TSRC, Tob. Sci. Res. Conf., 2016, 70, abstr. 17

Ex vivo effects of smokeless tobacco extracts on select immune responses

(1) RAI Services Company; (2) Wake Forest University Health Sciences; (3) R.J. Reynolds Tobacco Co.; (4) Eurofins Lancaster Labs Inc, Winston-Salem, NC, USA

Our previous work demonstrated that exposure to Tobacco Product Preparations (TPPs) from combustible tobacco cause cytotoxic and select immunosuppressive effects in PBMCs, and this ex vivo model is well suited to investigate the chronic immune suppression associated with smoking. However, the assessment of potential immunomodulatory effects of Smokeless Tobacco (ST) are hampered by solvent interference from Complete Artificial Saliva (CAS) in some ex vivo assays, which is often used to prepare ST extracts. To obviate such challenges, we report a method to prepare aqueous extracts of a CORESTA moist snuff reference product, 2S3 in phosphate buffer (ST/PB), and the relative effects of exposure of different TPPs were assessed. Limited chemical characterization of the ST/PB was performed, and biological effects were expressed as equi-nicotine units, based on the nicotine content of TPPs. Cytotoxicity was determined by 7AAD measurement in total PBMCs and H2AX levels were measured as an indicator of DNA damage via double-stranded breaks. We examined the effects of TPPs on the intracellular and secreted cytokines in PBMCs after lipopolysaccharide and PMA/ionomycin stimulation. We also determined the cytolytic effects of PBMCs exposed to different TPPs. The combustible TPPs were markedly more cytotoxic and potent suppressors of several immune functions evaluated in this ex vivo model. ST/CAS was a stronger suppressor of select cytokine levels and caused more DNA damage and cell death to a greater extent than ST/PB. In summary, ST/PB does not appear to interfere with the assays utilized. Combustible TPPs appear to compromise immune responses more than ST/PB. Therefore, the aqueous ST/PB may be more suitable to further investigate the effects of ST products on immune responses, compared to ST/CAS