Cigarette Smoke-induced Morphological Transformation of Bhas 42 Cells In Vitro

Abstract

In vitro cell transformation assays detect transformed cells that have acquired the distinct characteristics of malignant cells and thus model one stage of in vivo carcinogenesis. These assays have been proposed as surrogate models for predicting the non-genotoxic carcinogenic potential of chemicals. The Bhas 42 cell transformation assay, a short-term assay that uses v-Ha-ras-transfected Balb/c 3T3 cells, can detect the tumour promoter-like activities of chemicals, but has not previously been used with cigarette smoke. The particulate phase of cigarette smoke (total particulate matter [TPM]) is known to induce tumours in vivo in the mouse skin painting assay. Therefore, we investigated the ability of this Bhas cell assay to form morphologically transformed foci in vitro when repeatedly challenged with TPM from a standard research cigarette. TPM induced a dose-dependent increase in Type III foci, and a significant increase (up to 20-fold) in focus formation at moderately toxic concentrations between 5 and 60μg TPM/ml, with a peak at 20μg/ml. Three batches of TPM were tested in three independent experiments. Precision (repeatability and reproducibility) was calculated by using 0, 5, 10, and 20μg TPM/ml. Repeatability and reproducibility, expressed as the relative standard deviation obtained from the normalised slopes of the dose–response curves, were 17.2% and 19.6%, respectively; the slopes were 0.7402 ± 0.1247, 0.9347 ± 0.1316, and 0.8772 ± 0.1767 (increase factor*ml/mg TPM; mean ± SD); and the goodness of fit (r2) of the mean slopes, each derived from n = 6 repeats, was 0.9449, 0.8198, and 0.8344, respectively. This in vitro assay with Bhas 42 cells, which are regarded as already initiated in the two-stage paradigm of carcinogenesis (initiation and promotion), is able to detect cell transformation induced by cigarette smoke in a dose-dependent manner with a high sensitivity and good precision. Because this assay is fast and yields reliable results, it may be useful in product assessment, as well as for further investigation of the non-geno-toxic carcinogenic activity of tobacco smoke-related test substances.

Bhas 42 cell transformation activity of cigarette smoke condensate is modulated by selenium and arsenic

“…The results showed that while both initiator and promoter activities were detectable in CSC‐D, the water soluble fraction (CSC‐W) possessed only promotion activity. Consistent with a recent report [Weisensee et al, ], a dose‐dependent response could be detected for CSC‐D at microgram levels showing lower threshholds for the intiator and promotion activity at concentrations of 20 and 2.5 μg mL −1 , respectively. It should be noted that Bhas 42 cells are transfected with v‐HA‐ ras gene and are considered already initiated but still responsive to single initiator treatment [Asada et al, ] which may explain the higher CSC dose requirement for detecting initiator activity.…”

Comparative tumor promotion assessment of e‐cigarette and cigarettes using the in vitro Bhas 42 cell transformation assay

“…Conversely, we have shown that the particulate matter (ACM) from e‐cigarettes is negative in the Bhas 42 CTA when tested at concentrations up to 120 µg/mL (77 ng/mL nicotine). This study follows our own previous findings that the Bhas 42 CTA can distinguish between the potential promotion activity of a conventional cigarette and a tobacco heating product [Breheny et al, ], and supports the work of others who demonstrated the potential use of the assay in tobacco product assessment [Weisensee et al, ; Han et al, ].…”

“…While this assay was successful in identifying initiator and promoter activity of cigarette smoke TPM, the doseresponse curve of the assay for positive controls and TPM was not sufficiently robust to facilitate product comparisons. This in in contrast to the Bhas 42 CTA, which gave a robust dose-reponse to cigarette smoke TPM [Weisensee et al, 2013] and prompted our current investigation on its potential for product assessment and comparison purposes.…”

“…Prior to the initiation of promotion assay, a single cell growth assay, as illustrated in Figure , was carried out to identify concentrations with a relative toxicity of 50% and above. The promotion assay was carried out as shown in Figure and described previously [Weisensee et al, ]. In brief, 14,000 cells/well were seeded and maintained in six‐well plates for 3 days (day 3 to day 0).…”

“…In recent years, this assay has undergone a number of international validation studies and is now the subject of an OCED guidance document [Organisation for Economic and Cooperative Development, ]. Weisensee et al [] and Han et al [] have shown that Bhas 42 CTA could be useful in assessing the promoter activity of complex mixtures such as cigarette smoke. Their work showed that total particulate matter (TPM) from a reference cigarette induced cell transformation in a concentration‐dependent manner.…”