Environmental and Molecular Mutagenesis 45:258^270 (2005)

Review Article

Human Population Studies With Cytogenetic

Biomarkers: Review of the Literature
and Future Prospectives
Stefano Bonassi,1* Donatella Ugolini,1,2 Micheline Kirsch-Volders,3
Ulf Str˛mberg,4 Roel Vermeulen,5 and James D. Tucker6
1
Unit of Environmental Epidemiology and Biostatistics,
National Cancer Research Institute, Genoa, Italy
2
Dipartimento di Oncologia, Biologia e Genetica, University of Genoa,
Genoa, Italy
3
Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel,
Brussel, Belgium
4
Department of Occupational and Environmental Medicine, Lund University,
Lund, Sweden
5
Occupational and Environmental Epidemiology Branch,
National Cancer Institute, Rockville, Maryland
6
Department of Biological Sciences, Wayne State University, Detroit, Michigan

Cytogenetic biomarkers are by far the most fre-
quently used endpoints in human population stu-
dies. Their sensitivity for measuring exposure to
genotoxic agents and their role as early predictors
of cancer risk have contributed to this success. In
this article, we present an overview of the last
25 years of population studies with cytogenetic
biomarkers, describing the evolution of this
research and addressing the most promising inno-
vations for the future. The evaluation has been
restricted to the most popular assays, i.e., chromo-
somal aberrations (CAs) and micronucleus (MN),
which are considered to be causally related to
early stages of chronic diseases, especially can-
cer, and may therefore play a major role in pre-
vention. An extensive literature search covering
the period 1 January 1980 to 31 December
2003 was performed using the Medline/PubMed
database. A total of 833 population studies using
CAs and 434 using matched MN inclusion cri-
teria were included in the analysis. We report the
distribution of selected papers by year of publica-
tion, country, language, agents investigated, and
methods employed. The state of the art and future
prospects regarding cytogenetic techniques and
epidemiologic and statistical methods are dis-
cussed. The role of susceptibility and its potential
impact on genotoxic damage are discussed with
special attention to the effect of major genetic
polymorphisms on the baseline frequency of
CAs and micronuclei. Environ. Mol. Mutagen.
45:258–270, 2005. c 2005 Wiley-Liss, Inc.

Key words: chromosomal aberrations; micronucleus test; molecular epidemiology; review;

environmental exposure; risk assessment

INTRODUCTION
Cytogenetic biomarkers are the most frequently used
endpoints in human population studies. Their sensitivity
for measuring exposure to genotoxic agents and their role
as early predictors of cancer risk have contributed to this
success. The earliest reports date to the 1960s, when the
first studies on subjects occupationally exposed to geno-
toxic agents showed the great potential of this assay both
for occupational safety and for mechanistic studies
[Tough and Court-Brown, 1965; Evans and Scott, 1969].
A classic review article published by Ashby and
Richardson [1985] reviewed papers on chromosomal aber-
rations (CAs) and sister-chromatid exchanges (SCEs) pub-
lished between 1965 and 1984. Since then, this field has
matured and many changes have occurred, e.g., the use of
Grant sponsor: the Associazione Italiana per la Ricerca sul Cancro; Grant
sponsor: the European Union 5th FP; Grant number: QLK4-CT-2000-
00628, QLK4-CT-2002-02831, and QLRT-2001-02198.
*Correspondence to: Stefano Bonassi, Environmental Epidemiology and
Biostatistics, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna
Benzi, 10, 16132 Genova, Italy. E-mail: stefano.bonassi@istge.it
Invited article: 25th anniversary of Environmental and Molecular
Mutagenesis
DOI 10.1002/em.20115
Published online 1 February 2005 in Wiley InterScience (www.interscience.
wiley.com).

c

2005 Wiley-Liss, Inc.

fluorescence in situ hybridization (FISH) with whole chro-
mosome paints, which has nearly replaced traditional
Giemsa staining. The use of SCEs has progressively dis-
appeared from the scientific literature and the cytokinesis
block micronucleus (MN) assay has become almost as
popular as CAs.
The purpose of the present article is to review the last
25 years of population studies with cytogenetic biomar-
kers, describing the evolution of this area of research and
addressing the most promising innovations for the years
ahead. We restricted our evaluation to the most popular
assays that are considered representative of the early
stages of chronic diseases, especially cancer, and that
may therefore have a direct role in prevention, i.e., CA
and MN.
LITERATURE REVIEW OF HUMAN STUDIES BASED
ON CA AND MN (1980^2003)
An extensive literature search covering the period
1 January 1980 to 31 December 2003 was performed
using the Medline/PubMed database (National Library of
Medicine, National Institutes of Health, Bethesda, MD;
http://www.ncbi.nlm.nih.gov/PubMed). Key words were
selected from the medical subject headings (MeSH). For
those key words introduced into the MeSH thesaurus after
1980, we used previously indexed key words or common
research terms along with the PubMed free text tool. The
purpose of this search was to retrieve from the literature
papers reporting human population studies based on CA
or MN analysis and aimed at assessing the effects of a
specific exposure or condition. To be included in this
review, papers were required to have the following: an
evaluation of the effects of an environmental exposure
such as smoking, medical treatments, diet and dietary sup-
plementation, or host-related factors such as age, gender,
genotype, or a disease; a formal statistical analysis; and a
referent group.
To select papers according to the cytogenetic endpoint,
we used the key words ‘‘chromosome aberrations,’’ exist-
ing from 1968, ‘‘micronucleus test,’’ existing from 1989,
and ‘‘micronuclei,’’ existing from 1990. For prior years, a
free text search with terms ‘‘micronucleus’’ and ‘‘micro-
nuclei’’ was performed. The concept of environmental
exposure was defined as follows: environmental pollution
(MeSH) or environmental pollutants (MeSH) or carcino-
gens, environmental (MeSH) or occupational diseases
(MeSH) or occupational medicine (MeSH) or accidents,
radiation (MeSH; from 1995 to 2003) or accidents
(MeSH; from 1980 to 1994) or nuclear reactors (MeSH;
from 1980 to 1994) or radiation injuries (MeSH; from
1980 to 1994). Other terms were added to retrieve articles
concerning lifestyle and behavioral habits, i.e., smoking
(MeSH) or alcohol drinking (MeSH) or diet (MeSH) or
Human Population Studies 259
food (MeSH) or dietary proteins (MeSH) or vitamins or
age factors (MeSH) or sex factors (MeSH). Finally, arti-
cles indexed with ‘‘monosomy,’’ ‘‘trisomy,’’ ‘‘uniparental
disomy,’’ ‘‘sex chromosome aberrations,’’ ‘‘philadelphia
chromosome,’’ ‘‘chromosome disorders,’’ ‘‘animals,’’
‘‘plants,’’ ‘‘in vitro’’ (as MeSH terms), or ‘‘case reports,’’
‘‘news,’’ ‘‘review’’ (as publication-type terms) were
excluded from the review. Only articles with a published
abstract were selected. All information about individual
papers was obtained from the abstracts available in the
PubMed database. To ensure the retrieval of all the rele-
vant papers, the free text searching procedure was also
used, especially for studies on patient series (e.g., neo-
plasm, Parkinson and Alzheimer diseases, mononucleosis,
tuberculosis, hepatitis, influenza, celiac disease). These
procedures initially identified 2,100 publications on CA
and 700 on MN. After removing those papers not match-
ing the inclusion criteria, 833 publications for CA and
434 for MN were considered valid.
Publications were then characterized according to para-
meters available in the database, such as the language and
the agents investigated. The field defined as ‘‘ad’’ in the
PubMed system indicates the first author affiliation and
through this information we obtained the distribution of
publications by country. These data were introduced in
1988 and therefore older publications could not be evalu-
ated for this purpose. We also attempted to tabulate the use
of different cytogenetic methods, e.g., the cytokinesis-block
method for MN and FISH for CA, but these are largely
underreported in the database and the results of the search
were not considered reliable. For example, the key word
‘‘in situ hybridization, fluorescence,’’ was associated with
only 55 CA papers, which is clearly an underestimate.
The distribution of all articles included in our review
by endpoint, year of publication, agent investigated, and
methods used is reported in Table I. The increasing trend
for both endpoints evaluated is evident (Fig. 1). Data
referring to the year 2003 are reported in Table I but not
in Figure 1, since the inclusion of recent publications in
the PubMed database is often incomplete, which could
lead to misinterpretation of the figure.
The use of the CA assay in population studies had an
evident increase in late 1980s, reaching a mean of 40
papers published per year. This number is currently
approaching 50. The trend of papers investigating occupa-
tional exposure, which is the most common purpose of
CA studies, is similar to the overall trend while studies
on environmental pollution reached their peak in the
1990s and their number is now slowly decreasing. The
interest in diet, lifestyle, and the investigation of clinical
series is confirmed by the recent increase of these studies.
The trend of articles describing applications of the MN
assay reflects the methodological improvements of this
assay. In fact, the presence of the MN endpoint in biomo-
nitoring studies is detected only after 1985, when the
 260
Bonassi et al.

TABLE I. Distribution of Peer-Reviewed Articles on CA and Micronuclei Published From 1980 to 2003a
Chromosomal aberrations Micronuclei
Conditions investigated Conditions investigated Method
Year Articles Occupation Environment Lifestyle Other Articles Occupation Environment Lifestyle Other Lymphocytes Exfoliated epithelial cells Unspecified

1980 16 11 1 2 2 1 0 0 1 0 1 0 0
1981 17 11 1 1 3 0 0 0 0 0 0 0 0
1982 20 12 2 2 4 0 0 0 0 0 0 0 0
1983 17 12 1 1 3 3 2 0 2 1 2 1 0
1984 20 15 2 0 2 3 1 0 1 1 3 0 0
1985 15 13 1 1 0 0 0 0 0 1 0 0 0
1986 19 11 3 3 1 2 0 0 2 0 1 1 0
1987 18 11 2 3 2 5 2 0 4 1 3 2 0
1988 39 25 6 1 6 6 6 0 1 0 6 0 0
1989 32 19 9 4 1 8 4 0 4 0 5 1 2
1990 39 26 7 3 3 18 8 2 6 3 9 7 2
1991 42 21 13 2 6 17 13 1 2 1 16 1 0
1992 40 27 8 2 3 17 9 0 7 3 10 4 3
1993 34 17 16 5 3 17 10 3 4 2 13 2 2
1994 40 18 19 0 2 27 13 7 3 4 20 4 3
1995 42 21 15 1 5 25 10 4 7 5 18 4 3
1996 53 31 17 0 5 27 17 4 2 4 22 5 0
1997 39 20 13 4 4 39 18 12 6 2 24 8 7
1998 56 26 19 1 8 36 18 4 8 5 26 6 4
1999 39 21 15 5 3 41 23 6 3 10 33 4 4
2000 53 28 11 3 11 36 16 6 7 11 25 7 4
2001 56 33 15 0 6 38 21 3 1 11 27 7 4
2002 50 24 15 3 8 45 24 6 4 11 27 11 7
2003 37 18 11 4 5 23 7 4 3 8 15 2 6
Total 833 471 222 51 96 434 222 62 78 84 306 77 51
a
The sum of individual conditions investigated is slightly higher than the total because a few studies evaluated more than one condition.

Fig. 1. Distribution of articles using CA and micronuclei published from
1980 to 2002. X-axis: year of publication; Y-axis: number of articles.
seminal paper describing the cytokinesis-block method
was published [Fenech and Morley, 1985]. Since then, the
number of publications has increased dramatically. As
seen for CA, most MN studies evaluated occupational
exposures but the number of studies on diet, dietary sup-
plementation, pharmaceutical treatment, or groups of
patients is increasing. Most studies included in the review
evaluated micronuclei in peripheral blood lymphocytes,
although an increasing number of studies are performed
on exfoliated epithelial cells; more than 70% of these
papers were published in the last 10 years.
The geographical distribution of articles shows that the
largest contribution is by European countries, which pub-
lished 67% and 64% of all studies on CA and MN,
respectively. The individual countries that contributed
papers on CA are Russia (111), United States (64), Italy
(60), Germany (42), India (36), Czech Republic (34),
China (30), Croatia (29), United Kingdom (25), Finland
(21), Brazil (19), Sweden (18), and Poland (17). For MN,
the country that published most population studies is Italy
with 74 articles, followed by the United States (44),
Germany (28), China (26), Russia (24), Sweden (21), the
Netherlands (16), and France, Australia, Turkey, India,
Spain, Brazil, Finland, and Croatia (10 to 13 each).
The majority of papers selected for review were pub-
lished in English, but 18% of CA studies and 11% of MN
studies were published in national journals and were writ-
ten in the language native to that country. The most com-
mon language of these non-English papers was Russian.
The data describing the geographical distribution of
papers selected for review are reported in Table II.
Human Population Studies 261
Another interesting feature is the type of agents investi-
gated in these studies (Table III). For CAs, radiation is by
far the most commonly studied exposure with 258 publi-
cations, most (221) referring to ionizing radiation. Expo-
sure to organic chemicals was also frequently reported
(212 articles), especially benzene (27), styrene (26), ethy-
lene oxide (16), and polycyclic aromatic hydrocarbons
(PAHs; 14). Inorganic chemicals, mostly metals, were stu-
died in 137 articles, with uranium (19), chromium (14),
lead (14), and nickel (10) as the most frequent exposures.
Other common exposures included pesticides (58) and
antineoplastic drugs (29). In 36 papers, environmental
pollution was reported as a key word. There were also
eight studies on vehicle emissions. In 28 papers, the agent
investigated was not specified. Finally, the number of stu-
dies dealing with the Chernobyl accident is remarkable,
with 54 studies on environmental effects of exposure to
ionizing radiation and 44 on power plant personnel and
cleanup workers.
For MN analyses, organic chemicals led the list of
exposures investigated, with 21% of all studies. The
most frequently evaluated compounds of this class were
styrene (20), PAHs (19), benzene (13), ethylene oxide
(11), and formaldehyde (8). Inorganic chemicals were
studied in 65 papers, mostly chromium (9), arsenic (8),
lead (7), and mercury (6). Fifty papers have been pub-
lished on radiation, including four investigating the
effects of electromagnetic fields. Pesticides (32 studies),
antineoplastic drugs (16), and environmental pollution
(13) are also among the most frequently evaluated
exposures.
TWENTY-FIVE YEARS OF BIOMONITORING STUDIES
WITH CYTOGENETIC BIOMARKERS
From this extensive evaluation of the scientific litera-
ture, it is evident that cytogenetic biomarkers have been a
valuable tool for studying the most important occupa-
tional and environmental hazards to public health occur-
ring in the past few decades. The use of these biomarkers
received a strong validation after recent results from
cohort studies and nested case-controls, which showed
CA as a marker of cancer risk [Hagmar et al., 1998; Liou
et al., 1999; Bonassi et al., 2000, 2004; Smerhovsky
et al., 2001]. The use of valid biomarkers of risk in popu-
lations exposed to agents inducing genetic damage is the
most suitable approach for studying many modern
exposures, where the low doses or the complexity of
mixtures make traditional epidemiologic studies poorly
informative.
This increased interest in the use of cytogenetic bio-
markers in populations exposed to genotoxic agents has
led to great improvements in the methods employed. The
most important changes concern the assays themselves.
262 Bonassi et al.

TABLE II. Distribution of Articles Using CA and Micronuclei TABLE III. Distribution of Articles Using CA and
in Human Population Studies Published From 1988 to 2003 Micronuclei in Human Population Studies Published
by Geographical Area (According to the Affiliation of From 1980 to 2003 by Agents Investigated
the First Author) and From 1980 to 2003 by Language of
the Reporta Agents CA % MN %

CA % MN % Clinical series/treatment 82 8.2 62 12.1

Diet/alcohol drinking 21 2.1 49 9.6
Geographic area Drugs (occupational) 29 2.9 16 3.1
America, central and south 33 5.0 17 4.1 Environmental pollution 36 3.6 13 2.5
America, north 72 10.8 53 12.6 Fossil fuels 24 2.4 9 1.8
Asia, eastern 51 7.7 41 9.8 Heterocyclic compounds 9 0.9 4 0.8
Asia, western and southb 64 9.6 42 10.0 Inorganic chemicals 137 13.7 65 12.7
Europe, central and western 212 31.7 196 46.7 Manufactured materials 10 1.0 2 0.4
Europe, eastern 235 35.2 71 16.8 Mutagens (not otherwise specified) 28 2.8 18 3.5
Total 667 100.0 420 100.0 Organic chemicals 212 21.4 107 20.8
Pesticides 58 5.8 32 6.1
Language of the article Radiation 258 25.9 50 9.8
English 687 82.5 385 88.7 Smoking 30 3.0 29 5.7
Other languages 146 17.5 49 11.3 Other 63 6.3 57 11.1
Total 833 100.0 434 100.0 Totala 997 100.0 513 100.0
a a
The field indicating author affiliation was introduced in 1988; therefore, A number of studies investigated more than one agent.
papers published before that date could not be evaluated.
b
Including Australia and New Zealand.
that occurred many years ago and to characterize lower
levels of exposure than were possible with older cytoge-
Some endpoints such as SCE have been abandoned while
netic methods. Due to its widespread success, few investi-
new methods have become available. For example, with
gators now debate the utility of chromosome painting for
MN analysis, it is now possible to evaluate events includ-
assessing exposure to genotoxic agents. As the field of
ing gene amplification and chromosome rearrangements
molecular cytogenetics matures, it is appropriate to deter-
[Fenech et al., 2003]. A direct effect of technical
mine what must be done to improve our ability to evalu-
improvements is the need for epidemiologists to create
ate cytogenetic risks associated with environmental
appropriate and innovative study designs. In parallel, sta-
exposure in human populations.
tistical analyses have improved and new innovative analy-
Currently, most painting is performed with a small
tical and computational methods are available that allow
number of probes at a time, usually with just one color of
investigators to account for individual variability in
paint, but sometimes with two or three chromosomes
regression models, greatly increasing the power of these
labeled in each of a small number of colors [Chen et al.,
studies. Another improvement is the ability to include
2000; Jones et al., 2002]. Each additional probe in the
markers of individual susceptibility in the study design.
cocktail increases the proportion of the genome in which
This has widened the potential for research, since the
aberrations can be observed and also increases the frac-
presence of gene-environment interactions can now be
tion of all exchanges that can be detected. However, there
extensively studied and sensitive subjects or high-risk
are additional costs associated with purchasing each addi-
groups can be identified more efficiently.
tional paint in the cocktail as well as a concomitant
increase in the analysis time for each cell. Some years
EVOLUTION OF CYTOGENETIC TECHNIQUES ago, two groups reported the ability to paint each of the
FOR POPULATION STUDIES 24 human chromosomes in a unique color, thereby
enabling the identification of every interchromosomal
In recent years, molecular cytogenetic techniques have exchange in each cell. Spectral karyotyping (SKY)
seen continued use as a biomarker for assessing chromo- [Schrock et al., 1996] and mFISH [Speicher et al., 1996]
some damage in human populations. FISH with whole are currently enjoying widespread use in clinical cytoge-
chromosome paints has been the primary method of netics [Lestou et al., 2003; Tchinda et al., 2004].
choice for most investigators who wish to quantify and Although research applications have been more limited,
characterize chromosome damage from environmental or mFISH has been successfully employed to decipher
occupational exposures. The reasons for using chromo- complex chromosome rearrangements following acute in
some painting have been the speed of the assay and the vitro radiation exposure [Cornforth, 2001; Loucas and
ability to identify relatively stable events such as translo- Cornforth, 2001; Loucas et al., 2004]. Unfortunately, this
cations in parallel with the enumeration of unstable approach requires expensive probes and the analysis time
dicentrics. This combination of features has enabled per cell is substantially longer than when only a few chro-
investigators to evaluate recent exposures as well as those mosomes are painted. As a result, the technology of

painting every chromosome in its own color has not yet
been applied to the evaluation of a human population.
In addition to the interchromosomal exchanges com-
monly detected by chromosome painting, intrachromoso-
mal exchanges such as pericentric and paracentric
inversions occur and may form an important component
of risk evaluation. However, whole chromosome painting
is not helpful for recognizing inversions. Identifying intra-
chromosomal exchanges, especially paracentric inversions
that do not alter the chromosome arm ratios, requires the
use of chromosome bands. The bands may be natural,
e.g., G-bands, or synthetic, i.e., based on region-specific
partial chromosome paints that are hybridized simulta-
neously and labeled in multiple colors (mBANDs
[Horstmann and Obe, 2003; Chudoba et al., 2004]).
Molecular cytogenetics thus finds itself in the position
of having the analytical tools that are needed to make a
thorough determination of the types of chromosome
damage present in human populations. What currently
inhibits the application of these tools is the cost of the
reagents and the cost of labor. Even though the reagents
are expensive, the majority of the costs are associated
with labor. This means that much of the solution to the
problem of expensive analyses may be addressed by
automation. Diminishing the time that humans are
directly involved in the analysis will lead to substantial
improvements in the ability to quantify chromosomal
damage by enabling the evaluation of additional cells at
the same total cost per analysis, or by enabling the eva-
luation of additional subjects, thereby obtaining a more
refined estimate of the average population risk or expo-
sure level.
Automation of cytogenetic assays has been a goal for
many years. Some progress has been made, for example,
in the area of karyotyping and aberration detection [Huber
et al., 1995; Odawara et al., 1997]. However, fully auto-
matic analysis of chromosomes in metaphase has yet to
be accomplished. Automatic metaphase finding shows
substantial promise for enhancing throughput and conse-
quently for increasing the sensitivity of aberration identifi-
cation. Automated aberration identification poses an even
greater challenge. Some of the major issues are recogni-
tion and correct separation of touching chromosomes,
electronic duplication of overlapping chromosomes so that
each involved chromosome can be evaluated, accurate
identification of centromeres, and correct enumeration of
broken chromosomes and color junctions. Even the best
automated system may never replace a skilled observer,
but a system that could count the normal cells and conser-
vatively eliminate most of them from consideration as
normal cells could reduce the human effort involved in
scoring by as much as 90%. With such a system, the
observer would view a preselected set of cells that had
been enriched for abnormal cells and score those cells in
the usual manner.
Human Population Studies 263
An even more promising field is the automation of MN
scoring, which has resulted in the ability to evaluate large
numbers of cells quickly and efficiently. Flow cytometry
and slide-based image analyses have both been used with
considerable success. Nüsse and Marx [1997] described a
flow cytometric system for quantifying micronuclei in cul-
tured cells and in human lymphocytes. Although their
data correlate well with microscopy, a significant limita-
tion is that apoptosis levels need to be very low in these
cultures. Image analysis systems have been described for
scoring micronuclei in mammalian cell lines as well as
cultured human lymphocytes [Verhaegen et al., 1994;
Schunck et al., 2004]. These systems appear to be rapid
and to offer accurate and reproducible results. Smolewski
et al. [2001] describe a laser-scanning system that com-
bines the analytical capabilities of flow and image cyto-
metry, which was used to obtain unbiased estimates of
MN frequencies.
Automated analysis of mBANDed chromosomes would
present more significant challenges. Automation will per-
mit the analysis of more cells than would otherwise be
possible, with the result that the sensitivity for performing
biological dosimetry will be increased and the problem of
measurement error in large studies with multiple scorers
will be avoided.
In summary, the future of human population cytoge-
netics using molecular methods is bright. Computer-based
approaches for analysis of metaphase chromosomes for
aberration identification and enumeration and MN fre-
quency would significantly enhance our ability to perform
studies of putatively exposed human populations with a
significant potential for improving risk analyses.
INCORPORATING CYTOGENETIC BIOMARKERS
INTO EPIDEMIOLOGICAL STUDIES
CAs and micronuclei have value as biological dosi-
meters and more recently as predictors of risk in epide-
miological studies [Tucker et al., 1997; Bonassi et al.,
2004]. This section discusses study design in relation to
their use as intermediate biomarker endpoints in cross-
sectional and cohort studies and as intermediate endpoints
in relation to cancer risk.
Cross-sectional studies using CA and micronuclei as
intermediate endpoints have been used to evaluate the
DNA-damaging effects resulting from a wide range of expo-
sures in the diet and environment as well as from lifestyle
factors. These studies can be used to provide mechanistic
insights into well-established exposure-disease relationships
and to supplement suggestive but inconclusive evidence of
the carcinogenicity of an exposure. Further, these studies
can evaluate whether or not there is chromosomal damage
by new exposures or recent changes in lifestyle that have
not been present long enough to have been evaluated for
264 Bonassi et al.
their association with cancer [Rothman et al., 1995]. How-
ever, for CA and MN, the etiological fraction for cancer,
i.e., the proportion of the disease that must progress through
the marker, is almost certainly well below 1, suggesting cau-
tion in the interpretation of these studies [Schatzkin et al.,
1990]. Specifically, a positive association between an expo-
sure and CA or MN frequency may be informative, but a
null association does not rule out that the exposure is carci-
nogenic as that exposure may act through a mechanism not
reflected by induction of CAs or micronuclei.
A distinct advantage of the cross-sectional study is that
detailed and accurate information can be collected on cur-
rent exposure patterns, potential confounders, and effect
modifiers. As both CA and MN frequencies are not fixed
but change over time depending on the exposure pattern as
well as the age of the subjects [Hando et al., 1994; Nath
et al., 1995; Ramsey et al., 1995; Bolognesi et al., 1997;
Albertini et al., 2000], the information collected should
reflect the appropriate etiologically relevant time window.
For unstable CAs and micronuclei, this probably extends to
a few months before sample collection, while for transloca-
tions, this could extend to years [Tucker, 2002]. Further-
more, within cross-sectional studies, it is possible to
process samples for CA and MN analyses within a rela-
tively short period of time after collection, thereby increas-
ing cell viability and decreasing the potential for
measurement errors. However, processing blood samples
for CA and MN analyses is laborious and processing large
numbers of samples simultaneously can be problematic.
Until now, most studies have focused on risk identification
that, when there is sufficient contrast in exposure between
controls and exposed subjects, requires a relatively small
sample size (generally < 100 subjects). However, future stu-
dies focusing on dose-response relationships, especially at
low exposure levels or when considering gene-environment
interactions, will require a sample size of at least several
hundred subjects. In these settings, the logistics surrounding
blood processing may become complex and it might be
advisable to cryopreserve whole blood or isolated lympho-
cytes for future culturing. A recent study has shown the
recovery of viable lymphocytes from cryopreserved whole
blood [Hayes et al., 2002], while others have demonstrated
the successful cryopreservation of separated lymphocytes
[Kleeberger et al., 1999; Beck et al., 2001]. However, cryo-
preservation of blood has been shown to lead to an increase
of chromatid aberrations and to diminished cell viability
[Cheng et al., 2001], both of which can have an adverse
effect on the study outcome. An alternative favored by
many investigators involves freezing the prepared slides in
nitrogen gas and a desiccant, a process that preserves the
cells’ ability to be hybridized for at least several years.
The use of CAs and micronuclei in prospective cohort
studies has been very limited. Both biomarkers could theo-
retically be used as intermediate endpoints, but their main
application is more likely to be as independent measures to
validate the exposure assessment. Although cohort studies
have the theoretical advantage of collecting serial samples
over time, many large studies have been able to collect a
single biological sample. This poses some limitations for
the use of CAs and micronuclei as the majority of aberra-
tions reflect only the past several months of exposure,
which might not be relevant for either the exposure and/or
disease under consideration. The situation for transloca-
tions might, however, be better in this regard as they have
been shown to persist for decades [Tucker, 2002]. Further-
more, only a very limited number of cohort studies have
collected microscope slides containing cells in metaphase
or have cryopreserved blood samples, as the collection of
viable cells in population-based studies is challenging.
Cohort studies on CAs and micronuclei as predictors of
future cancer risk in human populations have until now
been performed in so-called ad hoc cohorts [Hagmar et al.,
1994; Bonassi et al., 1995; Smerhovsky et al., 2001].
These cohorts were assembled from many small biomoni-
toring studies and suffer from major problems due to dif-
ferences in analytical techniques and the lack of detailed
information on confounding factors and possible effect
modifiers [Bonassi et al., 2004]. Prospective cohort studies
that have collected cryopreserved blood samples provide a
unique chance to follow these earlier findings. Although
they suffer from the same limitation that assumptions need
to be made on the relevance of CA in peripheral blood
lymphocytes versus the target organ(s), and on the rele-
vance of a single measurement for classifying subjects,
they have the distinct advantage of providing detailed
information on lifetime exposure and confounding factors.
Furthermore, when samples of cases and controls are ana-
lyzed at the same time, many of the analytical issues are
avoided. In addition, the application of FISH techniques
will enable the study of relationships between comprehen-
sive measures of chromosomal damage and future cancer
risk. Nested case-control analyses remain the design of
choice for such studies.
STATISTICAL ANALYSIS OF DATA
Human population studies with cytogenetic biomarkers
such as CAs and micronuclei have addressed different
research issues. Here we evaluate some of these aspects
in order to describe statistical methods of particular rele-
vance for this area. Preliminary research questions deal
with subject characteristics such as age, gender, smoking
history, and how they can directly affect CAs and MN
frequencies or modify the effect of exposure to mutagens.
Multivariate modeling is usually required for these pur-
poses. A modeling approach implies that relevant effect
measures can be estimated with confidence intervals,
which are more useful than P values obtained from statis-
tical testing [Gardner and Altman, 1986; Bonassi et al.,

1994]. From a statistical modeling viewpoint, it is impor-
tant to recognize the type of outcome data. Data on CA
and MN frequencies reflect the number of aberrant or
micronucleated cells out of the total number of cells
scored, which may differ between subjects. Given the
total number of cells scored for each subject (commonly,
at least 100 metaphases for CAs—although by today’s
FISH standards 500–1,000 cell equivalents is much more
appropriate—and at least 1,000 interphase cells for micro-
nuclei), the observation of an aberrant or a micronu-
cleated cell is usually a rare event. Poisson regression
modeling is appropriate for analyzing the impact of the
independent variables on CA and MN frequencies
[Clayton and Hills, 1993]. One problem with Poisson
regression modeling, however, is overdispersion, i.e.,
extra-Poisson variation, meaning that the residual varia-
tion is larger than that implied by the Poisson distribution.
Overdispersion may well be observed with CA and MN
frequency as the outcome variable. To cope with overdis-
persion, one can model only the residual variance of the
outcome variable, rather than completely specify the resi-
dual distribution [Rothman and Greenland, 1998]. Alter-
natively, one can specify a residual distribution that
allows a broader range of variation for the outcome vari-
able, such as the negative binomial [Cameron and Trivedi,
1998].
An alternative approach might be to consider a binary
outcome of each single cell (aberrant or not; micronu-
cleated or not), realizing that such outcomes are not inde-
pendent events since they are clustered within each
subject. Logistic regression models with random effects
can handle outcome data that are correlated within sub-
jects [Bonassi et al., 1999]. In a more general framework,
generalized linear mixed models can be considered
[Piegorsch and Bailer, 1997]. Models exist that are more
well known but they are not appropriate in this setting.
Multivariate linear regression modeling using CA or MN
frequencies for a given number of cells as the outcome
for each subject is inappropriate because the underlying
model assumptions are likely to be violated. The assump-
tion of normally distributed residuals is often not fulfilled
since the outcome data are constrained to a relatively few
values that are small.
Besides the role of host factors, an emergent issue is
the effect of CA and MN frequencies on the incidence of
adverse events such as cancer. Here, outcome data usually
correspond to time to event for each subject, where the
date of cytogenetic testing is the start of follow-up. The
cytogenetic biomarker (CA or MN frequency) is the pre-
dictor variable of primary concern, though subject charac-
teristics such as age, gender, smoking history, or other
predictor variables may affect the adverse event inci-
dence. One feasible approach is to standardize the CA or
MN frequencies with respect to test-related variables such
as number of cells scored, culture time, and laboratory.
Human Population Studies 265
The subjects may then be placed into relevant CA and
MN categories, for example, into low-, medium-, and
high-frequency groups [Hagmar et al., 2004]. The out-
come data may then be stratified according to the CA or
MN categories as well as other predictor variables such as
gender and age at the time of testing. Of course, continu-
ous predictor variables must be categorized to stratify the
outcome data. The data in each stratum can be summar-
ized sufficiently by the number of person-years (i.e., accu-
mulated follow-up time) and by the number of subjects
with each adverse event. Poisson regression modeling can
then be employed to evaluate the effect of CA or MN fre-
quencies on adverse event incidence, controlling for the
other predictor variables [Clayton and Hills, 1993]. This
approach is analogous to the Poisson regression modeling
described above, where the subject-specific numbers of
aberrant/micronucleated cells together with the numbers
of scored cells provide the outcome data.
Alternatively, Cox’s regression modeling can be
employed [Clayton and Hills, 1993]. The Poisson and
Cox regressions generally produce similar results. Never-
theless, Cox’s regression allows for more flexible model-
ing since continuous predictor variables can be included
without being categorized into strata. Data from studies
with repeated cytogenetic testing of subjects can also be
analyzed by Cox’s regression. In the analysis of such
data, it is reasonable to consider CAs or micronuclei as
time-dependent predictor variables, which allows a subject
to change the CA or MN category during the follow-up in
accordance with the repeated-testing results.
The evaluation of long-term effects of high levels of
CAs or micronuclei in healthy subjects has also been
addressed in case-control studies by comparing incident
cancer cases and selected controls (i.e., subjects without
cancer) with respect to previous cytogenetic test results
and relevant confounders or effect modifiers such as
smoking history and occupational exposure [Bonassi
et al., 2000]. Logistic regression modeling is appropriate
for analyzing case-control data [Hosmer and Lemeshow,
1989].
INDIVIDUAL VARIABILITY OF RESPONSE TO
GENOTOXIC DAMAGE
The individual response to physical or chemical stress
may vary according to the function of the particular gene
combination that an individual has regarding absorption,
metabolism of chemical mutagens, DNA repair, cell death
(apoptosis/necrosis), cell cycle control, and immune res-
ponse. Methods for genotyping have become relatively
easy to perform and in vitro phenotyping approaches are
developing. It is therefore interesting to consider whether
the methods for assessing genetic susceptibility can be
implemented to predict risks for complex diseases and the
266 Bonassi et al.
daily practice of environmental or occupational biomoni-
toring that might become possible in the future.
Considering our limited understanding and the com-
plexity of the genotype-phenotype relationship, one can
wonder whether the genotype and/or the phenotype should
be investigated for the study of occupational diseases.
Genotyping has the advantage of being technically easy
and inexpensive while giving definitive answers that are
not influenced by the environment. However, genotyping
is more distal than phenotyping to the disease process
[Ahsan and Rundle, 2003]. The functional variation of
polymorphic genes is likely to have a subtle effect on can-
cer risk for individuals, but may have a large impact on a
population because the relevant genetic polymorphism(s)
may have high frequencies [Brennan, 2002]. Until now,
gene-environment interaction studies have been based on
relatively limited sample sizes and have analyzed only
one or several genes. Larger studies based on more genes
are needed to provide definitive answers.
Phenotyping is more proximal to the disease and inte-
grates the effects of multiple genes. However, the meth-
odologies are not yet fully validated and tend to be
expensive. From this comparison, one could conclude that
both genotyping and phenotyping are important but we
will describe here only those results obtained with geno-
typing, since few phenotyping data are available [Ahsan
and Rundle, 2003].
RESULTS FROM STUDIES ANALYZING LINK BETWEEN
GENOTYPE AND CYTOGENETIC BIOMARKERS
At the present time, it is difficult to perform a systematic
overview of papers reporting on the association between
genotype and cytogenetic biomarkers. The size of most
populations is usually too small to evaluate rare poly-
morphisms and different genotypes are selected in different
studies, precluding a meaningful compilation of the results.
Furthermore, in different populations, the frequencies of
the alleles for each genotype may vary widely. Finally,
statistical methods are often inappropriate for evaluating
possible confounding factors, and therefore negative find-
ings are not very instructive and are probably not fully
reported. To overcome these shortcomings, pooled analyses
are needed but these are not yet available.
Despite these limitations, many papers show the pre-
sence of interaction between CA or MN frequencies and a
selected number of genetic polymorphisms involved
in metabolic activation/deactivation (GSTM1, GSTT1,
GSTP1, EPHX1, CYP2E1) or in DNA repair (XRCC1 and
XRCC3). For the genes involved in DNA repair, the pre-
sence of at least one variant allele (Gln instead of Arg for
XRCC1, Met instead of Thr for XRCC3) is associated with
increased MN and CA frequencies [Au et al., 2003; Aka
et al., 2004; Godderis et al., 2004]. As far as metabolic
activation/deactivation is concerned, individuals with one
or more variant alleles for GSTP1 codons 105 and 114
had higher MN frequencies than wild-type individuals
[Laffon et al., 2003], while increased CA and MN fre-
quencies were consistently reported for GSTT1 null indi-
viduals [Vlachodimitropoulos et al., 1997; Sram et al.,
1998].
The results for other genotypes are not so clear. Some
studies on GSTM1 and EPHX1 found increased MN and
CA frequencies in null individuals compared to wild-type
individuals [Scarpato et al., 1997; Sram, 1998; Falck
et al., 1999; Knudsen et al., 1999; Salama et al., 1999,
2001; Karahalil et al., 2002; Pitarque et al., 2002; Cajas-
Salazar et al., 2003; Leopardi et al., 2003; Marcon et al.,
2003], while others found the opposite effect [Sram,
1998; Pluth et al., 2000; Norppa, 2001, 2004]. When indi-
viduals were classified into low, medium, or high activity
according to their genotype for codons 113 and 139 of
the EPHX1 gene, low-activity individuals were found to
have higher MN frequencies, while high-activity indivi-
duals had higher CA frequencies [Cajas-Salazar et al.,
2003]. Contrasting findings between CA and MN analyses
were reported for the CYP2E1 polymorphism, showing
wild-type individuals with higher MN frequencies
[Ishikawa et al., 2004] and lower CA frequencies [Abdel-
Rahman et al., 2001]. These inconsistent results could be
partially due to the different types of exposure in the dif-
ferent studies. Depending on whether the studied genes
are involved in either the activation or the deactivation of
the exposure under study, or are not involved at all, dif-
ferent results may be observed.
In summary, there is conclusive evidence for the influ-
ence of certain genotypes on cytogenetic biomarkers.
However, further investigations are needed to elucidate
the mechanisms involved.
EVOLUTION IN METHODS FOR ASSESSING INDIVIDUAL
SUSCEPTIBILITY
When identifying individuals at risk of developing can-
cer and considering the genetic complexities involved in
carcinogenesis, it is important first to identify the genes
responsible for initiation, promotion, and progression and
then to genotype them. Wacholder et al. [2004] have
emphasized the risks of overinterpretation of statistically
significant results, as well as the need to improve the
plausibility of findings from high-throughput studies
defining prior probability that associations between
genetic variants and disease are real.
The actual variants contributing to most complex diseases
are not known, but the most common type of variation in
DNA sequence is the single nucleotide polymorphism
(SNP), present at about 1 per 1,000 nucleotides in humans
[Kruglyak and Nickerson, 2001]. Thus, an essential step in

mapping complex traits is to determine which of the myriad
SNPs influence disease risk. Various high-throughput geno-
typing methods, including several that use microarrays, are
being developed to meet this need (for review, see Zhao
et al. [2004]). The next generation of SNP-typing micro-
arrays will need to improve genomewide or region-specific
SNP typing for association, presumably by using SNPs that
capture the variation in haplotype blocks, e.g., a distinct
combination of SNPs that occur together in a chromosome.
As the SNP collections and the microarrays for genotyping
improve further, association studies based on haplotype
blocks may become the method of choice for the genetic
analysis of complex human diseases.
Although SNP-based genomewide association studies
are conceptually straightforward, they still face technical
and statistical obstacles. Cheung and Spielman [2002] and
Botstein and Risch [2003] have reviewed this question.
First, reliable and efficient techniques still need to be
developed and validated. Nevertheless, microarrays are
already available to genotype a few thousand SNP mar-
kers that span the genome (see Affymetrix at www.
affymetrix.com and Illumina at www.illumina.com). Sec-
ond, it is not clear which are the most informative mar-
kers or what density will be needed to study associations;
the HapMap project recently launched by the U.S.
National Human Genome Project (www.genome.gov/
10001688) is expected to provide valuable information on
this issue [Couzin, 2002]. Third, the genotype data must
be analyzed with methods that can detect small contribu-
tions from several genes while dealing with errors that
arise when many markers are tested. Last, existing mole-
cular and statistical methods usually do not take into
account gene-gene or gene-environment interactions,
which are likely to have the key role in susceptibility to
complex polygenic diseases.
An alternative strategy is based on genes and
sequences. Here genotyping focuses on SNPs identified in
coding regions that terminate or alter amino acid
sequence, or disrupt splice sites, or occur in promotor
regions. The number of gene-related SNPs in the human
genome is estimated to be between 50,000 and 100,000.
Based on results from cloned Mendelian diseases, one
may prioritize amino acid replacements according to the
severity of the alteration and the degree of evolutionary
conservation [Botstein and Risch, 2003].
The routine use of genotyping techniques in biomoni-
toring studies remains in the future. Each SNP is a rare
event and the only way to include a marker of susceptibil-
ity in a biomonitoring study is to study very large human
populations. The cost of the arrays is the main limitation
to this approach and most studies published so far have
investigated few subjects. A more promising alternative
comes from studies aimed at identifying functional poly-
morphisms [Xi et al., 2004]. The availability of reliable
data on functional SNPs would considerably improve the
Human Population Studies 267
sensitivity and the efficiency of population studies not
only for CAs and micronuclei but for all biomarkers of
genetic damage.
CHALLENGES FOR THE FUTURE
From the issues addressed in this article, it is clear that
the field of cytogenetic biomarkers is extremely promising
and continues to grow. Two main factors are driving this
expansion. The first is technological innovation. Follow-
ing a decrease in the use of the older chromosome damage
biomarkers, the development of FISH whole chromosome
painting has transformed the applications of these assays
away from monitoring exposures toward their use as
highly specific tools capable of providing valuable
insights into early stages of chronic diseases, especially
cancer. The most remarkable example is the ability to use
FISH painting for studying reciprocal translocations,
which are key events in early stages of carcinogenesis for
hematological malignancies as well as for solid tumors
[Mitelman et al., 2004]. Similarly, the potential for study-
ing interphase cells has transformed the MN assay, which
is not only a rapid and easy test for genotoxicity but also
a tool for evaluating important events such as gene ampli-
fication. In addition, increased access to high-throughput
techniques can be expected to have a direct impact on
the use of cytogenetic biomarkers in population studies
[Albertini et al., 2004]. The most evident advantages
expected for the next few years are the improved reliabil-
ity of exposure assessment and early damage detection in
populations exposed either to low levels or to complex
mixtures of genotoxic agents. The identification of genetic
variants specifically linked to chromosome damage will
improve the sensitivity and the power of these studies.
These achievements will be facilitated by the cost reduc-
tion of molecular techniques and by the increased use of
automation, which will enable studies of larger popula-
tions, thereby increasing the sensitivity for detecting the
effects of exposure.
The second aspect that has contributed to the success
of cytogenetic biomarkers is their validation as predictors
of cancer. A causal link between CAs and the risk of can-
cer has been described in various cohort studies [Bonassi
et al., 2004]. A large international cohort study on the
association between MN frequency in healthy subjects
and cancer incidence has been underway for some years
[Fenech et al., 1999] and will be concluded soon. The
application of cytogenetic biomarkers for predicting the
risk of cancer in healthy subjects is a significant chal-
lenge. The large uncertainty of risk prediction at the indi-
vidual level and the complexity of ethical issues may
delay the design and planning of screening individuals for
cancer with these assays. Fortunately, it is feasible to
adopt validated biomarkers in prevention policies and in
268 Bonassi et al.
screening programs for populations at high risk due to
exogenous exposure or to genetic predisposition. These
endpoints are already mature enough to be applied to
cross-sectional studies to screen early carcinogenic effects
in populations exposed to known or suspected carcino-
gens. A few publications have started the discussion of
defining appropriate procedures of risk assessment based
on biomarker frequencies [Shatzkin et al., 1990; van Delft
et al., 1998]. The application of cytogenetic biomarkers to
screening individuals is of paramount importance since it
represents the necessary next step of establishing surveil-
lance in national regulations for occupational and environ-
mental safety.
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