopreventive oltipraz (1) followed by addition of CD(3)I and immediate cell lysis yields, by LC/MS analysis, three isotopomers of the methylated pyrrolopyrazine (2), a known human metabolite of oltipraz. The major isotopomer (58%) is the one containing two CD(3)- groups attached to the pendant sulfur atoms of the pyrrolopyrazine ring, the others containing one CD(3)- and one CH(3)- group or two CH(3)- groups. It is concluded from this that the unmethylated pyrrolopyrazine (4) is the major metabolite of oltipraz. Prodrugs 5 and 6, which have been shown to rapidly generate 4 in the presence of GSH at physiological pH, induce the phase 2 enzyme NQO1 in Hepa 1c1c7 cells with potencies on par with oltipraz itself: CD(NQO1) = 14.4 +/- 1.3, 20.1 +/- 4.6, and 23.6 +/- 1.6 microM for oltipraz, 5, and 6, respectively. Pretreatment of oltipraz, 5, and 6 in cell culture media with 1 mM GSH, which is shown to immediately convert 5 and 6 to 4, followed by incubation with Hepa 1c1c7 cells shows similar potencies for oltipraz and the (decomposed) produrgs, with CD(NQO1) = 18.0 +/- 4.4 microM for 5, 17.8 +/- 0.2 microM for 6, and 13.5 +/- 1.4 microM for oltipraz. Treatment with compound 6 of murine hepatoma cells containing a luciferase gene under the control of the antioxidant response element (ARE) from the mouse heme oxygenase (ho-1) gene elicits induction of luciferase activity, CD = 35.8 +/- 2.8 microM, somewhat greater than the potency than oltipraz itself. Western blots of nuclear proteins isolated from Hepa 1c1c7 cells and probed with anti-Nrf2 indicate that as compared to vehicle DMSO, compound 6 stimulates nuclear translocation of Nrf2 from the cytosol. From this study, it is concluded that the major metabolite of the cancer chemopreventive oltipraz is a phase 2 enzyme inducer of comparable potency that activates the ARE and initiates nuclear translocation of transcription factor Nrf 2.
Subject(s)
Enzyme Induction/drug effects , Pyrazines/metabolism , Animals , Blotting, Western , Cell Death/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Enzyme Induction/physiology , Glutathione/drug effects , Glutathione/metabolism , Glutathione Transferase/metabolism , Heme Oxygenase (Decyclizing)/metabolism , Luciferases/genetics , Methylation , Mice , NAD(P)H Dehydrogenase (Quinone)/pharmacology , NF-E2-Related Factor 2 , Prodrugs/metabolism , Pyrazines/chemistry , Pyrazines/pharmacology , Pyrroles/chemistry , Pyrroles/metabolism , Thiones/chemistry , Thiones/metabolism , Thiophenes , Time Factors , Trans-Activators/genetics , Trans-Activators/physiology , Transcription Factors/genetics , Transcription Factors/physiology , Transfection , Tumor Cells, CulturedABSTRACT
Epidemiological studies have shown that exposure to aflatoxin B(1) (AFB(1)) and concurrent infection with hepatitis B lead to a multiplicative risk of developing liver cancer. This chemical-viral interaction can be recapitulated in the tree shrew (Tupia belangeri chinensis). As an initial characterization of this model, the metabolism of AFB(1) in tree shrews has been examined and compared to a sensitive bioassay species, the rat. Utilizing LC/MS/MS, an unreported product, aflatoxin M(1)-N(7)-guanine (AFM(1)-N(7)-guanine), was detected in urine and hepatic DNA samples 24 h after administration of 400 microg/kg AFB(1). In hepatic DNA isolated from tree shrews, AFM(1)-N(7)-guanine was the predominant adduct, 0.74 +/- 0.14 pmol/mg DNA, as compared to 0.37 +/- 0.07 pmol/mg DNA of AFB(1)-N(7)-guanine. Conversely, in rat liver, 6.56 +/- 2.41 pmol/mg DNA of AFB(1)-N(7)-guanine and 0.42 +/- 0.13 pmol/mg DNA of AFM(1)-N(7)-guanine were detected. Rats excreted 1.00 +/- 0.21 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.29 +/- 0.10 pmol AFM(1)-N(7)-guanine/mg creatinine as compared to 0.60 +/- 0.12 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.69 +/- 0.16 pmol AFM(1)-N(7)-guanine/mg creatinine excreted by the tree shrew. Furthermore, tree shrew urine contained 40 times more of the hydroxylated metabolite, AFM(1), than was excreted by rats. In vitro experiments confirmed this difference in oxidative metabolism. Hepatic microsomes isolated from tree shrews failed to produce aflatoxin Q(1) or aflatoxin P(1) but formed a significantly greater amount of AFM(1) than rat microsomes. Bioassays indicated that the tree shrew was considerably more resistant than the rat to AFB(1) hepatocarcinogenesis, which may reflect the significant differences in metabolic profiles of the two species.
