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DT-diaphorase protects against menadione-induced oxidative stress
T J Chiou1, Y T Wang, W F Tzeng
1Department of Medicine, Veterans General Hospital-Taipei and School of Medicine, National Yang-Ming University, Taiwan, ROC.
Abstract:
To study the role of DT-diaphorase in menadione-mediated cytotoxicity, menadione-resistant cells were selected from P19 cells by stepwise increasing concentrations of menadione from 10 to 60, 120 or 300 microM without mutagenic pretreatment. Three isolated clones, K60, K120 and K300, were maintained in media containing 60, 120 or 300 microM menadione, respectively. The resistance of these cells to menadione, in order, was: K300 > K120 > K60 > P19 cells. K300 cells were the most resistant. Acquisition of resistance was associated with elevation in DT-diaphorase activity. Pretreatment of the resistant cells with 30 microM dicumarol at 37 degrees C for 30 min sensitized the resistant cells to menadione. When the resistant cells were maintained in the absence of menadione for 28 days, the resistance of K60 and K120 cells was lost. The lower degree of resistance was accompanied by a decrease in DT-diaphorase activity in the revertant cells. However, the resistance and the activity of DT-diaphorase in K300 cells were quite stable in the same period. These results support strongly that DT-diaphorase protects against menadione-induced oxidative stress.
Insights
DT-diaphorase protects against menadione-induced oxidative stress. Increased DT-diaphorase activity in resistant cells enhanced protection, while its loss reversed resistance, supporting its protective role.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Menadione (vitamin K3) induces cytotoxicity through oxidative stress.
- DT-diaphorase (also known as NQO1) is an enzyme involved in redox cycling.
- The specific role of DT-diaphorase in menadione resistance is not fully understood.
Purpose of the Study:
- To investigate the involvement of DT-diaphorase in cellular resistance to menadione.
- To determine if elevated DT-diaphorase activity confers protection against menadione-induced oxidative stress.
Main Methods:
- Selection of menadione-resistant P19 cell clones (K60, K120, K300) by stepwise exposure to increasing menadione concentrations.
- Assay of DT-diaphorase activity in parental and resistant cell lines.
- Assessment of menadione sensitivity following DT-diaphorase inhibition with dicumarol.
- Evaluation of resistance and DT-diaphorase activity stability in cells cultured without menadione.
Main Results:
- Menadione-resistant cell clones exhibited progressively higher DT-diaphorase activity, correlating with resistance levels (K300 > K120 > K60).
- Dicumarol, a DT-diaphorase inhibitor, sensitized resistant cells to menadione.
- Loss of menadione resistance in K60 and K120 cells upon withdrawal was associated with decreased DT-diaphorase activity.
- K300 cells maintained stable resistance and DT-diaphorase activity even after prolonged culture without menadione.
Conclusions:
- DT-diaphorase plays a significant protective role against menadione-induced oxidative stress.
- Elevated DT-diaphorase activity is a key mechanism conferring resistance to menadione.
- The findings highlight DT-diaphorase as a crucial enzyme in cellular defense against menadione toxicity.
