3.1 Mutagenic properties of AFB1-FapyGua in different sequence contexts
The single-stranded pSBL vectors containing AFB1-FapyGua in the AXC, CXC, TXC, and CXA (X = AFB1-FapyGua) sequence contexts were constructed and replicated in COS7 cells. The CGC sequence has been identified as a hot-spot for AFB1-induced G > T transversions, while the AGC, TGC sequences and especially the CGA sequence were less prone to these substitutions (Fedeles and Essigmann 2018). Following isolation of the progeny DNAs, individual clones were obtained by transformation ofE. coli DH5α cells and subjected to Sanger sequencing. The experiments were conducted in triplicate, beginning from transfection of COS7 cells. The sample size was fixed in advance at the level of ten clones per DNA isolate, excluding clones that either did not contain the insert sequences (~ 20%) or had alterations others than point mutations (Table 1 ).
These data revealed that regardless of the sequence contexts, AFB1-FapyGua was highly mutagenic, inducing base substitutions at frequencies of ~ 80-90% (Figure 2 ). The difference between sequence contexts with regard to non-mutagenic replication was insignificant. Similar to results of the prior study, in which the TXA sequence was utilized (Lin et al. 2014), mutational spectra were dominated by G > T transversions. In addition, G > A transitions and G > C transversions were observed in each of the four sequences tested. The levels of G > A transitions were higher in the AXC and CXA sequences relative to the CXC and TXC sequences, with difference observed for the AXC versus CXC sequence approaching significant level (p = 0.057). The level of G > C transversions was elevated in the CXA sequence; relative to the AXC sequence, the difference was significant (p = 0.047).