PROTOCOL

Buccal micronucleus cytome assay

Philip Thomas1, Nina Holland2, Claudia Bolognesi3, Micheline Kirsch-Volders4, Stefano Bonassi5, Errol Zeiger6, Siegfried Knasmueller7 & Michael Fenech1
1CSIRO

Human Nutrition, Adelaide, South Australia. 2School of Public Health, University of California, Berkeley, CA, USA. 3Unit of Environmental Carcinogenesis, National Cancer Research Institute, Genoa, Italy. 4Laboratory for Cell Genetics, Vrije Universiteit, Brussels, Belgium. 5Unit of Molecular Epidemiology, National Cancer Research Institute, Genoa, Italy. 6Errol Zeiger Consulting, Chapel Hill, NC, USA. 7Institute of Cancer Research, Inner Medicine I, Medical University Vienna, Austria. Correspondence should be addressed to P.T. (philip.thomas@csiro.au) or M.F. (michael.fenech@csiro.au). Published online 7 May 2009; corrected online 22 September 2011 (details online); doi:10.1038/nprot.2009.53

2009 Nature Publishing Group http://www.nature.com/natureprotocols

The Buccal Micronucleus Cytome (BMCyt) assay is a minimally invasive method for studying DNA damage, chromosomal instability, cell death and the regenerative potential of human buccal mucosal tissue. This method is increasingly used in molecular epidemiological studies for investigating the impact of nutrition, lifestyle factors, genotoxin exposure and genotype on DNA damage, chromosome malsegregation and cell death. The biomarkers measured in this assay have been associated with increased risk of accelerated ageing, cancer and neurodegenerative diseases. This protocol describes one of the current established methods for buccal cell collection using a small-headed toothbrush, the generation of a single-cell suspension, slide preparation using cytocentrifugation, xation and staining using Feulgen and Light Green for both bright eld and uorescence microscopic analysis. The scoring criteria for micronuclei and other nuclear anomalies are also described in detail. The protocol in its current form takes approximately 4 h to complete from the time of buccal cell collection to the generation of stained slides for microscopic analysis.

INTRODUCTION The regenerative capacity of tissues and organs in the body is and/or quids, medical treatments, such as radiotherapy as fundamental to healthy ageing. Regeneration is dependent on the well as occupational exposure to potentially mutagenic and/or number and division rate of the proliferating (basal) cells, their carcinogenic chemicals, and for studies of chemoprevention of genomic stability and their propensity for cell death1,2. These events cancer13,1820. With regard to exposure to radiation it has been can be studied in the buccal mucosa (BM), which is an easily shown by Moore et al.21 that the BMCyt system can detect a 16-fold accessible tissue for sampling cells in a minimally invasive manner increase in micronucleus (MN) frequency in oral cancer patients and does not cause undue stress to study subjects313. This method after completion of treatment with photons. The BM also has the is increasingly being used in molecular epidemiological studies to potential to be utilized to identify inherited genomic instability investigate the impact of nutrition, lifestyle factors, genotoxin such as Blooms Syndrome22. In addition, the BMCyt protocol has been used to measure exposure and genotype on DNA damage and cell death38. The use of cells from the BM (Figs. 13) provides a unique opportunity distinct differences between the cytome proles associated with to study the regenerative capacity of the epithelial tissue, which is of ectodermal Oral cavity origin in humans. The assay has been used Stratum corneum successfully both in our laboratory and in Stratum others to study DNA damage as measured granulosum by micronuclei (MNi) or by the use of uorescent probes to detect aneuploidy, Stratum chromosomal breaks and changes in telospinosum mere length913. The proportion of basal Stratum cells and cells undergoing cell death in BM germinativum is an indication of the regenerative capacity of this tissue. Connective tissue The BM provides a barrier to potential carcinogens that can be metabolized to generate potential reactive products14,15. Basal cell Karyorrhectic cell As up to 90% of all cancers appear to be Binucleated cell Condensed chromatin cell Basal cell with MN epithelial in origin, the BM could be used to monitor early genotoxic events as a result of Differentiated cell with NBUD Differentiated cell Pyknotic cell potential carcinogens entering the body through ingestion or inhalation13,16,17. Karyolytic cell Differentiated cell with MN Exfoliated buccal cells have been used non-invasively to successfully show the Figure 1 | Diagrammatic representation of a cross section of normal buccal mucosa. The mucosa of genotoxic effects of lifestyle factors such as healthy individuals illustrating the different cell layers and possible spatial relationships of the various tobacco smoking, chewing of betel nuts cell types are shown.

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Oral cavity normal ageing relative to that for premature ageing clinical outcomes such as Downs syndrome and Alzheimers disease. These Karyolytic studies highlight the potential diagnostic cells value of the cytome approach for determinDifferentiated cell and dying ing genome instability events and measurcell layer Condensed Karyorrhectic Pyknotic ing regenerative potential9,23. chromatin cell cell cell 721 The BM is a stratied squamous epithedays lium consisting of four distinct layers Differentiated cell Binucleated Differentiated cell Differentiated cell cell with MN with NBUD for cells 1,2,24,25 . The various cell types, (Fig. 1) to nuclear anomalies and possible spatial migrate interrelationships among the various cell from types in the BM, which are observed and Basal Basal Normal Binucleated cell cell Basal cell basal Basal cell scored in a BMCyt assay, are shown diawith NBUD with MN layer grammatically in Figures 13 (refs. 9,13). Basal cell layer to The stratum corneum, or the keratinized cell Basal cell oral layer, lines the oral cavity comprising cells that are constantly being shed as a result of cavity Stem cell Daughter stem cell wear and tear of the surface tissue. Below Connective tissue this layer, lies the stratum granulosum, or the granular cell layer, and the stratum Figure 2 | Sequential origins and spatio-temporal sequence of the various cell types in the BMCyt assay. spinosum, or the prickle cell layer, contain- Shown is the time frame of events from a stem cell to a basal cell and subsequent differentiation and cell ing populations of differentiated, apoptotic death and the different types of genetic damage that can occur. and necrotic cells. Beneath these layers are the rete pegs or stratum germinativum, containing actively dividing basal cells and basal stem cells, which used to measure aneuploidy by determining the frequency of nuclei produce progeny that differentiate and maintain the prole, with abnormal chromosome number10. Tandem probes have been structure and integrity of the BM1,13,26,27. Figure 2 shows the successfully applied to measure chromosome breaks in specic sequential spatiotemporal origins of the various cell types and important regions of the genome10,31,32. The use of such molecular the time frame from a daughter basal cell originating in the basal probes to gain further information on the genomic damage in layer to its eventual differentiation, migration and exfoliation at the buccal cells is shown in Figure 4. The methodology and concepts described in this protocol may be BM surface layer. The time frame for cellular migration from the basal layer to the keratinized surface layer is thought to range from applied to other types of exfoliated cells such as those of the 7 to 21 d, although experimental data investigating such migration bladder, nose and cervix but the morphological characteristics, rates are limited2,26,28. In this assay cells derived from the BM are harvested from the inside of a patients mouth using a small-headed toothbrush. The Buccal Cytome Model2008 cells are washed to remove the debris and bacteria, and a single-cell suspension is prepared and applied to a clean slide using a Normal genome cytocentrifuge. The cells are stained with Feulgen and Light Basal cell Green stain allowing both bright eld and permanent uorescence Normal Chromosome analysis that can be undertaken microscopically. Healthy normal breakage or loss cells can then be distinguished from those considered abnormal Apoptotic cell death based on the nuclear-to-cytoplasmic ratio and nuclear morphology (morphogenetic Gene amplification or induced by and texture (Fig. 3). Micronucleated genome damage) The BMCyt assay has been used to measure biomarkers of DNA Cytokinesis damage (micronuclei and/or nuclear buds), cytokinetic defects defect (binucleated cells) and proliferative potential (basal cell frequency) Necrotic and/or cell death (condensed chromatin, karyorrhexis, pyknotic Cell death? Nuclear bud and karyolytic cells). The following BMCyt protocol describes one Condensed of the current established methods for buccal cell collection, slide chromatin preparation, cellular and nuclear staining and scoring criteria. The various cell types and aberrations that are scored in the BMCyt assay are illustrated in Figure 3. The protocol can also make use of Karyolysis Binucleated Karyorrhexis Pyknosis molecular probes for DNA adduct, aneuploidy and chromosome break measures within the nuclei of buccal cells2931. It is possible to Figure 3 | Diagrammatic representation of the various cell types scored in use antibodies to measure DNA adducts such as those induced by the BMCyt assay. Analyses of various cell types is done on the basis of the polycyclic aromatic hydrocarbons in the nuclei of buccal cells29. scheme proposed by Tolbert et al.27 and recent observations in studies of the Furthermore, chromosome-specic centromeric probes have been effect of ageing9.

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sampling and scoring methods are neither properly described nor standardized for cells from these tissues. Other methods currently available for the BMCyt use tongue depressors, tooth picks and metal spatulas to collect buccal cells; however, the number of cells collected is usually less than those collected using a toothbrush3336. Buccal smears can be applied directly to a clean slide but invariably the cells are clumped and contain bacteria and debris that can hamper accurate cytological analysis upon staining (P.T. unpublished observation). Several different staining techniques for buccal cells have been reported9,33,37,38. However, DNA-specic stains such as acridine orange or propidium iodide are recommended in comparison with Romanowsky stains such as Giemsa or Leishmanns, as the former stains minimize the possibility of scoring false positives for DNA damage events such as micronuclei, which often occur with Romanowsky stains13. Experimental design When considering the study design for biomonitoring using cells of the BM, it is most important to outline a clear hypothesis and ensure that the study is sufciently powered to measure a biologically meaningful effect size. The important considerations for study design are detailed below. Important demographic and exposure variables. It is important to establish normal range values for the different buccal cytome biomarkers from healthy control subjects that have not been exposed to abnormal doses of genotoxins and cytotoxins. This allows an investigator to identify key variables affecting the observed frequency of the biomarkers such as age, gender, vitamin B status (particularly folate and vitamin B12), genotype and smoking status. Detailed information on genotoxin exposure (e.g., type of toxin, duration of exposure, commencing date of exposure relative to sampling date of buccal cells) is also required in order to achieve a meaningful interpretation of data. This may be useful in identifying a threshold dose and the time frame of exposure required for a particular genotoxin to produce a measureable change in BMCyt biomarkers. It is important in casecontrol studies that the groups are matched properly for these variables. For further information on the use of controls and study design in biomonitoring studies the reader is referred to Albertini et al.39. Table 1 provides a list of important variables to be recorded when using the BMCyt assay in epidemiological studies. Sampling time in acute and chronic exposure. As the buccal cells turn over every 721 d, it is theoretically possible to observe the genotoxic effects of an acute exposure approximately 721 d later13,21,40. However, additional sampling at later time points may be required depending on the extent to which cell division has been inhibited by the genotoxin. Ideally, repeat sampling, at least once every 7 d after acute exposure, should be performed for 28 d or more so that the kinetics and extent of biomarker induction can be thoroughly investigated. In the case of chronic exposure (e.g., due to habitual diet or alcohol consumption or smoking) it is recommended that multiple samples are taken at least once every 3 months to take into account seasonal variation. Uniformity of sampling. A circular expanding motion is used with toothbrush sampling to enhance sampling over a greater area
Quantify using image cytometry of spots by measuring area and optical density of spots Monosomic Normal diploid Chromosome specific centromeric probes Score visually or by automated spot counting trisomic Score visually or by automated spot counting

Fluorescent antibody to DNA adducts

Tandem probe labelling

Single break

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No breaks Two breaks

Figure 4 | Molecular probe methods for measuring DNA adducts, aneuploidy and chromosome breaks.

and to avoid continual erosion in a single region of the BM. This is performed on the inside of both cheeks using a different brush (for sampling left and right areas of the mouth) to maximize cell sampling and to eliminate any unknown biases that may be caused by sampling one cheek only. It is important to keep the sampling method constant as distribution of cell types could vary slightly depending on the method used. It is important to note that repeated vigorous brushing of the same area can lead to increased collection of cells from the less differentiated basal layer23. Table 2 shows the effect of repeated sampling on the results of the BMCyt assay. The results in Table 2 show that the frequency of basal cells and karyorrhectic cells tends to increase with repeated sampling (P trend o 0.001 and P 0.048 respectively), but no change is observed for other biomarkers in the BMCyt assay. Transport and storage. In some investigations buccal cells may have to be collected from a distant site. This could result in a delay of several days before samples are delivered to the laboratory for

TABLE 1 | Important variables to record when using the BMCyt assay in epidemiological studies.

          

Date of Birth Gender Body mass index Smoking status and history Alcohol consumption status and history History of cancer, cardiovascular disease and neurodegenerative disease Medication or recent illness (disease history) Inherited mutations that predispose to degenerative diseases listed above as well as accelerated ageing syndromes (e.g., BRCA1 and ATM mutations) Dietary habits measured using a validated food frequency questionnaire Exposure to chemical carcinogens and radiation e.g., X-ray Genotype
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TABLE 2 | Changes in the ratios of buccal cell types (mean, standard deviation and P values) following sequential buccal cell sampling.
Micronuclei Nuclear buds Basal cells Binucleated Condensed Karyorrhectic Pyknotic Karyolytic cells per 1,000 chromatin cells cells per 1,000 cells per 1,000 cells per 1,000 per 1,000 per 1,000 per 1,000 Sequential samples cells cells cells cells per 1,000 cells cells cells cells Sample 1 (0 min) 0.50 0.57 0.50 0.57 28.75 12.79 9.25 4.78 25.75 15.59 20.3 13.1 0.50 0.57 156.5 118.4 Sample 2 (90 mins) 0.50 0.57 0.25 0.50 51.5 7.9a 12.0 4.16 22.5 11.47 29.0 16.79 0.7 1.5 111.8 49.17 Sample 3 (270 mins) 0.0 0.0 0.25 0.50 59.5 6.75a 9.75 3.78 30.0 5.1 31.0 18.31 0.0 0.0 135.5 49.17 11.5 5.82 21.25 2.06 29.3 19.21 0.0 0.0 102.0 32.30 Sample 4 (360 mins) 0.50 0.57 0.25 0.50 65.5 5.19a Sample 5 (450 mins) 0.25 0.5 0.0 0.0 84.75 6.0a,b 9.0 1.83 31.0 13.29 34.3 23.16 0.0 0.0 129.3 25.25 ANOVA P values 0.6272 0.6363 o0.001 0.7968 0.6749 0.2320 0.5032 0.6696 P value for linear trend 0.5744 0.1742 o0.001 0.8841 0.6146 0.0489 0.1672 0.4729
Buccal cells were collected from volunteers (n 4, 2 male, 2 female, age range 4050 yrs) over ve different time points, 0, 90, 270, 360 and 450 mins. Buccal cells were collected as outlined in the cell sampling and preparation part of this manuscript. Slides were stained as outlined in the Feulgen staining steps of this protocol. Slides were classied and scored as outlined under the scoring criteria detailed in this protocol. Statistical analysis for mean, standard deviation and one-way ANOVA and linear P trends were performed using Graphpad PRISM (Graphpad inc., San Diego, CA). Signicance was accepted at P o 0.05. aDenotes a signicant difference from sampling time at 0 min. bDenotes a signicant difference from time 0, 90, 270 and 360 mins.

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analysis, which may cause sample deterioration. In this case, a reliable method for preserving samples before delivery is required and an appropriate procedure for this purpose is described in the Buccal Cell Collection option B in the PROCEDURE (Buccal cell collection). Effects of ltration. We have performed experiments to investigate whether the ltration process in the preparation of the singlecell suspension has adverse effects on cell population ratios. The larger cell aggregates that are ltered out show the same cellular population ratios as the eventual single-cell suspension used in the slide preparation for analysis. This means that removal of cell clumps by ltration, which facilitates slide scoring, does not select against a particular cell type, which would otherwise have had signicant effects on both data analysis and interpretation. Cell xation and nuclear staining. It is possible to use alternative xative solutions such as methanol:glacial acetic acid (3:1) or 80% methanol, but the staining of both cytoplasm and nuclei may be altered slightly relative to the use of ethanol:glacial acetic acid (3:1). One of the unique features of the Feulgen staining technique used in this protocol is that the DNA material appears as bright red in color when viewed under uorescence with a far-red lter (emission wavelength range of 580620 nm). This is important because cells identied as containing MNi or other anomalies (e.g., nuclear buds) on the bright eld can be conrmed as being positive by examining the cells under uorescence. Furthermore, the nuclear texture, which is essential in classifying condensed chromatin and karyorrhectic cells, is usually easier to discern using uorescence microscopy. This minimizes the incidence of false positives or false negatives, thereby giving a more accurate assessment of DNA damage and nuclear anomaly events. Staining artifacts. Earlier studies have shown that false-positive results in the MN frequency can be obtained as a result of using Romanowsky-type stains such as Giemsa, May-Grunwald Giemsa and/or Leishmanns, which leads to inaccurate assessment of DNA damage13,41. In a study investigating MN frequency in relation to the staining techniques in the BM of smokers against non-smokers, a 4- to 5-fold increase in MN frequency in smokers was found using Romanowsky stains, which are not DNA-specic41,42. However, when a specic DNA uorescent dye (e.g., 4, 6-diamidino-2phenylindole (DAPI), Feulgen or Acridine orange) was used there were no signicant differences between these groups41. Romanowsky
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stains have been shown to increase the number of false positives as they positively stain keratin bodies that are often mistaken for micronuclei and are therefore not appropriate for this type of analysis41. For these reasons, it is advisable to avoid Romanowsky stains in favor of DNA-specic uorescent-based stains such as propidium iodide, DAPI, Feulgen, Hoechst 33258 or Acridine Orange42. It is recommended that Feulgen be used because permanent slides can be obtained that can be viewed under both transmitted and/or uorescent light conditions. Criteria for identifying and scoring cell types in the buccal micronucleus cytome assay. The scoring criteria for the various distinct cell types and nuclear anomalies in the BMCyt assay are mainly based on those originally described by Tolbert et al.27. These criteria are intended for classifying buccal cells into categories that distinguish between normal cells and cells that are considered abnormal on the basis of cytological and nuclear features, which are indicative of DNA damage, cytokinetic failure or cell death. These criteria are summarized in Table 3 and photomicrographs of cell types and nuclear anomalies are shown in Figure 5. A more detailed description of the scoring criteria for the BMCyt assay cell types is outlined below. Normal basal cells have a larger nucleus-to-cytoplasm ratio than the differentiated buccal cells (Fig. 5a). Basal cells have a uniformly stained nucleus and are smaller in size and more oval in shape when compared to the more angular and at differentiated buccal cells. No DNA-containing structures apart from the nucleus are observed in these cells. The cytoplasm is typically stained a darker shade of green with Light Green compared to the differentiated cells. Normal differentiated cells (Fig. 5b) have a uniformly stained nucleus, which is oval or round in shape. They are distinguished from basal cells by their larger size and by a smaller nucleus-tocytoplasm ratio. No other DNA-containing structures apart from the nucleus are observed in these cells. These cells are considered to be terminally differentiated relative to basal cells, as no mitotic cells are observed in this population2. Cells with micronuclei (Fig. 5c,d) are characterized by the presence of both a main nucleus and one or more smaller nuclear structures called micronuclei (MNi). The micronuclei are round or oval in shape and their diameter should range between 1/3 and 1/16 of the main nucleus. MNi have the same staining intensity and texture as the main nucleus. Most cells with MNi will contain only one MN but it is possible to nd cells with two or more MNi. Baseline frequencies for micronucleated cells in the BM are usually

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TABLE 3 | Criteria for classication of BMCyt cell cytome assay cell types based on morphological features of cells stained with Feulgen/Light Green with reference to photomicrographs shown in Figure 5aj. Buccal cell type Basal Morphological features Large nucleus: cytoplasm ratio relative to differentiated cell Smaller and more oval than differentiated cells Uniformly stained nucleus Darker green cytoplasm relative to differentiated cell when viewed under transmitted light Smaller nuclear: cytoplasmic ratio More angular and atter than basal cells Uniformly stained round nucleus Contains both main nucleus and micronucleus Micronuclei are round or oval with similar stain intensity as main nucleus Micronuclei usually have 1/31/16 diameter of main nucleus Micronuclei must be located in cellular cytoplasm Scored in basal and differentiated cells only Main nucleus has a sharp constriction forming a bud Bud is attached to main nucleus Bud has similar staining intensity as main nucleus Bud diameter can be quarter to half nuclear diameter Cells contain two main nuclei Nuclei are of similar size and staining intensity Nucleus shows areas of aggregated chromatin Distinct areas of nucleus are more intensely stained Nucleus exhibits striated pattern Nucleus has extensive aggregated chromatin Nuclear fragmentation may be evident Cell has small shrunken nucleus Nucleus is uniformly and intensely stained Nucleus diameter is 1/32/3 diameter of normal nucleus Nucleus is depleted of DNA Nucleus is not stained by Feulgen Figure 5a

Differentiated

5b

Micronucleated

5c,d

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Nuclear bud

5e

Binucleated Condensed chromatin

5f 5g

Karyorrhectic Pyknotic

5h 5i

Karyolytic

5j

within the 0.52.5 MNi/1,000 cells range13. Cells with multiple MNi are rare in healthy subjects but become more common in individuals exposed to radiation or other genotoxic agents. The nuclei in micronucleated cells have the morphology of nuclei in normal cells. The MNi must be located within the cytoplasm of the cells. The presence of MNi is indicative of chromosome loss or fragmentation occurring during earlier nuclear division13,43. MNi are scored only in differentiated cells with uniformly stained nuclei. It is possible to score MNi in basal cells, but this is impractical owing to the low frequency of this cell type. Cells, which are pyknotic (i.e., shrunken nuclei), and have condensed chromatin or karyorrhectic nuclei (see below), are not scored for MNi. Cells with nuclear buds (Fig. 5e) contain nuclei with an apparent sharp constriction at one end of the nucleus suggestive of a budding process, i.e., elimination of nuclear material by budding. In the original Tolbert et al. publication27 these were referred to as broken egg cells. The nuclear bud (NBUD) and the nucleus are usually in very close proximity and appear to be attached to each other. The nuclear bud has the same morphology and staining properties as the nucleus; however, its diameter may range from a half to a quarter of that of the main nucleus. The mechanism leading to nuclear bud formation is not known but it may be related to the elimination of amplied DNA or DNA repair4447. Binucleated cells (Fig. 5f ) are cells containing two main nuclei instead of one. The nuclei are usually very close and may touch each

other and usually have the same morphology as that observed in normal cells. The signicance of these cells is unknown, but they are probably indicative of failed cytokinesis following the last nuclear division in the basal cell layer. It has recently been shown that chromosomal non-disjunction occurs with a higher frequency in binucleated cells that fail to complete cytokinesis, rather than in cells that have completed cytokinesis48. This mechanism identied recently is thought to be a cytokinesis checkpoint for aneuploid binucleated cells48. The binucleate:mononucleate cell ratio may therefore prove to be an important biomarker for identifying individuals with a cytokinesis failure caused by higher-than-normal rates of aneuploidy, such as that observed in Downs syndrome9,10. Buccal cells with condensed chromatin (Fig. 5g) show a roughly striated nuclear pattern in which the aggregated chromatin is intensely stained. Similar nuclear morphologies have also been shown in other cell types49,50. In these cells it is apparent that chromatin is aggregating in some regions of the nucleus while being lost in other areas. When chromatin aggregation is extensive the nucleus may appear to be fragmenting51. These cells may be undergoing early stages of apoptosis, although this has not been shown conclusively. These cells as well as karyorrhectic cells invariably result in fragmented nuclei, leading to eventual disintegration, and sometimes appear to contain bodies similar to MNi, but these are not scored as MNi in the assay as their origin cannot be accurately determined.
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Figure 5 | Images of the different cell types stained using Feulgen and Light Green scored in the BMCyt assay viewed by transmitted light or under uorescence with a far red lter; (a) basal cell; (b) differentiated cell; (c) early differentiated cell with micronucleus (arrow); (d) late differentiated cell with micronucleus (arrow); (e) differentiated cell with nuclear bud (arrow); (f) binucleated cell; (g) condensed chromatin cell; (h) karyorrhectic cell; (i) pyknotic cell; (j) Karyolytic cell. Upper panels light microscopy, lower panels uorescence microscopy. All images were taken at 1,000 magnication.

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Karyorrhectic cells (Fig. 5h) have nuclei that are characterized by more extensive nuclear chromatin aggregation relative to condensed chromatin cells. They have a densely speckled nuclear pattern indicative of nuclear fragmentation leading to the eventual disintegration of the nucleus51,52. These cells may be undergoing a late stage of apoptosis, but this has not been conclusively proven. These cells should not be scored for MNi in the assay. Pyknotic cells (Fig. 5i) are characterized by a small shrunken nucleus, with a high density of nuclear material that is uniformly but intensely stained51,52. The nuclear diameter is usually one- to two-thirds of a nucleus in normal differentiated cells. The biological signicance of the pyknotic cells and the mechanism leading to their formation are unknown, but it is thought that these cells may be undergoing a unique form of cell death; however, the precise mechanism remains unknown. They may represent an alternative mechanism of nuclear disintegration that is distinct from the process leading to the condensed chromatin and karyorrhectic cell death stages13,53. Karyolytic cells (Fig. 5j) are cells in which the nucleus is completely depleted of DNA and is apparent as a ghost-like image that has no Feulgen staining51,52. Therefore, these cells appear to have no nucleus and represent a very late stage in the cell death process. Scoring method. Slides should be coded before scoring by a person not involved in the experiment so that the person who scores the slides is not aware of the treatment conditions, individual or groups to which the cells on the slides belong. Slides are best examined at 1,000 magnication using a good-quality bright eld and/or uorescence microscope (e.g., Leica DMLB, Nikon Eclipse E 600). The BMCyt assay is amenable to automated scoring by image cytometry, but these systems, which could potentially improve applicability in population studies, have yet to be developed and validated54. The optimal way to score slides in the BMCyt assay is to rst determine the frequency of all the various cell types in a minimum of 1,000 cells. Following this step, the frequency of DNA damage biomarkers (MNi and NBUDs) is scored in a minimum of 2,000 differentiated cells. A ow diagram of the scoring procedure is shown in Figure 6. The frequency of MNi and NBUDS could be scored in basal cells, but this is impractical because of the very low frequency of these cells.
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From each slide, the following information should therefore be recorded: 1. The number of basal and differentiated cells per 1,000 cells scored. 2. The number of pyknotic, condensed chromatin, karyorrhectic and karyolytic cells per 1,000 cells. 3. The number of binucleates per 1,000 cells scored. 4. The number of MNi and NBUDs in at least 2,000 differentiated cells. Preferably, cells are scored using both bright eld and uorescence microscopy. Cells containing MNi or NBUDS on bright eld should be conrmed as being positive for these biomarkers by examining the cell under uorescence. The incidence of false positives can be minimized as DNA materials such as nuclei and MNi uoresce brightly red when viewed under uorescence with a far-red lter (emission, wavelength range of 580620 nm). Cell inclusions or debris that do not contain DNA are not expected to uoresce under these conditions. 5. The frequencies of the various cell types in the assay are represented either as the number of cells in a 1,000 cells or as a percentage. For example, should 187 basal cells be scored in 1,000 cells, then the frequency may be reported either as 187% or 18.7%.
SCORING PROCEDURE FLOW CHART

STEP 1 Score 1,000 cells to determine the frequency of these cell types: Basal Differentiated Binucleated Condensed chromatin Karyorrhectic Pyknotic Karyolytic

STEP 2 Score 2,000 differentiated cells for presence of MNi and NBUD

Figure 6 | Flow diagram describing scoring method.

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Information that should be included on a score sheet for the BMCyt assay is shown below: 1. Name of the person scoring the slides. 2. Code number of each slide. 3. Total number of buccal cells scored. 4. The number of basal cells and differentiated cells per 1,000 cells scored. 5. The number of pyknotic, condensed chromatin, karyorrhectic and karyolytic cells per 1,000 cells. 6. The number of binucleated cells per 1,000 cells scored. 7. The number of MNi and NBUDs in 2,000 differentiated cells.
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2.

3.

4.

This information is then used to calculate (a) the frequency of the various cell types per 1,000 cells and (b) the frequency of MNi and NBUDs in differentiated cells per 1,000 cells. A typical score sheet is shown in Supplementary Table 1 online. Statistical methods and power analysis All international guidelines for genotoxicity tests recommend evaluating the biological signicance of a result followed by the use of statistics, when appropriate. The biological signicance of an effect should be critically assessed, considering its size, dose response, reproducibility and plausibility. As regards the statistical analysis of these buccal cytome biomarkers, a few basic issues should always be considered and properly addressed. 1. The frequency of micronucleated cells should be used as the main endpoint; however, the scoring of the number of micronuclei may also be used as a separate measure to show whether cells with multiple micronuclei were present. Given the rarity of cells bearing more than a single micronucleus there are no substantial differences between the two approaches, although

5.

6.

7.

measuring the frequency of micronucleated cells is more stable and should be preferred. The frequency of MN and all the other endpoints must be expressed as the number of events per 1,000 cells, independent of the number of cells scored. The size of the study groups must be evaluated in advance according to the results of statistical power calculations. Unless the presence of NBUDs or other endpoints is a specic target of the study, power estimates should be based on MN frequency. Given the common report of a skewed distribution of damaged cells, the normal distribution of all biomarkers should be tested before statistical analysis. In the case of signicant departure from normality, necessary data transformation should be applied before analysis. The presence of statistically signicant differences in the occurrence of damaged cells between the study populations should always be tested with parametric or non-parametric univariate analyses. A threshold value of P o 0.05 is generally recommended. The control for a confounding variable and the evaluation of effect modiers should be performed using multivariate statistical modeling. Given the count nature of these biomarkers, Poisson (in absence of overdispersion), negative binomial or lognormal models can be applied. The point estimate of effect is generally a mean ratio, and an appropriate condence interval (usually 95%) should be provided. The presence of several possible endpoints poses a problem of multiple comparisons. If the study is oriented toward hypothesis testing an appropriate correction for multiple comparisons should be considered.

MATERIALS

. Tris-HCl (Sigma, cat. no. T-3253) . Ethylenediaminetetraacetic acid tetra (EDTA) sodium salt
(Sigma, cat. no. E5391) . Sodium chloride (Sigma, cat. no. S5886) . Isoton II (Coulter Electronics, cat. no. 8546719) . Sodium hypochlorite Solution (125 ml per liter) (Chemwell Products, cat. no. 1791) . Dimethyl sulfoxide (DMSO), hybrimax, sterileltered (Sigma, cat. no. D2650) . Ethanol (Ajax Finechem, cat. no. 214-2.5L) . Glacial acetic acid (Ajax Finechem, cat. no. A1-2.5L) ! CAUTION Acetic acid is corrosive, a respiratory irritant and can cause serious burns. Fixative should be prepared in a fume hood or similar extraction cabinet and the following personal protection used: Tyvek gown, double nitrile gloves, P2 dust mask and safety glasses. . 50% Ethanol made up with milli-Q deionized water (18.2 O resistivity) . 20% Ethanol made up with milli-Q deionized water (18.2 O resistivity) . 5 M Hydrochloric acid (HCl) (Merck, cat. no. 101255Y) ! CAUTION HCl is corrosive, a respiratory irritant and can cause serious burns. The acid should be handled in a fume hood or similar extraction cabinet and the following personal protection used: Tyvek gown, double nitrile gloves, P2 dust mask and safety glasses. . Schiffs reagent (Sigma, cat. no. S-5133) ! CAUTION Schiffs reagent is a skin, eye and respiratory irritant and should be handled in a fume hood or similar extraction cabinet and the following personal protection used: Tyvek gown, double nitrile gloves, P2 dust mask and safety glasses. . 0.2% (wt/vol) aqueous Light Green (Gurrs, cat. no. 06477) . DePex (or DPX) mounting medium (Merck, cat. no. 3197) . Polyethylene glycol (Merck, cat. no. 29577)

REAGENTS

. Small-headed toothbrushes (2-cm head length) (Supply SA, cat. no. . 30 ml yellow-topped polystyrene containers (Sarstedt, cat. no. 60.9922.918) . 10 ml graduated sterile pipettes (Falcon, cat. no. 7551) . Milli-Q water purication system (Millipore Milli-Q, Adelab Scientic) . Swinnex lter holders 25 mm (Millipore, cat. no. MILSX0002500) . Nylon net lters 100 mm (Millipore, cat. no. MILNYH02500) . Cell counter (e.g., Coulter Electronics model ZB1). A hemocytometer . TV-10 polystyrene tubes (Sarstedt, cat. no. 60.9921.829) . Sterile plugged Pasteur pipettes 900 (2223 cm) (Chase, cat. no. 93P) . Syringes 10 ml (Crown Scientic, cat. no. SS+10S) . Needles 18G (Crown Scientic, cat. no. 2525RA) . Counting vials 15 ml (Johns Diluent vials, cat. no. DSV002) . Cytocentrifuge (e.g., Shandon Cytocentrifuge, Thermo Electron Corporation) . Cytocentrifuge cupssupplied with instrument from Thermo Electron . Filter cardsShandon 300 100 , thick, white, boxes of 200 (Thermo Electron . Microscope slides, frosted end, 76 mm 26 mm (300 100 ), 1 mm thick
(HD Scientic MZO2 110, HD Scientic)wiped with alcohol and allowed to dry before use. . Coplin jars, unit holds 5 single 300 100 (75 mm 25 mm) slides vertically or 10 slides back-to-back. Screw cap is white linerless polypropylene, which reduces solvent evaporation (Biolab, cat. no. 355) . Polypropylene slide staining rack, accommodates up to 20 slides with removable snap-on handle (70 mm 86 mm 21 mm), (Pro Sci Tech) . Coverslips, no. 1, 22 mm 50 mm (HD Scientic, cat. no. MZ LD2250) . Hand homogenizer (Wheaton Scientic, 0.1 mm0.15 mm gauge) NATURE PROTOCOLS | VOL.4 NO.6 | 2009 | 831 Corporation) Corporation can be used if an electronic cell counter is not available. 85300012)

EQUIPMENT

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. Microscope with excellent optics for bright-eld and uorescence
examination of stained slides at 1,000 magnication (e.g., Leica DMLB, Leica; Nikon Eclipse 600, Nikon) . Slide storage boxes (Kartell, cat. no. 278) . Paralm (Sigma, cat. no. P7793-1EA) REAGENT SETUP Buccal cell buffer To prepare 1 liter of buccal buffer, weigh 1.6 g TrisHCl, 38.0 g EDTA and 1.2 g of sodium chloride, and dissolve in 600 ml of Milli-Q water. Thoroughly dissolve the salts and adjust the volume to 1,000 ml. Adjust pH to 7.0 and autoclave at 121 1C for 30 min. The buffer can be stored for up to 3 months in sealed bottles at room temperature (approximately 1822 1C). 5 M Hydrochloric acid Schiffs reagent is commonly used to detect aldehydes, producing a red coloration. As aldehydes are not usually found free in nuclei they are induced by the use of 5 M HCl, which converts some of the deoxyribose in the DNA to aldehydes, which can then be detected by Schiffs reagent. During staining a negative control slide is included, which is not treated with 5 M HCl. Following treatment with Schiffs reagent, no nuclear magenta coloration should be observed in the negative control slide. This control is included to determine the efcacy of the acid treatment, toward the artifactual generation of aldehydes and the relative degree of positive Schiffs nuclear staining in the test slides. In order to determine the concentration of laboratory-sourced HCl, the molarity is derived from the stated specic gravity, which may vary among suppliers. The specic gravity of the British Drug House (BDH) HCl that is used in our laboratory is 1.18 g ml1, which is the equivalent of 1,180 g liter1. The molecular weight of HCl is 36.5 g mol1. The molarity can therefore be calculated by dividing the specic gravity by the molecular weight, resulting in an acidic molarity of 32.3 M (1,180/36.5). However, the HCl acid assay specications detailed on the accompanying product sheet indicate a 37% (vol/vol) acidic solution. In order to calculate the true molarity one has to adjust this percentage value. Our stock acid solution is found to have a molarity of 12 M by multiplying the calculated initial value of 32.3 M by 0.37 (37% vol/vol). In order to prepare a 200 ml working solution of 5 M HCl from our stock solution the following calculation is performed: 200 ml (desired volume) 5 M (desired concentration)/stock solution (12 M). This results in 83.3 ml of acid being required in our 200 ml working solution. The 5 M HCl solution should be freshly prepared each time before use. ! CAUTION Add 83.3 ml of acid slowly to 116.7 ml of water and not the reverse. This is important to avoid an exothermic reaction resulting in the generation of heat and potential splashing. This procedure should be performed in a well-ventilated fume hood with appropriate safety precautions. Ethanol/glacial acetic acid xative Ethanol is mixed with glacial acetic acid in the ratio of 3:1. The xative should be freshly prepared each time. This procedure should be undertaken in a well-ventilated fume hood and personal protection should be used. ! CAUTION Acetic acid is corrosive, a respiratory irritant and can cause serious burns. A xative should be prepared in a fume hood or similar extraction cabinet and the following personal protection should be used: Tyvek gown, double nitrile gloves, P2 dust mask and safety glasses. Sodium hypochlorite solution 80 ml of 12.5% (vol/vol) sodium hypochlorite is made up to 1 liter using Milli-Q deionised water (18.2 O resistivity) to give a working solution of 1% (vol/vol). Store the solution for 1 week at room temperature (1822 1C). Light Green cytoplasmic stain 500 ml of Light Green cytoplasmic stain is prepared by dissolving 1 g of Gurr Light Green powder in 450 ml of Milli-Q water. When dissolved make up to 500 ml and lter through Whatman No. 1 lter paper. Store in the dark at room temperature, where it should remain active for a few years. Saccomannos xative The Saccomannos xative consists of 50% (vol/vol) ethanol and 2% (vol/vol) polyethylene glycol diluted in water. Use 20 ml of the xative per subject. The xative can be stored for up to 3 months at 4 1C. EQUIPMENT SETUP Cytocentrifuge cups must be clean, rinsed six times in distilled or deionized water and completely dried before assembly. Microscope slides should be wiped with alcohol and allowed to dry before use.

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PROCEDURE Buccal cell collection TIMING B10 min 1| Before buccal cell collection, the mouth of the subject should be rinsed twice thoroughly with 100 ml of water to remove excess debris. ! CAUTION Human samples should be considered as infectious and the appropriate safety precautions should be taken. The appropriate institutional research ethics committees should approve the studies using human participants.

2| Buccal cell samples can either be collected and processed fresh using option A or be stored and xed in Saccomannos xative and processed at a later date using option B. (A) Collection of buccal samples for fresh-cell analysis (i) For each subject prepare two 30-ml yellow-topped containers labeled LC (left cheek) and RC (right cheek), each containing 10 ml of buccal cell buffer. (ii) Gently but rmly rotate a small-headed toothbrush (2-cm head length) 10 times against the inside of the cheek wall in a circular motion starting from the middle and gradually increasing in circumference to produce an outward spiral effect. (iii) Place the head of each brush into its respective buffer container and rotate repeatedly such that the cells are dislodged and released into the buffer, thereby producing a cloudy suspension of buccal cells in the buccal cell buffer. The brushes are then discarded following sampling and are not reused. Store the cell suspensions in a buffer at 4 1C. (B) Collection of buccal samples for xed-cell analysis (i) For each participant prepare two 30-ml yellow-topped containers labeled LC (left cheek) and RC (right cheek), each containing 10 ml of Saccomannos xative. (ii) Gently but rmly rotate a small-headed toothbrush (2-cm head length) 10 times against the inside of the cheek wall in a circular motion starting from the middle and gradually increasing in circumference to produce an outward spiral effect. Use a different toothbrush for each cheek. ! CAUTION It is important to remember not to revisit the mouth with the same toothbrush, so as to avoid the introduction of the xative to the mucosal lining. Use a new toothbrush for resampling. (iii) The head of the brush is then placed into the xative container and rotated such that the cells are dislodged and released into the suspension. ? TROUBLESHOOTING
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(iv) Tightly seal the tops of the xative containers and cover in paralm to prevent leakage during transit from the remote collection location to the laboratory. (v) The containers are then returned to the laboratory for analysis by a reputed courier service, and the laboratory should be informed of their shipment and anticipated arrival date, so that they can be processed as soon as possible after receipt. When received, the cells are collected in Saccomanos xative and are treated in the same manner as fresh buccal cells that have been collected in buccal cell buffer (starting from step 3 in the Buccal cell harvesting and slide preparation section below). PAUSE POINT Buccal cells xed in Saccomannos solution can be stored at 4 1C for up to 7 d before preparing slides. Buccal cell harvesting and slide preparation TIMING B2 h 3| Transfer the fresh or xed cells collected from both the right and left cheeks into separate TV-10 centrifuge tubes. Centrifuge the cells for 10 min at 581g at room temperature. 4| Aspirate off the supernatant leaving approximately 1 ml of cell suspension and replace with 5 ml of buccal cell buffer. Briey vortex the cells. 5| Centrifuge the cells for 10 min at 581g, room temperature. Aspirate the supernatant and resuspend the cells in another 5 ml of buccal buffer. m CRITICAL STEP The best results are achieved after two washes in buccal cell buffer. This buffer helps to inactivate endogenous DNAases present in the oral cavity and to remove bacteria and cell debris that could complicate scoring. 6| Remove the supernatant and replace with 5 ml of fresh buccal cell buffer. 7| Vortex the cell suspension and then homogenize for 23 min using a hand-held tissue homogenizer to increase the number of single cells in suspension. 8| Pool the cells from the left and right cheek tubes into a 30 ml container before drawing the cells into a syringe using an 18G needle. 9| To remove large aggregates of unseparated cells, pass the cells into a TV-10 tube through a 100 mm nylon lter held in a Swinex holder. 10| Centrifuge the cells for 10 min at 581g at room temperature and remove the supernatant. Resuspend the cells in 1 ml of buccal cell buffer. 11| To count the cells using a Coulter Counter, set the instrument settings for counting human buccal cells (e.g., Coulter Counter Model ZB1; threshold: 8, attenuation: 1, aperture: 1/4, manometer: 0.5 ml). 12| Dilute 300 ml of the cell suspension into 15 ml of Isoton. 13| Perform the count in duplicate to determine the mean cell number. ? TROUBLESHOOTING 14| Dilute the cell suspension to 80,000 cells ml1 with buccal cell buffer. 15| To further aid in cellular disaggregation and obtain slide preparations with clearly separated cells, add 50 ml of DMSO per ml of cell suspension. 16| Preparation of cells on microscope slides can be carried out in option A using a cytocentrifuge, or option B, manually. ? TROUBLESHOOTING (A) Preparation and transfer of cells onto the microscope slides using a cytocentrifuge (i) Follow the cytocentrifuge manufacturers instructions for the assembly of the microscope slides, lter cards and cytocentrifuge cups within the cytocentrifuge rotor. Ensure that the microscope slides are labeled with a code that matches the donor cells. (ii) Resuspend the cells thoroughly using a Pasteur or Gilson pipette to disaggregate them. (iii) Add 120 ml of the cell suspension into the well of each sample cup. ! CAUTION Loading and cytocentrifugation of the cell culture sample must be carried out in an approved cytoguard safety cabinet to avoid the possibility of infectious disease transfer from the buccal cells. Appropriate safety protection including gloves must be worn. m CRITICAL STEP The required volume may need a slight adjustment depending on the concentration of cells in suspension and the optimal cell density for slide scoring; this needs to be determined empirically.
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(iv) Replace and lock the rotor lid and centrifuge the cells for 5 min, at 600 r.p.m., between 18 and 20 1C. (v) Upon completion of the spin place the rotor in a safety cabinet and follow the manufacturers instructions for opening each slide holder. (vi) Air-dry the microscope slides for exactly 10 min at room temperature. (vii) Fix the cells in a slide-staining rack containing 200 ml of ethanol: glacial acetic acid mix (3:1) for 10 min followed by further air-drying for 10 min at room temperature. (viii) Disinfect the cytocentrifuge cups in 1% (vol/vol) sodium hypochlorite solution for 30 min and then rinse thoroughly (six times) in Milli-Q water and allow to dry completely before additional use. (B) Manual preparation and transfer of cells onto the microscope slides (i) Fix the cells obtained in step 15 using the required volume of ethanol: glacial acetic acid, 3:1, to give a concentration of 80,000 cells ml1. (ii) Using a pipette, drop 120150 ml of cell suspension onto the clean, dry and appropriately labeled microscope slides, and allow to air-dry for 10 min before staining. Buccal cell staining for microscopy TIMING B1 h 45 min 17| Immerse the microscope slides with the xed cells for 1 min each in Coplin jars containing 50% (vol/vol) and 20% (vol/vol) ethanol. Wash the cells for 2 min in a Coplin jar containing Milli-Q water. 18| Place the slides in a Coplin jar containing 5 M HCl for 30 min and then rinse in running tap water for 3 min. 19| Place one slide in Milli-Q water instead of 5 M HCl for 30 min as a negative control to check for the efcacy of the 5 M HCl treatment. 20| Drain the slides, but do not allow them to dry out, and place them in a Coplin jar containing Schiff s reagent for 60 min in the dark at room temperature. 21| Rinse the slides in running tap water for 5 min and then rinse well in Milli-Q water. 22| Counter stain the cells by immersing in Coplin jars containing 0.2% (wt/vol) Light Green for 2030 s and rinse well in Milli-Q water. 23| To blot away any residual moisture, immediately place the slides facedown onto Whatman no. 1 lter paper. m CRITICAL STEP Do not apply any pressure or rub on the cell spots as this will dislodge cells. 24| Place the slides on a slide tray and allow them to dry for about 1015 min. 25| Examine the efciency of staining and the density of the cells at 100 and 400 magnication. ? TROUBLESHOOTING 26| Leave the slides to dry completely for at least 30 min before applying coverslips. ! CAUTION Apply the coverslips in a fume hood to avoid inhalation of organic solvent in DePex and leave the slides in the hood until completely dry. Wear nitrile gloves when applying DePex medium. PAUSE POINT Slides can be left overnight at room temperature to dry but should be covered to prevent dust from settling on them. 27| Place the slides to be coverslipped on tissue paper and set out one coverslip alongside each. 28| Put two large drops of DePex (use a plastic dropper) on each of the coverslips in the approximate area corresponding to the cell spots. 29| Invert the slide and place on the coverslip. Allow the DePex to spread. Turn the slide so that the coverslip is on top, and press the coverslip gently to expel any excess DePex and air bubbles. m CRITICAL STEP Press the coverslip gently, as it may break. Ensure that the spots do not have air bubbles over them. 30| Wipe excess DePex from the edges of the slide and ensure that the medium or glass does not cover any of the frosted label area, as a coding label will not stick on the DePex. 31| Place the slides on a tray and leave overnight in the fume hood to dry. 32| Store the slides in slide boxes at room temperature.
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33| Observe the slides using transmitted light microscopy; the nuclei and the micronuclei are magenta in color, whereas the cytoplasm will be pale blue/green (see Fig. 5). In negative controls (i.e., no 5 M HCl treatment) the nuclei will not be stained with magenta color. ? TROUBLESHOOTING 34| The cells can also be viewed under uorescence with a far-red lter because Feulgen stained DNA appears bright red in color under these conditions. Microscopy TIMING B6080 min/slide 35| Using the scoring criteria described in Table 3 count a minimum of 1,000 cells, and determine the frequency of each cell type in the sample, e.g., basal and differentiated cells (see Table 3 and Fig. 5). ? TROUBLESHOOTING 36| Using the scoring criteria described above (Table 3), record the number of cells with MNi and NBUDs in a minimum of 2,000 differentiated cells (see Fig. 5ce). ? TROUBLESHOOTING TIMING Steps 1 and 2, buccal cell collection: B10 min Steps 316, buccal cell harvesting and slide preparation: B2 h Steps 1734, buccal cell staining for microscopic analysis: B1 h 45 min Steps 35 and 36, scoring of stained slides: B6080 min/slide ? TROUBLESHOOTING Troubleshooting advice can be found in Table 4.
TABLE 4 | Troubleshooting advice. Step 2B(iii) 13,16 Problem Leaking sample in transit Insufcient cell count Possible reason Cracked lid or not tightened Insufcient pressure applied with toothbrush Solution Ensure lid is tightened and seal well with paralm Obtain a second sample or concentrate the cell suspension into 240 ml. Apply 120 ml to each well producing 1 slide with 2 spots if using cytocentrifuge. This consists of two consecutive steps If the cell density is too high or sparse, concentrate or dilute the cell suspension as necessary by centrifuging the cells and resuspend in a larger or smaller volume of buffer and repeat the cell harvesting and staining steps. Check settings on coulter counter Use Saccomannos xative if cells cannot be processed on the same day and/or if collected in remote location from laboratory. Storage temperature in buffer or Saccomannos should not exceed 4 1C Examine cell under both transmitted light and under uorescence because nuclear texture and DNA staining intensity is better under uorescence. Otherwise, make new slide preparations from residual cells stored in buccal cell buffer or Saccomannos xative. Make a fresh batch of Schiffs reagent and check concentration of HCl

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25

Low or excess cell density on cytocentrifuge slide

Inaccurate cell count

33

Buccal cell deterioration evident when viewing slides

Use of inappropriate buffer to store cells and/or high storage temperature

35,36

Uncertainty in classication of cell types on presence of MNi and NBUD

Poor Feulgen staining on slide preparation

Abbreviations: MNi, Micronuclei; NBUD, Nuclear bud.

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TABLE 5 | Typical BMCyt results for cells collected from healthy young and old subjects.
Basal cell no. Diff cell no. BN cell no. PYK cell no. (a) Young controls n 30 (mean age 22.47 2.2 yrs, range 1826 yrs) Minimum 5.00 460.00 2.00 0.00 25% percentile 12.00 629.00 8.00 2.00 Median 22.00 715.00 10.00 2.00 75% percentile 37.00 788.50 14.00 5.00 Maximum 91.00 895.00 27.00 10.00 Mean 27.40 712.90 11.63 3.23 s.d. 21.36 112.70 5.49 2.35 s.e. 3.89 20.58 1.00 0.43 Lower 95% CI of mean 19.43 670.80 9.58 2.35 Upper 95% CI of mean 35.37 755.00 13.69 4.11 CC cell no. 13.00 30.50 45.50 60.50 103.00 46.67 20.53 3.74 39.00 54.33 KYL cell no. 43.00 109.00 181.00 256.50 441.00 187.90 108.60 19.83 147.30 228.50 KHC cell no. 1.00 4.00 6.50 12.00 44.00 9.36 8.71 1.59 6.11 12.62 NBUD no. 0.00 0.00 1.00 1.00 4.00 0.93 1.11 0.20 0.51 1.34 MN frequency 0.00 0.00 0.00 0.00 4.00 0.30 0.83 0.15 0.01 0.61

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(b) Older controls n 30 (mean age 68.13 2.8 yrs, range 6475 yrs) Minimum 15.00 373.00 2.00 25% percentile 57.50 570.50 8.50 Median 92.50 651.50 14.00 75% percentile 128.50 737.50 21.00 Maximum 187.00 902.00 42.00 Mean 93.53 655.90 15.80 s.d. 44.97 116.20 9.01 s.e. 8.21 21.22 1.64 Lower 95% CI of mean 76.74 612.50 12.43 Upper 95% CI of mean 110.30 699.30 19.17

0.00 0.00 1.00 3.50 8.00 1.96 2.10 0.38 1.17 2.75

11.00 32.50 64.50 84.00 155.00 65.10 34.76 6.34 52.12 78.08

14.00 74.50 104.50 135.00 270.00 113.60 56.44 10.30 92.56 134.70

2.00 30.00 48.50 68.00 137.00 51.50 33.29 6.07 39.07 63.93

0.00 0.00 1.00 2.00 4.00 1.16 1.34 0.24 0.66 1.66

0.00 0.00 1.00 3.00 6.00 1.43 1.56 0.28 0.84 2.01

Results shown are per 1,000 cells, i.e., %. Diff, Differentiated; BN, Binucleated; PYK, Pyknotic; CC, Condensed Chromatin; KYL, Karyolytic; KHC, Karyorrhectic; MN, Micronucleus; NBUD, Nuclear bud; s.d., standard deviation; s.e., standard error; CI, condence interval.

ANTICIPATED RESULTS The anticipated results with the buccal micronucleus cytome assay (BMCyt) are dependent on the level of exposure and potency of genotoxic or cytotoxic agents, genetic background and the age and gender of the donor cells being tested. Detailed typical results for healthy young and old participants, who are not abnormally exposed to genotoxins, are shown in Table 5. The table includes the minimum and maximum value, 25th and 75th percentiles, the median, mean, standard deviation (s.d.), standard error, and lower 95% and upper 95% condence interval for each BMCyt biomarker in young and old controls. The BMCyt assay has as yet not been approved for diagnostic use and is applied only for research purposes at this stage. However, it is reasonable to consider individual values that are beyond two s.d of the mean for the biomarker measured as likely to be abnormal when comparing results within the same age group. For example, on the basis of the data in Table 5, a young adult with a micronucleus frequency value of 4% would be considered to have an abnormally high micronucleus frequency, given that the mean for the group is 0.3 and the s.d. is 0.8. The frequency of the buccal cytome biomarkers in the assay may vary with respect to their relative frequencies depending on the rate of ageing, as has been shown in recent studies on Downs syndrome and Alzheimers disease, in which the frequency of basal cells was signicantly lower as compared with the controls9,23. Most buccal cell studies have only evaluated changes in micronucleus frequencies in relation to biomonitoring, lifestyle and dietary factors and have not scored the remaining cytome biomarkers to show a more comprehensive evaluation. Anticipated changes in micronucleus frequency in the buccal cells may be large in the case of ionizing radiation exposure. Following radiotherapy, MNi frequency increased 16-fold as compared with the baseline levels21.
Note: Supplementary information is available via the HTML version of this article. ACKNOWLEDGMENTS We are grateful to the numerous volunteers whose donation of buccal cell samples over the many years has enabled us to achieve the renement of this protocol. We are particularly indebted to Professor Hans Stich who pioneered this eld of research and whose vision enabled the evolution of this protocol. Published online at http://www.natureprotocols.com/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Shojaei, A.H. Buccal mucosa as a route for systemic drug delivery: a review. J. Pharm. Pharmaceut. Sci. 1, 1530 (1998). 2. Squier, C.A. & Kremer, M.J. Biology of oral mucosa and esophagus. J. Natl. Cancer Inst. Monogr. 715 (2001). 3. Sarto, F., Tomanin, R., Giacomelli, L., Iannini, G. & Cupiraggi, A.R. The micronucleus assay in human exfoliated cells of the nose and mouth: application to occupational exposures to chromic acid and ethylene oxide. Mutat. Res. 244, 345351 (1990). 4. Machado-Santelli, G.M., Cerqueira, E.M., Oliveira, C.T. & Pereira, C.A. Biomonitoring of nurses handling antineoplastic drugs. Mutat. Res. 322, 203208 (1994). 5. Burgaz, S. et al. Urinary cyclophosphamide excretion and micronuclei frequencies in peripheral lymphocytes and in exfoliated buccal epithelial cells of nurses handling antineoplastics. Mutat. Res. 439, 97104 (1999). 6. Stich, H.F., Rosin, M.P. & Vallejera, M.O. Reduction with vitamin A and betacarotene administration of proportion of micronucleated buccal mucosal cells in Asian betal nut and tobacco chewers. Lancet 1, 12041206 (1984). 7. Titenko-Holland, N. et al. Quantication of epithelial cell micronuclei by uorescence in situ hybridization (FISH) in mortuary science students exposed to formaldehyde. Mutat. Res. 371, 237248 (1996).

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8. Ozkul, Y., Donmez, H., Erenmemisoglu, A., Demirtas, H. & Imamoglu, N. Induction of micronuclei by smokeless tobacco on buccal mucosa cells of habitual users. Mutagenesis 12, 285287 (1997). 9. Thomas, P., Harvey, S., Gruner, T. & Fenech, M. The buccal cytome and micronucleus frequency is substantially altered in Downs syndrome and normal ageing compared to young healthy controls. Mutat. Res. 638, 3747 (2007). 10. Thomas, P. & Fenech, M. Chromosome 17 and 21 aneuploidy in buccal cells is increased with ageing and in Alzheimers disease. Mutagenesis 23, 5765 (2007). 11. Surralles, J. et al. Molecular cytogenetic analysis of buccal cells and lymphocytes from benzene-exposed workers. Carcinogenesis 18, 817823 (1997). 12. Titenko-Holland, N., Jacob, R.A., Shang, N., Balaraman, A. & Smith, M.T. Micronuclei in lymphocytes and exfoliated buccal cells of postmenopausal women with dietary changes in folate. Mutat. Res. 417, 101114 (1998). 13. Holland, N. et al. The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: the HUMN project perspective on current status and knowledge gaps. Mutat. Res. 659, 93108 (2008). 14. Vondracek, M. et al. Cytochrome P450 expression and related metabolism in human buccal mucosa. Carcinogenesis 22, 481488 (2001). 15. Spivack, S.D. et al. Gene-environment interaction signatures by quantitative mRNA proling in exfoliated buccal mucosal cells. Cancer Res. 64, 68056813 (2004). 16. Cairns, J. Mutation selection and the natural history of cancer. Nature 255, 197200 (1975). 17. Rosin, M.P. The use of the micronucleus test on exfoliated cells to identify anti-clastogenic action in humans: a biological marker for the efcacy of chemopreventive agents. Mutat. Res. 267, 265276 (1992). 18. Stich, H.F., Curtis, J.R. & Parida, B.B. Application of the micronucleus test to exfoliated cells of high cancer risk groups: tobacco chewers. Int. J. Cancer 30, 553559 (1982). 19. Stich, H.F. & Rosin, M.P. Quantitating the synergistic effect of smoking and alcohol consumption with the micronucleus test on human buccal mucosa cells. Int. J. Cancer 31, 305308 (1983). 20. Stich, H.F. et al. Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with beta-carotene and with beta-carotene plus vitamin A. Int. J. Cancer 42, 195199 (1988). 21. Moore, L.E., Warner, M.L., Smith, A.H., Kalman, D. & Smith, M.T. Use of the uorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells. Environ. Mol. Mutagen 27, 176184 (1996). 22. Rosin, M.P. & German, J. Evidence for chromosome instability in vivo in Bloom syndrome: increased numbers of micronuclei in exfoliated cells. Hum. Genet. 71, 187191 (1985). 23. Thomas, P., Hecker, J., Faunt, J. & Fenech, M. Buccal micronucleus cytome biomarkers may be associated with Alzheimers disease. Mutagenesis 22, 371379 (2007). 24. Masters, B.R., Gonnord, G. & Corcuff, P. Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a exible confocal microscope. Journal of Microscopy 185, 329338 (1997). 25. Veiro, J.A. & Cummins, P.G. Imaging of skin epidermis from various origins using confocal laser scanning microscopy. Dermatology 189, 1622 (1994). 26. Squier, C.A., Johnson, N.W. & Hopps, R.M. Human Oral Mucosa: Development, Structure and Function 744 (Blackwell Scientic, Oxford, United Kingdom, 1976). 27. Tolbert, P.E., Shy, C.M. & Allen, J.W. Micronuclei and other nuclear anomalies in buccal smears: methods development. Mutat. Res. 271, 6977 (1992). 28. Harris, D. & Robinson, J.R. Drug delivery via the mucous membranes of the oral cavity. J. Pharm. Sci. 81, 110 (1992). 29. Van Schooten, F.J. et al. Effects of oral administration of N-Acetyl-L-cysteine: a multi-biomarker study in smokers. Cancer Epidemiol. Biomarkers Prev. 11, 167175 (2002). 30. Schwartz, J.L. et al. Oral cytology assessment by ow cytometry of DNA adducts, aneuploidy, proliferation and apoptosis shows differences between smokers and non-smokers. Oral Oncology 39, 842854 (2003). 31. Ramirez, M.J., Surralles, J., Galofre, P., Creus, A. & Marcos, R. FISH analysis of 1cen1q12 breakage, chromosome 1 numerical abnormalities and centromeric content of micronuclei in buccal cells from thyroid cancer and hyperthyroidism patients treated with radioactive iodine. Mutagenesis 14, 121127 (1999). 32. Moore, L.E., Titenko-Holland, N. & Smith, M.T. Use of uorescence in situ hybridization to detect chromosome-specic changes in exfoliated human bladder and oral mucosa cells. Environ. Mol. Mutagen. 22, 130137 (1993). 33. Pastor, S. et al. Biomonitoring of four European populations occupationally exposed to pesticides: use of micronuclei as biomarkers. Mutagenesis 18, 249258 (2003). 34. Titenko-Holland, N., Jacob, R.A., Shang, N., Balaraman, A. & Smith, M.T. Micronuclei in lymphocytes and exfoliated buccal cells of postmenopausal women with dietary changes in folate. Mutat. Res. 417, 101114 (1998). 35. Schad, C.R. et al. Application of uorescent in situ hybridization with X and Y chromosome specic probes to buccal smear analysis. Am. J. Med. Genet. 66, 187192 (1996). 36. Casartelli, G. et al. Micronucleus frequencies in exfoliated buccal cells in normal mucosa, precancerous lesions and squamous cell carcinoma. Anal. Quant. Cytol. Histol. 22, 486492 (2000). 37. Bagwe, A.N. & Bhisey, R.A. Occupational exposure to tobacco and resultant genotoxicity in bidi industry workers. Mutat. Res. 299, 103109 (1993). 38. Desai, S.S., Ghaisas, S.D., Jakhi, S.D. & Bhide, S.V. Cytogenetic damage in exfoliated oral mucosal cells and circulating lymphocytes of patients suffering from precancerous oral lesions. Cancer Lett. 109, 914 (1996). 39. Albertini, R.J. et al. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety. Mutat. Res. 463, 111172 (2000). 40. Kassie, F., Darroudi, F., Kundi, M., Schulte-Hermann, R. & Knasmuller, S. Khat (Catha edulis) consumption causes genotoxic effects in humans. Int. J. Cancer. 92, 329332 (2001). 41. Nersesyan, A., Kundi, M., Atee, K., Schulte-Hermann, R. & Knasmuller, S. Effect of staining procedures on the results of micronucleus assays with exfoliated oral mucosa cells. Cancer Epidemiol. Biomarkers Prev. 15, 18351840 (2006). 42. Casartelli, G. et al. Staining of micronuclei in squamous epithelial cells of human oral mucosa. Anal. Quant. Cytol. Histol. 19, 475481 (1997). 43. Fenech, M. & Morley, A.A. Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation. Mutat. Res. 161, 193198 (1986). 44. Fenech, M. & Crott, J.W. Micronuclei, nucleoplasmic bridges and nuclear buds induced in folic acid decient human lymphocytes-evidence for breakagefusion-bridge cycles in the cytokinesis-block micronucleus assay. Mutat. Res. 504, 131136 (2002). 45. Shimizu, N., Itoh, N., Utiyama, H. & Wahl, G.M. Selective entrapment of extrachromosomally amplied DNA by nuclear budding and micronucleation during S phase. J. Cell Biol. 140, 13071320 (1998). 46. Shimizu, N., Kamezaki, F. & Shigematsu, S. Tracking of microinjected DNA in live cells reveals the intracellular behavior and elimination of extrachromosomal genetic material. Nucl. Acids Res. 33, 62966307 (2005). 47. Nersesyan, A.K. Nuclear buds in exfoliated human cells. Mutat. Res. 588, 6468 (2005). 48. Shi, Q. & King, R.W. Chromosome nondisjunction yields tetraploid rather than aneuploid cells in human cell lines. Nature 437, 10381042 (2005). 49. Frankfurt, O.S. & Krishan, A. Identication of apoptotic cells by formamideinduced DNA denaturation in condensed chromatin. J. Histochem. Cytochem. 49, 369378 (2001). 50. Strange, R., Li, F., Saurer, S., Burkhardt, A. & Friis, R.R. Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115, 4958 (1992). 51. Wyllie, A.H. Cell Death: A New Classication Separating Apoptosis from Necrosis 934 (Chapman and Hall Ltd, London, 1981). 52. Tolbert, P.E., Shy, C.M. & Allen, J.W. Micronuclei and other nuclear anomalies in buccal smears: a eld test in snuff users. Am. J. Epidemiol. 134, 840850 (1991). 53. Chen, C. et al. Cytogenetic damage in buccal epithelia and peripheral lymphocytes of young healthy individuals exposed to ozone. Mutagenesis 21, 131137 (2006). 54. Neher, A., Ofner, G., Appenroth, E. & Gschwendtner, A. High-resolution image cytometry on smears of normal oral mucosa: a possible approach for the early detection of laryngopharyngeal cancers. Head & Neck 26, 694700 (2004).

2009 Nature Publishing Group http://www.nature.com/natureprotocols

NATURE PROTOCOLS | VOL.4 NO.6 | 2009 | 837

CORRIGENDUM

Corrigendum: Buccal micronucleus cytome assay

Philip Thomas, Nina Holland, Claudia Bolognesi, Micheline Kirsch-Volders, Stefano Bonassi, Errol Zeiger, Siegfried Knasmueller & Michael Fenech
Nat. Protoc. 4, 825837 (2009); doi:10.1038/nprot2009.53; published online 7 May 2009; corrected online 22 September 2011

In the version of this article initially published, in the REAGENTS SETUP section (page 832), the proportions of salts shown in the Buccal cell buffer section were incorrect. 1.2 g EDTA and 37.2 g of sodium chloride should have read 38.0 g EDTA and 1.2 g of sodium chloride. The errors have been corrected in the HTML and PDF versions of the article.

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