Coexposure to benzo[a]pyrene and UVA induces DNA damage: First proof of double‐strand breaks in a cell‐free system

Abstract

DNA damage induced by solar ultraviolet (UV) radiation plays an important role in the induction of skin cancer. Although UVA constitutes the majority of solar UV radiation, it is less damaging to DNA than UVB. The DNA damage produced by UVA radiation, however, can be augmented in the presence of a photosensitizer. We previously used benzo[a]pyrene (BaP), an environmental carcinogenic polycyclic aromatic hydrocarbon, as an exogenous photosensitizer, and demonstrated that combined exposure to BaP and UVA resulted in DNA double-strand breaks (DSBs) in cultured Chinese hamster ovary (CHO-K1) cells. In this study, we investigated whether coexposure to BaP and UVA induces DSBs in a cell-free system and whether reactive oxygen species (ROS) were involved in the generation of the DSBs. DSBs were induced by the coexposure both in the cell-free system (in vitro) and in CHO-K1 cells (in vivo), but not by treatment with BaP or UVA alone. DSB induction in vitro required higher doses of UVA and BaP than were required in vivo, suggesting that the mechanism of DSB induction differed. A similar difference in efficiency also was observed in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by coexposure to BaP and UVA in vitro and in vivo. A singlet oxygen ((1)O2) scavenger (NaN3) effectively inhibited the production of DSBs and 8-oxodG, suggesting that (1)O2 is a principal ROS generated by BaP and UVA both in vitro and in vivo. Furthermore, repair-deficient xrs-5 cells were more sensitive to coexposure with BaP and UVA than were CHO-K1 cells, but the two cell lines were equally sensitive to the combined treatment in the presence of NaN3. This result suggested that the cell death produced by coexposure to BaP and UVA was at least partly due to the DSBs generated by (1)O2. Our findings indicate that coexposure to BaP and UVA effectively induced DNA damage, especially DSBs, which results in phototoxicity and possibly photocarcinogenesis.

Coexposure to benzo[a]pyrene and UVA induces phosphorylation of histone H2AX

“…In the experiment using a scavenger of singlet oxygen ( 1 O 2 ), sodium azide (NaN 3 ) was added 15 min before the UVA irradiation and the cells were irradiated in the presence of NaN 3 . The conditions for UVA irradiation were as described previously [16,17].…”

“…DSBs were detected using a biased sinusoidal field gel electrophoresis (BSFGE) system (Atto Co., Japan) as described previously [16,17]. In brief, cells treated with BaP and UVA were suspended in 1% lowmelting agarose and solidified immediately after treatment.…”

“…Although the induction of c-H2AX by general DSB inducers such as ionizing radiation and anti-cancer drugs has been well established, there are no reports on the induction of c-H2AX by environmental carcinogens. We expected BaP under UVA irradiation to induce phosphorylation of H2AX because coexposure to BaP plus UVA was shown to induce DSBs in our previous study [16,17].…”

“…Several groups have demonstrated that coexposure to PAHs and UVA significantly augmented DNA damage, such as single strand breaks [11,12] and the formation of 8-oxo-7,8-dihydro-2 0 -deoxyguanosine [13][14][15]. In addition, we recently showed that coexposure to the most commonly studied PAH, benzo[a]pyrene (BaP), and UVA induced DNA double strand breaks (DSBs) [16,17]. Among the many forms of DNA damage, DSBs are considered the most serious threat to the cell, and when not repaired or misrepaired, can result in mutations or chromosomal rearrangements and eventually cell death [18].…”

A study of the phototoxic effect of Benzo[a] Pyrene on bacteria

“…Photodegradation of PAHs may be due in addition to UVA rays as well as visible light, and this is especially true in the case of PAHs with a higher content of aromatic rings, which are photosensitized upon radiation with a wavelength >400 nm [32][33][34].…”