Induction of SCE by DNA cross-links in human fibroblasts exposed to 8-MOP and UVA irradiation.

To study the SCE-inducing effect of psoralen cross-links in the DNA of normal, human fibroblasts, cell cultures were exposed to PUVA (0.2-1 micrograms of 8-MOP per ml, followed by UVA irradiation at 0.04 J/cm2) and carefully washed to remove non-covalently bound psoralen. Some cell cultures were then given a second dose of UVA (1.1 J/cm2), either immediately after PUVA or 1-3 days later. By this type of treatment, cells with different proportions of DNA cross-links are obtained. The initial PUVA treatment will mainly give rise to psoralen monoadducts and only few cross-links in the DNA, and the second UVA irradiation will convert a number of the psoralen monoadducts into cross-links. SCE analysis was carried out on cells grown for 2 cell cycles in the presence of BrdUrd (10 mumoles/1). PUVA treatment alone did not induce an increase in the SCE frequency, whereas a clear increase of SCE was observed in cells treated with PUVA immediately followed by the second UVA dose. This PUVA + UVA-induced increase of SCE was also observed after incubation of the cells for 3 days at confluency, as well as when a period of 3 days at confluency was introduced between the PUVA exposure and the second irradiation with UVA. In contrast, the SCE frequency gradually returned to the normal level when PUVA + UVA-treated cells were allowed to proliferate for 1-2 days, or when a proliferation period of 2-4 days was introduced between the PUVA exposure and the second irradiation. Because the SCE frequency was not changed by the initial PUVA treatment but markedly increased by PUVA + UVA, it is concluded that psoralen cross-links are considerably more effective at inducing SCE than monoadducts. The results also indicate that SCE-inducing PUVA damage is removed very slowly if at all from the DNA of confluent cells. In contrast, repair functions that eliminate cross-links as well as monoadducts seem to become activated during cell proliferation.