Characterization of carbadox-induced mutagenesis using a shuttle vector pSP189 in mammalian cells

Carbadox, a quinoxaline 1,4-dioxide derivative, is a known mutagen with its functional mechanism yet to be well defined. In the present study we used a shuttle vector assay in vitro to uncover the functional details of carbadox-induced mutagenesis in mammalian cells. The plasmid DNA of a shuttle vector pSP189 was treated with different doses of carbadox at 37 degrees C for 1 or 2h with or without the presence of S9. The target gene SupF in the plasmid was sequenced after replication in Vero cells followed by amplification in Escherichia coli MBM7070 to evaluate mutation frequency. DNA sequencing analysis of recovered carbadox-induced mutations revealed 76.3% single base substitution, 7.9% single base insertion, 10.5% single base deletion and 5.3% large fragments deletion. All single base substitutions occurred at G:C base pairs, among which transversion and transition occurred at a 2:1 ratio. The mutations did not occur randomly in the supF gene, but had sequence specificity and hotspots instead: most substitutions were detected at the nucleotide N in a 5'-NNTTNN-3' sequence; 75% of base insertions were seen in the 5'-TCC-3' sequence; whereas all large fragments deletions occurred in the 5'-ANGGCCNAAA-3' sequence. Nucleotide 129, 141 and 155 in the supF gene of plasmid pSP189 were identified as the hotspots for carbadox-induced mutations that accounted for 65% of all single base substitutions. We conclude that carbadox and its metabolites induce sequence-specific DNA mutations at high frequencies, therefore its safe usage in animal husbandry should be seriously considered.