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The Tryptophan-Derived Endogenous Aryl Hydrocarbon Receptor Ligand 6-Formylindolo[3,2-b]Carbazole Is a Nanomolar UVA Photosensitizer in Epidermal Keratinocytes.

Park SL, Justiniano R, Williams JD, Cabello CM, Qiao S, Wondrak GT - J. Invest. Dermatol. (2014)

Bottom Line: Array analysis revealed pronounced potentiation of cellular heat shock, endoplasmic reticulum stress, and oxidative stress response gene expression observed only upon FICZ/UVA cotreatment.FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment.Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, College of Pharmacy, Arizona Cancer Center, University of Arizona, Tucson, Arizona, USA.

ABSTRACT
Endogenous UVA chromophores may act as sensitizers of oxidative stress underlying cutaneous photoaging and photocarcinogenesis, but the molecular identity of non-DNA key chromophores displaying UVA-driven photodyamic activity in human skin remains largely undefined. Here we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct and endogenous high-affinity aryl hydrocarbon receptor (AhR) agonist, acts as a nanomolar photosensitizer potentiating UVA-induced oxidative stress irrespective of AhR ligand activity. In human HaCaT and primary epidermal keratinocytes, photodynamic induction of apoptosis was elicited by the combined action of solar-simulated UVA and FICZ, whereas exposure to the isolated action of UVA or FICZ did not impair viability. In a human epidermal tissue reconstruct, FICZ/UVA cotreatment caused pronounced phototoxicity inducing keratinocyte cell death, and FICZ photodynamic activity was also substantiated in a murine skin exposure model. Array analysis revealed pronounced potentiation of cellular heat shock, endoplasmic reticulum stress, and oxidative stress response gene expression observed only upon FICZ/UVA cotreatment. FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment. Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin.

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UVA-induced photodynamic activity of FICZ is observable in primary human epidermal keratinocytes, reconstructed epidermis, and mouse skin(a)Human dermal Hs27 fibroblasts were exposed to the isolated or combined action of FICZ (100 nM) and UVA (6.6 J/cm2) or remained untreated (control). After 24 h, viability was assessed as in Figure 3b; bar graph: FICZ dose response relationship of UVA-induced phototoxicity (UVA 6.6 J/cm2). (b) Gene expression changes at the mRNA level after exposure to the isolated or combined action of FICZ (100 nM) and UVA [6.6 J/cm2; 6 h after treatment; n=3, mean ± SD; (p<0.05)]. (c) Primary human epidermal keratinocytes (HEKs) exposed and analyzed as described in Figure 4a. (d) Gene expression changes in HEKs treated and analyzed as in Figure 4b. (e) Epidermal reconstruct (Epiderm™) specimens were cultured in growth medium supplemented with or without FICZ (100 nM; 6 h). After culture, UVA (6.6 J/cm2) or mock-irradiation occurred. After 24 h, reconstructs were processed for IHC; arrowheads: localization of apoptotic basal keratinocytes (H&E: pycnotic/eosinophilic features, IHC: cleaved procaspase 3). (f) SKH-1 hairless mice (n=3 per treatment group) were exposed to the isolated or combined action of topical FICZ (1 mM) and UVA (6.6 J/cm2) radiation. 48 h after treatment, H&E and IHC (Hsp70) analysis revealed photodynamic effects including epidermal necrosis (‘n’) and re-epithelialization (‘re’). Per treatment group, representative images taken from three repeat samples are displayed (scale bars = 25 μm; right insert, panel F: 50 μm).
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Figure 4: UVA-induced photodynamic activity of FICZ is observable in primary human epidermal keratinocytes, reconstructed epidermis, and mouse skin(a)Human dermal Hs27 fibroblasts were exposed to the isolated or combined action of FICZ (100 nM) and UVA (6.6 J/cm2) or remained untreated (control). After 24 h, viability was assessed as in Figure 3b; bar graph: FICZ dose response relationship of UVA-induced phototoxicity (UVA 6.6 J/cm2). (b) Gene expression changes at the mRNA level after exposure to the isolated or combined action of FICZ (100 nM) and UVA [6.6 J/cm2; 6 h after treatment; n=3, mean ± SD; (p<0.05)]. (c) Primary human epidermal keratinocytes (HEKs) exposed and analyzed as described in Figure 4a. (d) Gene expression changes in HEKs treated and analyzed as in Figure 4b. (e) Epidermal reconstruct (Epiderm™) specimens were cultured in growth medium supplemented with or without FICZ (100 nM; 6 h). After culture, UVA (6.6 J/cm2) or mock-irradiation occurred. After 24 h, reconstructs were processed for IHC; arrowheads: localization of apoptotic basal keratinocytes (H&E: pycnotic/eosinophilic features, IHC: cleaved procaspase 3). (f) SKH-1 hairless mice (n=3 per treatment group) were exposed to the isolated or combined action of topical FICZ (1 mM) and UVA (6.6 J/cm2) radiation. 48 h after treatment, H&E and IHC (Hsp70) analysis revealed photodynamic effects including epidermal necrosis (‘n’) and re-epithelialization (‘re’). Per treatment group, representative images taken from three repeat samples are displayed (scale bars = 25 μm; right insert, panel F: 50 μm).

Mentions: UVA-induced photodynamic activity of low nanomolar concentrations of FICZ was also observed when human dermal Hs27 fibroblasts were exposed to the combined action of FICZ and UVA (6.6 J/cm2) (Figure 4a), accompanied by changes at the mRNA level indicative of stress response gene expression (HMOX1, DDIT3, HSPA6; Figure 4b). In accordance with earlier reports that document constitutive attenuation of AhR signaling in dermal fibroblasts, we observed that FICZ exposure (FICZ only or FICZ/UVA combination) failed to upregulate CYP1A1 mRNA levels indicating again that FICZ photodynamic effects occur in the absence of AhR-mediated signaling.


The Tryptophan-Derived Endogenous Aryl Hydrocarbon Receptor Ligand 6-Formylindolo[3,2-b]Carbazole Is a Nanomolar UVA Photosensitizer in Epidermal Keratinocytes.

Park SL, Justiniano R, Williams JD, Cabello CM, Qiao S, Wondrak GT - J. Invest. Dermatol. (2014)

UVA-induced photodynamic activity of FICZ is observable in primary human epidermal keratinocytes, reconstructed epidermis, and mouse skin(a)Human dermal Hs27 fibroblasts were exposed to the isolated or combined action of FICZ (100 nM) and UVA (6.6 J/cm2) or remained untreated (control). After 24 h, viability was assessed as in Figure 3b; bar graph: FICZ dose response relationship of UVA-induced phototoxicity (UVA 6.6 J/cm2). (b) Gene expression changes at the mRNA level after exposure to the isolated or combined action of FICZ (100 nM) and UVA [6.6 J/cm2; 6 h after treatment; n=3, mean ± SD; (p<0.05)]. (c) Primary human epidermal keratinocytes (HEKs) exposed and analyzed as described in Figure 4a. (d) Gene expression changes in HEKs treated and analyzed as in Figure 4b. (e) Epidermal reconstruct (Epiderm™) specimens were cultured in growth medium supplemented with or without FICZ (100 nM; 6 h). After culture, UVA (6.6 J/cm2) or mock-irradiation occurred. After 24 h, reconstructs were processed for IHC; arrowheads: localization of apoptotic basal keratinocytes (H&E: pycnotic/eosinophilic features, IHC: cleaved procaspase 3). (f) SKH-1 hairless mice (n=3 per treatment group) were exposed to the isolated or combined action of topical FICZ (1 mM) and UVA (6.6 J/cm2) radiation. 48 h after treatment, H&E and IHC (Hsp70) analysis revealed photodynamic effects including epidermal necrosis (‘n’) and re-epithelialization (‘re’). Per treatment group, representative images taken from three repeat samples are displayed (scale bars = 25 μm; right insert, panel F: 50 μm).
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Related In: Results  -  Collection

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Figure 4: UVA-induced photodynamic activity of FICZ is observable in primary human epidermal keratinocytes, reconstructed epidermis, and mouse skin(a)Human dermal Hs27 fibroblasts were exposed to the isolated or combined action of FICZ (100 nM) and UVA (6.6 J/cm2) or remained untreated (control). After 24 h, viability was assessed as in Figure 3b; bar graph: FICZ dose response relationship of UVA-induced phototoxicity (UVA 6.6 J/cm2). (b) Gene expression changes at the mRNA level after exposure to the isolated or combined action of FICZ (100 nM) and UVA [6.6 J/cm2; 6 h after treatment; n=3, mean ± SD; (p<0.05)]. (c) Primary human epidermal keratinocytes (HEKs) exposed and analyzed as described in Figure 4a. (d) Gene expression changes in HEKs treated and analyzed as in Figure 4b. (e) Epidermal reconstruct (Epiderm™) specimens were cultured in growth medium supplemented with or without FICZ (100 nM; 6 h). After culture, UVA (6.6 J/cm2) or mock-irradiation occurred. After 24 h, reconstructs were processed for IHC; arrowheads: localization of apoptotic basal keratinocytes (H&E: pycnotic/eosinophilic features, IHC: cleaved procaspase 3). (f) SKH-1 hairless mice (n=3 per treatment group) were exposed to the isolated or combined action of topical FICZ (1 mM) and UVA (6.6 J/cm2) radiation. 48 h after treatment, H&E and IHC (Hsp70) analysis revealed photodynamic effects including epidermal necrosis (‘n’) and re-epithelialization (‘re’). Per treatment group, representative images taken from three repeat samples are displayed (scale bars = 25 μm; right insert, panel F: 50 μm).
Mentions: UVA-induced photodynamic activity of low nanomolar concentrations of FICZ was also observed when human dermal Hs27 fibroblasts were exposed to the combined action of FICZ and UVA (6.6 J/cm2) (Figure 4a), accompanied by changes at the mRNA level indicative of stress response gene expression (HMOX1, DDIT3, HSPA6; Figure 4b). In accordance with earlier reports that document constitutive attenuation of AhR signaling in dermal fibroblasts, we observed that FICZ exposure (FICZ only or FICZ/UVA combination) failed to upregulate CYP1A1 mRNA levels indicating again that FICZ photodynamic effects occur in the absence of AhR-mediated signaling.

Bottom Line: Array analysis revealed pronounced potentiation of cellular heat shock, endoplasmic reticulum stress, and oxidative stress response gene expression observed only upon FICZ/UVA cotreatment.FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment.Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, College of Pharmacy, Arizona Cancer Center, University of Arizona, Tucson, Arizona, USA.

ABSTRACT
Endogenous UVA chromophores may act as sensitizers of oxidative stress underlying cutaneous photoaging and photocarcinogenesis, but the molecular identity of non-DNA key chromophores displaying UVA-driven photodyamic activity in human skin remains largely undefined. Here we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct and endogenous high-affinity aryl hydrocarbon receptor (AhR) agonist, acts as a nanomolar photosensitizer potentiating UVA-induced oxidative stress irrespective of AhR ligand activity. In human HaCaT and primary epidermal keratinocytes, photodynamic induction of apoptosis was elicited by the combined action of solar-simulated UVA and FICZ, whereas exposure to the isolated action of UVA or FICZ did not impair viability. In a human epidermal tissue reconstruct, FICZ/UVA cotreatment caused pronounced phototoxicity inducing keratinocyte cell death, and FICZ photodynamic activity was also substantiated in a murine skin exposure model. Array analysis revealed pronounced potentiation of cellular heat shock, endoplasmic reticulum stress, and oxidative stress response gene expression observed only upon FICZ/UVA cotreatment. FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment. Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin.

Show MeSH
Related in: MedlinePlus