Cisplatin-DNA adduct determination in the hepatic albumin gene as compared to whole genomic DNA.
A quantitative time-resolved fluorometriC PCR-stop assay has been used to determine cisplatin-DNA adducts in a 1.612 kb region and a polymorphic 1.85 kb region of the rat liver albumin gene containing parts of exons B and C and all of the BC intron. The values were compared to adducts in the whole rat liver genome determined by atomic absorbance spectrometry (AAS). Initial validation of the PCR-stop assay involved modification of purified rat liver DNA in vitro to desired levels by incubation with different concentrations of cisplatin. In these DNA samples, cisplatin-DNA adduct levels determined in the 1612 base pair fragment by PCR-stop assay were shown to be similar to those determined in the whole genomic DNA by AAS. In freshly isolated primary rat hepatocytes cultured for 2 h with 50, 75, 100, and 150 microM cisplatin, adduct levels determined by the PCR-stop assay were similar to those measured by AAS. Cultured MH1C1 rat hepatoma cells, which express albumin, had a polymorphism in the rat albumin gene such that the fragment amplified with the same primers was about 1.85 kb (13% larger). When MH1C1 cells were exposed to 5, 15, 25, 50, and 75 microM cisplatin for 24 h, 50% cell kill was at 21.0 +/- 5.5 microM cisplatin. For doses of 15-75 microM cisplatin, the cisplatin-DNA adduct levels in this fragment, measured by PCR-stop assay, were about one-half of those in the whole genomic DNA measured by AAS. In addition, MH1C1 cells exposed to 150 microM cisplatin for 4 h and subsequently incubated with fresh medium for 24 h showed no change in adduct level in whole genomic DNA during this time but showed a 29% adduct removal in the 1.85 kb fragment. The data demonstrate that this 1.85 kb region containing expressed regions of the albumin gene has undergone both less adduction and more rapid adduct removal, as compared to the MH1C1 genome as a whole.
Zhang
Z
Z
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA. Zhangz@DC37A.NCI.NIH.GOV
Poirier
M C
MC
D000418:Albumins / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002945:Cisplatin / Q000032:analysis*; D004247:DNA / Q000032:analysis*; D018736:DNA Adducts / Q000032:analysis*; D005260:Female; D016678:Genome; D008099:Liver / Q000737:chemistry*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D014407:Tumor Cells, Cultured
D000418:Albumins; D018736:DNA Adducts; D004247:DNA; D002945:Cisplatin