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4-(Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen in cigarette smoke, while ultraviolet (UV) irradiation from sunlight is a major factor for causing skin aging and skin cancer. However, little is known about the effects of the interaction between NNK and UV light on the induction of DNA damage and oxidative stress. In this study, we incubated human white blood cells (WBCs) with NNK, followed by irradiating the cells with ultraviolet A (UVA) (320-380 nm), and we measured DNA strand breaks (by the Comet or single-cell gel electrophoresis assay), lipid peroxidation (as thiobarbituric acid-reactive substances, TABRS), and the levels of intracellular reactive oxygen species (ROS). We found that preincubation with 1.0 mM NNK, followed by UVA irradiation (7.6 kJ/m2) synergistically increased DNA damage, lipid peroxidation, and the level of intracellular ROS in WBCs, while NNK or UVA alone had little or no effect. Electron spin resonance spectroscopic analyses showed that NNK plus UVA enhanced the UVA-induced generation of singlet oxygen but not hydroxyl radicals. In addition to ROS, bioactivation of NNK by cytochromes P450 (CYP) to form reactive NNK intermediates may also be involved in the synergistic damage to WBCs by NNK plus UVA. This is evidenced by the synergistic increase in N7-methylguanine (7-mGua), a major DNA adduct produced by NNK. Overall, the present study demonstrates that exposure of WBCs to both NNK and UVA synergistically increases DNA damage and lipid peroxidation and that such effects involve enhanced generation of ROS, especially singlet oxygen, and activation of NNK to 7-mGua by CYP. The results imply that NNK is a phototoxic agent.