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Mutation Research 659 (2008) 93–108
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Mutation Research/Reviews in Mutation Research
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Review
The micronucleus assay in human buccal cells as a tool for biomonitoring
DNA damage: The HUMN project perspective on current status and
knowledge gaps
Nina Holland a,1,*, Claudia Bolognesi b,1,**, Micheline Kirsch-Volders c, Stefano Bonassi d, Errol Zeiger e,
Siegfried Knasmueller f, Michael Fenech g
a
School of Public Health, University of California, Berkeley, CA, USA
Unit of Environmental Carcinogenesis, National Cancer Research Institute, Genoa, Italy
c
Laboratory for Cell Genetics, Vrije Universiteit, Brussel, Belgium
d
Unit of Molecular Epidemiology, National Cancer Research Institute, Genoa, Italy
e
Errol Zeiger Consulting, Chapel Hill, NC, USA
f
Institute of Cancer Research, Medical University, Vienna, Austria
g
CSIRO Human Nutrition, Adelaide, Australia
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 5 October 2007
Received in revised form 24 March 2008
Accepted 25 March 2008
Available online 11 April 2008
The micronucleus (MN) assay in exfoliated buccal cells is a useful and minimally invasive method for
monitoring genetic damage in humans. This overview has concluded that although MN assay in buccal
cells has been used since the 1980s to demonstrate cytogenetic effects of environmental and
occupational exposures, lifestyle factors, dietary deficiencies, and different diseases, important
knowledge gaps remain about the characteristics of micronuclei and other nuclear abnormalities, the
basic biology explaining the appearance of various cell types in buccal mucosa samples and effects of
diverse staining procedures and scoring criteria in laboratories around the world. To address these
uncertainties, the human micronucleus project (HUMN; see http://www.humn.org) has initiated a new
international validation project for the buccal cell MN assay similar to that previously performed using
human lymphocytes. Future research should explore sources of variability in the assay (e.g. between
laboratories and scorers, as well as inter- and intra-individual differences in subjects), and resolve key
technical issues, such as the method of buccal cell staining, optimal criteria for classification of normal
and degenerated cells and for scoring micronuclei and other abnormalities. The harmonization and
standardization of the buccal MN assay will allow more reliable comparison of the data among human
populations and laboratories, evaluation of the assay’s performance, and consolidation of its world-wide
use for biomonitoring of DNA damage.
ß 2008 Published by Elsevier B.V.
Keywords:
Biomarkers
Biomonitoring
Environmental exposure
Occupational exposure
Cancer
Precancerous lesion
Exfoliated cell
Oral epithelia
Genotoxicity
Vitamin deficiency
Smoking
Scoring criteria
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Measurement of MN in exfoliated buccal cells: a brief history and rationale . . . .
Biomonitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Occupational and environmental exposures . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
MN and radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
MN frequencies in buccal cells of patients with cancer and other diseases
3.4.
Lifestyle and host factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author at: 733 University Hall, Berkeley, CA 94720-7460, USA. Tel.: +1 510 455 0561; fax: +1 510 643 5426.
** Corresponding author.
E-mail addresses: [email protected], [email protected] (N. Holland), [email protected] (C. Bolognesi).
1
These authors contributed equally to the work.
1383-5742/$ – see front matter ß 2008 Published by Elsevier B.V.
doi:10.1016/j.mrrev.2008.03.007
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N. Holland et al. / Mutation Research 659 (2008) 93–108
94
4.
5.
6.
7.
Methodological aspects affecting identification of MN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Collection of buccal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Slide preparation and staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Scoring criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Number of cells scored; power of the test to detect an increase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A proposed validation study to examine slide preparation and scoring variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Study design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Positive control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Sampling method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.
Slide preparation method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.
Scoring criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6.
Number of cells to be scored . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.7.
Cytome approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Addressing knowledge gaps with the buccal cell assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Effect of cell division kinetics on MN frequency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Improved knowledge of the biology and structure of buccal cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.
Variables that affect MN frequency in buccal cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.
Correlation with MN frequency in other cell types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5.
Does the MN frequency in buccal cells predict risk for cancer or other chronic diseases? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
The human micronucleus (HUMN) project (http://www.humn.org), established in 1997, is an international collaborative
program aimed at studying the micronucleus (MN) frequency in
human populations, and assessing the effects of protocol and
scoring criteria on the values obtained. The initial focus of the
project was the analysis of MN in peripheral lymphocytes from
unexposed and exposed individuals, primarily because this was a
well-established human test system at the time the project began.
The ultimate goals of the HUMN project are to standardize
methodologies, define baseline MN frequency rates, characterize
the effects of demographic, genetic, and methodological factors on
these frequencies, and to determine whether the MN frequencies
in different tissues are predictive of cancer risk [1]. These
objectives have been achieved for lymphocytes by providing a
detailed description of the scoring criteria [2] and by assessing
sources of variability for the cytokinesis-block MN assay through a
validation effort undertaken by 34 laboratories from 21 countries
[3]. More recently, results from the prospective analysis of the
database of more than 6700 subjects from 20 laboratories
representing 10 countries, confirmed that an elevated MN
frequency in human lymphocytes is predictive of an increased
risk of cancer [4].
Genomic damage is probably the most important fundamental
cause of developmental and degenerative disease. It is also well
established that genomic damage is produced by environmental
exposure to genotoxins, medical procedures (e.g. radiation and
chemicals), micronutrient deficiency (e.g. folate), lifestyle factors
(e.g. alcohol, smoking, drugs, and stress), and genetic factors such
as inherited defects in DNA metabolism and/or repair [5–23]. It is
essential to have reliable and relevant minimally invasive
biomarkers to improve the implementation of biomonitoring,
diagnostics, and treatment of diseases caused by, or associated
with, genetic damage. The MN assay in exfoliated buccal cells is
potentially an excellent candidate to serve as such a biomarker.
The collection of buccal cells is arguably the least invasive
method available for measuring DNA damage in humans,
especially in comparison to obtaining blood samples for lymphocyte and erythrocyte assays, or tissue biopsies. The buccal cell MN
assay was first proposed in 1983 [6] and continues to gain
popularity as a biomarker of genetic damage in numerous
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applications (Fig. 1). More than 40 laboratories from many
countries either have used, or are currently using this assay, and
the number of articles published annually is steadily increasing.
Different issues related to the buccal cell MN assay were reviewed
in several publications over the last decade [18,19,23–25].
However, most of these articles focused primarily on either broad
evaluation of non-invasive methods for biomonitoring [24,26], the
associations of micronuclei (MNi) and other markers with cancer
in various types of exfoliated cells [19], or are limited to effects of a
specific type of exposure, such as smoking and smokeless tobacco
[25] or formaldehyde [23]. More importantly, the identification
and relative importance of confounding variables affecting the MN
frequency in buccal cells has not been adequately addressed and
quantified. The predictive value of the buccal MN frequency for
cancer risk has also not been studied in a comprehensive manner.
Finally, the assay protocol has not been standardized in such a way
that results from different laboratories can be easily compared or
combined.
The HUMN project is an effective vehicle for the development
and implementation of an international collaborative validation
effort to bring together the various buccal MN databases, and to
identify and quantify the key variables affecting this biomarker. In
addition, an inter-laboratory slide-scoring exercise could be
undertaken to evaluate the intra- and inter-laboratory variability
in the scoring of buccal cell MN, similar to the approach
successfully used by the HUMN project for the MN assay in
lymphocytes [2,3].
Fig. 1. Increase in the number of laboratories and publications reporting the use of
the MN assay in buccal cells.
N. Holland et al. / Mutation Research 659 (2008) 93–108
The aims of this paper are: (a) to provide an overview of the
current status of the MN assay in buccal cells, (b) to identify the
most important knowledge gaps in the basic biology that affect
the expression of MNi and other nuclear abnormalities, and (c)
to compare the strengths and limitations of the existing
techniques for collection, staining, and scoring of buccal cells.
This overview is designed to help to focus the HUMN buccal cell
project on the key areas of concern, and those in need of further
development.
2. Measurement of MN in exfoliated buccal cells: a brief history
and rationale
Micronuclei originate from chromosome fragments or whole
chromosomes that lag behind at anaphase during nuclear division
[1,27]. The MN index in rodent and/or human cells has become one
of the standard cytogenetic endpoints and biomarkers used in
genetic toxicology in vivo or ex vivo. In humans, MNi can be easily
assessed in erythrocytes, lymphocytes, and exfoliated epithelial
cells (e.g. oral, urothelial, nasal) to obtain a measure of genome
damage induced in vivo. The MN assay can be performed in buccal
and other exfoliated cells originating from rapidly dividing
epithelial tissue without the need for ex vivo nuclear division, so
that the cell cultures required for cytogenetic assays based on
analysis of metaphase chromosomes, such as chromosome
aberrations and sister chromatid exchanges, are not needed. There
is precedence for this because, while the majority of MN studies
with lymphocytes in the HUMN database used cytochalasinblocked proliferating lymphocytes [1], some investigators assessed
MNi in non-proliferating lymphocytes during interphase to
measure MN expressed during in vivo nuclear division of their
parent cells [28]. The general genotoxicity results by both methods
of MN analysis are comparable.
Buccal cells are the first barrier for the inhalation or ingestion
route and are capable of metabolizing proximate carcinogens to
reactive products [29–32]. Approximately 90% of human cancers
originate from epithelial cells [33]. Therefore, it could be argued
that oral epithelial cells represent a preferred target site for early
genotoxic events induced by carcinogenic agents entering the body
via inhalation and ingestion.
95
The oral epithelium is composed of four strata of structural,
progenitor, and maturing cell populations, including the lamina
propria (connective tissue), the basal cell layer (stratum basale),
prickle cell layer (stratum spinosum), and the keratinized layer at
the surface (Fig. 2). A series of finger-like structures called ‘‘rete
pegs’’ project up from the lamina propria into the epidermal layer
producing an undulating basal cell layer effect. The oral epithelium
maintains itself by continuous cell renewal whereby new cells
produced in the basal layer by mitosis migrate to the surface
replacing those that are shed. The basal layer contains the stem
cells that may express genetic damage (chromosome breakage or
loss) as MNi during nuclear division. The daughter cells, which may
or may not contain MN, eventually differentiate into the prickle
cell layer and the keratinized superficial layer, and then exfoliate
into the buccal cavity. Some of these cells may degenerate into cells
with condensed chromatin, fragmented nuclei (karyorrhectic
cells), pycnotic nuclei, or completely lose their nuclear material
(karyolitic or ‘‘ghost’’cells) [5]. In rare cases, some cells may be
blocked in a binucleated stage or may exhibit nuclear buds (also
known as ‘‘broken eggs’’ in buccal cells [5]), a biomarker of gene
amplification (Fig. 3). These biomarkers of genome damage (e.g.
MNi, nuclear buds) and cell death (e.g. apoptosis, karyolysis) can be
observed in both the lymphocyte and buccal cell systems, and thus
provide a more comprehensive assessment of genome damage
then only MNi in the context of cytotoxicity and cytostatic effects
[5,27].
In the early studies from the 1980s, exfoliated buccal mucosa
cells were used to evaluate the genotoxic effects of betel nuts and
quids and of chewing tobacco [34–39]. Most studies showed higher
MN frequencies at the site within the oral cavity where the quid or
tobacco mixture was kept compared to the opposite, control, site.
The MN assay in buccal cells was also used to study cancerous and
precancerous lesions and to monitor the effects of a number of
chemopreventive agents [40–42]. It is notable that the first studies
of Stich and Rosin conducted between 1983 and 1984 had higher
baseline MN frequencies than subsequent studies. This may have
been due to a lack of defined scoring criteria and a relatively small
number of scored cells (in some cases less than 500). Since then,
published biomonitoring studies using the MN assay in buccal
mucosa cells have investigated the effects of multiple factors
Fig. 2. Structure and differentiation of oral epithelium. (A) Photomicrograph of section through healthy oral mucosa showing the various layers of cells in the oral epithelium.
The oral epithelium is a stratified squamous epithelium. It consists of five layers: (i) keratinized layer at the surface, (ii) prickle cell layer (or stratum spinosum), (iii) basal layer
(or stratum basal), (iv) rete pegs, (v) lamina propria (connective tissue). Reproduced from http://www.eastman.ucl.ac.uk/cal/ulcerspath/healthy.htm with permission from
the Eastman Dental Institute. (B) Schematic of buccal mucosa cell layers and turnover.
N. Holland et al. / Mutation Research 659 (2008) 93–108
96
Fig. 3. Buccal MN assay cytome model. Schematic diagram of different types of buccal cells and the possible mechanisms for their origin. The majority of these cells have a
large cytoplasm relative to the nucleus, and the shape of the cell is angular rather than spherical; the exception is basal cells. Genomic instability or genotoxic insult in the
basal cells leads to chromosome breakage/loss and MN formation. Some cells with genome damage may be eliminated via the apoptotic process. The daughter cells from the
basal layer differentiate into ‘‘prickle cells’’ which are eventually differentiated into the flattened and keratinized surface mucosal cells which exfoliate from the surface of the
oral lining. Each of these cell types may contain MNi to varying extents. The molecular mechanisms leading to the various cell death events, and their inter-relationships, are
unknown.
including environmental and occupational exposures, radiotherapy, chemoprevention, vitamin supplementation trials, lifestyle
habits, cancer, and other diseases.
3. Biomonitoring
3.1. Occupational and environmental exposures
In the last 15–20 years the MN assay has been applied to
evaluate chromosomal damage for biological monitoring of human
populations exposed to a variety of mutagenic and carcinogenic
chemical or physical agents (Table 1). A broad range of baseline MN
frequencies has been reported (0.05–11.5 MN/1000 cells) with the
majority of values between 0.5 and 2.5 MN/1000 cells. There is no
clear pattern of the variations among laboratories from different
countries. Many studies report a statistically significant elevation
of MN levels in exposed individuals compared to control groups,
although the observed effects are relatively small, ranging between
1.1- and 4-fold [43–47], and many other studies report changes
that were not statistically significant [48–50]. A 12-fold increase in
MN frequency was observed in mortician students after a 3-month
embalming course compared to pre-exposure levels [51]. This
increase is unusually large, but has been confirmed by an
independent analyses using a different staining procedure,
Table 1
Micronucleus frequency in exfoliated oral mucosa cells
Exposure/lifestyle factors
Study
subjects/
control
Baseline,
MN cells/
1000 cells
Fold
difference
vs. control
Cells
scored/
subject
Staining
methoda
Scoring
criteriab
Year
Country
Reference
Exposure
Type
Antineoplastic
drugs
Hospital staff
Hospital staff
Day-care hospital nurses
Pharmacy technicians
Ward nurses
25/25
25/14
12/30*
5/30
13/30*
1.68
0.80
0.46
0.46
0.46
2.1*
2.0*
1.9
1.1
2.0
1000
1000
2000
2000
2000
1
1
4
4
4
3/4
3/4
4
4
4
1994
1999
2005
2005
2005
Brazil
Turkey
Italy
Italy
Italy
[43]
[44]
[137]
[137]
[137]
Areca nut only
(no tobacco)
Chewers
15/10
1.93
3.8*
1000
1
3
1992
India
[138]
N. Holland et al. / Mutation Research 659 (2008) 93–108
97
Table 1 (Continued )
Exposure/lifestyle factors
Study
subjects/
control
Baseline,
MN cells/
1000 cells
Fold
difference
vs. control
Cells
scored/
subject
Staining
methoda
Scoring
criteriab
Year
Country
Reference
Exposure
Type
Arsenic
Drinking water
Drinking water
Drinking water
Drinking water
Drinking water
Drinking water
Glass factory workers
Smelter workers
9/8
33/32
19/13
45/21
163/150
105/102
200/165
72/83
0.30
0.58
0.65
0.77
1.28
2.74
2.10
0.50
5.7*
4.0*
3.4*
6.7*
4.6*
1.2
7.2*
2.0*
NR
1000
3000
1000
3000
2000
2000
2000
1
1
1
1
1
2
3
1
4
4
4
4
4
6
7
4
1997
1997
2001
2002
2004
2005
2006
2007
Chile
Mexico
Mongolia
India
India
Chile
India
Poland
[124]
[14]
[56]
[54]
[139]
[20]
[55]
[140]
Dioxin
Chemical fertilizer plant
(controls live 1–3 km away)
Chemical fertilizer plant
(controls live 5–8 km away)
15/16
0.56
0.6
2000
5
4
2001
Russia
[72]
15/14
0.46
0.7
2000
5
4
2001
Russia
[72]
Ethylene oxide
Hospital sterilization unit
Hospital workers
Chemical plant
9/50
10/10
75/22
0.51
0.36
2.70
0.9
1.6
1.2
3000
3000
1000
1
1
1
3
3
3
1990
1991
1994
Italy
Sweden
Brazil
[8]
[141]
[118]
Formaldehyde
Mortician students
Mortician students, all MN
Centromere negative MN
Anatomy class students
Anatomy and pathology staff
29/29
19/19
13.0*
3.3*
9.0*
1.5*
2.2*
1500
1500
1
6
4
6
1993
1996
USA
USA
[51]
[13]
25/25
28/18
0.05
0.60
0.1
0.57
0.33
NR
3000
7
1
3
3/4
1997
2002
China
Turkey
[58]
[59]
Battery shop workers
10/10
1.00
3.3*
2000
1
4/6
2003
Brazil
[62]
Car painters
10/10
1.10
3.1*
2000
1
4/6
2003
Brazil
[62]
Ozone
Chamber, 200 ppb
15/15
1.25
0.7
3000
3
6
2006
USA
[22]
PAHc
Child laborers
(working in engine
repair shops)
Taxi drivers
Traffic police
34/28
0.50
1.4
3000
1
3/4
1999
Turkey
[45]
17/20
15/20
0.30
0.30
4.0*
3.3*
3000
3000
1
1
3/4
3/4
1999
1999
Turkey
Turkey
[45]
[45]
PAH, colorants, toxic gases
Photocopy machine operators
98/90
3.80
1.7*
2000
3
NR
2004
India
[64]
Pesticide mixture
Greenhouse workers
Agricultural workers
Exposed workers
Agricultural workers
64/50
50/66
84/65
228/213
1.77
1.73
1.98
2.12
1.0
0.8
0.8
1.0
2000
2000
2000
2000
2
2
2
2
NR
6
6
6
2000
2001
2002
2003
[111]
[50]
[49]
[112]
Pesticide manufacturing unit
Floriculturists
54/54
30/30
3.20
3.80
3.9*
2.7*
2000
3000
2
1
NR
2
2006
2000
Spain
Greece
Hungary
Hungary,
Poland, Greece
India
Mexico
Beedi smokers
Cigarette smokers
Chewers of betel quid
with tobacco
Gudakhu users (for >20 years)
Powdered tobacco
(snuff users)
Chewers of tobacco
mixed with lime
Chewers of tobacco
mixed with lime
Maras powder
Powdered tobacco
(snuff users)
25/25
15/14
27/35
1.36
8.40
2.59
1.3*
2.4*
1.9*
1500
1500–3000
1000
4
1
5
7
3
1
2004
1997
1991
India
Turkey
India
[142]
[16]
[94]
102/56
20/13
3.50
1.90
5.9*
2.9*
1000
1000
1
1
3
3
1992
1993
India
India
[92]
[93]
27/35
2.59
2.0*
1000
5
1
1991
India
[143]
20/15
1.90
3.1*
1000
1
3
1993
India
[93]
15/25
15/38
8.40
1.58
*
2.2
2.4*
1500–3000
3000
1
1
3
3
1997
1991
Turkey
USA
[16]
[10]
Shoe factory
workers (exposed
to solvent-based
adhesives)
Shoe manufacture
29/25
0.31
1.9
2000
1
4/6
2005
Brazil
[74]
21/18
0.33
1.8*
3000
1
3/4
2002
Turkey
[59]
0.70
0.96
*
5000
5000
2
1
2
2
1997
1997
Germany
Germany
[144]
[144]
Lead paints, solvents,
benzene
Tobacco
Tobacco (smokeless)
Toluene, hexane, acetone,
methyl-ethyl-ketone
2-Trans-hexenol
Banana growers
Hexanol rinse of 10 ppm
7/7
7/7
2.6
2.7*
[65]
[66]
Naturally present in fruit (i.e. banana).
a
Staining techniques: (1) Feulgen/Fast Green; (2) DAPI, (3) Giemsa, (4) Acridine orange, (5) Acetorseine, (6) FISH, (7) Wrights.
b
Scoring criteria: (1) Basic, (2) Stich and Rosen [36], (3) Sarto et al. [9], (4) Tolbert et al. [10], (5) Livingston et al. [128], (6) Titenko-Holland et al. [95], (7) Countryman [149],
NR = not reported.
c
PAH = polycyclic aromatic hydrocarbons.
*
Statistically significant at p < 0.05 according to the authors.
98
N. Holland et al. / Mutation Research 659 (2008) 93–108
propidim iodide with pancentromeric FISH labeling [13]. Significantly higher frequencies of MN have also been observed in
exfoliated buccal cells from people exposed to organic solvents,
antineoplastic agents, diesel derivatives, polycyclic aromatic
hydrocarbons, lead-containing paints and solvents, and drinking
water contaminated with arsenic [14,43–46,52–67]. Studies on the
effects of exposure to pesticide mixtures, however, generally show
no increases, with the exception of a study involving a small group
of floriculturists in Mexico in whom a 2.6-fold increase in MN
frequency was observed [66], and a 3.9-fold increase reported for
workers at the pesticide manufacturing plant [65]. The lack of
effect can be attributed to the relatively weak genotoxicity of most
modern pesticides and the dermal, rather than oral, route of
exposure [68–70]. Negative results were also obtained in subjects
exposed to chromium, ethylene oxide, nickel, benzene, and other
chemicals [8,15,47,61,71–74]. Recent studies have also suggested
genotoxicity and cytotoxicity of urban air pollution and ozone
during the summer season, particularly in places with high
ambient levels [22,75,76]. Overall, although the link between
exposures and increases in MN frequencies is strong for many
chemicals and exposure conditions, further efforts are warranted
to build a reliable database on the effects of common exposures,
such as pesticide mixtures and air pollution, on induction of MN in
buccal cells.
3.2. MN and radiation
Ionizing radiation plays an important role in the treatment of
many neoplasias, but it also produces genetic damage. As a
consequence, secondary tumors may develop years after the
primary tumor treatment. Several studies evaluated MNi in buccal
cells of patients undergoing radiotherapy in the head and neck
region. The most striking increase in cytogenetic damage (150–
300 MN/1000 cells) was observed in an early study of three
patients exposed to a cumulative dose of 3400–4000 cGy [6].
However, these results may have been influenced by the inclusion
of degenerated cells, given that criteria for distinguishing viable
and degenerating cells were not yet established at the time. Other
authors reported 68 MNi/1000 cells after 2000 cGy [9] and 16 MN/
1000 cells after treatment with 1000 cGy for 3 weeks [10]. This
range of doses and treatment periods then raises the question of
the time-course and dose-response for the induction of MN in
buccal cells. This issue was carefully addressed in a study by Moore
et al. [77] (Fig. 4) in which a more than 16-fold increase in MN
frequency was observed shortly after initiation of radiotherapy,
followed by return to baseline 12 weeks later and 3 weeks after
cessation of the treatment. An interesting aspect of this study was
the use of pancentromeric labeling that allowed differentiation
between centromere-positive and -negative MNi. At the peak of
the radiation-induced cytogenetic response, approximately 90% of
MNi contained acentric fragments [77], which reflect chromosome
breakage events. In a more recent study, an increase in MN
frequency in a group of head-and-neck cancer patients undergoing
radiotherapy was evident both in buccal cells and peripheral
lymphocytes [78]. In this study a significant decrease in buccal
mucosa cells with MNi was observed beginning 30 days from the
last treatment. Cao et al. [79] found that the cytokinesis-blocked
MN assay in peripheral blood lymphocytes was more sensitive
than the buccal MN assay for monitoring genetic damage in nine
nasopharyngeal cancer patients undergoing radiotherapy. It was
reported that radiation-sensitive oral tumors showed higher MN
levels in exfoliated cells after radiation therapy than the more
radiation-resistant ones, and the assay can be used as a predictor of
tumor radiosensitivity [80]. However, this approach requires
further confirmation. Data from such radiation exposure studies
have helped to determine the kinetics of appearance and
disappearance of MNi in buccal mucosa and to define the optimal
sampling time. Whether the observed kinetics of MN expression
following radiotherapy is applicable to other genotoxic exposures
that affect nuclear division in the basal cell layer of the buccal
mucosa remains unclear.
3.3. MN frequencies in buccal cells of patients with cancer and other
diseases
Biomonitoring of the changes in patients with diagnosed
diseases or pathological changes that may lead to the development
of cancer and other illnesses is becoming increasingly popular, and
may be the most rapidly growing area of application of the MN
assay to epithelial cells. The MNi in buccal mucosa cells were used
to study preneoplastic effects by collecting the cells directly from
the affected tissues (Table 2). The MN frequencies in patients with
oral lesions such as oral submucosal fibrosis, oral leukoplakia and
oral lichen planus were increased relative to healthy subjects, but
no significant differences in MN rates were observed among the
various patient groups [81]. Another study of untreated cancer
patients observed increased genomic instability in somatic cells
(blood lymphocytes and exfoliated buccal epithelia) in comparison
to healthy control subjects [82]. It was suggested that the MNi in
buccal mucosa may predict cancer risk for the upper aerodigestive
Fig. 4. Dynamics of total MN and centromere-negative (MN ) in buccal cells after radiotherapy. Modified from Moore et al. [77]. Data points show the frequency of MN cells at
different times during and after 9 weeks of radiation therapy in the oral region. Columns show percentage of MN cells that were centromere negative by FISH with
pancentromeric labeling, indicating chromosomal breakage. Approximately 2000 cells were scored at each time period. The MN+/MN ratio at 12 weeks was not determined.
N. Holland et al. / Mutation Research 659 (2008) 93–108
99
Table 2
Micronucleus frequency in exfoliated oral mucosa cells of patients with cancer and precancerous conditions
Type of cancer
Study subjects/
exposure control
Baseline, MN
cells/1000 cells
Upper digestive tract
Oral submucous fibrosis
Oral leukoplakia
Oral lichen planus
Squamous cell carcinomas, upper
aerodigestive track
Oral leukoplakia
Untreated breast cancer patients
First degree female relatives of
breast cancer patients
Oral carcinoma
Breast
Cervix and corpus uteri
Various untreated tumors
Precancerous oral lesions
Oral cancer
38/37
68/20
18/20
14/20
44/98
1.30
1.90
1.90
1.90
9.00
16/98
30/46
60/46
9.00
2.70
2.70
30/30
12
36
37/43
29/60
24/60
1.13
0.93
0.93
0.60
1.6
1.6
a
b
*
# cells scored/
subject
Stain a
Scoring
criteriab
Country
Year
Reference
2.0
6.1*
5.7*
6.2*
2.3*
1000
1000
1000
1000
1000
1
3
3
3
3
2
3
3
3
1
France
India
India
India
Germany
1987
1996
1996
1996
2000
[145]
[81]
[81]
[81]
[83]
2.2*
12.6 *
6.1*
1000
1000
1000
3
3
3
1
NR
NR
Germany
India
India
2000
2000
2000
[83]
[84]
[84]
2000
2000
2000
2000
2000
2000
1
1
1
5
3
3
4
2
2
4
NR
NR
Brazil
Armenia
Armenia
Mexico
India
India
2002
2004
2004
2004
2007
2007
[98]
[85]
[85]
[61]
[146]
[146]
Fold-difference
vs. control
4.5*
2.23*
2.2–3.2*
1.3
1.2*
1.6*
Staining techniques: (1) Feulgen/Fast Green; (2) DAPI, (3) Giemsa, (4) Acridine Orange, (5) Acetorseine.
Scoring criteria: (1) Basic, (2) Stich and Rosen [36], (3) Sarto et al. [9], (4) Tolbert et al. [10], NR = not reported.
Statistically significant at p < 0.05 as defined by the authors.
tract, including premalignant stages such as oral leukoplakia [83].
A significantly higher frequency of buccal cells with MN was found
in breast and uterus cancer patients and their first-degree relatives,
compared to controls [84,85]. At this time it remains unclear
whether an elevated frequency of MNi in certain tissue, such as
oral epithelia, would be predictive of increased risk of future cancer
say only for oral cavity, limited to upper digestive tract epithelia, or
may be projected for various cancers in other parts of the body.
This gap of knowledge will be an important focus of future HUMN
projects.
Several publications reported MN frequencies in patients with
DNA repair deficiencies with varied results. For example, patients
with Louis–Bar syndrome did not have elevated MN frequency [86]
while both Ataxia telangiectasia and Bloom’s syndrome exhibited
increased genome instability in patients (homozygous) compared
to heterozygous subjects [87,88]. A site-specificity was observed
for Xeroderma pigmentosum patients, with a higher MN frequency
in cells from the dorsal tip of tongue, possibly due to greater light
exposure [89].
Recently, a number of papers applied the MN assay and cytome
approach [132] in buccal epithelia. In patients with Alzheimer’s
disease there was no significant increase in the MN frequency but
the frequencies of basal cells, condensed chromatin cells and
karyorrhectic cells were lower than in matched controls [147].
Down syndrome was associated with a 733% increase in MNi in
comparison to younger healthy controls, and the MN frequency
was 78.5% higher than in older controls [148]. A statistically
significant decrease in pycnotic, karyolytic and condensed
chromatin cells was also observed. An increase in MN frequency
in buccal cells was reported for Diabetes mellitus with the patients
having double the level of genetic damage in comparison to
matched controls [109] and for treated pediatric patients with
ulcerative colitis in comparison with controls or children with
Crohn’s disease [108].
3.4. Lifestyle and host factors
Fig. 5 summarizes the distribution of positive and negative
findings with respect to the effect of age, sex, smoking, and alcohol
consumption on buccal cell MN frequency. Although many studies
report the age and sex of the study subjects, only a fraction of these
studies were able to establish a statistically significant effect by
gender [14,90] or by age [16,47,67]. It is interesting that in two
studies men had a slightly higher MN frequency in buccal cells than
women. These results are in contrast to data showing higher MN
frequencies in lymphocytes of women and older subjects [3]. In the
study of 120 healthy subjects from Poland, neither age or sex were
significantly associated with MNi [127]. However, smokers on
Fig. 5. Baseline distribution of MN frequency: role of host and lifestyle factors.
100
N. Holland et al. / Mutation Research 659 (2008) 93–108
average had almost triple the MN frequency compared to nonsmokers. Yet other publications report no difference between
smokers and non-smokers or men and women [82]. Thus, the
potential contribution of gender and age in the frequency of MN in
buccal cells warrants further investigation.
Lifestyle factors that are associated with genetic damage
include smoking, alcohol consumption, and diet, especially
vitamin deficiencies and supplementation [1,91]. The majority
of the studies reporting a significant increase in MN in buccal
mucosa cells related to a risk of oral cancer were performed in subgroups of subjects with specific lifestyle habits, i.e. chewers of betel
quids (areca nut, betel leaves, slaked lime and tobacco) from India,
Taiwan and Philippines; reverse smokers (who hold the lit end of
the cigarette inside their mouths) from India and Philippines; snuff
dippers from Canada; users of Khaini tobacco (tobacco mixed with
slaked lime) from India, and other similar practices [10,92–94].
Results in these studies may be affected by overestimates of the
MN frequency because both smoking and chewing of tobacco
mixtures are known to cause nuclear degeneration and appearance
of MN-like bodies in exfoliated cells, which may be confused with
MNi. The increased frequency of cells with degenerating nuclei
may occur due to normal cytotoxic processes and may not
necessarily be associated with clastogenic or aneugeneic DNA
damage [5,95–97]. It is important for monitoring and risk
assessment to distinguish cell death events from genome damage
in viable epithelial cells both in terms of biological dosimetry and
for evaluating cancer risk.
In several studies of lifestyle factors, however, it was difficult to
differentiate the effect of alcohol from that of smoking. For
example, neither alcohol or smoking, alone, increased the MN
frequency in buccal cells, but a synergistic effect of smoking and
alcohol was evident, with up to a 5.5-fold increase relative to nonsmoker and non-drinker controls [36]. A prospective study
comparing alcoholics with oral or oropharyngeal carcinoma to
healthy subjects who did not consume any alcohol found a
significant association between MN frequency and the age of the
beginning and cessation of drinking, and the duration of alcohol
consumption [98]. This apparent synergistic interaction of alcohol
consumption and smoking, and the relative contribution of each
exposure, is another aspect of buccal cell monitoring that requires
further study.
A number of micronutrients, including beta-carotene and other
vitamins, have been shown to significantly decrease MN levels
(1.4–4-fold) in healthy tobacco users, as well as in individuals with
precancerous lesions [7,99–101]. A study of heavy smokers in the
Netherlands found a statistically significant decrease in MN
frequency when N-acetylcysteine was administered as chemopreventive agent [101]. The effect of chemoprevention on the
remission of specific lesions such as leukoplakia, and on the
prevention of new lesions, can be observed after treatment, and it
is accompanied by a corresponding decrease in MN frequency
[102,103]. These results suggest that decreases in MN frequency in
established precancerous lesions, which are likely to be genomically unstable, may be indicative of a reversion to a more normal
phenotype, but whether these changes are causal or coincidental
remains unclear. Other micronutrients, such as retinol, riboflavin,
zinc, and selenium, however, failed to reduce the MN frequency in
a study carried out in China in areas with a high incidence of
oesophageal cancer [104]. There was no effect of drinking water in
women from two regions in Turkey with high or low boron [105].
In contrast, a reduced MN frequency was associated with green tea
consumption in patients with oral leukoplakia [106]. Declines in
MN frequencies were reported in children and women who
received controlled folate supplementation [107,108] and in
patients with diabetes [109]. It is not clear whether the observed
reductions in the MN frequencies in these studies were due to a
decrease in chromosomal instability as a result of supplementation, or to a modified basal cell proliferation that altered the
kinetics of MN expression.
4. Methodological aspects affecting identification of MN
Methodological factors that can affect the levels of MNi in
buccal cells include differences in cell collection (timing and
implements used), fixation and staining techniques, selection and
number of cells counted, and the scoring criteria for MNi and other
nuclear anomalies in normal and degenerated cells. Many of these
methodological factors for buccal cells overlap with the MN assay
in lymphocytes, but the differences in tissues may contribute to the
variability. Thus, the data reported by the HUMN on MN assay in
human lymphocytes [3] can be applied to buccal cells with caution,
although a special validation study for buccal cells is needed. It is
difficult to tease apart the effects of these factors or their
combinations based on the available studies in different laboratories. Many laboratories have their preferred protocols and only a
handful of attempts have been made to compare modifications of
the procedure within the same study [95–97,110]. These are
discussed in more detail in Section 4.2. The main sources of
variability among the laboratories may lie in the scoring criteria
and staining procedures used. The effects of these factors on MN
scoring in the buccal assay have not been properly evaluated or
quantified.
4.1. Collection of buccal cells
Exfoliated buccal mucosa cells can be collected using a wooden
tongue-depressor, a metal spatula, or a cytobrush moistened with
water or buffer to swab or gently scrape the mucosa of the inner
lining of one or both cheeks. A few studies have used toothpicks or
toothbrushes [17,49,50,56,90,111,112]. In some cases, buccal cells
have also been collected from the inner side of the lower lip and
from the palate; the variability in MN frequency between these
areas was minimal for control subjects as reported in earlier
studies [35,113]. In contrast, MN frequency was significantly
higher in epithelia of the pharynx of adolescent girls from Mexico
City in comparison to their cheek [114]. Cytobrushes appear to be
most effective for collecting large numbers of buccal cells.
Casartelli et al. [115] observed that MN frequencies were higher
when cells were collected by vigorous, rather than by light,
scraping, suggesting a decreasing MN frequency gradient from
basal to superficial layers of mucosa.
The turnover rate for the appearance of MN in exfoliated buccal
cells in an otherwise normal cell after exposure to an acute
genotoxic event, such as ionizing radiation, is estimated to be a
minimum of 5–7 days. Inter-individual variation during this timecourse of MN expression, however, has been observed so that peak
expression of MN may be delayed up to 21 days [6,8,77]. Thus,
multiple sample times are required to identify optimal timing
between 7 and 21 days after exposure because peak expression
may vary depending on the effects of the particular DNA damage or
chromosomal exposure on basal cell turnover rate. It is possible
that certain genotoxic exposures could cause an inhibition or an
enhancement of the basal cell proliferation and thus confound the
kinetics of MN expression.
4.2. Slide preparation and staining
In many MN studies, buccal cell smears have been prepared by
spreading the cells on a clean slide. In a number of studies the
cytobrush used to collect buccal cells was shaken in a centrifuge
N. Holland et al. / Mutation Research 659 (2008) 93–108
tube containing saline solution (Hank’s basic or other buffer
solution) to release the cells, and the tube is then centrifuged to
wash the cells in a buffer solution [17,37,43,47,51,54,60,67,
72,95,111,116–118] or a fixative [56]. This washing procedure
helps to remove bacteria and cell debris, which confound the
scoring. Cells are then transferred to slides either by careful
dropping with pipette or by cytocentrifugation followed by
fixation. Commonly used fixatives include 80% methanol, absolute
ethanol, or a methanol–glacial acetic acid mixture. The stringency
of the fixative and the condition of the cells before fixation (air
dried or fresh) may affect cell integrity and the preservation of
normal and degenerated cells with MN and other abnormalities.
Several staining methods have been used, although DNAspecific stains are preferred for staining nuclei, MN, and other
nuclear anomalies in buccal exfoliated cells. Feulgen-Fast Green
(FFG) staining is favored by many investigators because of its DNA
specificity and a clear transparent appearance of the cytoplasm
which enables easy identification of MNi (Table 1). MNi stained
with FFG have been examined under light microscopy in the
majority of the studies, although it is possible to examine these
slides under fluorescence. One shortcoming of this staining
method is that it is relatively lengthy and may lead to the
underscoring of MNi [96]. Some studies used fluorescent dyes such
as DAPI [104,111,112], acridine orange (AO) [73,119], Hoechst [96],
and propidium iodide (PI) [15,77,95,96]. May-Grunwald Giemsa
(Giemsa) stain has been used at different concentrations (2–10%)
in several laboratories [22,81,83,84,86,104,120]. Some of the
studies reported increased frequencies of MNi with Giemsa
staining and suggest the possibility that cellular structures
resembling MNi, such as keratohyalin granules or bacteria, can
lead to false positive results [96,97]. Keratohyalin granules are
reported to occur in cells of the granular layer of interfollicular
epidermis of the skin [121] and are predominantly composed of
the 400-kDa protein profillagrin and, therefore, possibly distinguishable from MNi using immunohistochemical methods. However, no specific data about the commonality of these granules or
their frequency in oral epithelia is currently available.
Another factor that can interfere with MN scoring is contamination by the bacteria that are commonly found in the mouth
[95,110]. Bacteria can be differentiated from MNi by their
characteristic shape, smaller size, color, staining intensity, and
their presence upon and between buccal cells on the slide. Another
common confounding issue is the small dye granules that may
sometimes resemble MNi but usually have a slightly different
refractility and color intensity. These factors need to be taken into
consideration when optimizing the staining protocol to minimize
the possibility of scoring these artifacts.
A wide range of baseline MN values have been reported,
regardless of the staining method used (Table 1). The conclusions
differed in five studies that attempted a systematic comparison of
two or more staining methods for buccal cells. For example, FFG
staining was compared with PI, which also served as a counterstain
for pancentromeric fluorescent probes following in situ hybridization (FISH) [95]. In this study the fluorescent dye PI performed
equally well as FFG, but provided an additional advantage of
identifying the mechanism of MN formation through centromeric
FISH labeling. Casartelli et al. evaluated the strengths and
limitations of three staining procedures (Hoechst, PI, and Giemsa),
and suggested that Hoechst was the easiest and most reliable for
identifying buccal cell MN [96]. A more recent paper that assessed
four staining procedures (Giemsa, FFG, DAPI, and acridine orange),
concluded that Giemsa is more likely to lead to false-positive
identifications of MNi and recommended FFG as a preferable
staining method [97]. Another recent study compared Giemsa with
the Papanicolaou (Pap) stain for buccal cell analysis under field
101
conditions [110] and concluded that the Pap stain was the
preferred method of detecting MN in oral epithelia. FFG was
compared with acridine orange in the study of oral exfoliated
subjects with leukoplakia and squamous cell carcinoma [122], and
the authors reported that in their hands fluorescent staining
(acridine orange) was more sensitive for MN detection than FFG.
The possibility that Giemsa staining could lead to the misidentification of artifacts as MNi, because it may not distinguish
true MNi from profillagrin or keratohyalin granules also needs to
be investigated. Because these studies came to different conclusions, it is apparent that the stain, staining procedure, and
laboratory scoring procedures, may all contribute to the variability
seen. Studies are needed to determine whether some MNi and
nuclei may lose DNA through karyolysis while maintaining the
protein structure of chromatin and the nuclear envelope, so that
they would still be detectable by stains that are not DNA-specific.
Therefore, a systematic study investigating the performance
characteristics and the extent of correlations among the MN
frequencies determined with various staining methods currently
in use is urgently required so that an optimal staining protocol can
be determined. Because FFG has been shown to be the most
consistent, and is DNA-specific, it would be advisable to suggest
this staining method as the interim standard against which other
methods should be validated.
4.3. Scoring criteria
Another important aspect of the MN assay in buccal mucosa
cells is the criteria for identifying and scoring the cells. The first
publications of Stich and Rosin [7,34,35,40,99] referred to the wellestablished basic criteria for MN that were initially described by
Heddle [123]. However, criteria for identifying cells for inclusion
into the MN frequency count were not provided. Other authors
refer to the Heddle criteria as such, or with minor modifications
(Table 1). The criteria developed by Tolbert et al. [5,10] for
choosing the cells are the most widely used. They consist of the
following parameters for cell inclusion in the cells to be scored: (a)
intact cytoplasm and relatively flat cell position on the slide; (b)
little or no overlap with adjacent cells; (c) little or no debris; and
(d) nucleus normal and intact, nuclear perimeter smooth and
distinct. The suggested criteria for identifying MN are: (a) rounded
smooth perimeter suggestive of a membrane; (b) less than a third
the diameter of the associated nucleus, but large enough to discern
shape and color; (c) Feulgen positive, i.e. pink in bright field
illumination; (d) staining intensity similar to that of the nucleus;
(e) texture similar to that of nucleus; (f) same focal plane as
nucleus; and (g) absence of overlap with, or bridge to, the nucleus.
Tolbert et al. [10] outlined all the specific nuclear alterations
that should be taken into account when deciding to include or
exclude cells for determining the frequency of MN cells, and
provided evidence that other nuclear anomalies are at least as
common as micronucleation and, therefore, have the potential for
misclassification. An updated diagram, based on the original
scheme published by Tolbert et al. [5] of the various cell types and
nuclear anomalies that can be observed as the cells migrate to the
surface of the buccal mucosa is shown in Fig. 3. Common cell types
associated with the differentiation process are shown, including
cells with pycnotic, condensed chromatin, karyorrhectic, karyolitic
(‘‘ghost’’ cells), nuclear bud (or ‘‘broken egg’’) and binucleated cells.
Alternately, these cells could also represent responses to cytotoxicity or be secondary to genotoxic events. The processes
underlying the frequency and appearance of these structures are
not yet fully understood. Despite these drawbacks, a large number
of publications refer to these criteria [16,43–46,54,56,67,
71,72,75,81,120,124–127]. Less detailed criteria, similar to those
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N. Holland et al. / Mutation Research 659 (2008) 93–108
of Tolbert’s but without dividing the cells into three categories of
certainty of identification, have also been used [9,102,104,
107,117,128,129]. Results from studies that do not report scoring
criteria are more difficult to evaluate [73,84,106,119], which is
why this information should be requested by peer-reviewers and
editors to improve the quality and coherence of MN publications. A
systematic comparison and optimization of scoring criteria,
however, has yet to been performed.
4.4. Number of cells scored; power of the test to detect an increase
The number of buccal mucosa cells to be scored in order to
obtain statistically significant results needs to be addressed. The
first studies from the 1980s [6,34,35] evaluated a relatively low
number of cells (500). Later, Tolbert et al. [10] recommended the
scoring of at least 1000 cells, with an increase to 2000–3000 if
fewer than 5 micronucleated cells were observed after counting
1000 cells. Most published studies have scored between 1000 and
3000 cells (Table 1), although it has been suggested that 10,000
cells may be needed to observe a statistically significant, 50%
increase, in MN frequency [130]. This approach would require
automation, especially in biomonitoring studies that include a
large number of subjects. However, the power to detect
statistically significant results also depends on the baseline MN
level in the study population. The factors that may modulate
baseline MN levels in buccal cells are discussed elsewhere in this
review. The frequencies of the various cell types illustrated in Fig. 3
should be determined independently and concurrently during the
scoring as these markers may also be correlated with exposure to
genotoxins. Further research is needed to quantify these various
cell types and nuclear anomalies in buccal cells, and to establish
their relationship with MN frequency and exposure to genotoxins.
5. A proposed validation study to examine slide preparation
and scoring variables
The HUMN project’s experience with the assessment of the
intra- and inter-individual variability of the lymphocyte MN assay
using an international validation project and slide-scoring exercise
[2,3] can serve as an example for developing a strategy for
understanding and validating the buccal cell MN procedures. Fig. 6
shows the different factors contributing to the variability of the
human lymphocyte MN assay that was developed by the HUMN
validation project; it is evident that, in this assay, there is only a
very small fraction of unexplained variance (3.1%) in the MN
frequency [3]. These values were obtained through the use of a
careful study design which took the main explanatory variables
into consideration, and also because of prior agreement and
standardization of the scoring procedures among participating
laboratories. It should be noted that the participating laboratories
represented a wide range of prior experience with the assay. This
inter-laboratory agreement was possible because scoring criteria
with detailed photos illustrating each endpoint were prepared for
the participants before the exercise [2] and later published along
with its results [3]. The main response variable, i.e. dose of
genotoxin (in this case, irradiation, which would be expected to
produce less inter-laboratory variability than chemical treatment)
accounts for the largest portion of the variance (65%). The
contribution of the laboratory to the MN frequency variance
was much smaller (4.6%), and the intra-scorer variability and effect
of staining method each contributed less than 1% of the total
variance. The situation with the buccal cell MN assay may be
somewhat different because a wider range of cell collection, slide
preparation, and staining methods are currently being used, and
Fig. 6. Factors affecting variability when scoring MN in human lymphocytes.
Reproduced with permission from Fenech et al. [3]. MN frequency in human
lymphocytes was evaluated by 51 scorers from 34 laboratories in 21 countries. Cell
cultures were established using peripheral blood of one donor and irradiated with 1
or 2 Gy of gamma rays. Spot is referring to two round areas separately scored for
each slide. Methods of staining and criteria of scoring are described in detail in Ref.
[3].
there is a larger variability in scoring procedures among the
different laboratories.
HUMN committee recently sent out an invitation to the
prospective collaborators to contribute to HUMNXL project (XL
subscript was designed to emphasize the focus of this HUMN
initiative on the MN assay in exfoliated buccal cells). A detailed
questionnaire was mailed to over 200 researchers who have
published papers using the buccal cell MN assay, participated in
the HUMN Workshops over last 10 years, or contacted HUMN
committee through the HUMN website or personal communications. We anticipate that MN data for several thousands subjects
will be incorporated in the database to be used for analysis of
factors of variability, and later for establishing predictive value of
the MN frequency in buccal cells for tissue-specific or generalized
cancer risk. These documents are available on the HUMN website
(http://www.humn.org).
5.1. Study design
The next step of the HUMNXL will be to conduct a specially
designed validation exercise focused primarily on the effects of
staining and scoring procedures. The process of the initial slide
generation for HUMNXL buccal MN project may be more
complicated than it was for the lymphocyte MN validation
exercise. In the latter case, blood cells from the same donor were
used for control and irradiated cultures, and the half of the slides
were stained in a single laboratory by a standard Giemsa method
[2]. The remainder of the fixed slides were stained in each of the
participating laboratories by their method of choice. Because most
of the laboratories had one or two scorers involved in the exercise
and only few research groups had four participants, the scope of
the intra-laboratory scoring analysis remained manageable. Each
scorer spent approximately 2 days to analyze six slides using two
separate, delineated areas (spots) on each slide. However, for the
buccal cell study, it may be necessary to collect cells from a number
of subjects, because only a limited number of cells can be obtained
from each subject at one time without introducing a possible
variability of the depth of exfoliation or the timing of collection. In
N. Holland et al. / Mutation Research 659 (2008) 93–108
order to standardize the slides for the scoring exercise, cells from
these several subjects can be pooled together and thoroughly
mixed before placing them randomly on a sufficient number of
slides for the distribution to the participating laboratories. Initially,
the slides could be generated and stained by a standard method
(e.g. FFG) in one laboratory, with the provision that a portion of the
slides will be available for staining in each of the participating
laboratories using their preferred method. All slides would be
coded before distribution to the scorers in different laboratories.
The background MN frequency in buccal cells is relatively low
(0.1%); therefore the large number of cells that will have to be
scored for sufficient statistical power of analysis may impose a
significant demand on time and resources of participating
laboratories. However, if the assay is to have any validity as a
biomarker of exposure or effect, sufficient cells would have to be
scored to allow for a stringent statistical analysis of the data.
This initial stage of assay validation will focus on (a) addressing
the effect of different staining methods on the results, and (b)
determining the extent of variability in slide scoring among scorers
in the same laboratories, and among laboratories. This latter
exercise would only be performed after detailed scoring criteria are
established and distributed, as described in Section 5.5. A
subsequent phase of the validation study may include a
simultaneous collection of samples in different locations from a
designated number of subjects in order to perform a sufficiently
powered study aimed at determining the effect of geographic,
ethnic, and lifestyle variability, as well as sampling variability. The
slides used in such a study would be prepared, stained and scored
according to a previously standardized protocol developed and
endorsed by the HUMNXL project participants.
5.2. Positive control
The choice of a positive control (comparable to in vitro
irradiation that was employed for lymphocyte project) will present
a challenge for the buccal cell MN validation project. One of the
possibilities is to use cells from patients undergoing X-ray
treatment in the head/neck area during the period most likely
to lead to significant MN induction, as was discussed above.
However, it may be difficult to generate sufficient, identical
positive control slides to provide to all participating laboratories.
Another alternative is to use specimens from patients with a DNA
repair disease, such as Bloom syndrome, which have been shown
to have an elevated buccal cell MN frequency [131]. Before these
positive control slides are used, however, a pilot scoring exercise
will be needed to assure that significantly increased MN levels can
be observed.
5.3. Sampling method
It will be necessary to design a protocol to allow the scoring of
the ratios of basal, differentiated, and degenerated cells, and to
determine whether the buccal cell MN frequency is affected by
these ratios because (a) sampling method and inter-individual
differences may alter ratios of basal to differentiated and
degenerating cells, and (b) MNi in the degenerating cells may be
lost due to lysis or degradation. This potentially confounding factor
could be addressed by alternating the timing of collection after
controlled exposure, and by variation of intensity of ‘‘brushing’’ of
the cheek lining.
5.4. Slide preparation method
The effect of staining method on the MN frequency in various
buccal cell types needs to be determined conclusively. A standard
103
protocol for slide preparation, fixation, and staining for both light
and fluorescence microscopy will have to be developed and
validated. Alternative methods of staining should be validated
against the better-established FFG method, which allows scoring of
slides under both transmission and fluorescence microscopy. It is
evident that methods that could also stain other structures that
resemble MNi, such as keratohyalin granules, need to be
discouraged or should be required to include a process (e.g.
immunohistochemical stain to profillagrin) that allows these
structures to be distinguished from true MNi.
5.5. Scoring criteria
A standard set of scoring criteria, together with matching
photomicrographs and line diagrams for the buccal MN assay,
similar to those developed for the cytokinesis-block lymphocyte
MN assay [2], is required to standardize scoring and minimize
inter-laboratory differences. These criteria should be designed to
enable (a) easy and unbiased classification of the various cell types,
as outlined in Fig. 3, and (b) identification of those cells that
contain one or more ‘‘true’’ MNi. Because ‘‘true’’ MNi are originally
expressed in basal cells and may be retained in normal
differentiated cells but lost in degenerating cells (i.e. condensed
chromatin, karyorrhectic, pycnotic, and karyolytic cells), and cells
with degenerating (apoptotic) nuclei may generate structures that
look similar to MNi, it is essential that MN frequencies in these
different cell types are reported separately.
5.6. Number of cells to be scored
A statistical assessment of the optimal number of buccal cells to
be scored is required because of the low spontaneous MN
frequency in these cells. Currently, most of the published reports
use 1000–3000 cells. It is anticipated that more precise results
would be obtained with increasing numbers of cells, up to a point
where the additional precision gained would not be worth the
additional effort. Automated scoring of MN in buccal cells could
potentially increase feasibility of scoring of 10,000 cells or more,
thus significantly increasing the power of analysis. An acceptable
minimum number of basal and normal differentiated cells needs to
be established for determining MN frequency, and an adequate
coefficient of variation for repeat measurements needs to be
established.
5.7. Cytome approach
Experience with the cytokinesis-block lymphocyte MN assay
has shown that a ‘‘cytome’’ approach based on scoring not only MN
frequency but also other genome damage markers (e.g. nuclear
buds, etc.), dead or degenerated cells, as well as the proliferation
index, assures a more comprehensive measure of cytotoxic and
genotoxic effects and provides important mechanistic insights
[132]. In this regard, the development of strict scoring criteria and
visual examples of various types of exfoliated buccal cells for all
participating scorers becomes essential.
6. Addressing knowledge gaps with the buccal cell assay
Although it would be wonderful to present a detailed plan for all
future stages of the HUMNXL at this time, we view the proposed
overview as a work in progress, to be developed following
discussions among the collaborators, including the prospective
participants. Because there are several different aspects of the
assay to be addressed, the ‘‘plan’’ of attack will evolve as the project
progresses based on information developed from the initial
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N. Holland et al. / Mutation Research 659 (2008) 93–108
activities and from new information generated by the scientific
community as a consequence of this article. There are many
remaining gaps of knowledge related to biomonitoring using the
MN assay in buccal cells that will be addressed in next sections,
including the effects of cell kinetics, a better understanding of the
buccal cells biology and structure, effects of various host and
lifestyle factors, correlation of the MN data between buccal and
other cell types, and a predictive value of the MN frequency for
cancer and other diseases.
6.1. Effect of cell division kinetics on MN frequency
MN expression occurs only in cells that have completed nuclear
division following the genotoxic insult, and the proportion of those
cells in the overall cell population affects the observed MN
frequency. The relationship between the scorable MN frequency
and the proportion of dividing cells among the basal cells is not
known, but may be altered by division kinetics in response to an
environmental challenge or a genetic defect in cell cycle
checkpoints. The modeling of the effect of altered cell division
kinetics in basal cells on the MN frequency in buccal cells is a
necessary area of research.
6.2. Improved knowledge of the biology and structure of buccal cells
A better understanding of the molecular events underlying the
cytogenetic and cytological changes in basal and differentiated
buccal cells in response to environmental and nutritional stress is
needed. This would help to clarify whether other structures in
these cells (such as keratohyalin granules) are increased concomitantly with MN formation. Insufficient information is available about metabolic capacity of buccal cells and persistence of
MNi in the epithelial stem cells.
6.3. Variables that affect MN frequency in buccal cells
A systematic and adequately powered investigation of key
variables such as age, gender, genotype, season, diet, oral hygiene
(e.g. hydrogen peroxide in tooth paste) and dental health (e.g. high
number of missing teeth, periodontal status, etc.), lifestyle,
smoking, alcohol, and other recreational drugs, needs to be
performed to identify the variables that have to be controlled.
6.4. Correlation with MN frequency in other cell types
A number of studies used two or three cell types from the same
subjects to assess MN frequency [15,51,107,112]. However, it
remains unclear whether the MN frequency in buccal cells would
be predictive of MN frequency in other cell types such as
lymphocytes. Further, in addition to a direct contact exposure in
the mouth, a possibility of MN induction in buccal cells resulting
from systemic exposure throughout blood stream should be
considered. This knowledge is important to determine whether
one of these measurements can be a surrogate for the other. Until
this is known, it may be necessary to biomonitor both buccal and
blood cells, and determine the extent to which one can extrapolate
observations between the two cell types and integrate the damage
from a variety of concurrent exposures.
6.5. Does the MN frequency in buccal cells predict risk for cancer or
other chronic diseases?
It is well-established that chromosome aberrations in human
lymphocytes predict a risk of cancer [133–136], and the same
association was recently confirmed for MNi in lymphocytes [4].
However, it remains unknown whether an elevated MN frequency
in buccal cells is predictive of cancer risk, and whether the
relationship is primarily site-specific (oral cavity), or extends to
overall cancer risk. A prospective study would help to validate the
use of the buccal MN assay as a biomarker of cancer risk in addition
to being a biomarker of exposure.
7. Summary and future directions
Although many studies have consistently shown a statistically
significant increase in the buccal cell MN frequency in human
populations exposed to genotoxic agents, or a decrease as a result
of micronutrient supplementation or chemoprevention, the
magnitude of changes is usually relatively small. Different
confounding factors influencing the MN frequency in peripheral
lymphocytes, such as gender, age, and lifestyle habits, have been
considered for the buccal cell MN assay. However, the majority of
studies failed to demonstrate any influence of age or sex, although
few of the studies have included a sufficiently broad age range, and
some were underpowered for these effects. Only heavy smoking
and other forms of tobacco consumption have been associated
with oral malignancies. The low baseline MN frequency in buccal
cells may amplify the statistical problems in the scoring, but at the
same time provide a low background level against which induced
genotoxic effects may be more readily observed. Despite the
considerable potential of the buccal MN assay for biomonitoring,
the diversity of possible methodological variables, and their impact
on assay performance, could hinder consistency among laboratories with regard to measuring the effects of dietary, lifestyle, and
genetic factors.
In this context it is mandatory that a standardization of the
buccal cell MN protocol and of the scoring criteria be undertaken,
and that an inter-laboratory calibration exercise be organized. This
would enable data from different laboratories and different
countries to be more comparable and eliminate or minimize
uncertainty due to methodological variables. In addition, the
development of automated scoring systems is encouraged as a
critical advancement for high-throughput and statistically powerful analysis.The HUMNXL project plans to address some of the
important issues listed above in consultation with key scientists in
this field.
The discussions about the best approach to carry forward the
buccal cell MN project took place at the HUMN workshop during
the 5th International Conference on Environmental Mutagenesis in
Human Populations that was held in Antalya, Turkey, on 21 May
2007. The workshop was attended by 70 representatives from
various laboratories, universities, private companies and government departments from around the world. A list of participants
and their email addresses was collected during the meeting for
future contacts.
The aims of the workshop were to: (1) discuss current state of
knowledge on the buccal MN assay; (2) identify important gaps of
knowledge regarding theory, biology and methods used for buccal
cell analyses; (3) decide on a plan of action to resolve the key
methodological and knowledge gap issues; (4) explore pooling
databases to determine most important variables affecting the
assay. Presentations were followed by an open discussion on
priorities, prospects and plans for a collaborative study to improve
and standardize the buccal MN assay. There was an energetic
discussion on various aspects of the assay reflecting the urgency and
importance of resolving methodological issues that would enable a
harmonized application of this important method. A detailed report
about this workshop is available at the HUMN website.
It was decided to initiate first phases that include creation of the
protocol for collection of the datasets and publication of the
N. Holland et al. / Mutation Research 659 (2008) 93–108
detailed method paper based on the most commonly used
protocols and scoring criteria as soon as possible. The draft of
this paper will be available for an open review by experts outside of
the HUMN committee before its submission to Nature Protocols.
The first stage in collecting already available datasets from
prospective collaborators was initiated in March 2008. An
invitation and a detailed questionnaire that will enable investigators to characterize factors of variability for the MN assay in
buccal cells were mailed to over 200 researchers all over the world,
and are also available at http://www.humn.org.
The next steps of the HUMNXL plan for the buccal cell MN assay
include international validation efforts, starting with the currently
planned scoring and staining validation exercise followed by
establishing the predictive value of this assay for cancer and other
diseases. Based on our previous experience with the MN assay in
human lymphocytes, these efforts will require combined efforts of
dozens of collaborators from many countries and will be carried
forward over the course of several years. Expression of interest for
participation in this project and responses to the HUMNXL
questionnaire should be send to the HUMN website contact list
(http://www.humn.org).
Acknowledgements
This research was partially supported by the following grants:
NewGeneris (FOOD-CT-2005-016320-2) an integrated research
project operating within the European Union 6th Framework
Program, Priority 5: ‘‘Food Quality and Safety’’; ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a
network of excellence (FOOD-CT-2005-513943) operating within
the same EU Framework Program, Priority 5; from the Packard
Foundation, Kacyra Foundation and Crohn’s and Colitis Foundation
of America; NIH (M01 RR01271 and K24 DK60617), NIH grants R01
60689 and M01RR00083-41, and the UC Toxic Substances Research
and Teaching Program. Contribution of the Associazione Italiana
per la Ricerca sul Cancro (AIRC) and assistance with the paper
preparation by Dr. C. Chen and N. Shaikh are gratefully acknowledged.
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