Mutation Research 511 (2002) 73–86

Review
Biomarkers in molecular epidemiology studies
for health risk prediction
Stefano Bonassi a , William W. Au b,∗
a Department of Environmental Epidemiology, National Cancer Research Institute, Genoa, Italy
b Department of Preventive Medicine and Community Health, The University of Texas Medical Branch,
700 Harborside Drive, Galveston, TX 77555-1110, USA
Received 17 September 2001; received in revised form 10 December 2001; accepted 12 December 2001

Abstract
The field of molecular epidemiology is very promising, as sophisticated techniques are being developed to address etiology,
genetic susceptibility and mechanisms for induction of disease. The use of biomarkers plays a key role in these investigations
because the information can be used to predict the development of disease and to implement disease prevention programs.
However, as emphasized by Frederica P. Perera, the field is strewn with studies either that failed to use validated biomarkers or
whose designs did not adequately consider the biology of the endpoints, and the availability of validated biomarkers of health
risk is still limited. In this review, we have briefly described the usefulness of certain biomarkers for the documentation of
exposure and early biological effects, with special concern for the prediction of cancer. An emphasis is placed on understanding
the biological and health significance of biomarkers. By building reliable biomarker databases, a promising future is the
integration of information from the genome programs to expand the scientific frontiers on etiology, health risk prediction and
prevention of environmental disease. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Biomarkers; Molecular epidemiology; Chromosome aberrations; Micronuclei; FISH; HPRT; Gene mutation; Population studies;
Health risk assessment

1. Introduction [1,2]. For example, the results from these investi-

The traditional epidemiological technique has
always been the hallmark approach to demonstrate
associations between exposure to hazardous subs-
tances and development of disease such as cancer.
With respect to cancer, the endpoints for such inves-
tigations are usually mortality and disease incidence.
However, such traditional approach has its limitations
∗ Corresponding author. Tel.: +1-409-772-1545;
fax: +1-409-772-9108.
E-mail address: William.au@utmb.edu (W.W. Au).
gations are applied to the entire population for im-
plementation of disease prevention activities without
regards to inter-individual variations in response to
the exposure. Since such variations play a significant
role in determining who is more likely to be affected,
these factors need to be incorporated into studies
to improve the prediction of environmental disease
[3]. Furthermore, with more stringent regulations on
environmental exposure and with automation of haz-
ardous processes, e.g. in the workplace, the exposure
concentrations and the availability of exposed indi-
viduals may be too small for conducting meaningful

1383-5742/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 4 2 ( 0 2 ) 0 0 0 0 3 - 0
74 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86
traditional epidemiological investigations. Therefore,
a variety of modifications have been integrated into
the traditional approach to increase the sensitivity and
specificity of epidemiological studies.
Among the many approaches that have been used
to improve the epidemiological protocol, the one
proposed by Perera and Weinstein [4] has attracted
many followers. They suggested the incorporation of
laboratory analytical techniques with traditional epi-
demiological surveys to elucidate the biochemical or
molecular basis of disease etiology. Since then, many
such studies have been conducted to provide useful
analytical data such as internal exposure doses and
biological effects. Examples are, the measurement of
DNA adducts, chromosome aberrations (CAs) and
gene mutations in exposed populations. Thus, the new
field of molecular epidemiology was born [5–7] and
the biological endpoints used in these studies became
known as biomarkers [8,9].
Fig. 1. Causal pathway from initiation to the occurrence of disease.
2. Usefulness of biomarkers
A major objective of molecular epidemiologi-
cal investigations is to provide reliable and specific
information regarding the etiology and mechanism of
disease processes for disease prevention. The possi-
bility to use a biomarker to substitute classical end-
points, such as disease incidence or mortality is the
most promising feature and one that is most likely to
affect public health. The use of events that are on the
direct pathways from the initiation to the occurrence
of disease to surrogate the disease incidence is a very
appealing approach, which is currently investigated
in different fields (Fig. 1).
A great deal of research is currently ongoing in the
field of clinical trials. The development of therapies
for chronic diseases often requires long-term studies
to evaluate the impact on well-defined clinical end-
points, such as recurrence of cancer or survival, and
 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86 75

this aspect has created the need for surrogate end-
points that can be measured in a shorter period of time.
Scientific literature has reported examples of useful
biomarkers such as CD4 cell counts and HIV viral load
for clinical trials of anti-AIDS treatments [10], and as
a matter of facts the US Food and Drug Administration
has regulated the approval of some new drug products
based on the results on surrogate endpoints [11].
The use of biomarkers in clinical trials requires a
robust linkage or correlation with a clinical endpoint,
especially when studies are performed for regulatory
approval. A strong evidence of association is gener-
ally achieved when pre-clinical stages of disease are
concerned, and therefore the use of these endpoints
for preventive purposes has limited use. To gain the
most leverage in time, and then to offer the greatest
potentiality to preventive intervention, the biomarkers
should represent a biological event occurring in the
early stages of the disease, although the price to pay is
a reduction of the association between the biomarker
and the clinical outcome [12].
The different interpretation of biomarkers in various
field of biomedical research is reflected by an un-
certain nomenclature. Biomarkers used to predict
classical endpoints are often defined surrogate end-
points, intermediate endpoints, early-outcome pre-
dictors, or just predictors. A simple differentiation
between biomarkers used as an alternative outcome
in clinical trials, i.e. surrogate endpoints, and in pop-
ulation studies for preventive aims, i.e. intermediate
endpoint, early-outcome predictors, is acceptable for
most purposes. Most authors agree that the concept
of causation, i.e. laying in the direct pathway from
initiation to the onset of the disease, is not strictly
necessary for considering biomarkers as predictors,
although biomarkers that are part of the process
eventually leading to the disease are perceived as
more relevant [13]. In the event when misleading
biomarkers are used, the etiology and mechanism of
the disease process will not be understood correctly.
Consequently, effort for the development of disease
prevention strategies will be misdirected.
3. Biomarkers of exposure
An important piece of the information to gather for
molecular epidemiology study is the documentation
of exposure. Unfortunately, exposure information is
frequently the weakest component of epidemiological
investigations. Traditionally, exposure information is
gathered via questionnaire and historical information
such as employment records and monitoring data.
Whenever possible, a more precise way to document
the exposure such as the use of biomarkers of expo-
sure should be adopted. Some examples of exposure
biomarkers are listed in Table 1.
Among these biomarkers, the first two categories
(the measurement of chemicals and their metabolites,
and of adducts to macromolecules in body fluid) are
highly sensitive and specific to the exposure. More
importantly, if the data have been collected appro-
priately, the information can be used to calculate the
internal exposure doses to the chemicals and to deter-
mine the dose–response relationship. The measure-
ment of adducts is particularly useful for identifying
the chemicals that form bulky adducts with macro-
molecules (e.g. protein and DNA adducts). Although
the presence of protein and DNA adducts can be
readily measured, the latter has become very popular
because the presence of specific DNA adducts is more
likely to be associated with the development of the
disease [14,15]. On the other hand, the effects from
chemicals that produce small adducts is difficult to
measure at the DNA level but may be possible at the
protein level, e.g. benzene [16].

Table 1
Examples of biomarkers of exposure to mutagenic agents in human populations
Biomarkers of exposure Characteristics

Chemicals and metabolites Highly sensitive and specific to chemicals; allows determination of internal exposure doses
Adducts to macromolecules Highly sensitive and specific to chemicals that bind to proteins and DNA; associate
with further consequences
DNA strand breaks Sensitivity and specificity not validated yet; significance of DNA strand breaks for
health consequences uncertain
SCEs Sensitive to chemical exposure but unknown biological significance
76 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86
Table 2
Fate of biomarkers of exposure in blood and urine
Biomarkers Estimated percent detectable at post-exposure daysa
Day 1 Day 10
Exposure to a persistent toxic substance
Blood metabolite 77 64
Albumen adduct 151 84
Hemoglobin adduct 118 51
Lymphocyte DNA adduct 82 41
Exposure to a non-persistent toxic substance
Blood metabolite 4 0
Albumen adduct 170 80
Hemoglobin adduct 132 63
Lymphocyte DNA adduct 80 45
a Detectable level shortly after exposure is set as 100% [17].
For many types of biomarkers the most important
consideration is the stability of the biomarkers with
respect to time after the exposure. Table 2 shows
some examples regarding the stability of biomarkers
of exposure for a persistent and a non-persistent toxic
substance [17]. For a persistent toxic substance, the
concentration of blood metabolites will decrease con-
sistently but slowly with time compared to exposure
to a non-persistent chemical. The concentrations of
albumin and hemoglobin at Day 1 after the exposure
can be higher than that detected immediately after
exposure. Then, the concentrations decline with time.
DNA adducts in lymphocytes may be more stable
than protein adducts, but still decline with time. It
should be emphasized that this example is based on
an acute exposure condition. The situation is more
complicated with chronic exposures. In addition,
these biomarkers, like most others, are of limited use
for documenting previous exposures. For previous or
chronic exposures, historical information may have
to be used or archived specimens may need to be
analyzed in order to supplement the investigation.
Among the biomarkers of exposure, DNA adducts
have been clearly shown to be relevant to the disease
process in prospective studies. The best examples can
be drawn from studies on the etiology of liver cancer
[18–21]. It has been well documented that infec-
tion with hepatitis B virus (HBV) and exposure to a
food-borne mutagen, aflatoxin (AFB1 ), are the most
important risk factors for liver cancer. Using speci-
mens that have been collected from individuals before
Day 100 Day 1000
50 13
18 4
9 2
6 2
0 0
15 0
10 0
10 0
they developed liver cancer, the AFB1 -N7 -guanine
adduct was found to be significantly predictive of
liver cancer among the study population. The pres-
ence of DNA adducts was supported by the detection
of AFB1 metabolites in the collected urine. Further-
more, the presence of DNA adducts interacted with
the infection to significantly increase the risk for liver
cancer. Such validated data led to the development of
disease prevention program that targeted the reduction
of dietary AFB1 and the vaccination against HBV.
Another good example can be drawn from the study
of p53 gene mutation as a key mechanism of lung
carcinogenesis. It is well known that cigarette smoking
is the most important risk factor for lung cancer and
that more than half of the tumors have mutated p53
gene. The mutations are concentrated in a few exons of
the gene [22]. Therefore, it becomes a crucial question
to ask which chemicals in cigarette smoke may induce
the specific p53 mutation to initiate lung cancer. In an
in vitro study, benzo[a]pyrene was found to form DNA
adducts and to induce mutations at precisely the same
sites as found in mutated p53 in lung tumor [23].
Another biomarker of exposure is the measure-
ment of DNA strand breaks using a variety of assays.
The determination of exposure-induced DNA strand
breaks, e.g. the single cell gel electrophoresis assay,
has advantages and shortcomings [24]. When stable
cell lines are used in vitro to screen toxic chemicals,
the sensitivity and cost effectiveness of these assays
are clearly demonstrated. On the other hand, the use
of such assays in population studies has produced
 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86 77

highly variable results [24]. Several confounding fac-
tors that can have profound impact on the outcome of
the studies have been identified. Examples are the sta-
bility of the DNA strand breaks during transportation
and storage of specimens, the source of cells for the
determination of strand breaks (lymphocytes versus
polymorphonuclear leukocytes; G0 versus G1 cells)
and the relationship of the observed DNA strand
breaks with biological processes (DNA breakage,
apoptosis, other forms of cell death) [25]. Variations
in these factors between the exposed and control
populations can affect the assay results, thus gen-
erating false positive or false negative observations.
Compared with the measurement of DNA adducts,
the DNA strand break assays have not been vigor-
ously validated. Therefore, the value of the assay for
population studies needs to be carefully evaluated.
A variation of the DNA strand break assay is the
measurement of reduction of DNA strand breaks with
respect to time. The hypothesis is that the reduction
is due to the repair of breaks. However, the hypoth-
esis needs to be validated using molecular analysis
based on the measurement of DNA repair activities.
Furthermore, other confounding factors need to be
considered. For example, is the reduced DNA strand
breaks caused by the eliminated of cells with exten-
sive strand breaks? On the other hand, the significance
of the observation needs to be interpreted carefully.
For example, is the reduction in strand breaks due to
correct or incorrect rejoining of the DNA molecules?
The sister chromatid exchange (SCE) assay has
been popular in the past because the assay is more
sensitive than the traditional CA assay for determin-
ing exposure to mutagenic chemicals. However, since
the biological significance of the SCE event is still
elusive, the enthusiasm for the assay became less
during the last few years.
4. Biomarkers of early biological effects
Exposure to mutagenic chemicals can cause damage
to cellular macromolecules. The damage will gene-
rally stimulate the affected cells to mount responses.
The responses may involve the repair of the damage.
Incorrect repair will most certainly cause health con-
sequences. Therefore, a variety of biomarkers of early
biological effects have been developed and tentatively
validated. Some examples are shown in Table 3.
The CA assay is the most extensively used and best
validated biomarker of early biological effects in pop-
ulation studies [25]. The assay can be used with con-
fidence for detecting exposure to ionizing radiation
[26] because the radiation induces chromosome-type
aberrations such as dicentrics that are easily detected.
The induction of dicentric is so specific to radiation
exposure that the collected data is frequently used to
calculate exposure doses with confidence. Exposure to
chemicals usually induces chromatid-type aberrations
such as breaks and exchanges. The ability of cells
having CA to survive has been investigated. From
studies of individuals with birth defects, it is clearly
shown that individuals with CA, e.g. small deletions,
and gain or loss of certain chromosomes, can survive.
In addition, chromatid-type exchanges can be con-
verted into chromosome-type translocations and these
subsequent cells are expected to survive for a long
time [27]. Cells with chromosome abnormalities can
lead to the development of cancer as indicated by the
presence of chromosome deletions and translocations
in the majority of cancer cells [28].
It has been difficult to use the traditional CA
assay to distinguish the damage from one chemical
to another except in a few cases. Certain chem-
icals such as bleomycin and neocarcinostatin are
known as radiomimetic agents because they induce

Table 3
Examples of biomarkers of early biological effects from exposure to mutagenic agents in human populations
Biomarkers of early biological effects Characteristics

CAs Distinguish radiation from chemical effects; show specificity to certain chemicals;
predict survivability of cells with certain CAs
MN Distinguish chromosome breaks from aneuploidy; survivability of damaged cells uncertain
HPRT gene mutation Detect somatic mutation of a hemizygous gene in the X-chromosome; identify clonal
origin of mutated cells; predictability of mutation in other genes in somatic cells uncertain
Glycophorin A mutation Detect somatic mutation in progenitor erythrocytes in bone marrow; needs heterozygous
donors for investigation
78 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86
chromosome-type instead of chromatid-type aberra-
tions [29,30]. Another example is mitomycin C that
induces mainly chromatid exchanges around the hete-
rochromatid regions of chromosomes [31]. Recently,
with the incorporation of molecular techniques, it is
possible to elucidate chemical-specific induction of
CAs (to be discussed later).
A drawback of the traditional CA assay has been the
labor-intensive procedure. Therefore, several molec-
ular techniques have been developed to enhance the
usefulness of the assay. The most significant improve-
ment is the use of the fluorescence in situ hybridiza-
tion (FISH) procedure to detect chromosome-specific
aberrations. Chromosome-specific probes that are
labeled with fluorescent dyes are used to hybridize to
chromosomes for the determination of chromosome
rearrangements and chromosome region-specific
breaks [32,33]. The former technique is known as
whole chromosome painting and the latter the tandem-
probe assays. Furthermore, the former is most useful
for detecting damage from exposure to ionizing ra-
diation [34] and the latter for exposure to chemicals
such as pesticides [35].
The original tandem-probe assay was developed
to detect damage at the heterochromatic region of
chromosome 1. Although this region may be prone to
damage from exposure to mutagens, the significance
of such damage to biological consequences remains
to be determined. Therefore, several scientists have
developed alternative probes to detect damage in eu-
chromatic regions of chromosomes where functional
genes are located. Using probes that can recognize the
breakage–translocation region between chromosomes
5 and 7, the abnormality that is frequently observed in
benzene-induced leukemia, benzene-exposed work-
ers were found to have significantly high frequency
of blood cells containing the specific abnormality
compared to unexposed controls [36]. Using cells in
culture, it was found that cells resistant to chemi-
cals that can form bulky adducts exhibited chromo-
some instability whereas cells resistant to alkylating
chemicals that form small adducts exhibited mi-
crosatellite instability [37]. The latter is associated
with mismatch DNA repair defects. Therefore, these
molecular chromosome studies show the way for a
new direction towards identifying chemical-specific
and biologically-relevant CA. These observations to-
gether with the finding that CA are predictive of the
development of cancer (to be described later) indi-
cate that the CA is the most important biomarker of
effects, especially for predicting health risk.
The micronuclei (MN) assay is a variation of the
CA assay. Instead of detecting the abnormality at the
metaphase stage of the cell cycle, MN are detected
after the cells have progressed pass the first cell cycle.
The major advantage of this assay over the tradi-
tional CA assay is that it can detect aneuploidy events
[25,38]. In addition, it is much less labor-intensive than
the traditional CA assay. On the other hand, the MN as-
say has shortcomings. Since the cells need to survive at
least one nuclear division, some of the damaged cells
may have been lost. Although the association between
the frequency of MN in binucleated cells and long
term biological consequences has not been adequately
elucidated yet, a number of theoretical evidences
support a potential predictive role for this biomarker
[39].
The HPRT assay is the most frequently used soma-
tic cell mutation assay in population studies [40]. By
assaying the recombination regions of the genome
among the mutant clones, the clonal origin of the
mutated cells can also be determined. With this tech-
nique, it was found that the high mutant frequencies
for some individuals were due to clonal expansion of
only a few mutant cells rather than the induction of
a large number of mutated cells [40]. The assay is
used frequently for the detection of mutation in ex-
posed populations [41,42]. However, the significance
of these observations towards risk assessment has not
been determined yet because the extrapolation of mu-
tation in the hemizygous HPRT gene to diploid genes
(e.g. the p53 tumor suppressor gene) and to somatic
mutation related diseases remain to be elucidated
[43,44].
The glycophorin mutation assay is another well-
recognized somatic cell mutation assay. Whereas the
HPRT gene mutation assay detects damage in post-
thymic lymphocytes, the glycophorin assay detects
damage in the bone marrow cells [45]. The former
makes use of the hemizygous state of the HPRT gene
whereas the latter utilizes heterozygous individuals.
In a side-by-side comparison with the FISH and the
HPRT assays in population studies, the glycophorin
assay is not as sensitive as the other assays [46,47].
This may be due to the fact that the glycophorin assay
is based on a different mechanism of genotoxicity, i.e.
 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86
loss of alleles [48]. However, the need to use heterozy-
gous individuals among the study populations limits
the use of this assay to large populations only.
5. Biomarkers for health risk
The ultimate goal of using biomarkers in molecular
epidemiological studies is to be able to predict health
risk. Two issues are crucial in the application of pre-
dictive biomarkers to public health policies. The first
is dealing with the meaning of altered levels of pre-
dictive biomarkers at individual level. A conservative
and traditional approach is that of considering risk
predictions valid only at group level. This interpreta-
tion allows cutting down the effect of inter-individual
variability and reduces the variability due to technical
parameters. On the other hand, we have learned from
epidemiology that variability is a fundamental source
of information. In addition, differences among indi-
vidual should not be viewed as a nuisance but should
be seen as useful hints in the hypothesis generation
and as an enhanced possibility to apply preventive
measures in subsets of high risk subjects. The second
crucial aspect is the validation issue. A biomarker
must be validated before it can be used for health risk
assessment, especially as far as regulatory aspects
are involved. Despite the characterization of valid
biomarkers is a leading priority in environmental
research, defining validity is troublesome.
Validity is a general concept that refers to a range
of characteristics of the biomarker, and an impressive
amount of literature has been published on the con-
cept of biomarker validity and the various aspects of
the validation process [49,50]. To provide a more gen-
eral and practical approach to the validation of those
biomarkers that have been identified as components
or correlate of the diseases pathway, the process can
be summarized in three steps starting from the devel-
opment of the biomarker, to its characterization, and
Table 4
Examples of biomarkers of health risk
Biomarkers of health risk Characteristics
CAs Biological dosimeter for ionizing radiation; predict cancer incidence and mortality
Cancer gene mutation/expression Relevant to disease initiation and progression
79
to the final step of longitudinal studies [15]. In the
process of validating biomarkers as early predictors
of disease, assessment of the relationship between
the frequency of a biomarker and the incidence of
disease is based on longitudinal studies, with the gold
standard being prospective cohort studies. Although
this approach is costly and time consuming, it has
the great advantage of studying subjects who are pre-
sumably healthy at the time of testing. This feature
makes prospective studies the design of choice when
a biomarker is transient or affected by the disease
because they avoid reverse causality bias. Yet cohort
studies are not widely conducted in cancer research,
primarily because of the lack of biomarkers with a
history long enough to provide a sufficient number
of events, deaths, or incident cases during follow
up [25].
Only few biomarkers have undergone the vigorous
validation process and one is the frequency of chro-
mosomal aberrations as an early predictor of cancer
risk. Examples of some biomarkers that are predictive
of health effects are shown in Table 4.
The presence of a causal association between the
frequency of CA in healthy subjects and the risk of
cancer has been hypothesized since the beginning
of the century. Though the evidence supporting this
association has accumulated along many years, only
in the last decade have studies in Nordic countries,
Italy, Taiwan and Czech Republic demonstrated that
the presence of CA is predictive of cancer incidence
and mortality. A more detailed description will be
provided in Section 6.
There are a lot of interests in detecting the presence
of mutated cancer genes or the abnormal expression
of these genes as a prediction of cancer develop-
ment. A challenge is in the selection of appropriate
surrogate tissues using non-invasive collection proce-
dures [51]. For example, mutation in K-ras has been
detected in brochoalveolar cells, sputum and blood
of cancer patients [52–55]. The application of the
80 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86
approach in normal individuals before they develop
cancer is promising and should be initiated.
6. Prediction of cancer using chromosome
aberrations
The hypothesis that CAs are associated with can-
cer and can possibly act as an intermediate step of
the causal pathway is supported by many reports.
Evidence can be summarized in four major argu-
ments: (1) chromosome rearrangements play an im-
portant role in the activation of protooncogenes and
inactivation of tumor suppressor genes; (2) subjects
with a congenital disease, such as Fanconi’s anemia
or AT, are characterized by abnormally high CA rates
and increased incidence of malignancies; (3) alter-
ations of the karyotype have been found in all types
of neoplastic cells and are often highly specific for
particular diagnostic categories; (4) there is a ten-
dency for carcinogenic chemicals to be clastogenic
and that clastogenicity tends to be associated with
known human carcinogens. All the evidence concern-
ing the role of CAs in the carcinogenic process also
applies for PBL, on the understanding that the extent
of genetic damage in these cells reflects similar events
in the target tissue [56].
The extensive use of this assay has resulted in the
accumulation of valuable data in many laboratories.
This has enabled the examination of the potential
association between previously measured CA fre-
quency and subsequent cancer outcome. An associa-
tion between high CA frequency and increased cancer
incidence was originally detected in a collaborative
project of 10 Nordic cytogenetic laboratories [57].
No association could be shown for SCEs or MN. An
independent study among 10 laboratories in Italy,
based on cancer mortality data, arrived at the same
conclusion [58].
The two cohorts were recently updated and exam-
ined together. The results supported the findings that
CAs, but not SCEs or MN, are predictive of cancer
risk [59]. Furthermore, a case-control study nested
within the two cohorts indicated that this association
is not merely a reflection of smoking or occupa-
tional exposure to carcinogens, but is similarly seen
in apparently unexposed subjects [60]. A group of
occupational hygienists re-evaluated the occupational
histories of cases (all cancers) and controls (four for
each case), and also reconstructed the smoking his-
tory of these subjects. Thus, these results suggested
that a high CA level as such is predictive of increased
cancer risk, irrespective of the cause for the induction
of CA.
Aside from the Italian–Nordic collaboration, other
studies addressing the cancer predictivity of cytoge-
netic biomarkers in humans have been published in
Taiwan and more recently in the Czech Republic. A
small group of 22 cancer cases and 22 control resi-
dents of an endemic black-foot area in Taiwan have
been studied with a nested case-control design; the
results suggested that chromosome-type CAs, but not
chromatid-type CAs or SCEs, predict cancer risk [61].
More recently, a large cohort study, based on 37,775
person-years has substantially confirmed the other
European findings, finding an overall hazard ratio
(HR) of 1.6 (95% CI = 1.01–2.37) and higher risks
in the sub-groups of radon workers (HR = 8.0; 95%
CI = 2.42–26.1) [62]. Other projects are ongoing,
and a new cohort study, to be performed in central
and eastern European countries, is in its preliminary
phase.
Common criticisms to these data refer to the use of
surrogate tissues to estimate events occurring at the
target organs and of unstable type of CA since cells
carrying these aberrations are not expected to sur-
vive. Regarding the first criticism, studies on cancer
patients showed that CAs in blood and target cells
correlate well [56]. Regarding the second criticism,
unstable CA has been reported to be a good rep-
resentation of stable CA [27]. In addition, unstable
exchanges can be converted into stable translocations,
thus allowing the abnormal cells to survive. The cor-
relation between unstable CAs and translocations in
a study published by Verdorfer et al. [63] was eval-
uated. Based on non-cancer patients, the correlation
was excellent (Fig. 2; R = 0.75 P < 0.05).
What is noteworthy is that the prediction based on
unstable aberrations in blood cells is less specific and,
therefore, the hypothesis of an under-evaluation of the
strength of the CA–cancer association is plausible.
Future studies using modern technologies based on
the constellation of techniques dealing with the FISH
have the potential to confirm this point.
The evidence of association between an increased
level of CA in healthy subjects and the risk of cancer
 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86
Fig. 2. Correlation between unstable CA and translocations [63].
repeatedly described by the studies published so far
has generated some ethical and practical problems. In
fact, when an increased level of CA is reported in a
group this group should be considered at risk of can-
cer and preventive measures should be undertaken as
soon as possible. This is not easy to be realized when
economic aspects are involved, e.g. when a company
has to spend money to improve working conditions,
or residents near a chemical waste site should be re-
located to a safer place. These considerations focus
on the need of stronger scientific evidence before
information on biological markers can really affect
public health policies.
A good example of how these results can be applied
is the estimation of the cancer risk in a small group of
subjects exposed to a large variety of radiation, i.e. the
astronauts. The presence of various types of chromo-
some damages in these subjects after space missions
have been reported since the first flights [64]. Now,
using these data and the results from European stud-
ies on the association between CA and increased risk
of cancer, a relative risk of 1.25 has been calculated
for astronauts taking part in a space mission [65]. The
association between the exposure to radiation and the
risk estimate is in good agreement with data from the
Hiroshima database. Despite the intrinsic limitation of
this exercise, the introduction of more effective mea-
sures for the safety of astronauts during flights should
be taken into serious consideration.
81
7. Enhancement of biomarker database
A typical limitation of molecular epidemiology
studies, due to logistical difficulties, is the limited size
of study groups. A recent review by Gordon et al. [66]
has calculated that the statistical power of published
studies on genetic susceptibility ranges from 20 to
70%, and then only a few studies have an accept-
able power to detect a real difference. To overcome
this situation, larger studies are designed, although
quite often the low frequency of biological events or
the high cost of the assay has made it impossible to
have studies properly sized. From the experience of
clinical trials the use of re-evaluation of the existing
evidence has turned out to be an efficient and suitable
approach to extract information from a great amount
of small studies.
The typical approach to the re-evaluation of pub-
lished data is meta-analysis, and this approach has
been used since many years in clinical trials as
well as in many other fields. Pros and cons of this
methods are well known and a large extent of liter-
ature is available [67]. More recently, the use of the
pooled analysis has been proposed as an alternative to
meta-analysis. This is an alternative way to summa-
rize data in the observational studies, pooling individ-
ual records and re-analyzing the data. This approach,
although more costly, gives some advantages over the
classical meta-analysis of published data, mainly the
possibility to perform sub-group analysis including
dose–response curves, to evaluate other covariates,
not to mention the creation of international network
of collaboration [68].
Possible enhancements of biomarkers currently
used in human studies deal with the optimisation of
resources and the exploitation of the available infor-
mation. A suitable example of this approach is an in-
ternational collaborative project on the micronucleus
assay: Human MicroNucleus (HUMN) project—
international database comparison for results with
the cytokinesis-block micronucleus assay in human
lymphocytes.
Since the development of the cytokinesis-block
micronucleus method [38], which allowed MN to
be scored specifically in cells that completed nu-
clear division, this assay has been used extensively
to evaluate the presence and the extent of chromo-
somal damage in human populations. The MN test
82 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86
provides a reliable measure of chromosome break-
age and loss, at lower cost and with less work than
chromosomal aberrations, and a further potential for
this assay is the possible use as a predictor of ad-
verse health effects. As with other cytogenetic assays,
the MN assay is affected by a large extent of unex-
plained variability and limited information is avail-
able on the effects of the laboratory method, subject
life-style, as well as on the role of individual sus-
ceptibility.
In order to address these issues and to provide
a network of laboratories working with this assay,
the international collaborative HUMN study was
launched in September 1997. Among the main goals
of HUMN were the comparison of protocols currently
in use to assess whether, and to what extent, such
variations might affect the baseline MN frequencies.
The comparison of baseline MN frequencies from
the various laboratories and a determination of the
relationships of these frequencies to subject factors
will allow the evaluation of the effect of some key
subject variables (e.g. age, gender, life-style, ex-
posure to genotoxic agents) affecting baseline MN
frequencies in peripheral blood lymphocytes. Other
factors such as the analysis of variables not specifi-
cally evaluated in single studies, the effect of differ-
ent laboratory protocols and methods, the analysis
of rare characteristics in healthy subjects can be
elucidated.
Finally, and even more promising, the availability
of a large database of individual data will permit a
prospective cohort study, linking the accumulated
micronucleus frequency data from each laboratory
with the incidence of common diseases, as demon-
strated by the example presented before of studies on
the association between CA and the risk of cancer.
Currently, the database of the HUMN project includes
7000 subjects from 25 laboratories worldwide dis-
tributed. More detail on the HUMN project and on
the CBMN assay can be found in [39,69] or in the
website http://www.HUMN.org.
The use of a pooled re-evaluation of published
data is the most informative approach to exploit the
information produced by a constellation of studies
and that cannot be used in other ways. The increase
of methodological research in the field could even-
tually lead to the definition of guidelines, although
a number of recommendations and examples on the
planning and conducting of these studies are already
available in the literature.
8. Applications and future direction
The most obvious new direction for molecular epi-
demiological studies is in dealing with the availability
of new technologies. New information coming from
the genome-derived methods can be of paramount
importance in human studies. Since the role of sus-
ceptibility genes can be more efficiently investigated,
their influence on specific effects of exposures and
response to genotoxic agents are being elucidated.
Among coke oven workers, those with the GSTM1
null susceptibility genotype appeared to be sensitive
to the exposure conditions because they had signif-
icantly increased polyaromatic hydrocarbon DNA
adducts [70]. Cigarette smokers with the NAT2 rapid
acetylator genotype had significantly high frequency
of stable CA, suggesting that these individuals may
be at increased risk for lung cancer [71]. Lung can-
cer patients had significantly higher frequency of
CA than the controls, especially among the patients
who had the susceptibility GSTM1 and/or GSTT1
genes [72]. Other studies have provided support re-
garding the important role of polymorphic GSTs on
modifying the toxicity of environmental and occupa-
tional chemicals [73,74]. With in vitro studies, the
exposure conditions can be precisely controlled for
better elucidation of the cause-effect relationship. In
these studies, the GSTM1 and GSTT1 null genotypes
were found to be significantly associated with the
induction of CA by cigarette smoke-specific chemi-
cals, benzo[a]pyrene and NNK [75,76]. Furthermore,
dose-dependent effect was clearly documented. Stud-
ies like these will permit a better insight in the
mechanism of actions of chemical substances. These
considerations, concerning the so-called toxicoge-
nomics are even more valid when the contribution
of proteomics is considered as well. However, the
contribution of high throughput technologies to tox-
icology is nowadays substantially theoretical, since
studies of groups of exposed individuals are not avail-
able and therefore no evidence has been produced
about the association between data on gene expres-
sion or proteins and classical markers of exposure
or early effect. This is the first step of a validation
 S. Bonassi, W.W. Au / Mutation Research 511 (2002) 73–86
process as population biomarkers, which is not started
yet.
9. Conclusion
Many scientists have enthusiastically accepted the
field of molecular epidemiology. The future of this
field is promising, as sophisticated analytical tech-
niques are being developed to determine smaller and
smaller amount of environmental chemicals and of
biological activities. However, one must consider the
relevance of a minuscule amount of chemical and/or
biological activities on health consequences carefully.
Similarly, one must consider the value of molecular
epidemiology studies based on vigorous evaluation
of their experimental designs [77]. As emphasized by
Frederica P. Perera, the field is strewn with studies
either that failed to use validated biomarkers or whose
designs did not adequately consider the biology of the
endpoints [78], and the validation of biomarkers of
health risk is a leading priority. By building reliable
biomarker databases, a promising future is the inte-
gration of information from the genome programs to
expand the scientific frontiers on etiology, health risk
prediction and prevention of environmental disease.
Acknowledgements
This work was supported by grants funded by
the Associazione Italiana per la Ricerca sul Cancro
(AIRC) and the European Union Fifth FP (QLRT-
2000-00628) to SB; and by the National Institute
of Environmental Health Sciences Center Grant
ES06676 and the Sealy Center for Environmental
Health and Medicine, University of Texas Medical
Branch to WA.
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