Abstract During the last few years, there has been in- creasing interest in buccal epithelial cells for cytogenetic evaluation of different materials. In the present study, the use of these cells and peripheral lymphocytes for cytoge- netic evaluation of chlorhexidine digluconate (CHX) with comet assay (single cell gel electrophoresis, or SCGE) is reported. This technique detects DNA strand breaks in individual cells in alkaline conditions. Thirteen volunteers were requested to rinse their mouths with 0.12% CHX solution for 18 days. Buccal epithelial cells and peripheral blood lymphocytes were obtained from all participants at baseline and the end of the experimen- tal period. One hundred cells per subject were analysed for the DNA damage. A statistical increase was observed in the damaged buccal and blood cells after the CHX ap- plication. The mean grade of damage in buccal cells was statistically different from that in blood cells. Due to minimal absorption of chlorhexidine into the tissues and low concentrations of free chlorhexidine in the oral cavi- ty, the DNA damage produced by chlorhexidine in lym- phocytes was lower than in buccal epithelial cells. As chlorhexidine does not accumulate in the body, the fre- quencies of DNA damage could be transient. Detected DNA damage after CHX use might be the indication of an earlier effect, before DNA repair begins, and could be reversible. Keywords Chlorhexidine · Comet assay · DNA damage · Human monitoring · Mouthrinse Introduction Chlorhexidine digluconate (CHX) is a symmetrical cat- ionic molecule consisting of two 4-chlorophenyl rings and two biguanide groups connected by a central hexa- methylene chain. The initial use of CHX in dentistry in- cluded washing operation sites and disinfecting root ca- nals. In the late 1960s, CHX’s antiplaque properties were reported and, since then, the ability of CHX to reduce plaque accumulation has been documented [13]. The most common preparation of CHX contains digluconate salt because of its high water solubility. Although the clinical benefits of CHX have been proved in several trials [3, 7, 9, 27, 31], even in low con- centrations, it was reported to be toxic for a variety of cell types such as gingival fibroblasts, epithelial cells, macrophages, neutrophils, and red blood cells in culture [1, 2, 6, 8, 11, 12]. Moreover, in an animal study, topical application of CHX was reported to result in its penetra- tion through the epithelial barrier, causing tissue damage [10]. Withrow et al. [32] reported CHX to be toxic for mouse lymphoma cells. When these cells were exposed to ultraviolet A radiation, CHX was reported to be muta- genic. Furthermore, the breakdown products of CHX, i.e., parachloroaniline, have also been considered to be mutagenic [5, 15, 30]. Some doubts over the long-term use of CHX mouthrinse arose due to the reports on cyto- toxic effects. Single cell gel electrophoresis (SCGE) is a technique that has been extensively used in fundamental DNA re- pair studies, biomonitoring of the population, and DNA damage evaluations [16, 17, 22, 23]. This technique in- volves embedding cells in agarose gel on microscope slides and lysing with detergent and high salt. The slides are then soaked in an alkaline solution to allow cleavage of DNA at alkali-labile sites. During electrophoresis un- der alkaline conditions, cells with damaged DNA display increased migration of DNA from the nucleus towards the anode. Broken DNA migrates farther in the electric field, and the cell then resembles a “comet” with a brightly fluorescent head and a tail region [16]. K. Eren · N. Özmeriç ( ✉ ) Department of Periodontology, Faculty of Dentistry, Gazi University, 8. cadde 84. sokak 06510, Emek Ankara, Turkey e-mail: nozmeric1@superonline.com Tel.: +90-533-2306640, Fax: +90-312-2121646 S. S ¸ardas ¸ Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey Clin Oral Invest (2002) 6:150–154 DOI 10.1007/s00784-002-0168-1 ORIGINAL ARTICLE Kaya Eren · Nurdan Özmeriç · Semra S ¸ ardas ¸ Monitoring of buccal epithelial cells by alkaline comet assay (single cell gel electrophoresis technique) in cytogenetic evaluation of chlorhexidine Received: 27 September 2001 / Accepted: 28 April 2002 / Published online: 25 July 2002 © Springer-Verlag 2002