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

The mutagenic potential of titanium dioxide nanoparticles (TiO2-NPs) of an average size 30.6 nm was investigated using beta-galactosidase (lacZ) gene complementation in plasmid pUC19/lacZ(-) Escherichia coli DH5 alpha system. Plasmid pUC19 was treated with varying concentrations of TiO2-NPs and allowed to transfect the CaCl2-induced competent DH5 alpha cells. The data revealed loss in transformation efficiency of TiO2-NP5 treated plasmids as compared to untreated plasmid DNA in DH5 alpha host cells. Induction of multiple mutations in alpha-fragment of lacZ gene caused synthesis of non-functional beta-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 147 nt 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 DH5 alpha lacZ gene alpha-complementation system, as a novel genetic approach for determining the mutagenic potential, and specificity of manufactured NPs and nanomaterials. (C) 2012 Elsevier B.V. All rights reserved.