Use of β-galactosidase (lacZ) gene α-complementation as a novel approach for assessment of titanium oxide nanoparticles induced mutagenesis

Abstract

The mutagenic potential of titanium dioxide nanoparticles (TiO2-NPs) of an average size 30.6nm was investigated using β-galactosidase (lacZ) gene complementation in plasmid pUC19/lacZ−Escherichia coli DH5α system. Plasmid pUC19 was treated with varying concentrations of TiO2-NPs and allowed to transfect the CaCl2-induced competent DH5α cells. The data revealed loss in transformation efficiency of TiO2-NPs treated plasmids as compared to untreated plasmid DNA in DH5α host cells. Induction of multiple mutations in α-fragment of lacZ gene caused synthesis of non-functional β-galactosidase enzyme, which resulted in a significant number of white (mutant) colonies of transformed E. coli cells. Screening of mutant transformants based on blue:white colony assay and DNA sequence analysis of lacZ gene fragment clearly demonstrated TiO2-NPs induced mutagenesis. Multiple alignment of selectable marker lacZ gene sequences from randomly selected mutants and control cells provided a gene specific map of TiO2-NPs induced mutations. Mutational analysis suggested that all nucleotide changes were point mutations, predominantly transversions (TVs) and transitions (TSs). A total of 32 TVs and 6 TSs mutations were mapped within 296 nucleotides (nt) long partial sequence of lacZ gene. The region between 102 and 147nt within lacZ gene sequence was found to be most susceptible to mutations with nine detectable point mutations (8 TVs and 1 TSs). Guanine base was determined to be more prone to TiO2-NPs induced mutations. This study suggested the pUC19/E. coli DH5αlacZ gene α-complementation system, as a novel genetic approach for determining the mutagenic potential, and specificity of manufactured NPs and nanomaterials.