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.
Murli
H
H
Covance Laboratories, Vienna, Virginia 22182, USA. hema.murli@covance.com
Aardema
M
M
Lawlor
T
T
Spicer
C
C
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000633:toxicity*; D000818:Animals; D016466:CHO Cells; D002746:Chlorpromazine / Q000633:toxicity; D002869:Chromosome Aberrations; D006224:Cricetinae; D008730:Methoxsalen / Q000633:toxicity; D009153:Mutagens / Q000633:toxicity*; D013472:Sunlight
D009153:Mutagens; D015127:9,10-Dimethyl-1,2-benzanthracene; D002746:Chlorpromazine; D008730:Methoxsalen