Abstract
Polychlorinated biphenyls (PCBs) and their metabolites are environmental pollutants that are known to have adverse health effects. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of 4-monochlorobiphenyl (PCB3, present in the environment and human blood) is toxic to human skin keratinocytes, and breast and prostate epithelial cells. This study investigates the hypothesis that 4-ClBQ-induced metabolic oxidative stress regulates toxicity in human keratinocytes. Results from Seahorse XF96 Analyzer showed that the 4-ClBQ treatment increased extracellular acidification rate, proton production rate, oxygen consumption rate and ATP content, indicative of metabolic oxidative stress. Results from a q-RT-PCR assay showed significant increases in the mRNA levels of hexokinase 2 (hk2), pyruvate kinase M2 (pkm2) and glucose-6-phosphate dehydrogenase (g6pd), and decreases in the mRNA levels of succinate dehydrogenase (complex II) subunit C and D (sdhc and sdhd). Pharmacological inhibition of G6PD-activity enhanced the toxicity of 4-ClBQ, suggesting that the protective function of the pentose phosphate pathway is functional in 4-ClBQ-treated cells. The decrease in sdhc and sdhd expression was associated with a significant decrease in complex II activity and increase in mitochondrial levels of ROS. Overexpression of sdhc and sdhd suppressed 4-ClBQ-induced inhibition of complex II activity, increase in mitochondrial levels of ROS, and toxicity. These results suggest that the 4-ClBQ treatment induces metabolic oxidative stress in HaCaT cells, and while the protective function of the pentose phosphate pathway is active, inhibition of complex II activity sensitizes HaCaT cells to 4-ClBQ-induced toxicity.
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Abbreviations
- 4-ClBQ:
-
1-(4-Chlorophenyl)-benzo-2,5-quinone
- 2-NBDG:
-
2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose
- BSO:
-
l-Buthionine sulfoximine
- DHEA:
-
Dehydroepiandrosterone
- ECAR:
-
Extracellular acidification rate
- mBBr:
-
Monobromobimane
- METC:
-
Mitochondrial electron transport chain
- MFI:
-
Mean fluorescence intensity
- NBT:
-
Nitro-blue tetrazolium chloride
- OCR:
-
Oxygen consumption rate
- OXPHOS:
-
Oxidative phosphorylation
- PCBs:
-
Polychlorinated biphenyls
- PCB3:
-
4-Monochlorobiphenyl
- PEG-SOD:
-
Polyethylene glycol-superoxide dismutase
- PPP:
-
Pentose phosphate pathway
- PPR:
-
Proton production rate
- ROS:
-
Reactive oxygen species
- SDH:
-
Succinate dehydrogenase
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Acknowledgments
We thank Professors Larry W. Robertson and Hans J. Lehmler in the Occupational and Environmental Health at The University of Iowa for providing us with 4-ClBQ compound and the staff at the Flow Cytometry. We thank Ms. Amanda L. Kalen for technical support with the Glucometer assay, Dr. Netanya Y. Spencer for assistance with the G6PD antibody, and Dr. Jaimee C. Eckers for DHEA. This study was supported by National Institute of Environmental Health and Sciences (NIEHS P42ES013661) and National Institute of Health (NIH 2R01CA111365). Core facilities were supported in part by the Holden Comprehensive Cancer Center, P30CA086862. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government.
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The authors declare they have no actual or potential competing financial interests.
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204_2014_1407_MOESM1_ESM.eps
Suppl. Figure 1 4-ClBQ treatment increases ECAR, PPR, and OCR in HaCaT cells. Representative profile plots of ECAR (a), PPR (b), and OCR (c) in control and 1.0 μM 4-ClBQ-treated cells at 24 h of treatments. (EPS 1467 kb)
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Xiao, W., Sarsour, E.H., Wagner, B.A. et al. Succinate dehydrogenase activity regulates PCB3-quinone-induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes. Arch Toxicol 90, 319–332 (2016). https://doi.org/10.1007/s00204-014-1407-3
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DOI: https://doi.org/10.1007/s00204-014-1407-3