The mutagenic assessment of mainstream cigarette smoke using the Ames assay: A multi-strain approach

Abstract

Salmonella typhimurium strains TA1535, TA1537, TA97, TA102 and TA104 were assessed for their suitability and use in conjunction with a Vitrocell(®) VC 10 Smoking Robot and 3R4F reference mainstream cigarette smoke. Little information exists on TA97, TA104, TA1535, TA1537 and TA102 using an aerosol 35mm spread-plate format. In this study, TA1535 and TA1537 were considered sub-optimal for use with a scaled-down format, due to low spontaneous revertant numbers (0-5 revertants/plate). In the context of a regulatory environment, TA97 is deemed an acceptable alternative for TA1537 and was therefore selected for whole smoke exposure in this study. However, there is no acceptable alternative for TA1535, therefore this strain was included for whole smoke exposure. TA1535, TA97, TA102 and TA104 were assessed for mutagenic responses following exposure to cigarette smoke at varying concentrations (using diluting airflow rates of 1.0, 4.0, 8.0 and 12.0L/min), and exposure times of 24 and 64min. A positive mutagenic response to cigarette smoke was observed in strain TA104 at both the 24 and 64min time points, in the presence of S-9, at the highest smoke concentration tested (1.0L/min diluting airflow). The three remaining strains were found to be unresponsive to cigarette smoke at all concentrations tested, in the presence and absence of metabolic activation. Cigarette smoke particulate deposition was quantified in situ of exposure using quartz crystal microbalance technology, enabling data to be presented against an associated gravimetric mass (μg/cm(2)). Finally, data obtained in this study were combined with previously published Ames data for TA98, TA100, YG1024, YG1042 and Escherichia coli (WP2 uvrA pKM101), generated using the same 35mm methodology. The combined data-set was used to propose an aerosol testing strategy, based on strain compatibility with the whole smoke aerosol, whilst maintaining the essence of the regulatory guidelines for the standard Ames assay.

Bacterial mutagenicity assays: Vehicle and positive control results from the standard Ames assay, the 6‐ and 24‐well miniaturized plate incorporation assays and the Ames II™ assay

“…When miniaturising the plate incorporation assay, the low number of revertants per well for TA1537 resulted in vehicle control values that were near to or at zero (preventing the calculation of a fold‐increase in response to mutagenic chemicals). This observation was also noted by others for these strains when they miniaturized the Ames assay [Thorne et al, ]. The TA97a strain exhibits a higher number of revertants per well in vehicle control cultures compared with TA1537, even in smaller plate formats (Table ).…”

Development, qualification, validation and application of the Ames test using a VITROCELL® VC10® smoke exposure system

“…Exposure systems such as the VITROCELL ® VC10 ® have been developed for use in combination with biological endpoints to detect potential interactions at the ALI or AAI for a variety of inhaled materials including tobacco smoke [ [44] , [45] , [46] , [47] , [48] , [49] ], e-cigarettes [ 50 ], nanomaterials [ [51] , [52] , [53] ], therapeutic aerosols [ 54 ], airborne chemicals and pollutants [ 31 , [55] , [56] , [57] ]. In addition, the VITROCELL ® VC10 ® system has been shown to be a robust and reproducible in vitro method through an inter-machine comparison of tobacco smoke particle deposition using six independent smoke exposure systems [ 44 ].…”

Hyperforin Exhibits Antigenotoxic Activity on Human and Bacterial Cells

“…In the Ames test, we investigated the potential antimutagenic effects of HF on three bacterial strains of S. typhimurium (TA97, TA98, and TA100). The strain TA97 enables the detection of frameshift mutagens [ 24 ]. 9-aminoacridine, which was used as diagnostic mutagen for this strain, interacts with GC base-pairs and induces frameshift mutations due to DNA intercalation [ 25 ].…”