Diesel exhaust (DE) is a major airborne pollutant in urban areas. In this study, we estimated the systemic effect of diesel exhaust inhalation by investigating mutations in extraplumonary organs such as the testis and liver. gpt delta Transgenic mice carrying the guanine phosphoribosyltransferase (gpt) transgene for the detection of mutations in genomic DNA were exposed to inhalation of 3 mg m-3 diesel exhaust (as suspended particulate matter) for 12 or 24 weeks. Compared to the control mice, DE resulted in a 2.0-fold increase in mutant frequency in the testis of mice that were exposed to DE for 24 weeks (inhaled group, 1.17×10-5; control group, 0.57×10-5), but not in the testis of mice exposed for 12 weeks (0.61×10-5). The mutant frequency in the lungs was 2.6-fold higher in mice exposed to DE for 24 weeks than the control group, but it was not elevated in the liver (0.67×10-5). In the testis, the major mutations on the gpt gene were G:C→T:A transversions, 1 base deletions and G:C→A:T transitions, while the major mutation in the lung was G:C→A:T transitions. The mutations on nucleotide nos. 402, 406, 409 and 416-418 in the gpt gene in testis seemed to be characteristic of DE inhalation in the testis. Our results suggest that inhalation of diesel exhaust is genotoxic to the testis as well as respiratory organs.
The mutagenic specificity of N-nitrosotaurocholic acid (NO-TCA) in human cells was investigated using supF shuttle vector plasmids. The plasmids pMY189 were treated with NO-TCA in vitro and introduced into normal fibroblasts (WI38-VA13) and nucleotide excision repair (NER)-deficient cells (XP2OS(SV)) for replication. The background mutation frequency of the supF gene was 4.1× 10-4 and 2.0×10-4 after replication in normal and NER-deficient cells, respectively. The mutation frequency increased 5 and 15 times in normal and NER-deficient cells, respectively, after the treatment of the plasmid with 50 mg/mL of NO-TCA. The higher mutation frequency in NER-deficient cells indicates that the DNA damage induced by NO-TCA is repaired by NER. Base sequence analysis of 101 and 94 plasmids with mutations in the supF gene propagated in normal and NER-deficient cells, respectively, revealed that the majority of the mutations were base substitutions (about 89 and 90%) and the rest were deletions and insertions (about 11 and 10%) in both cell lines. About half of the mutant plasmids contained a single base substitution. Of the single base substitutions, the most frequent mutations were G:C to A:T transitions (about 37 and 36%), followed by G:C to C:G transversions (about 31 and 28%) in both cell lines. The mutations were not distributed randomly but were located at several hot spots in the supF gene, and almost all hot spots were at G:C sites. These observations accord with previous findings that NO-TCA forms DNA adducts with dC and induces G:C to A:T base substitution in Salmonella typhimurium TA100.
While offering the promise of therapeutic use in the future, RNA interference (RNAi) technology is useful to knock down genes posttranscriptionally. We attempted to knock down severe acute respiratory syndrome (SARS)-corona virus (Cov) genes using small interfering RNA (siRNA) employing siRNA-expression vectors and synthetic double-stranded RNA (dsRNA) as a model for effective siRNA design. First, we selected three target sites without mutations among 15 SARS-Cov strains using a prediction algorithm and constructed three siRNA-expression vectors. Using a pGL3 vector, we constructed three corresponding model target vectors to the firefly luciferase gene (Fluc), to which the model targets were connected. Using Renilla luciferase gene (Rluc) as the internal control, the three siRNA vectors knocked down the targets, providing effective target sequences. Almost identical results were obtained when Rluc was integrated into the pGL3-Fluc target vector. Next, effective structures of synthetic double-stranded RNA (dsRNA) were investigated using two targets. In all, six RNAs per target were synthesized: complementary sense and antisense 19-mer core RNAs; sense 21-mer RNAs having a 2-nucleotide (nt) match or unmatch overhang at the 3'-end; and antisense 21-mer RNAs having a 2-nt match or unmatch overhang at the 3'-end. The six RNAs provided nine species of dsRNAs (a blunt 19-mer duplex, a total of 4 19-mer/21-mer duplexes with a match or unmatch 2-nt overhang at the 3'- end of the sense or antisense strand, 4 21-mer/21-mer duplexes with match or unmatch 2-nt overhang at both ends) in combination. Targets were sense or antisense sequences. Generally, 19-mer/21-mer dsRNA with a match 2-nt overhang at the 3'- end of the antisense strand showed the highest activity, irrespective of the thermodynamic stabilities at terminal ends, suggesting that the 2-nt overhang is more critical than thermodynamic stabilities to select the antisense strand to the RNA-induced silencing complex (RISC).
Translesion DNA synthesis (TLS) is a tolerance pathway of replication block caused by DNA lesions. To measure the efficiency and fidelity of TLS in human cells, we established a shuttle vector assay by modifying of a bacterial TLS assay system. The assay consists of transfection of DNA repair-deficient human cells with a plasmid possessing a single DNA adduct, and transformation of indicator bacteria with plasmids extracted from the cells. We show that plasmid replication was suppressed to 1/9 by a single aminobiphenyl-dG adduct, and the mutant frequency of TLS-operated plasmids was 0.31, of which the major mutation (78%) was G to T transversion. The results demonstrate that this assay is applicable in practice for investigating TLS in human cells.