Letter to the Editor Comment on a letter written by Ribeiro DA ‘‘Risk assessment of oral cancer in patients with pre-cancerous states of the oral cavity using micronucleus test and challenge assay’’ [Saran R, Tiwari RK, Reddy PP, Ahuja YR. Oral Oncol 2008;44:354–60] Sir, Upon carefully reading a recent letter written by Ribeiro DA 1 with comments on the methodology used in the study ‘‘Risk assessment of oral cancer in patients with pre-cancerous states of the oral cavity using micronucleus test and challenge assay’’ [Saran R, Tiwari RK, Reddy PP, Ahuja YR. Oral Oncol 2008;44:354–60], we decided to contribute to the topic by pre- senting a modification of the comet assay methodology used in our recent research. 2,3 Such a modified method proposes improve- ments that enabled us to overcome several shortcomings of the co- met assay in the study of buccal cells (e.g. low cell viability, saprophyte bacterial contamination and difficult cell lysis due to highly resistant specialized cellular membrane). 4 In his letter, Ribeiro DA 1 suggests that the single cell gel (comet) assay, as it is commonly performed, may not be suitable for geno- toxicity assessment in human oral mucosa cells, since only a small number of cells from oral mucosa samples yield comets that can be analyzed by current methods. This is due to the fact that oral cells maintained massive DNA damage with subsequent disintegration, and that majority of oral mucosa cells which do form comets, are probably leukocytes as already reported by Osswald et al. 5 Contrarily, other authors 4,6 did not report such a result, proba- bly because the mouth was thoroughly rinsed prior to sampling and disposal of the first scrapping. By washing out the mouth thoroughly three times with tepid water prior to sampling, we managed to remove the majority of dead exfoliated cells. Conse- quently, our samples contained 90% epithelial cells and only 10% leukocytes which conforms with the results of Pinhal et al. 4 Similar results on lower leukocyte frequencies upon mouth washing were obtained by Endler et al. 6 that reported on samples comprising 75% epithelial cells and 25% leukocytes. Moreover, upon sampling we stirred the brushes in 5 ml of RPMI (RPMI 1640 Medium, Gibco-Invitrogen, Carlsbad, CA, USA), liquid (with L-Glutamine, 25 mM HEPES), Fetal Bovine Serum (Gib- co-Invitrogen, Carlsbad, CA, USA) and Penicillin–Streptomycin solution (Sigma–Aldrich, Munich, Germany). The samples were transported to the laboratory for further processing within 30 min to increase cell viability and prevent saprophyte bacterial contamination of oral buccal cells, which ultimately lead to low efficiency of the SCGE assay. Such a procedure yielded high cell viability values and prevented saprophyte bacterial contamination, which might be correlated with a high efficiency of SCGE assay. Oral cells are generally highly resistant to lyses. 1,4 However, possible cell disruption induced by trypsin during sample prepara- tion 7 , might lead to an misinterpretation of final comet assay results. Accordingly, we avoided an use of trypsin or proteinase K-for sample preparation. For the single cell gel (comet) assay, the cells were therefore re-suspended in a chilled buffer pH 7.5 [0.075 M NaCl (Kemika, Zagreb, Croatia) and 0.024 M Na 2 EDTA (Sigma–Aldrich, Munich, Germany)] and macerated with a glass stick on ice for 2 min. Na 2 EDTA was employed for inhibiting nucle- ase activity and DNA damage. Standard procedures including elec- trophoresis followed by neutralization and staining were employed upon cell lysis. Slides were analyzed by using Leitz Orthoplan epifluorescence microscope (Wetzlar, Germany) at a magnification 250Â followed by analysis with the Comet Assay IV image analysis system (Perceptive Instruments Ltd. Suffolk, Hal- stead, UK). One hundred randomly selected cells (50 cells on each of two replicate slides) for each subject were scored. DNA damage was evaluated as percentage of DNA in the tail (%DNA) and tail length, which was measured from the centre of the comet head. 2,3 Ribeiro 1 points out that The International Comet Assay Expert Group recommends the use of image analysis of comets, with the measurement of parameters such as percentage of DNA in the tail (percent migrated DNA) and/or tail moment (fraction of migrated DNA multiplied by some measure of tail length). Differ- ent authors are in line with this recommendation 1,8,9 as these parameters were shown to give a clear indication of the appear- ance of the comet, and in addition are linearly related to the DNA break frequency over a wide range of levels of damage. However, Dhawan and Anderson 10 report on the IWGT (Inter- national Workshops on Genotoxicity Testing) consensus stating that image analysis is preferred but not required. Indeed, percent- age of DNA in tail is more linearly related to the dose than the tail moment and therefore easier to intuitively understand. The tail moment values may vary if different image analysis systems are employed which might lead to quantitative differences and poor comparison of inter-laboratory data. The percentage of DNA in the tail is considered to be the easiest comparable value between laboratories. No consensus has been reached stating that the per- centage of DNA in tail should be the only parameter used. How- ever, if measurements of tail moment are used, then percentage of DNA in the tail and tail length data must be additionally provided. 10 Heavily damaged cells potentially representing dead or dying cells (commonly referred as head hogs) were not included in our study of buccal cells. 2,3 However determining their frequency may be useful for data interpretation. 10 We hope that our experience might clarify some of the issues encountered by researchers while performing comet assay on buc- cal cells. Inclusion of experimental steps for sample preparation re- ported in our studies might lower shortcomings in buccal cells damage analyses by comet assay. We believe that advantages that include minimal invasiveness during sampling and easy availabil- ity of these cells are worthy of further optimization of the method and its wider use for preliminary genotoxicity screening. 1368-8375/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2011.01.007 Oral Oncology 47 (2011) 777–778 Contents lists available at ScienceDirect Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology