Photoclastogenicity-an improved protocol, its validation, and investigation of the photogenotoxicity of DMBA

An improved protocol was developed to detect light-induced clastogenic photoproducts in Chinese hamster ovary (CHO) cells. Dishes (60 mm) containing cells and the test material or vehicle control in 3 mL of phosphate-buffered saline were exposed to light using a SUNTEST CPS solar simulation unit. Importantly, cells were exposed at about 25 cm from the light source, thereby allowing a short exposure time of 2 min. With this exposure the assay was conducted with lids removed during the UV exposure with minimal risk of contamination. After preliminary experiments an exposure of 165.6 mJ/cm(2) UVA: 17.0 mJ/cm(2) UVB was selected for treatments with the different phototoxins. Under these exposure conditions about 10-15% aberrant cells were induced in vehicle control cultures with no or minimal cytotoxicity. The well-known photoclastogens 8-methoxypsoralen (8-MOP) and chlorpromazine (CLZ) were tested. In agreement with published data, 8-MOP and CLZ were clastogenic (lowest observed effect level, LOEL, was 0.0159 microg/mL and 1.03 microg/mL, respectively). In the absence of UV, 8-MOP was clastogenic at a much higher concentration (LOEL 251 microg/mL without UV vs. 0.0159 microg/mL with UV) while CLZ was negative up to a toxic concentration of 35 microg/mL. 7,12-Dimethylbenz[a]anthracene (DMBA), which is photomutagenic in bacteria, was clastogenic at > or =0.005 microg/mL with UV light (without S9) and at > or =2.53 microg/mL with S9 (without UV light). These results demonstrate the utility of the protocol for the detection of photoclastogenicity and expand the characterization of DMBA's photogenotoxic activity.