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Mutagenesis Advance Access originally published online on June 14, 2005
Mutagenesis 2005 20(4):305-310; doi:10.1093/mutage/gei042
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© The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oupjournals.org

Wavelength dependent responses of primary human keratinocytes to combined treatment with benzo[a]pyrene and UV light

Rebecca A. Crallan, Eileen Ingham1 and Michael N. Routledge*

Molecular Epidemiology Unit, Centre for Epidemiology and Biostatistics, Leeds Institute for Genetics, Health and Therapeutics and 1The School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The major risk factor for skin cancer is exposure to UV radiation from sunlight, but other environmental exposures may also play a role in combination with UV. We have studied the effects of combined exposure of primary human skin cells in vitro to UVA, UVB or UVC with benzo[a] pyrene (BaP), an environmental carcinogen. Normal human keratinocytes were exposed to 5 µM BaP for 24 h followed by either 1 kJ/m2 UVA, 100 J/m2 UVB or 10 J/m2 UVC. Only BaP + UVA caused increased cell death. BaP or UVA alone did not induce significant DNA damage as measured by comet assay but combined exposure induced 35.1 ± 6.0% tail DNA, compared with 9.7 ± 1.3% tail DNA in control cells. After including the Fapy-DNA glycosylase enzyme incubation step to detect oxidized purines, % tail DNA increased another 11.2 ± 2.9%. Combined exposure of BaP and UVB did not increase damage in the comet assay without Fapy-DNA glycosylase, but in the presence of this enzyme % tail DNA increased by 9.3 ± 2.2%. BaP + UVB also abrogated the UVB-induced cell cycle G2 arrest. BaP + UVC had no effect on the keratinocytes compared with each treatment alone. These results show a wavelength-dependent difference in the effects of combined exposure on normal human keratinocytes. Both UVA and UVB damage can be enhanced by BaP pre-exposure, although the effects seen with UVA were greater. These findings are important to understanding the role of UVA and UVB in skin carcinogenesis and may have implications for recommended sun exposure limits, especially in polluted areas.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Skin cancer is the most common form of cancer in the UK. The vast majority (>90%) are non-melanoma skin cancers (NMSCs), either basal or squamous cell carcinomas that arise from keratinocytes in the epidermis of the skin. Over 60 000 new cases are diagnosed each year, an incidence that is increasing (1Go,2Go) (http://www.cancerresearchuk.org/aboutcancer/reducingyourrisk/sun_uv). Cumulative sunlight exposure has been shown by numerous epidemiological and animal studies to be the most important risk factor (3GoGo–5Go), but exposure to pollution, cigarette smoke and occupational chemicals also plays a significant role in the development of NMSCs (6GoGoGo–9Go).

UV radiation is the carcinogenic component of sunlight. The sun emits UVA (320–400 nm), UVB (280–320 nm) and UVC (<290 nm), as well as visible light. UVC is absorbed by molecular oxygen (O2) in the atmosphere and most of the UVB is absorbed by the ozone layer (O3); consequently, 95% of UV reaching the earth's surface is UVA. All wavelengths of UV can induce DNA damage, although different spectra of lesions are associated with different wavelengths. UVC and UVB are very efficient at producing cyclobutane pyrimidine dimers (CPDs) and (6-4)photoproducts (10Go). UVA is poorly absorbed by DNA and induces much lower levels of CPDs, but generates reactive oxygen species (ROS), resulting in oxidative DNA damage (11Go–12Go). Despite these differences, the relative contribution of UVA versus UVB to NMSC development is still not clear; although the majority of CPDs induced by sunlight and sunlamps in cultured cells are caused by UVB radiation (13Go) and GC->AT transitions are the most common mutations found in NMSCs (14Go), UVA exposure alone can cause skin cancer (15Go). Higher levels of UVA than UVB penetrate to the basal layer of the epidermis, and mutations from oxidative damage may significantly contribute to photocarcinogenesis (16Go,17Go). UV radiation also contributes to carcinogenesis through interactions with other cellular components, such as cell signalling molecules (18Go) or immunoregulatory proteins, by causing cell injury and inflammation (19GoGo–21Go).

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon and an environmental pollutant found in cigarette smoke, burnt food and smoke from the burning of fossil fuels. BaP can be absorbed by human skin (22Go) and metabolized, producing, amongst other molecules, the ultimate carcinogen BaP diol epoxide (BPDE) (23Go,24Go). BPDE binds to DNA, most commonly to guanines (25Go), forming bulky adducts that induce GC->TA transversions (26Go). BaP has been implicated in the development of skin cancer when combined with UV exposure. Combined tobacco smoke and sunlight exposure is associated with squamous cell carcinomas (27Go), and psoriasis patients given coal tar therapy (which contains BaP) followed by UVB radiation have an increased risk of skin cancer (28Go).

These observations suggest that combined exposure of human cells to BaP and UV radiation may be much more carcinogenic than would be expected from the effects of each agent alone, and this hypothesis is supported by recent investigations. We have previously shown that binary exposure of plasmid DNA to BPDE followed by UV radiation caused a synergistic increase in single-strand breaks and mutation frequency compared with the equivalent individual treatments (29Go,30Go); and exposure of lung epithelial cells to BaP followed by UV caused a more-than-additive increase in DNA damage compared with each agent alone (31Go). However, the effects of UV in combination with BaP have yet to be investigated in the relevant cell type for skin carcinogenesis. Therefore, the aim of this study was to elucidate the effects of UVA, UVB and UVC radiation, alone and in combination with BaP exposure, on normal human keratinocytes (NHKs) in vitro, by measuring cell proliferation, DNA damage, and cell cycle perturbations.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
NHKs were isolated from paediatric foreskin samples, obtained with informed parental consent and Local Ethics Committee approval, using a protocol adapted from Walters et al. (32Go). Foreskins were collected in Hank's Balanced Salt Solution (HBSS) containing 1% (v/v) penicillin/streptomycin (Invitrogen, Paisley, UK) and stored at 4°C. Samples were washed three times in HBSS, dissected into small pieces and incubated with 2 mg/ml dispase (Invitrogen) for 1 h at 37°C. The epidermis was peeled from the dermis, finely minced in 1x trypsin–EDTA (0.05% trypsin, 0.53 M EDTA, Invitrogen) and incubated for 15 min at 37°C. Trypsin was deactivated with 10 mg/ml trypsin inhibitor (TI, Sigma, Poole, UK) and cells harvested by centrifugation at 100 g for 5 min. The isolated NHKs were resuspended in 5 ml defined keratinocyte serum-free medium (KSFM, Invitrogen), counted using a haemocytometer and seeded onto 25 cm2 tissue culture flasks (Nunc, Fisher Scientific, Leicester) coated with 0.67 µg/cm2 collagen (type IV, Sigma) at a density of at least 1 x 106 cells/flask. NHKs isolated in a similar manner and pooled from at least three donors were also purchased from Invitrogen. NHKs were cultured in KSFM at 37°C and 5% CO2 in air and the medium was changed every 2–3 days. Sub-confluent cultures were passaged using 1 x trypsin–EDTA, collected into KSFM containing 2.5 mg/ml TI, harvested by centrifugation and re-seeded at a 1:3 ratio in KSFM.

Cell based assays were repeated on at least three NHK cultures established from independent donors.

Cell treatments
Sub-confluent NHK cultures were exposed to 1–50 µM BaP (Sigma), dissolved in dimethyl sulfoxide (DMSO) and diluted in KSFM (final concentration of DMSO <0.1%), for 24 h under normal culture conditions. For treatment of cells with UV, culture medium was replaced with phosphate-buffered saline (PBS) and culture dishes placed on an ice block. UVA exposure (365 nm) was carried out with a UVL-26 lamp (UVP, Cambridge, UK) at a dose rate of 2 mW/cm2 for 50 s to 25 min; UVB (312 nm) and UVC (254 nm) irradiations were carried out with a LF206MS UV lamp (UVP) at dose rates of 0.2 mW/cm2 for 5–50 s and 0.1 mW/cm2 for 2–10 s, respectively. For combined exposures, cells were exposed to 5 µM BaP for 24 h, washed twice with PBS and then exposed to 1 kJ/m2 UVA, 100 J/m2 UVB or 10 J/m2 UVC in PBS.

MTT cell viability assay
A quantitative determination of cell viability was based on the ability of metabolically active cells to convert yellow formazan MTT salt to insoluble blue crystals. NHK cells were seeded into collagen-coated 96-well plates at 6 x 103 cells/cm2, which had been previously determined to allow NHK cells to reach confluency after 7–10 days. Cell viability was assessed before treatment (T0) and at various time-points after treatment, up to 7 days, by adding 0.5 mg/ml MTT (Sigma) to each well and incubating at 37°C for 4 h. The formazan crystals were dissolved in DMSO and the absorbance at 570 nm was measured on a Multiskan Spectrum plate reader (Thermo Labsystems).

Cell cycle analysis by flow cytometry
The cell cycle profile of NHK cells was analysed by measuring the DNA content of nuclei stained with propidium iodide (33Go), using DNA Staining Reagent kit (Sigma). Actinomycin D (5 nM) was added to the cells as a positive control for G1 arrest. Attached cells were lifted and combined with floating cells 16 h after treatment and stained following the manufacturer's protocol. Briefly, 1 x 105 cells were washed twice with citrate buffer, digested with trypsin and RNAse then stained with propidium iodide for 10 min at RT. The DNA content of 10 000 cells/sample was analysed using a FACSCaliber flow cytometer and CELLQuest software (Becton Dickinson, Oxford, UK).

Comet assay
For comet analysis of DNA damage, 1 x 106 cells/ml KSFM were collected immediately after treatment and viability of the collected cells was assessed by trypan blue exclusion, to ensure that viability amongst the assayed cells was sufficiently high (at least 70%) for analysis in the comet assay. Cell suspension (60 µl) was added to 300 µl 1% (w/v) low-melting-point agarose (Sigma) in PBS kept at 37°C. Gel solution (162 µl) was pipetted onto duplicate microscope slides coated with 1% (w/v) normal-melting point agarose, flattened with a coverslip and placed on ice for 30 s. Once the agarose had set, the coverslips were removed and the slides were immersed in lysis solution [2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 1% (w/v) N-lauryl sarcosine, 1% (v/v) Triton and 10% (v/v) DMSO] at 4°C for at least 1 h. Slides were transferred to running buffer (0.3 M NaOH, 1 mM EDTA, pH 13) at 4°C for 40 min to allow DNA to unwind, subjected to electrophoresis at 1 V/cm for 20 min, neutralized in 0.4 M Tris, pH 7.5, for 5 min, air-dried and stained with 25 µl of 20 mg/ml ethidium bromide (Sigma). Slides were stored on damp tissue paper at 4°C until scoring. Comet formation was detected by fluorescent microscopy and quantified by computer assisted image analysis (Komet analysis software version 4, Kinetic Imaging, Nottingham, UK). Fifty nuclei/slide were randomly selected and percentage tail DNA measured. To detect oxidized purines, nuclei were incubated with Fpg enzyme (a kind gift from Andrew Collins) after lysis: slides were washed 3 x 5 min in enzyme buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA), whereafter 50 µl Fpg (1:3000; final concentration 0.16 µg/ml) or 50 µl enzyme buffer was added to each slide with a coverslip and incubated at 37°C for 30 min prior to unwinding. The assay was completed and analysed as described above.

32P-postlabelling of DNA adducts
Genomic DNA was extracted from NHK cells after 24 h incubation with 5 µM BaP (Qiagen blood and cell culture DNA mini kit, Qiagen, Crawley, UK). DNA (5 µg) was analysed by 32P-postlabelling, as previously described (29Go). Radioactivity was visualized using a FLA-5000 phosphorimager (Fujifilm).

Statistical analysis
Results are expressed as means of three replicates ±95% confidence intervals (CIs), unless stated otherwise. Percentage data were first transformed (arcsin transformation) before analysis by ANOVA using Instat (version 3.0, Graphpad, San Diego).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Cell viability
Cell survival was measured for 7 days after exposure to BaP and UV, alone and in combination, using the MTT viability assay (Figure 1). Exposure to 5 µM BaP for 24 h caused a decrease in cell viability for the duration of the exposure, followed by recovery to control levels. UV exposure alone slightly reduced the growth rate of NHKs over the 7 day period, with cultures reaching 75% of the control population viability. After combined exposure to 5 µM BaP and 100 J/m2 UVB or 10 J/m2 UVC, no further effect on viability was observed. However, combined exposure of NHKs to 5 µM BaP followed by 1 kJ/m2 UVA caused a dramatic reduction in viability to 60% of the initial cell density and <10% of the final control cell viability, with no recovery within 7 days. These cells retained normal keratinocyte morphology but were resistant to trypsin.



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  		Fig. 1.. Cell viability over 7 days measured by MTT assay. Cells were seeded at low density and viability measured before treatment (0) and on days 1, 2, 4 and 7 after addition of BaP. BaP was added to cells in six replicate wells for 24 h. After removal, cells were washed twice with PBS before exposure to UV in PBS. Data expressed as mean ± SD (n = 6) from one cell line, representative of three independent cell lines. Solid black line with diamonds, untreated NHKs; solid black line with crosses, 5 µM BaP; dotted line with closed triangles, 1 kJ/m2 UVA; dotted line with open triangles, 5 µM BaP +1 kJ/m2 UVA; dashed line with closed ellipses, 100 J/m2 UVB; dashed line with open ellipses, 5 µM BaP + 100 J/m2 UVB; solid gray line with closed rectangles, 10 J/m2 UVC; solid gray line with open rectangles, 5 µM BaP + 10 J/m2 UVC.

 
Cell cycle
The cell cycle profile was measured by labelling NHK cell nuclei with propidium iodide and measuring DNA content by flow cytometry. Untreated NHK cultures had a proliferating cell cycle profile with ~50% of cells in G1, 20% in S phase and 30% in G2 (Figure 2). Exposure to BaP or UVA had no effect on cell cycle, but 100 J/m2 UVB and 10 J/m2 UVC caused a significant accumulation of nuclei in G2 to 47.7% (±1.8%, P = 0.0017) and 42.6% (±2.2%, P = 0.0128), respectively. Pre-exposure to 5 µM BaP did not affect the UVC-induced G2 delay (39.6 ± 3.3%, P = 0.0392), but the UVB-induced delay was abolished (34.1 ± 1.0%, not significant compared with control).



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  		Fig. 2.. Cell cycle profiles generated by propidium iodide labelling of nuclei, with increasing DNA content along the x-axis versus number of nuclei on the y-axis. (A) untreated NHKs; (B) 1 kJ/m2 UVA; (C) 100 J/m2 UVB; (D) 10 J/m2 UVC; (E) 5 mM BaP; (F) 5 µM BaP + 1 kJ/m2 UVA; (G) 5 µM BaP + 100 J/m2 UVB; (H) 5 µM BaP + 10 J/m2 UVC.

 
DNA damage in NHKs
Using the nuclease P1 enhanced 32P-postlabelling assay, we demonstrated formation of DNA adducts in NHKs after 24 h exposure to 10 µM BaP (Figure 3). The pattern of radioactivity from NHK genomic DNA was similar to the pattern obtained from plasmid labelled with BPDE as a positive control. The ‘hotspot’ on each chromatogram probably represents the major adduct BPDE-N2-deoxyguanine. No adducts were detected in DNA extracted from unexposed NHKs.



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  		Fig. 3.. 32P-postlabelling of DNA. Chromatogram A shows plasmid DNA reacted with BPDE in vitro as a positive control. Chromatogram B shows the pattern of adducts detected in genomic DNA extracted from NHKs immediately after exposure to 10 µM BaP for 24 h. Chromatogram C shows NHK genomic DNA without BaP exposure as a negative control.

 
DNA damage (single and double strand breaks, alkali-labile sites) in NHKs was measured by the comet assay. Doses that caused little or no detectable DNA damage were chosen from preliminary dose–response studies. Exposure to 5 µM BaP for 24 h or 1 kJ/m2 UVA did not increase tail formation above background levels (<10% tail DNA), whereas 100 J/m2 UVB and 10 J/m2 UVC induced a small, but significant, increase in percentage tail DNA to 17.9 ± 5.1% and 13.8 ± 2.6% respectively (Figure 4). Exposure to 5 µM BaP followed by 1 kJ/m2 UVA radiation caused a very significant increase in percentage tail DNA to 35.1% (±2.5%, P = 0.0018); this effect was not observed if the treatment order was reversed (data not shown). BaP pre-exposure also increased tail formation after UVB radiation but to a much lesser extent than UVA, and BaP pre-exposure had no effect on tail formation by UVC radiation. Although combined exposure to BaP followed by UVA caused a dramatic reduction in viability, the majority of dead cells were resistant to trypsin and were not harvested for comet analysis. Trypan blue exclusion of harvested cells showed that 76.6% (±13.6%) were viable after combined treatment.



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  		Fig. 4.. Mean percentage tail DNA in NHKs analysed by the comet assay illustrating the effects of UV with and without BaP exposure on DNA damage. Data expressed as mean ± 95% CI of three cell lines. *P = 0.0015 compared with 1 kJ/m2 UVA alone, {dagger}P = 0.0379 and {ddagger}P = 0.0449 compared with unirradiated control, by ANOVA.

 
Oxidative DNA damage
Incubation of comet slides with Fpg enzyme enabled the detection of oxidized purines, by conversion of the base damage into single strand breaks. The increase in percentage tail DNA after Fpg incubation compared with slides incubated with enzyme buffer alone is shown in Figure 5. There was a small increase in percentage tail DNA in control cells owing to basal levels of oxidative damage. After combined exposure of NHKs to 5 µM BaP followed by 1 kJ/m2 UVA, there was a significant increase of 11.2 ± 2.9% tail DNA compared with UVA exposure alone (P = 0.0034). Combined exposure to 5 µM BaP followed by 100 J/m2 UVB also caused a significant increase of 9.3 ± 2.2% tail DNA (P = 0.0011). Pre-exposure to BaP did not significantly increase the levels of oxidative lesions detected after UVC radiation.



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  		Fig. 5.. Increase in percentage tail DNA after Fpg incubation. Exposure to 5 µM BaP followed by 1 kJ/m2 UVA or 100 J/m2 UVB caused significantly increased tail formation compared with each agent alone. Data expressed as mean ± 95% CI of three cell lines. *P = 0.0034, {dagger}P = 0.0011 compared with UV radiation alone, by ANOVA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Human epithelial cells are exposed in vivo to a myriad of genotoxic agents, but limited consideration has been given to the effect of combined exposures to these agents. Here we have investigated the combined effects of two common mutagens, BaP and UV light, on NHKs in vitro and showed that the effects of combined exposure differ, depending on the wavelength of the UV source. The most dramatic effects were observed with UVA, the principal component of sunlight. NHKs in vitro were highly sensitive to UVA radiation after BaP exposure, and the marked reduction in viability was accompanied by increased resistance to trypsin, an effect which has previously been described in breast carcinoma cells (34Go). Enhanced cytotoxicity after coexposure to BaP and UVA has been reported in human skin fibroblasts (35Go) and CHO cells (36Go). Binary exposure to BaP followed by UVA also caused significantly enhanced DNA damage, both strand breaks and oxidative lesions, even though each agent alone did not induce any detectable effects. Increased ROS production and oxidative lesions after combined BaP/UVA treatment have been described in several human cell types in vitro (35Go,37Go,38Go), in mouse skin in vivo (39Go) and in calf thymus DNA (40Go). However, many of these studies looked at simultaneous, rather than sequential, BaP/UVA exposure. Our results suggest that the presence of BaP or BPDE-adducts synergistically enhances the induction of DNA damage by UVA light, leading to cell death, and may play a role in development of mutations in human skin cells. Indeed, combined exposure of mouse embryonic fibroblasts to non-mutagenic doses of BaP and UVA was recently demonstrated to enhance mutation frequency (41Go).

The mechanism of this enhancement, however, is still unclear. BPDE–DNA adducts formed during the 24 h incubation with BaP may be photosensitive, leading to DNA strand breakage on exposure to UVA light, which has been shown to occur in oligonucleotides containing a BPDE-N2-guanine adduct (42Go). Alternatively, as BaP absorption lies within the UVA and visible light spectrum and BaP has been shown to be photomutagenic (43Go), BaP, or metabolites of it, remaining inside the keratinocytes after washing may become activated by UVA light and subsequently cause damage.

In contrast to UVA, UVB had no effect on cell viability and did not increase strand breakage in human keratinocytes after BaP pre-exposure. This contradicts a previous study that reported enhanced DNA damage by the comet assay in bovine retinal pigment epithelial cells after combined exposure to BaP and UVB (44Go). BaP/UVB treatment did, however, induce increased oxidative DNA damage compared with BaP or UVB exposure alone. This observation could be important in understanding the increased incidence of skin cancer in psoriasis patients who received coal tar therapy followed by UVB irradiation as treatment for their disorder.

Both UVB and UVC induced G2 arrest in NHKs, as previously described (45Go,46Go). Interestingly, the UVB-induced G2 arrest of keratinocytes was abrogated by BaP pre-exposure. BaP has been reported to have ‘stealth’ properties and can avoid inducing cell cycle arrest (47Go,48Go): in this instance, it appeared that BaP inhibited the recognition of UVB-induced DNA damage, which could have significant long-term effects on the genetic stability of the cells.

The response of NHKs to UVC was not affected by BaP pre-exposure. This is surprising as we have previously shown that plasmid exposed to BPDE followed by UVC has a greater increase in single strand breaks and mutation frequency than plasmid treated with BPDE and UVB (29Go), and lung epithelial cells treated with BaP followed by UVC have enhanced DNA damage (31Go). These differences may be due to cell-specific properties, which is why it is important to investigate the effects of genotoxin exposure in the relevant cell type.

The effects of combined exposure of NHKs to BaP and UV light are wavelength-dependent. BaP and UVA induced considerable cell death and DNA damage. This finding could be of particular significance due to the prevalence of UVA in sunlight and the penetration of UVA to the basal layers of the skin, especially as the dose used in this investigation is equivalent to ~3 min of sun exposure on a sunny summer's day in northern Europe (49Go). BaP and UVB exposure increased the induction of oxidative damage and bypassed UVB-induced G2 cell cycle arrest. UVB is also present in sunlight, and the dose used in this study equates to just over 1 min of sunlight exposure. The mechanisms of these responses are unclear but the importance of studying the effects of agents in combination is highlighted. Our results also have implications for the relative contributions of UVA and UVB to skin cancer development. UVB is more efficient at causing CPDs than UVA; for example, Woollons et al. (13Go) showed that in cultured keratinocytes the majority (75%) of CPDs are induced by the 0.8% UVB content in a UVA sunlamp. However, the upper layers of human skin in vivo will filter out more UVB than UVA and may effect these relative contributions. The data presented here suggest that UVA may have a much more prominent role in skin carcinogenesis because it interacts with other agents, like BaP, in the environment. Thus, ‘safe’ sun exposure recommendations may need to be reduced, especially in polluted areas, in order to prevent skin cancer development. Also, as BaP exposure caused oxidative damage after both UVA and UVB radiation, the inclusion of anti-oxidants in sun cream, which has been suggested to have anti-ageing benefits, may further contribute to skin cancer prevention.


   Acknowledgments
 
We thank Andrew Collins for the gift of Fpg enzyme. This work was funded by Yorkshire Cancer Research; and supported by a Yorkshire Cancer Research grant L299 awarded to M.N.R. and E.I.


   Notes
 
* To whom correspondence should be addressed. Tel: +44 113 3437763; Fax: +44 113 3436603; Email: medmnr{at}leeds.ac.uk


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received on March 23, 2005; revised on May 19, 2005; accepted on May 20, 2005.


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