Expression of an adenovirus encoded reporter gene and its reactivation following UVC and oxidative damage in cultured fish cells

Abstract

Purpose: Recombinant human adenovirus, AdCA35lacZ, was used to examine expression of a reporter gene and its reactivation following UVC (200-280 nm) and oxidative damage in fish cells.

Materials and methods: AdCA35lacZ is a recombinant nonreplicating human adenovirus, which expresses the beta-galactosidase (beta-gal) reporter gene. UVC light produces DNA damage repaired by nucleotide excision repair (NER). In contrast, methylene blue plus visible light (MB+VL) produces oxidative DNA damage, mainly 8-oxoguanine, that is repaired by base excision repair (BER). We examined expression of the reporter gene and host cell reactivation (HCR) of the UVC-treated and MB+VL-treated reporter gene in fish cells.

Results: AdCA35lacZ infection of Chinook salmon cells (CHSE-214), eel cells (PBLE) and four rainbow trout cell lines (RTG-2, RT-Gill, RTS-34st and RTS-pBk), but not zebrafish (ZEB) or carp (EPC) cells resulted in expression of beta-gal. HCR of UVC-treated AdCA35lacZ in fish cells varied from that obtained in NER-deficient xeroderma pigmentosum group A fibroblasts to greater than that for NER-proficient normal human fibroblasts. HCR of UVC-treated AdCA35lacZ correlated with beta-gal expression levels for untreated AdCA35lacZ. Exposure of cells to fluorescent light (400-700 nm) increased expression of the undamaged reporter gene in normal human fibroblasts and in all fish cells except PBLE and increased HCR of the UVC-damaged reporter gene in fish cells but not in human fibroblasts. HCR of the MB + VL-treated reporter gene was similar to that in human cells for PBLE, CHSE-214, RTG-2 and RTS-pBk, but was reduced in RT-Gill and RTS-34st cells.

Conclusions: These results indicate the detection of functional photoreactivation (PR) of UVC-induced DNA damage in fish cells but not in normal human fibroblasts and a link between NER and transcription of the reporter gene in the fish cells in the absence of PR. We show also efficient BER of the reporter gene in several fish cell lines.