UV-induced CYP1A1 gene expression in human cells is mediated by tryptophan
Introduction
Many effects of UV radiation in human skin have been characterized, including the production of reactive oxygen species, the alterations of cytoskeleton and DNA damage which eventually leads to skin cancer. In experimental studies exposure to UV radiation results in enhanced transcription of a wide variety of genes [1], [2], [3], [4], [5], [6], some of which are early cellular responses and whose role is to protect cells against DNA damage [6]. Some other recently discovered genes are involved in the regulation of biological rhythms, as has been shown in Neurospora, Drosophila and in mice exposed to fluorescent light [7], [8]. Although a number of UV-inducible genes have been intensively studied, very few studies have been conducted to assess the effects of UV radiation on genes coding for drug and carcinogen metabolizing enzymes.
Cytochrome P4501A1 (CYP1A1) is a heme-containing monooxygenase which metabolizes a variety of xenobiotics, drugs as well as some dietary compounds. CYP1A1 is highly inducible by a number of toxic environmental compounds, such as dioxins and polycyclic aromatic hydrocarbon (PAH) [9], [10]. The CYP1A1 induction is in general regulated by a cytosolic receptor, the so called Ah receptor. Ligands binding to the Ah receptor causes conformational changes and the release of the heat shock 90 protein. The liganded receptor is transferred to the nucleus and interacts with its partner, the Ah receptor nuclear translocator (Arnt) protein. This heterodimer binds to dioxin responsive element (DRE) sequences located in the 5′-flanking region of the Ah receptor responsive genes and activates gene transcription [11]. Most studies on the toxicological consequences of CYP induction have been focused on the effects of the dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and on the pathway by which substrate molecules are bioactivated to reactive and genotoxic compounds. The physiological role of the Ah receptor system is however not known.
In this respect it is of interest that in vivo studies have shown that UV light induces CYP1A1 enzyme activity in the skin of neonatal rats [12] and in the liver of mice [13]. The latter result was recently confirmed by the same research group, indicating that rat hepatic 7-ethoxyresorufin O-deethylase activity (EROD) was induced by UVB irradiation of the skin [14]. A seasonal variation in CYP1A1 activity in human lymphocytes has been reported by Paigen et al. [15]. They showed that maximal enzyme activity occured during late summer and early fall. High winter values from a few subjects were explicable by vacations or stays in more light intensive areas. These results indicate that exposure of human skin to UV radiation, primarily as a result of sunlight, may cause alterations in the activity of cutaneous and extracutaneous P4501A1. The mechanism of CYP1A1 induction by UV is not fully understood and needs to be elucidated. It is not possible that the enzyme induction in the animal liver is due to a direct effect of the radiation. More likely, the UV-radiation gives rise to stable substance(s) in the skin or in the cellular components of the peripheral blood which are circulated and responsible for the CYP1A1 induction in the animal liver via binding to the Ah receptor.
Tryptophan is the most strongly near-UV absorbing amino acid and acts as an endogenous photosensitizer to enhance UV-induced biological effects. We have earlier found that UV-oxidation of tryptophan gives rise to substances with extremely high Ah receptor binding affinity, and these compounds were suggested to be the endogenous ligands for the Ah receptor [16]. The most active Ah receptor binding compound derived from photooxidizied tryptophan was recently identified as 6-formylindolo[3,2-b]carbazole (FICZ) [17] (Fig. 1). FICZ was also shown to induce aryl hydrocarbon hydroxylase (AHH) activity in rat hepatoma H4IIE cells [18]. Therefore, we hypothesized that tryptophan photoproducts formed in the skin or other tissues following UV-irradiation might explain the CYP1A1 induction by UV light.
Induction of CYP1A1 gene expression by the tryptophan photoproduct FICZ in a human keratinocyte cell line (HaCaT) and primary human blood mononuclear cells were shown in an earlier study [19]. In the present study, the induction of CYP1A1 gene expression by UV was investigated in the same human cell types in the presence and absence of tryptophan during UV irradiation and in tryptophan pretreated cells. In addition, a mutant mouse Hepa-1 cell line with much reduced level of Ah receptor was applied in this study to see whether UV and FICZ cause CYP1A1 gene expression via the Ah receptor signaling pathway.
Section snippets
Materials
l-tryptophan was purchased from Aldrich Chemical, Germany and purified with recrystallisation from ethanol. The tryptophan photoproduct FICZ was produced by UV-irradiation of aqueous solutions of tryptophan and purified as described earlier [16]. β-Naphthoflavone (BNF) was purchased from Aldrich Chemical, USA. Dimethylsulfoxide (DMSO) was from Merck, Germany. RNeasy Total RNA Kit and QIAshredder were from QIAGEN. RNase-free DNaseI was purchased from Boehringer Mannheim. SuperScript™ II RNase H−
UV exposure and cytotoxicity
The UV spectrum delivered by the Philips TL20W/12RS lamps was characterized at nine different measuring points. The spectral distribution was 45.5% UVA, 54.2% UVB and 0.3% UVC and the mean flux density of the UVB at a distance of 20 cm was 2.2 mW/cm2 and the UVA mean flux density was 0.5 mW/cm2. In order to expose cells to 10 mJ/cm2 UV the exposure time was set at 4.5 s.
The UV doses for the irradiation of HaCaT cells and lymphocytes were chosen according to literature data [22], [23] as well as
Discussion
Induction of CYP1A1 activity by UV light had been observed earlier in animal studies [12], [13]. In the present study, we characterized the effect of UV light on CYP1A1 gene expression in human and mouse cells and the relationship between UV and FICZ, a photo-oxidation product of tryptophan. The CYP1A1 mRNA levels were enhanced by UV irradiation in the presence of tryptophan (Fig. 2, Fig. 3), indicating that tryptophan mediates the UV-induced expression and that an inducer may be formed from
Acknowledgements
We thank Dr Oliver Hankinson for kindly providing mouse Hepa-1 cell lines and Dr Norbert Fusenig for human keratinocyte HaCaT cell line. We deeply appreciate the expert UV flux density measurements performed by Dr Torkel Fisher and Dr Jouni Surakka. This work was supported by the Swedish Foundation for Strategic Environmental Research, the Swedish Council for Work Life Research, the Swedish National Environmental Protection Agency and Swedish Match.
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