Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells

https://doi.org/10.1016/j.bbrc.2009.12.113Get rights and content

Abstract

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process.

Section snippets

Materials and methods

Chemicals. DCIP, menadione, NADPH and UDPGA were purchased from Sigma–Aldrich (St. Louis, MO). TransIT®-LT1 was purchased from Mirus Bio LLC., (Madison, WI). pIRESpuro3, pTRE2hyg and 293 Tet-Off™ cell line (HEK293) were purchased from Clontech Laboratories, Inc. (Mountain View, CA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCF-DA) was purchased from Molecular Probes, Inc. (Eugene, OR). [3H]menadione (185 GBq/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA). Menadiol,

Establishment of stably transfected HEK293 cell lines

In this study, we established three HEK293 cell lines, namely, the parent HEK293 stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with DOX-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and of UGT1A10 (HEK293/NQO1/UGT1A10). In each cell line, NQO1 activity toward DCIP and menadione was significantly higher than in the HEK293 wild-type cell line. NQO1 activity did not differ significantly among the established cell lines (Table 1). In addition, we assessed the effect of DOX

Discussion

We previously suggested that menadiol, which is formed from the NQO1-mediated two-electron reduction of menadione, is not a detoxification pathway because menadiol undergoes the reformation of menadione with concomitant formation of ROS. Consequently, glucuronide conjugates remain as reduced species and therefore their formation results in detoxification of menadione [15]. In the present study, to evaluate the role of NQO1 and UGTs in menadione detoxification pathways, we established HEK293

References (26)

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