Introduction Tobacco smoke is considered to be a major risk factor in the development of many dis- eases, and its association with periodontal disease has been shown in a number of stud- ies (1). However, data demonstrating a link between tobacco smoke and injury to oral tissues have not yet appeared in the litera- ture. The exposure of gingival tissues to ciga- rette smoke for long or short periods is undoubtedly a major factor in the assess- ment of biological damage and has been reported in the literature. Some studies have analysed patients who have been smoking for two years, while others have considered patients with a 20-year history of smoking. Many authors agree that a significant clini- cal effect is seen when the use of tobacco has been greater than ten cigarettes per day for at least two years (2). Current studies for the assessment of the adverse effects of tobacco on health are often conducted in vivo with animal models. For example, mice have been employed to exam- ine the influence of nicotine on leucocytes in the microcirculation (3), while the long-term ATLA 27, 449–459, 1999 449 In Vitro Testing of the Responses of Human Gingival Fibroblasts and L-929 Cells to Nicotine Gabriela Ciapetti, 1 Giulia Remiddi, 1 Franca Savioli, 1 Giuseppe Monaco, 2 Giacomo Ori 2 and Luigi Checchi 2 1 Laboratorio di Biocompatibilitá dei Materiali da Impianto, Istituti Ortopedici Rizzoli, via di Barbiano 1/10, 40136 Bologna, Italy; 2 Clinica Odontoiatrica, Università degli Studi Bologna, via S. Vitale 59, 40125 Bologna, Italy Summary — Tobacco smoke is considered to be a major risk factor in the development of car- diac diseases and lung cancer. It has also been shown that periodontitis is more prevalent and more severe in smokers than in non-smokers. Nicotine, the major pyridine alkaloid in tobacco, has been shown to participate in periodontal disease, exerting both local and systemic effects. In the present study, the effects of nicotine (6μg/ml, 60μg/ml and 600μg/ml) on human gingival fibroblasts (HGF) were assessed by using various exposure protocols. The responses of HGF cultures obtained from smokers and non-smokers were compared to those found when using a continuous cell line (L-929). Neutral red uptake (NRU) and the measurement of DNA content with bis-benzimide dye were used to assess cell viability and cell number, respectively. NRU was the most sensitive technique for the detection of cytotoxic effects. L-929 cells were found to be affected by nicotine in the NRU assay, with a strong cytotoxic effect with 600μg/ml nicotine, and a “response” with 60μg/ml nicotine when prolonged or double challenge was applied. Non- smoker HGF and smoker HGF reacted to nicotine in different ways, depending on the concen- trations and the exposure times used, but had identical reactions following double exposure. With the Hoechst DNA assay, 600μg/ml nicotine was found to affect the growth of non-smoker HGF after long or repeated exposure, while smoker HGF were affected only by repeated expo- sure; growth of L-929 cells was not affected. It was concluded that HGF from smokers are able to sustain higher concentrations of nicotine without adverse effects than are non-smoker HGF and L-929 cells. If this occurs in vivo, nicotine would not be considered to be a major toxicant to HGF in smokers. Key words: human gingival fibroblasts, HGF, nicotine, NRU assay, Hoechst DNA assay.