2,3,7,8-Tetrachlorodibenzo-p-dioxin-Mediated Production of Reactive Oxygen Species Is An Essential Step in the Mechanism of Action to Accelerate Human Keratinocyte Differentiation

Abstract

Chloracne is commonly observed in humans exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); yet, the mechanism of toxicity is not well understood. Using normal human epidermal keratinocytes, we investigated the mechanism of TCDD-mediated enhancement of epidermal differentiation by integrating functional genomic, metabolomic, and biochemical analyses. TCDD increased the expression of 40% of the genes of the epidermal differentiation complex found on chromosome 1q21 and 75% of the genes required for de novo ceramide biosynthesis. Lipid analysis demonstrated that eight of the nine classes of ceramides were increased by TCDD, altering the ratio of ceramides to free fatty acids. TCDD decreased the expression of the glucose transporter, SLC2A1, and most of the glycolytic transcripts, followed by decreases in glycolytic intermediates, including pyruvate. NADH and Krebs cycle intermediates were decreased, whereas NAD(+) was increased. Mitochondrial glutathione (GSH) reductase activity and the GSH/glutathione disulfide ratio were decreased by TCDD, ultimately leading to mitochondrial dysfunction, characterized by decreased inner mitochondrial membrane potential and ATP production, and increased production of the reactive oxygen species (ROS), hydrogen peroxide. Aryl hydrocarbon receptor (AHR) antagonists blocked the response of many transcripts to TCDD, and the endpoints of decreased ATP production and differentiation, suggesting regulation by the AHR. Cotreatment of cells with chemical antioxidants or the enzyme catalase blocked the TCDD-mediated acceleration of keratinocyte cornified envelope formation, an endpoint of terminal differentiation. Thus, TCDD-mediated ROS production is a critical step in the mechanism of this chemical to accelerate keratinocyte differentiation.

Current skin symptoms of Yusho patients exposed to high levels of 2,3,4,7,8-pentachlorinated dibenzofuran and polychlorinated biphenyls in 1968

“…Dioxins and other halogenated polycyclic aromatic hydrocarbons bind to aryl hydrocarbon receptor (AhR), which is abundantly expressed in keratinocytes and sebocytes (Ju et al, 2011;Kennedy et al, 2013). AhR plays a pivotal role in epidermal homeostasis (Furue et al, 2014).…”

“…AhR plays a pivotal role in epidermal homeostasis (Furue et al, 2014). Ligand-specific activation of AhR induces hyperkeratinization by upregulating the expression of epidermal differentiation complex genes (e.g., filaggrin, loricrin and involucrin) (Kennedy et al, 2013). Highly lipophilic dioxins appear to accumulate in and are excreted via sebaceous glands and sebum (Iida et al, 1999), which may involve their efficient excretion from the body (Matsumoto et al, 2013).…”

Loss of Multidrug Resistance–Associated Protein 1 Potentiates Chronic Doxorubicin-Induced Cardiac Dysfunction in Mice

“…The protein concentrations of heart ventricle homogenates were determined with the bicinchoninic acid protein assay. The measurement of GSH and GSSG by high-performance liquid chromatography was performed exactly as described (Deng et al, 2015) and used by others (Yamazaki et al, 2008(Yamazaki et al, , 2012Yamazaki and Villarreal, 2011;Kennedy et al, 2013;Barajas-Espinosa et al, 2014). The use of authentic GSH and GSSG to develop the standard curves ensured quantitation of the correct entities by high-performance liquid chromatography, thus validating the assay.…”

Elevated Glutathione Is Not Sufficient to Protect against Doxorubicin-Induced Nuclear Damage in Heart in Multidrug Resistance–Associated Protein 1 (Mrp1/Abcc1) Null Mice

“…The HPLC assay for GSH and GSSG is based on that developed by Senft et al (2000), and is used widely (Yamazaki et al, 2008(Yamazaki et al, , 2012Yamazaki and Villarreal, 2011;Kennedy et al, 2013;Barajas-Espinosa et al, 2014). Mouse heart homogenate was diluted with 0.5 volume of buffer A containing 15% metaphosphoric acid and mixed thoroughly, followed by centrifugation at 12,000 rpm for 15 minutes at 4°C.…”