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.

FIG. 1.

TCDD modulates the expression of genes critical to the formation of the cornified layer. (A) Depiction of the microarray analysis of the EDC, located on human chromosome 1q21, following treatment with TCDD. The level of RNA corresponding to the genes in bold print (normal and italics) had increased expression in response to TCDD (24h). The genes in italic bold print confirmed previously published increases in expression (Sutter et al., 2011). The RNAs of the remaining genes were not significantly altered by TCDD. (B)–(D) qRT-PCR was used to verify the FC values obtained in microarray analysis for the indicated genes following TCDD treatment (24h). Levels of RNA (mean [n = 4] ± SD) are expressed in units relative to the DMSO control, given a value of 1. *p < 0.05 indicates a significant difference between the control and treated samples.

FIG. 2.

TCDD enhances sphingolipid biosynthesis in NHEKs. (A) The sphingolipid metabolism pathway adapted from the KEGG pathway database. Bold outlined boxes indicate RNAs significantly increased, and gray boxes indicate RNAs significantly decreased, in response to TCDD (24h) based on microarray analysis. Note: DEGS2 and LASS3 were significant by qRT-PCR analysis, but not in the analysis of the microarray data. (B) A representative HPTLC plate using a ceramide development system of lipid extracts from control or TCDD-treated cultures of NHEKs (72h). (C) Quantitation using densitometry of the indicated lipid bands (mean [n = 3] ± SD). *p < 0.05 indicates a significant difference between the control and treated samples.

FIG. 3.

TCDD impairs mitochondrial function. (A) Microarray analysis was performed using IPA. Depiction indicates TCDD-mediated (24h) mitochondrial dysfunction in NHEKs. Green indicates decreased levels of RNA (treated/control); red indicates increased levels. Coloration of the ETC complexes indicates regulation of genes within the complex which may or may not be individually depicted. Individual genes are listed in Supplementary table S4. IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. (B) and (C) NHEKs were treated for the indicated times. (B) IMM potential was measured as described in the Materials and Methods section (mean [n = 8] ± SD). (C) Levels of cellular ATP were measured using CL as described in the Materials and Methods section (mean [n = 4] ± SD), expressed as the percent of the 24h control sample. *p < 0.05 indicates a significant difference between the time-matched control and treated samples. (D) NHEKs were treated with or without TCDD for 72h in the presence or absence of the indicated AHR antagonists. Levels of cellular ATP were measured as in (C), above; a indicates a significant difference between the control and TCDD-treated samples; b indicates a significant difference between TCDD alone-treated samples and the TCDD plus AHR antagonist-treated samples, p < 0.05.

FIG. 4.

TCDD increases mitochondrial oxidative stress. (A, left) qRT-PCR was used to verify TCDD-mediated (24h) decreases observed in microarray analysis of mitochondrial GSR. Levels of RNA (mean [n = 4] ± SD) are expressed in units relative to the DMSO control, given a value of one. (A, right) Mitochondrial GSR activity was measured following TCDD treatment (24h) as described in the Materials and Methods section (mean [n = 3] ± SD). (B) Levels of mitochondrial GSH (left) and GSSG (right) were measured following TCDD treatment (24h) as described in the Materials and Methods section (mean [n = 3] ± SD). (C) Levels of mitochondrial H₂O₂ were measured following TCDD treatment (24h) using CL as described in the Materials and Methods section (mean [n = 3] ± SD). *p < 0.05 indicates a significant difference between the control and treated samples.

FIG. 5.

TCDD decreases glycolysis. (A) The glycolytic pathway adapted from the KEGG pathway database. Gray boxes indicate decreased RNA expression by TCDD (24h) based on microarray analysis (Supplementary table S4); white boxes were not significant. Gray circles indicate the biochemical products that were decreased by TCDD, based on the metabolomic data presented in (C). White circles indicate the biochemical products that were not measured. (B) Validation of transcript levels for SLC2A1 and the glycolysis pathway. NHEKs were treated with or without TCDD for 24h in the presence or absence of the indicated AHR antagonists. Levels of RNA were measured by qRT-PCR. a indicates a significant difference between the control and TCDD-treated samples; b indicates a significant difference between TCDD alone- and the TCDD plus AHR antagonist–treated samples; c indicates a significant difference between control and AHR antagonist–treated samples, p < 0.05. Note that HK1, GPI, TPI1, GAPDH, and PGK1 were decreased significantly in response to TCDD as measured by qRT-PCR analysis, in comparison to the microarray analysis. (C) Quantitation by LC/GC-MS of the glycolytic pathway intermediates following treatment for the indicated times with control or TCDD. (D) Quantitation by LC/GC-MS of citrate, succinate, NADH, and NAD⁺, following treatment for the indicated times with control or TCDD. *p < 0.05 indicates a significant difference between the time-matched control and treated samples (n = 5). ND indicates that the compound was not detected in at least three of the five samples in a group.

FIG. 5.

TCDD decreases glycolysis. (A) The glycolytic pathway adapted from the KEGG pathway database. Gray boxes indicate decreased RNA expression by TCDD (24h) based on microarray analysis (Supplementary table S4); white boxes were not significant. Gray circles indicate the biochemical products that were decreased by TCDD, based on the metabolomic data presented in (C). White circles indicate the biochemical products that were not measured. (B) Validation of transcript levels for SLC2A1 and the glycolysis pathway. NHEKs were treated with or without TCDD for 24h in the presence or absence of the indicated AHR antagonists. Levels of RNA were measured by qRT-PCR. a indicates a significant difference between the control and TCDD-treated samples; b indicates a significant difference between TCDD alone- and the TCDD plus AHR antagonist–treated samples; c indicates a significant difference between control and AHR antagonist–treated samples, p < 0.05. Note that HK1, GPI, TPI1, GAPDH, and PGK1 were decreased significantly in response to TCDD as measured by qRT-PCR analysis, in comparison to the microarray analysis. (C) Quantitation by LC/GC-MS of the glycolytic pathway intermediates following treatment for the indicated times with control or TCDD. (D) Quantitation by LC/GC-MS of citrate, succinate, NADH, and NAD⁺, following treatment for the indicated times with control or TCDD. *p < 0.05 indicates a significant difference between the time-matched control and treated samples (n = 5). ND indicates that the compound was not detected in at least three of the five samples in a group.

FIG. 6.

Antioxidants block the effect of TCDD to increase keratinocyte differentiation. (A) CEs were isolated after treatment, control or TCDD for 72h in the presence or absence of one of the following antioxidants: DPPD (2.5µM), quercetin (10µM), or catalase (500U/ml). a indicates a significant difference between the control and TCDD-treated samples (mean [n = 4] ± SD); b indicates a significant difference between TCDD alone- and the TCDD plus antioxidant–treated samples, p < 0.05. (B) CEs were isolated after treatment, control, or TCDD for 72h in the presence or absence of one of the antioxidants, NAC (1.25mM), catalase (500U/ml), PEG-catalase (1000U/ml), or the AHR antagonist, CH223191 (1μM). a indicates a significant difference between the control and TCDD-treated samples (mean [n = 4] ± SD); b indicates a significant difference between TCDD alone- and the TCDD plus antioxidant- or antagonist-treated samples, p < 0.05.