Cytotoxicity and Mutagenicity of Narrowband UVB to Mammalian Cells

Abstract

Phototherapy using narrowband ultraviolet-B (NB-UVB) has been shown to be more effective than conventional broadband UVB (BB-UVB) in treating a variety of skin diseases. To assess the difference in carcinogenic potential between NB-UVB and BB-UVB, we investigated the cytotoxicity via colony formation assay, genotoxicity via sister chromatid exchange (SCE) assay, mutagenicity via hypoxanthine phosphoribosyltransferase (HPRT) mutation assay, as well as cyclobutane pyrimidine dimer (CPD) formation and reactive oxygen species (ROS) generation in Chinese hamster ovary (CHO) and their NER mutant cells. The radiation dose required to reduce survival to 10% (D₁₀ value) demonstrated BB-UVB was 10 times more cytotoxic than NB-UVB, and revealed that NB-UVB also induces DNA damage repaired by nucleotide excision repair. We also found that BB-UVB more efficiently induced SCEs and HPRT mutations per absorbed energy dosage (J/m²) than NB-UVB. However, SCE and HPRT mutation frequencies were observed to rise in noncytotoxic dosages of NB-UVB exposure. BB-UVB and NB-UVB both produced a significant increase in CPD formation and ROS formation (p < 0.05); however, higher dosages were required for NB-UVB. These results suggest that NB-UVB is less cytotoxic and genotoxic than BB-UVB, but can still produce genotoxic effects even at noncytotoxic doses.

Keywords: DNA damage; HPRT; SCE; broadband UVB; cytotoxicity; narrowband UVB.

Figure 1

Spectrum of each UV light source. Three light devices were used in this study: (a) Broadband UVB-Westinghouse Sunlamp (Broad spectrum 280–350 nm, 63.6 J/m² per minute); (b) Narrowband UVB-Phillips TL01 (311 nm peak, 82.8 J/m² per minute); (c) UVC-germicidal lamp (254 nm peak, 58.2 J/m² per minute). (d) Merge of UV spectrum. Light spectrum was confirmed with spectroradiometer Aseq-LR1. (e) Filtration effect of petri dish cap either on (+) or off (−) with conventional broadband ultraviolet-B (BB-UVB) irradiation. The dose rate (J/m²) of each light device was measured with a UVP-UVX radiometer with UVC and UVB probes.

Figure 2

UV exposure cell survival curves. (a) Ultraviolet C (UVC)-exposed cells; (b) Broadband UVB (BB-UVB)-exposed cells; (c) Narrowband UVB (NB-UVB)-exposed cells. Black circles indicate wildtype CHO10B2 cells. Red circles indicate XPG mutant UV135 cells. Error bars indicate standard errors of the means from as many as three independent experiments.

Figure 3

Representative image of sister chromatid exchange (SCE) after NB-UVB 500 J/m²: Red arrows indicate sister chromatid exchanges.

Figure 4

Genotoxicity and mutagenicity following UV exposure in CHO10B2 cells. (a) Sister chromatid exchange (SCE) frequency following UVC, BB-UVB, and NB-UVB at increasing dosage. (b) Hypoxanthine phosphoribosyltransferase (HPRT) mutation frequency following UVC, BB-UVB, and NB-UVB at increasing dosage. Error bars indicate standard errors of the means from at least three independent experiments.

Figure 5

UV light formation of cyclobutane pyrimidine dimers (CPDs) in CHO10B2 cells at noncytotoxic and cytotoxic dosages. (a) UVC-exposed cells; (b) Broadband-UVB-exposed cells without petri dish cap off during exposure; (c) Broadband-UVB-exposed cells with petri dish cap on during exposure; (d) Narrowband-UVB-exposed cells. Error bars indicate standard error of the mean of at least three independent experiments. * Indicates statistically significant differences compared to control (p < 0.05), one-way ANOVA followed by Turkey’s Multiple Comparison Test.

Figure 6

Oxidative stress following 1 h of UV light exposure of CHO10B2 cells. Error bars indicate standard error of the mean of at least three independent experiments. * Indicates statistically significant differences compared to control (p < 0.05), one-way ANOVA followed by Turkey’s Multiple Comparison Test.

Figure 7

Summary of CPD formation vs. cytotoxicity and genotoxicity. (a) CPD formation vs. cytotoxicity; (b) CPD formation vs. genotoxicity.