Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan.
Photochem Photobiol Sci. 2018 Apr 18;17(4):404-413. doi: 10.1039/c7pp00348j.
The amount of photolesions produced in DNA after exposure to physiological doses of ultraviolet radiation (UVR) can be estimated with high sensitivity and at low cost through an immunological assay, ELISA, which, however, provides only a relative estimate that cannot be used for comparisons between different photolesions such as cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproduct (64PP) or for analysis of the genotoxicity of photolesions on a molecular basis. To solve this drawback of ELISA, we introduced a set of UVR-exposed, calibration DNA whose photolesion amounts were predetermined and estimated the absolute molecular amounts of CPDs and 64PPs produced in mouse skin exposed to UVC and UVB. We confirmed previously reported observations that UVC induced more photolesions in the skin than UVB at the same dose, and that both types of UVR produced more CPDs than 64PPs. The UVR protection abilities of the cornified and epidermal layers for the lower tissues were also evaluated quantitatively. We noticed that the values of absorbance obtained in ELISA were not always proportional to the molecular amounts of the lesion, especially for CPD, cautioning against the direct use of ELISA absorbance data for estimation of the photolesion amounts. We further estimated the mutagenicity of a CPD produced by UVC and UVB in the epidermis and dermis using the mutation data from our previous studies with mouse skin and found that CPDs produced in the epidermis by UVB were more than two-fold mutagenic than those by UVC, which suggests that the properties of CPDs produced by UVC and UVB might be different. The difference may originate from the wavelength-dependent methyl CpG preference of CPD formation. In addition, the mutagenicity of CPDs in the dermis was lower than that in the epidermis irrespective of the UVR source, suggesting a higher efficiency in the dermis to reduce the genotoxicity of CPDs produced within it. We also estimated the minimum amount of photolesions required to induce the mutation induction suppression (MIS) response in the epidermis to be around 15 64PPs or 100 CPDs per million bases in DNA as the mean estimate from UVC and UVB-induced MIS.
通过一种名为 ELISA 的免疫学检测方法，可以高度敏感且低成本地估算出 DNA 在暴露于生理剂量的紫外线辐射（UVR）后产生的光损伤数量。然而，该方法只能提供相对估计值，无法用于比较不同类型的光损伤，如环丁烷嘧啶二聚体（CPD）和嘧啶（6-4）嘧啶酮光产物（64PP），也无法基于分子水平分析光损伤的遗传毒性。为了解决 ELISA 的这一缺点，我们引入了一组经 UVR 暴露、校准的 DNA，其光损伤数量预先确定，并估算了经 UVC 和 UVB 暴露的小鼠皮肤中产生的 CPD 和 64PP 的绝对分子数量。我们证实了之前的研究结果，即在相同剂量下，UVC 在皮肤中产生的光损伤比 UVB 更多，并且两种类型的 UVR 产生的 CPD 比 64PP 更多。我们还定量评估了角质层和表皮层对下层组织的 UVR 保护能力。我们注意到，ELISA 中获得的吸光度值并不总是与损伤的分子数量成正比，尤其是对于 CPD，这提醒我们不要直接使用 ELISA 吸光度数据来估算光损伤数量。我们进一步使用我们之前在小鼠皮肤中进行的研究的突变数据，估算了 UVC 和 UVB 在表皮和真皮中产生的 CPD 的致突变性，发现 UVB 在表皮中产生的 CPD 的致突变性是 UVC 的两倍以上，这表明 UVC 和 UVB 产生的 CPD 性质可能不同。这种差异可能源于 CPD 形成的依赖于波长的甲基 CpG 偏好。此外，无论 UVR 来源如何，CPD 在真皮中的致突变性都低于表皮，这表明真皮中存在更高的效率来降低其内部产生的 CPD 的遗传毒性。我们还估计了在表皮中引起突变诱导抑制（MIS）反应所需的最小光损伤数量，约为 15 个 64PP 或 100 个 CPD 每百万碱基 DNA，这是 UVC 和 UVB 诱导的 MIS 的平均值估计。
