Uncertainties related to the assignment of a toxic equivalency factor for 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin

The Toxic Equivalency Factor (TEF) approach is a methodology that assigns relative toxicity values to structurally related chemicals in comparison to a reference chemical. For the polychlorinated dibenzo-p-dioxins (PCDDs), the reference is the most potent congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here, we critically review the literature on the effects of a weak PCDD, 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin (OCDD), and describe the uncertainties of assigning its TEF. PCDDs, including OCDD, are less potent in human cell models compared to the rat models from which the TEF are estimated. This lack of concordance is even more pronounced with the weaker congeners such as OCDD. Furthermore, OCDD is also likely to compete with TCDD for binding to cytochrome P4501A2 (CYP1A2), effectively decreasing the hepatic tissue/fat ratio of TCDD. Overall, the predictive value of TEFs would be improved by incorporating into this number the relative sensitivity of human cell responses compared to rodent responses, by determining the toxicological effects of altering the tissue distribution of dioxin-like compounds through competition for CYP-binding sites, and by understanding the mechanism of cancer causation of any dioxin and whether this mechanism is conserved in humans and at equivalent doses. © 2005 Elsevier Inc. All rights reserved

Combined treatment with sodium butyrate and PD153035 enhances keratinocyte differentiation

Epidermal growth factor (EGF) receptor (EGFR) signalling is a critical determinant of keratinocyte proliferation and differentiation in both normal and diseased skin. Here we explore the effects of combined treatment with the differentiation-promoting agent sodium butyrate (SB) and the EGFR inhibitor (EGFRI) PD153035 on terminal differentiation of normal human epidermal keratinocytes (NHEKs). Cells treated with SB showed increased expression of the levels of mRNA and protein of the differentiation markers filaggrin and transglutaminase 1. Cotreatment with EGF significantly blunted these effects of SB. Combined treatment with SB and PD153035 alleviated these inhibitory actions of EGF, resulting in improved effects of decreased cell growth and increased terminal differentiation, relative to the individual treatments. These results indicate that the combined use of a differentiation-promoting agent and an EGFR inhibitor may offer an additional approach to the management of hyperproliferative skin diseases. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Contributions of nitric oxide to AHR-ligand-mediated keratinocyte differentiation

Activation of the aryl hydrocarbon receptor (AHR) in normal human epidermal keratinocytes (NHEKs) accelerates keratinocyte terminal differentiation through metabolic reprogramming and reactive oxygen species (ROS) production. Of the three NOS isoforms, NOS3 is significantly increased at both the RNA and protein levels by exposure to the very potent and selective ligand of the AHR, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Inhibition of NOS with the chemical N-nitro-l-arginine methyl ester (l-NAME) reversed TCDD-induced cornified envelope formation, an endpoint of terminal differentiation, as well as the expression of filaggrin (FLG), a marker of differentiation. Conversely, exposure to the NO-donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), increased the number of cornified envelopes above control levels and augmented the levels of cornified envelopes formed in response to TCDD treatment and increased the expression of FLG. This indicates that nitric oxide signaling can increase keratinocyte differentiation and that it is involved in the AHR-mediated acceleration of differentiation. As the nitrosylation of cysteines is a mechanism by which NO affects the structure and functions of proteins, the S-nitrosylation biotin switch technique was used to measure protein S-nitrosylation. Activation of the AHR increased the S-nitrosylation of two detected proteins of about 72 and 20 kD in size. These results provide new insights into the role of NO and protein nitrosylation in the process of epithelial cell differentiation, suggesting a role of NOS in metabolic reprogramming and the regulation of epithelial cell fate

Regioselective 2-hydroxylation of 17β-estradiol by rat cytochrome P4501B1

Previous work demonstrated that human cytochrome P4501B1 (CYP1B1) forms predominantly 4-hydroxyestradiol (4-OHE2), a metabolite which is carcinogenic in animal models. Here, we present results from kinetic studies characterizing the formation of 4-OHE2 and 2-hydroxyestradiol (2-OHE2) by rat CYP1B1 using 17β-estradiol (E2) as a substrate. Km and Kcat values were estimated using the Michaelis-Menten equation. For rat CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 0.61 ± 0.23 and 1.84 ± 0.73 μM; the turnover numbers (Kcat) were 0.23 ± 0.02 and 0.46 ± 0.05 pmol/min/pmol P450; and the catalytic efficiencies (Kcat/Km) were 0.37 and 0.25, respectively. For human CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 1.22 ± 0.25 and 1.10 ± 0.26; the turnover numbers were 1.23 ± 0.06 and 0.33 ± 0.02; and the catalytic efficiencies were 1.0 and 0.30, respectively. The turnover number ratio of 4- to 2-hydroxylation was 3.7 for human CYP1B1 and 0.5 for rat CYP1B1. These results indicate that, although rat CYP1B1 is a low Km E2 hydroxylase, its product ratio, unlike the human enzyme, favors 2-hydroxylation. The Ki values of the inhibitor 2,4,3′,5′-tetramethoxystilbene (TMS) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.69 and 0.78 μM, respectively. The Ki values of 7,8-benzoflavone (α-NF) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.01 and 0.02 μM, respectively. The knowledge gained from this study will support the rational design of CYP1B1 inhibitors and clarify results of CYP1B1 related carcinogenesis studies performed in rats. © 2006 Elsevier Inc. All rights reserved

Hypoxia-inducible factor 1α protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses to reduced O(2) availability in mammals, including angiogenesis, erythropoiesis, and glycolysis. HIF-1 activity is controlled by the O(2)-regulated expression of the HIF-1α subunit. Under nonhypoxic conditions, HIF-1α protein is subject to ubiquitination and proteasomal degradation. Here we report that missense mutations and/or deletions involving several different regions of HIF-1α result in constitutive expression and transcriptional activity in nonhypoxic cells. We demonstrate that hypoxia results in decreased ubiquitination of HIF-1α and that missense mutations increase HIF-1α expression under nonhypoxic conditions by blocking ubiquitination

2,3,7,8-tetrachlorodibenzo-p-dioxin increases the expression of genes in the human epidermal differentiation complex and accelerates epidermal barrier formation

Chloracne is commonly observed in people exposed to dioxins, yet the mechanism of toxicity is not well understood. The pathology of chloracne is characterized by hyperkeratinization of the interfollicular squamous epithelium, hyperproliferation and hyperkeratinization of hair follicle cells as well as a metaplastic response of the ductular sebum secreting sebaceous glands. In vitro studies using normal human epidermal keratinocytes to model interfollicular human epidermis demonstrate a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated acceleration of differentiation and increase in gene expression of several prodifferentiation genes, including filaggrin (FLG). Here, we demonstrated that the TCDD-activated aryl hydrocarbon receptor (AHR) bound a small fragment of DNA upstream of the transcriptional start sites of the FLG gene, containing one of two candidate xenobiotic response elements (XREs). Reporter assays using the promoter region of FLG containing the two putative XREs indicated that the increase in this messenger RNA (mRNA) was due to TCDD-mediated enhanced transcription, which was lost when both XREs were mutated. As FLG is part of the human epidermal differentiation complex (EDC) found on chromosome 1, we measured mRNAs from an additional 18 EDC genes for their regulation by TCDD. Of these genes, 14 were increased by TCDD. Immunoblot assays demonstrated that the proteins of FLG as well as that of another prodifferentiation gene, small proline rich protein 2, were increased by TCDD. In utero exposure to TCDD accelerated the formation of the epidermal barrier in the developing mouse fetus by approximately 1 day. These results indicate that the epidermal permeability barrier is a functional target of the TCDD-activated AHR. © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved

Stability of RNA structural motifs and its influence on editing efficiency by adenosine deaminases

We propose a novel method to estimate editing efficiency by adenosine deaminases that act on RNA (ADARs). The method employs the notion of stability of secondary structure in the vicinity of edited sites during transcription. Such an analysis of \u27dynamic\u27 structural motifs of RNA is important because as a pre-spliced RNA is being transcribed and elongated, its entire structure, and thus its local structures, may change drastically. Our simulation showed that the stability of structures in the vicinity of edited sites correlates moderately highly with editing efficiency of edited sites recently established in laboratory experiments. Copyright © 2010 Inderscience Enterprises Ltd

EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes

Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear trans- locator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell differentiation, as measured by the formation of cornified envelopes. We show that this acceleration is mediated by the AHR; also, that dioxin increases the expression of several genes known to be regulated by ARNT, which have critical roles in the cornification and epidermal barrier function of the skin. Importantly, we demonstrate that all of these responses are opposed by ligand-activation of the EGF receptor (R), an important regulator of keratinocyte cell fate. In the CYP1A1 enhancer, EGFR activation prevents recruitment of the p300 coac- tivator, although not affecting the binding of the AHR or ARNT. The total cellular level of p300 protein does not decrease, and overex- pression of p300 relieves EGFR-mediated repression of transcription, indicating that p300 is a critical target for the repression of the AHR complex by EGFR signaling. These results provide a mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin is able to disrupt epidermal homeostasis and identify EGFR signaling as a regulator of the AHR. This signaling may modulate the incidence and severity of chloracne and be of therapeutic relevance to human poisonings by dioxin