Evaluation method for the cytotoxicity of cigarette smoke byin vitro whole smoke exposure

July 29, 2013


Xiang Li, Cong Nie, Pingping Shang, Fuwei Xie, Huimin Liu, Jianping Xie

Key Laboratory of Tobacco Chemistry, Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou 450001, China

With the development of direct exposure technology at the air/liquid interface, the toxicological responses of fresh WS can be evaluated. The whole smoke of the 3R4F reference cigarette and two experimental cigarettes were tested on Chinese hamster ovary (CHO) cells to evaluate the endpoints Ames Salmonella reversemutagenicity assay and in vitro micronucleus assay for genotoxicity and neutral red uptake (NRU)assay for cytotoxicity.


An in vitro whole smoke (WS) exposure method was established to evaluate the toxicological effectsof fresh cigarette smoke using the VITROCELL®system associated with the neutral red uptake (NRU)cytotoxicity assay. The VITROCELL®system is a newly representative culture and exposure system forin vitro studies of gases or complex mixtures. The impacts of two factors on cytotoxicity measurements ofcigarette smoke were investigated using this WS exposure system. The factors include synthetic air expo-sure and optimal time to perform the NRU assay after smoke exposure. Results showed that synthetic airexposure used in the system did not significantly alter cell survival; 24 h after smoke exposure appearedto be an optimal time-point to assess the cytotoxicity of cigarette smoke. A clear dose–response relation-ship between smoke exposure and cell viability was demonstrated using this system, and the evaluationmethod was sensitive to distinguish the differences in smoke-induced cytotoxic effects from differentcigarettes. In addition, we tried converting the values of EC50from WS exposure testing into the values inunit used in total particulate matter (TPM) testing for a purpose of comparison, and the data indicate thatthe cytotoxicity of smoke measured by WS exposure is greater than that measured by TPM exposure.


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