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Published on: July 28, 2010
Quinone toxicity in DT-diaphorase-efficient and -deficient colon carcinoma cell lines
J M Karczewski1, J G Peters, J Noordhoek
1Department of Toxicology, University of Nijmegen, The Netherlands. J.Karczewski@toxi.kun.nl
Abstract:
The human colon carcinoma cell lines Caco-2 and HT-29 were exposed to three structurally related naphthoquinones. Menadione (MEN), 1,4-naphthoquinone (NQ), and 2,3-dimethoxy-1,4-naphthoquinone (DIM) redoxcycle at similar rates, NQ is a stronger arylator than MEN, and DIM does not arylate thiols. The Caco-2 cell line was particularly vulnerable to NQ and MEN and displayed moderate toxic effects of DIM. The HT-29 cell line was only vulnerable to NQ and MEN after inhibition of DT-diaphorase (DTD) with dicoumarol, whereas dicoumarol did not affect the toxicity of quinones to Caco-2 cells. DTD activity in the HT-29 and Caco-2 cell lines, as estimated by the dicoumarol-sensitive reduction of 2,6-dichlorophenolindophenol, was 393.7 +/- 46.9 and 6.4 +/- 2.2 nmol NADPH x min(-1) x mg protein(-1), respectively. MEN depleted glutathione to a small extent in the HT-29 cell line, but a rapid depletion similar to Caco-2 cells was achieved when dicoumarol was added. The data demonstrated that the DTD-deficient Caco-2 cell line was more vulnerable to arylating or redoxcycling quinones than DTD-expressing cell lines. Exposure of the Caco-2 cell line to quinones produced a rapid rise in protein disulphides and oxidised glutathione. In contrast to NQ and DIM, no intracellular GSSG was observed with MEN. The relatively higher levels of ATP in MEN-exposed cells may account for the efficient extrusion of intracellular GSSG. The reductive potential of the cell as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction was only increased by MEN and not with NQ and DIM. We conclude that arylation is a major contributing factor in the toxicity of quinones. For this reason, NQ was the most toxic quinone, followed by MEN, and the pure redoxcycler DIM elicited modest toxicity in Caco-2 cells.
Insights
Naphthoquinones like menadione (MEN) and 1,4-naphthoquinone (NQ) are toxic to colon cancer cells, especially those deficient in DT-diaphorase (DTD). Arylation, not just redox cycling, is a key factor in quinone toxicity.
Area of Science:
- Cell Biology
- Toxicology
- Biochemistry
Background:
- Naphthoquinones are a class of compounds with diverse biological activities.
- Cellular defense mechanisms, such as DT-diaphorase (DTD), play a role in mitigating quinone toxicity.
- Understanding the differential toxicity of related quinones is crucial for drug development and risk assessment.
Purpose of the Study:
- To investigate the toxicity of three related naphthoquinones (Menadione, 1,4-naphthoquinone, 2,3-dimethoxy-1,4-naphthoquinone) in human colon carcinoma cell lines.
- To elucidate the role of DT-diaphorase (DTD) activity and arylation in naphthoquinone-induced cellular damage.
- To compare the mechanisms of toxicity, including redox cycling and glutathione depletion.
Main Methods:
- Exposure of Caco-2 and HT-29 colon cancer cell lines to Menadione (MEN), 1,4-naphthoquinone (NQ), and 2,3-dimethoxy-1,4-naphthoquinone (DIM).
- Assessment of DT-diaphorase (DTD) activity using dicoumarol inhibition and spectrophotometric assays.
- Measurement of glutathione levels, protein disulfides, oxidized glutathione (GSSG), and cellular ATP levels.
- Evaluation of reductive potential using MTT assay.
Main Results:
- Caco-2 cells, deficient in DTD, were more sensitive to NQ and MEN than HT-29 cells, which express DTD.
- 1,4-naphthoquinone (NQ) was identified as a potent arylating agent and the most toxic quinone, followed by MEN.
- DIM, a poor arylator, exhibited moderate toxicity, primarily through redox cycling.
- Quinone exposure led to increased protein disulfides and oxidized glutathione (GSSG) in Caco-2 cells.
- MEN exposure resulted in higher ATP levels and efficient GSSG extrusion in Caco-2 cells.
Conclusions:
- Arylation is a significant contributor to the toxicity of naphthoquinones.
- DTD-deficient cells are more vulnerable to the toxic effects of arylating and redoxcycling quinones.
- The study highlights the differential toxicity mechanisms of structurally related quinones based on their chemical properties and cellular DTD activity.
