Mutation Research 770 (2016) 140–161

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Review

Biomonitoring of humans exposed to arsenic, chromium, nickel,

vanadium, and complex mixtures of metals by using the micronucleus
test in lymphocytes
Balasubramanyam Annangia , Stefano Bonassib , Ricard Marcosa,c,* , Alba Hernándeza,c,*
a
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Spain
b
Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Via di Val Cannuta, 247, 00166 Rome, Italy
c
CIBER Epidemiología y Salud Pública, Instituto de Salud Carlos III, Madrid, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Various metals have demonstrated genotoxic and carcinogenic potential via different mechanisms. Until
Received 17 July 2015 now, biomonitoring and epidemiological studies have been carried out to assess the genotoxic risk to
Received in revised form 15 February 2016 exposed human populations. In this sense, the use of the micronucleus assay in peripheral blood
Accepted 1 March 2016
lymphocytes has proven to be a useful tool to determine increased levels of DNA damage, as a surrogate
Available online 6 March 2016
biomarker of cancer risk. Here we review those biomonitoring studies focused on people exposed to
arsenic, chromium, nickel, vanadium and complex mixtures of metals. Only those studies that used the
Keyword:
frequency of micronuclei in binucleated (BNMN) cells have been taken into consideration, although the
Metals
Biomonitoring
inclusion of other biomarkers of exposure and genotoxicity are also reflected and discussed. Regarding
Micronucleus arsenic, most of the occupational and environmental biomonitoring studies find an increase in BNMN
Peripheral blood lymphocytes among the exposed individuals. Thus, it seems conclusive that arsenic exposure increases the risk of
exposed human populations. However, a lack of correlation between the level of exposure and the
increase in BNMN is also common, and a limited number of studies evaluated the genotype as a risk
modulator. As for chromium, a BNMN increase in occupationally exposed subjects and a correlation
between level of exposure and effect is found consistently in the available literature. However, the quality
score of the studies is only medium-low. On the other hand, the studies evaluating nickel and vanadium
are scarce and lacks a correct characterization of the individual exposure, which difficult the building of
clear conclusions. Finally, several studies with medium-high quality scores evaluated a more realistic
scenario of exposure which takes into account a mixture of metals. Among them, those which correctly
characterized and measured the exposure were able to find association with the level of BNMN. Also,
several genes associated with DNA damage repair such as OGG1 and XRCC1 were found to influence the
exposure effect.
ã 2016 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2. Quality scoring of the evaluated studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
3. Meta-analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4. Arsenic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4.1. Human exposure to arsenic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4.2. Health effects of arsenic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4.3. Biomonitoring of human populations using the MN assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
4.3.1. Studies on people occupationally exposed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
4.3.2. Studies on people environmentally exposed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

* Corresponding author at: Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Edifici Cn, Campus de Bellaterra,
Cerdanyola del Vallès, Barcelona 08193, Spain.
E-mail addresses: ricard.marcos@uab.es (R. Marcos), alba.hernandez@uab.es (A. Hernández).

http://dx.doi.org/10.1016/j.mrrev.2016.03.003
1383-5742/ã 2016 Elsevier B.V. All rights reserved.
 B. Annangi et al. / Mutation Research 770 (2016) 140–161 141

4.4. Meta-analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

4.5. Summing up and potential gaps . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
5. Chromium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
5.1. Human exposure to chromium . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
5.2. Health effects of chromium . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
5.3. Biomonitoring of human populations using the MN assay . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
5.3.1. Studies on people occupationally exposed . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
5.4. Summing up and potential gaps . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
6. Nickel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
6.1. Human exposure to nickel . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
6.2. Health effects of nickel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
6.3. Biomonitoring of human populations using the MN assay . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
6.4. Summing up and potential gaps . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
7. Vanadium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
7.1. Human exposure to vanadium . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
7.2. Health effects of vanadium . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
7.3. Biomonitoring of human populations using the MN assay . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
7.4. Summing up and potential gaps . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
8. Complex mixtures of metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
8.1. Human exposure to metal mixtures . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
8.2. Health effects of metals exposure . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
8.3. Biomonitoring of people exposed to mixtures of metals using the MN assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
8.4. Summing up and potential gaps . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
9. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

1. Introduction general pattern of the genotoxic/carcinogenic mode of action of

Heavy elements are widely distributed in the Earth’s crust. They
comprise a defined group of elements that include transition
metals and some metalloids. Soil erosions by wind and water
dissolution are natural mechanisms involved in the environment
resulting in the occurrence of heavy elements in air and water
matrices. Other natural phenomena, such as volcanic eruptions,
can also contribute to the presence of metals in the environment
[1]. Nevertheless, anthropogenic activities, such as mining and
smelting, act as primary sources of metal spread by polluting wide
areas, thus acting as important sources of human exposure [2]. For
this reason, areas covering mining, foundries and smelters, as well
as other metal-based industrial operations, are considered highly
polluted zones. Workers involved in such activities or people living
in surrounding areas are considered to be highly metal-exposed
human groups [3,4].
Metals are of great importance in our daily life, and mankind as
we know it would not have evolved if not for their existence. Their
frequent use makes their omnipresence and a constant source of
human exposure. This close relationship between humans and
metals suggests that several metals are biologically considered
essential nutrients, necessary for the correct functioning of various
biochemical and physiological processes [5]. Cobalt, copper,
chromium, iron, magnesium, manganese, molybdenum, nickel,
selenium and zinc belong to the group of essential metals, but their
presence in the body below or above certain concentrations can
also lead to disease states [6]. In fact, for most metals, exposure has
been linked to the induction of adverse health effects as well as to
different human pathologies. From bacteria to humans, metal
exposure has been associated with cell damage located in different
organelles and membranes, with the proven capability of reaching
the nucleus and interacting with DNA [7]. From this point of view,
different metal compounds are considered genotoxic and carcino-
genic [8–11].
The genotoxic and carcinogenic potential of metals is mainly
dependent on their state of oxidation, as it affects their uptake,
intracellular transport, distribution and bioavailability [12]. A
metals comprises the following steps: (1) Induction of oxidative
stress and damage to cellular components, including DNA; (2)
interference with DNA repair systems, resulting in genomic
instability and (3) interruption of cell growth and proliferation
via signalling pathways and deregulation of oncogenes or tumour
suppressor genes [12,13].
Many studies have been conducted on human populations
exposed to metals to determine the induction of biomarkers of
genotoxicity. Unfortunately, in many cases, the exposure consists
of complex mixtures of metals, making it difficult to assign the
observed effects to one particular metal compound. This is true in
the case of exposure to air pollution particles, for instance, where
part of their genotoxic effects can be attributed to their metal
contents [14]. A similar situation occurs in smelting operations,
where metals other than the predominant one can be found at
considerable concentrations in the area [15,16]
In addition to the epidemiological studies aiming to determine
the carcinogenic risk associated with metal exposure, the
biomonitoring of human populations exposed to potential
xenobiotic agents gives us valuable information regarding expo-
sure biomarkers and biomarkers of effect [17]. Among the
biomarkers of effect, those used to detect DNA damage – primary
or fixed at the gene or chromosome level – are of prime
importance. Some of these biomarkers are considered surrogate
biomarkers of cancer risk, particularly the frequency of micro-
nuclei in peripheral blood lymphocytes (PBL) [18].
The micronucleus (MN) assay is regarded as a sensitive and
simplistic method to perform. A MN is formed as a result of
chromosomal breakage due to misrepair of DNA lesions or loss of
chromosomal segregation due to mitotic errors [18]. The possible
causative factors in MN formation are induction of oxidative stress,
exposure to clastogens or aneugens, genetic defects in cell cycle
checkpoints or DNA repair genes, deficiency of essential cofactors
in DNA metabolism and chromosomal segregation [19–24].
Evaluation of MN frequency in PBL using the cytokinesis blocked
micronucleus (CBMN) assay has been widely employed to measure
chromosomal or genetic damage in human populations resulting
142 B. Annangi et al. / Mutation Research 770 (2016) 140–161
from exposure to genotoxic agents or in genetically susceptible
subjects [25]. Apart from measuring genomic damage, the CBMN
assay is also useful for measuring mitotic dysfunction and cell
death by necrosis and apoptosis [26,27]. These advantages led to
the development of the CBMN cytome assay, wherein cells are
analysed for viability, mitotic status and DNA damage status [26].
The high reliability and low cost of this method led to the global
adoption of this biomarker for genetic damage studies in vitro and
in vivo [28].
Here we review published studies investigating the use of PBL
to determine the levels of micronuclei in people environmentally
or occupationally exposed to metals. The exposures evaluated in
this paper comprise arsenic, chromium, nickel and vanadium, as
well as people exposed to metal mixtures.
2. Quality scoring of the evaluated studies
The studies included in this review result from an intensive
search using different databases, mainly MedLine/PubMed data-
base (www.ncbi.nlm.nih.gov/PubMd). In addition to their presen-
tation/discussion in the text, details of these studies are also
included in tabulated forms. In each of the included tables aspects
such as number of subjects, age, gender ratio, fold-change in
micronucleus frequency in the lymphocyte CBMN assay and in
other DNA damage assays (e.g. comet assay, buccal MN assay) were
indicated.
Furthermore, a “quality” scoring criteria has also been
indicated. This quality score was based on the following
parameters: (i) number of subjects in control; (ii) number of
subjects in exposed group (iii) age-matching, (iv) gender-match-
ing, (v) smoking status-matching, (vi) alcohol intake-matching,
(vii) nutritional intake matching (viii) appropriate measurement of
chemical exposure depending on the chemical exposure being
investigated. For each, a total quality scoring (TQS) value was
obtained. Values were categorized as low (9–15), medium (16–20),
high (21–26) and excellent (27).
3. Meta-analysis
A meta-analysis was carried out only for the biomonitoring
studies carried out in human populations exposed environmen-
tally or occupationally to arsenic. For each study included in the
review mean MN frequency in the groups of subjects exposed to
arsenic and in the control groups were considered, together with
their standard deviation and the number of individuals investi-
gated. The source of exposure, i.e. occupational or environmental
(drinking water) was evaluated as potential effect modifier. Given
the well-known variability of the outcome measure we decided to
standardize the results of the studies to a uniform scale before
combine them. The standardized mean difference (SMD) expresses
the size of the intervention effect in each study relative to the
variability observed in that study. The overall intervention effect
can be difficult to interpret as it is reported in units of standard
deviation rather than in MN frequency or risk ratio, however
assuming similar standard deviation distribution in the two groups
SMD can be transformed in a (log) odds ratio, approximately as
follows:
p complications and malignancy stages [37]. In the preclinical
lnOR ¼ pffiffiffiSMD
3
The presence of heterogeneity among studies was measured
with the Chi squared test, and the inconsistency index (I2) – a
measure of the percentage of the variability in effect estimates that
is due to heterogeneity rather than sampling error – was evaluated
[29,30]. Random and fixed effect modeling for the effect of
exposure to arsenic and forest plots were realized with the
statistical software STATA 12.
4. Arsenic
Arsenic is an ubiquitous element sharing properties with both
metals and non-metals and, consequently, it is classified as a
metalloid. In the environment arsenic can be found in both
inorganic and organic forms.
Its presence into the environment is due to both anthropogenic
and natural sources. The natural weathering of arsenic-rich
minerals and its consequent presence in drinking water is a major
natural source of arsenic exposure. In addition, human industrial
activities such as mining and fossil fuel combustion also contribute
to its released into the environment [31].
4.1. Human exposure to arsenic
Although arsenic is distributed worldwide, there are certain
areas that contain notably high levels in soils and waters as a
consequence of their geological constitution. This occurs in
certain regions of Asia, including West Bengal, Bangladesh and
some parts of China as well as in the area comprising northern
Chile and Argentina and the region including northern Mexico
and the southern USA. The environmental presence of high levels
of arsenic due to natural sources or anthropogenic activities
imposes an important health concern due to the important
pathologies associated with exposure. To avoid such undesirable
effects the European Union (EU), the United States (US) and the
World Health Organization (WHO) have established a value of
10 mg As/L as the maximum contaminant level for total arsenic in
potable water [11].
Once into the body, inorganic arsenic is rapidly absorbed and
distributed in blood and tissues where it is bio-transformed,
mainly in the liver, to its organic methylated forms. Excretion
occurs via urine although some of the ingested inorganic arsenic
and its metabolites are retained in the body, mainly in keratin-
rich biological derivatives of the ectoderm, such as hair and nails
[32].
4.2. Health effects of arsenic
Arsenic has been classified as a human carcinogen by the IARC
[11]. Chronic exposure to arsenic has been associated with an
increased incidence of cancer in different organs including lung,
skin, urinary bladder and liver, as well as with other pathologies
involving skin lesions, vascular diseases, peripheral neuropathy
and diabetes [33–35]. The indicated effects are observed after low
to moderate levels of arsenic exposure (10–300 mg/L). In this
context it must be emphasized that it is estimated that an about
100 million populations all around the world are exposed to
arsenic levels more than 50 mg/L. Although this exposure occurs
mainly via drinking water, this can also occur through industrial
processes [36].
Health effects of arsenic exposure can differ according the type
of exposure (chronic or acute) and they are distinctly divided into
four stages. These correspond to preclinical, clinical, internal
stage, although there are no clinical symptoms elevated levels of
arsenic and its metabolites are detected in urine. At the clinical
stage dermatological symptoms (melanosis and keratosis, among
others) are detected. The next stage supposes the alteration of
different organs such as eyes, lung, liver and muscles. Finally, at
the malignant stage there is the development of tumor and other
complications in various organs eventually causing death [37].
 B. Annangi et al. / Mutation Research 770 (2016) 140–161
Cancer as well as the other health effects induced by arsenic can
be caused by several of the mechanisms proposed for cancer
formation. Aberrant cell proliferation, altered DNA methylation,
oxidative damage, genotoxicity and co-carcinogenesis have been
widely demonstrated in in vitro and in vivo studies. This has been
the subject of exhaustive revisions, as observed in the literature
[38–42]. Fig. 1 show the cell processes that can be affected by
arsenic exposure, and the human diseases resulting from the
exposure.
4.3. Biomonitoring of human populations using the MN assay
Different biomonitoring studies looking for increased levels of
genotoxic damage have been carried out in people occupationally/
environmentally exposed to arsenic using different biomarkers,
including the frequency of micronuclei in PBL [43]. According to
the study subjects and type of exposure, these studies can be
grouped in two categories: those evaluating people occupationally
exposed and those evaluating people environmentally exposed via
arsenic-contaminated drinking water. A summarized description
of the biomonitoring studies carried out in people exposed to
arsenic that used the micronucleus assay in PBL as endpoint is
presented in Table 1.
4.3.1. Studies on people occupationally exposed
With respect to occupational exposure, four studies have been
published so far. Two investigations included workers in a copper
mine in northern Chile [44,45], another was carried out in copper
smelter industries in southwestern Poland [46] and the last was
conducted in people indirectly exposed to copper smelting
activities while living near the industry in Bulgaria [47]. The
studies conducted in northern Chile comprised a total of 207
copper mine workers who were occupationally exposed to
different arsenic levels, according to their work activities. Exposure
levels were measured as total content of arsenic in the urine and
this value ranged from 23 mg/L (in workers from a mine with low
levels of arsenic in the ground) to 136 mg/L (in smelters using
Fig. 1. Graphic representation of human exposure to arsenic. Its metabolism, cell processes, and the human diseases provoked are indicated.
143
minerals extracted from a very arsenic-rich ground area). In spite
of these differences in the levels of internal arsenic exposure, no
differences in the frequency of binucleated cells with micronuclei
(BNMN) were observed (8.08 vs. 8.50) [44]. Interestingly, a
retrospective study of these workers focused on the role of the
AS3MT gene as a potential modulating factor [45]. It is important to
point out that arsenic(III) methyltransferase (AS3MT) has been
demonstrated to be the key enzyme in the metabolism of arsenic
by catalysing the methylation of arsenite and monomethyl arsonic
acid (MMA) to form methylated arsenic species, which have higher
toxic and genotoxic potentials than the parent compounds [48,49].
This study demonstrated the important role of the Met287Thr
polymorphism in arsenic-related effects, as those individuals
carrying the variant allele showed a higher frequency of BNMN
(10.9  1.0 vs. 8.1  0.4) and a significantly higher genotoxic risk
[OR, 95CI; 3.4 (1.6–5.2)] [43]. These results confirm previous
findings obtained in the same group of exposed workers showing
that carriers of the variant allele (287Thr) accumulate 4.63% more
MMA in the urine and present 3-fold increased odds of excreting
levels of MMA over the standard [50].
A study carried out in Poland determined the genotoxic effects
of heavy elements on workers from three different smelting plants.
Significant effects were observed in the frequency of BNMN
(7.96  4.28 vs. 3.47  1.70). The study also evaluated the frequency
of MN in buccal cells, showing a significant increase (0.98  0.76 vs.
0.50  0.52). However, there was no significant association
between genetic damage and exposure levels as occurred in the
study involving workers in northern Chile. The use of FISH as an
additional measure to determine the origin of micronuclei
revealed the presence of clastogenic and aneugenic effects, the
clastogenic effects being slightly (but not significantly) more
pronounced in the smelter workers [46]. The last study in people
exposed to arsenic by living in the surroundings of a copper
smelting plant was a pilot case-control study in which only five
exposed women and four controls were analysed. Results showed
high levels of arsenic in hair (3.18 vs. 0.07 ppm), as well as in nails
(6.72 vs. 0.35 ppm), in the exposed women. This exposure was
144 B. Annangi et al. / Mutation Research 770 (2016) 140–161

Table 1
Results of L-CBMN and other genotoxicity assay studies in various cell types of arsenic exposed persons.

Exposure subjects, country Number of participants Exposure Exposure Number of binucleated cells TQS Results Ref.
sex (age) measurement period (BNCs) evaluated in L-CBMN and
(type and value) assay and stain comments
Copper mine workers, E: 105 m (46.20) Total urinary As 17 years 1000 BNCs 18/27 $ [44]
North of Chile C: 50 m (40.74) E: 136 mg/L (E group) Giemsa medium E: 8.08
IC: 52 m (51.85) C: 23 mg/L C: 8.50
IC:63 mg/L IC: 9.96
Copper mine workers, E: 207 m (46.30) Total urinary As E1:20.35 1000 BNCs 19/27 $ [45]
North of Chile classified in 3 groups E1: 182.6 mg/L y Giemsa medium E1: 8.1
(69 m) according to E2: 70.3 mg/L E2:21.69 E2: 9.7
exposure levels E3: 21.2 mg/L y E3: 8.3
E3:17.20 " (1.34) AS3MT Met287Thr
y (T/C) polymorphism:
10.9 (T/T) vs 8.1 (TC + CC)
Workers from three copper E: 72 m (42.2) Total urinary As n.s. 1000 BNCs 19/27 L-CBMN " (2.3) 7.96vs [46]
smelters in South-Western C: 83 m (37.9) E: 54.04 mg/L Giemsa medium 3.47
Poland C: 11.01 mg/L U-MN " (1.9)
0.98vs 0.50
People living near a copper E: 5f (30–58) As in hair (ppm) n.s. 1000 BNCs 16/27 " (3.1) [47]
smelter in Srednogorie, Bulgaria C: 4f (39–58) E: 3.18 Giemsa medium 45.0 vs14.5
C: 0.07
As in nails
(ppm)
E: 6.72
C: 0.35
Living in an area with high content E: 39 As content in n.s. 1000 BNCs 17/27 L-CBMN " (5.5) 38 vs6.9 [50]
of As in water in the North- (15f + 24 children) water Giemsa medium SCE $
Western of Argentina (32.6, 11.1) E: 200 mg/L 5.0 vs5.0
C: 31 C: 0.7 mg/L
(11f + 20 children)
(30.7, 11.6)
Living in an area with high content E: 106 (24m + 82f) As in water n.s. 1000 BNCs 20/27 " (1.21) [55]
of As in water in the North of Chile (39.5) E: up750 mg/L Giemsa medium 14.44 vs11.96
C: 111 (42m + 69f) C: 2 mg/L
(37.7) As in nails
E: 10.15 mg/g
C: 3.57 mg/gL
Living in an As contaminated area E: 30m + 15f As content in 11 years 2000 BNCs 14/27 L-CBMN " (12.05) 6.39 [56]
in C: 17m + 4f water Exposed Giemsa low vs 0.53
West Bengal, India (between 15 to 60) E: 368.11 mg/L show skin B-MN " (6.7)
C: 5.49 mg/L lesions 5.15 vs 0.77
U-MN " (10.2)
5.74 vs 0.56
Living in an As contaminated area E: 163 (35.2) As content in n.s. 2000 BNCs 19/27 L-CBMN " (5.63) 9.34 vs [57]
West Bengal, India (86m + 77f) water Giemsa medium 1.66
C: 154 E: 214.72 mg/L 3000 B-cells B-MN " (4.64)
(88m + 66f) C: 9.20 mg/L 1000 U-cells 5.94 vs 1.28
(33.6) U-MN " (4.71)
6.65 vs 1.41
Living in an As contaminated area E: 422 As content in n.s. 2000 BNCs 19/27 L-CBMN " (4.49) 9.13 vs [58]
West Bengal, India (218m + 204f) water Giemsa medium 2.03
classified according skin lesions (39.9) E: 170 242 mg/ 3000 B-cells B-MN " (3.36)
C: 102 L 1000 U-cells 5.62 vs 1.67
(51m + 51f) C: 7.16 mg/L U-MN " (3.53)
(39.2) 6.01 vs 1.70
CA " (4.28)
9.08 vs 2.12
Living in an As contaminated area E: 159 (38.6) Five years n.s. 2000 BNCs 19/27 L-CBMN #(3.12) [59]
West Bengal, India C: 156 (39.1) follow-up Giemsa medium 11.11vs3.55
Total urinary As: 2000 U-cells First vs second
First sample sampling
E: 274.87 mg/L U-MN # (3.06
C: 27.62 mg/L 5.97 vs 1.95
Second sample First vs second
E: 121.9 mg/L sampling
C: 24.8 mg/L

E—exposed; C—control, IC—internal control, B—buccal cells; L—lymphocytes; MN—micronuclei; L-CBMN—lymphocyte cytokinesis-block micronucleus assay; SCE—
lymphocyte sister chromatid exchange assay; B-MN—buccal micronucleus assay; U-MN—urothelial micronucleus assay, n.s.—not specified, m—male, f—female, y—years; h—
hours, TQS—total quality study score "—significant increase, #—significant decrease, number following arrow in brackets indicates fold increase/decrease, $—no effect.
 B. Annangi et al. / Mutation Research 770 (2016) 140–161
found to be associated with an increased frequency of BNMN (45.0
vs. 14.5) [47].
4.3.2. Studies on people environmentally exposed
With regard to populations that are environmentally exposed to
arsenic, six studies have been reported so far. Two were carried out
in the north of Chile/Argentina and four in West Bengal, India. In
north western Argentina, the study involved women and children
living in a highly polluted area with arsenic levels in the water up to
200 mg/L [51]. This group was compared with a similar control
group living in a noncontaminated area (0.70 mg/L). Children were
included in the study because they are considered different from
adults in terms of arsenic exposure, metabolism and response [52].
The observed levels of arsenic in urine and nails were 10 and 30
times higher, respectively, in the exposed group. Results indicated
that exposed women and children had higher BNMN frequencies
than controls (41 vs. 8.5 and 35 vs. 5.6, respectively). Children had
lower BNMN than women (both in the exposed and control
groups), a result that did not demonstrate a relatively higher
sensitivity of children to arsenic. FISH analyses were carried out in
chromosomal metaphases of 12 exposed individuals, showing a
significant increase in MN originated from aneuploidy. Contrarily
to what has been reported by other authors [53,54], no significant
differences were observed here when the frequency of sister-
chromatid exchanges (SCE) was taken as a biomarker of genotoxic
effects. The study describes equivalent values for exposed
individuals and controls: 5.7 and 4.4 SCE for exposed women
and children, respectively, vs. 5.5 and 4.6 for control women and
children, respectively.
At the other side of the Andes in the Atacama desert in Chile, a
biomonitoring study included people from five different small
villages (40 men and 71 women), and the individual exposure
levels were measured as the content of arsenic in nails [55]. This
value was significantly higher (10.15 mg/g) in the exposed group
than in the control group (106 individuals from Concepcion area,
23 men and 83 women; 3.57 mg/g). In this case, the frequency of
BNMN in the exposed group (14.44) was significantly higher than
the value observed in the controls (11.96). When ethnicity was
taken into account, no differences were observed between
Atacameños and Caucasians; and no associations were observed
either between the levels of arsenic in nails or the BNMN values.
As previously indicated, all of the biomonitoring studies
reported in Asia were carried out in the West Bengal region by
the same research group. The first study included a small number
of exposed persons (30 men and 15 women) with cutaneous signs
of arsenicism living in a contaminated district (368 mg/L arsenic in
tap water) [56]. The levels of BNMN in this group (6.39) were
significantly higher than the observed values in the control group
(0.53). The control group consisted of 21 healthy individuals living
in a noncontaminated district (5.49 mg/L arsenic in tap water) and
without signs of arsenicosis. In this study, the frequency of
micronuclei in buccal and bladder cells was also evaluated,
showing higher values in the exposed group (5.15 and 5.74,
respectively), than in the controls (0.77 and 0.56, respectively).
Exposure levels were confirmed by analysing the arsenic content of
urine, nails and hair, obtaining values of 24.45 mg/L, 12.58 mg/g and
6.97 mg/g, respectively. These levels were significantly higher than
those observed in the control group (4.88 mg/L, 0.51 mg/g and
0.34 mg/g, respectively). This study was later extended by
increasing the number of subjects [57], all of them from one of
the four districts in which the previous study was conducted. The
exposed group manifested cutaneous signs of arsenicism, such as
hyperpigmentation, hypopigmentation, raindrop pigmentation,
palmoplantar hyperkeratosis or ulcerative lesions. These lesions
were associated with the high content of arsenic in drinking water
(214.72 mg/L) in the district from which they were recruited. These
145
high environmental levels of arsenic translated to individual
exposure, as indicated by the levels observed in urine (165.5 mg/L
in exposed vs. 10.5 mg/L in controls), nails (6.9 mg/g in exposed vs.
0.5 mg/g in controls) and hair (4.1 mg/g in exposed vs. 0.3 mg/g in
controls). The frequency of BNMN was significantly higher in the
exposed (9.34) than in the control group (1.66). Buccal and
urothelial cells were also used to determine the frequency of MN.
In both cases, samples from exposed individuals showed
significantly higher MN values than controls (5.94 vs. 1.28 and
6.65 vs. 1.41 for buccal and urothelial cells, respectively).
Nevertheless, when the MN values from the three cell types were
associated with the environmental levels of arsenic in drinking
water, no significant associations were obtained. The authors
suggested that this lack of association was likely due to differences
in the quantity of water intake and the duration of arsenic
exposure in the study participants, as well as to interindividual
variations in susceptibility.
Another study by the same group increased the sample size
further and the final exposed group constituted 422 individuals
classified according to the presence of arsenicosis symptoms: 244
skin-symptomatic and 178 asymptomatic [58]. When biomarkers
of exposure were determined in water, urine, nails and hair, all
measurements in the symptomatic and nonsymptomatic exposed
individuals were significantly higher than in the controls;
nevertheless, no differences were observed between symptomatic
and nonsymptomatic individuals. When the levels of MN were
determined in lymphocytes and buccal and urothelial cells, in all
cases, the exposed symptomatic and nonsymptomatic individuals
showed significantly higher values than the controls. Interestingly,
symptomatic subjects showed higher MN values for lymphocytes
(9.13), buccal (5.62) and urothelial (6.01) cells than nonsympto-
matic individuals (6.3, 3.5 and 4.1, respectively). Further genotox-
icity studies were carried out in this group using chromosome
aberrations (CA) in lymphocytes as biomarkers. In this case, results
were similar to those reported for MN. Again, the CA values
observed in symptomatic individuals were significantly higher
than those observed in nonsymptomatic individuals. Since only a
small percentage (15%-20%) of exposed individuals showed skin
lesions, the study looked for the modulating effect associated with
the GSTT1, GSTM1 and GSTP1 genetic backgrounds. Thus, allelic
frequencies were determined by carrying out association studies.
No differences in allelic variants in the GSTT1 and GSTP1 genes were
observed between symptomatic and nonsymptomatic individuals.
Nevertheless, the incidence of the GSTM1 null allele was
significantly higher in the asymptomatic group. Those individuals
carrying the normal GSTM1 allele carried a significantly higher risk
of arsenic-induced skin lesions (OR: 1.73; 95% CI: 1.24–2.22). Since
the frequency of MN was increased in both symptomatic and
nonsymptomatic subjects, these results would indicate that,
although asymptomatic individuals are subclinically affected, they
are still at significant risk of arsenic-induced genotoxicity.
The last report is a cross-sectional study of people showing
arsenicosis symptoms and unexposed individuals [59]. People
were sampled during two different periods separated by five years.
The aim of the study was to determine whether decreases in
exposure levels modify the levels of genetic damage. Decreases in
arsenic exposure (190.1 mg/L to 37.94 mg/L) were associated with a
significant decrease in the BNMN frequency (11.11  2.5 in the first
sampling period vs. 3.55  1.48 in the second period). This decrease
was not observed in the control group (2.39  0.81 vs. 2.06  0.81).
The incidence of micronuclei was also analysed in urothelial cells
and the same behaviour was observed. Thus, the frequency of
micronuclei declined significantly in the exposed group between
the two sampling periods (5.97  1.52 vs. 1.95  1.10). Nevertheless,
no changes were observed in the control group (1.45  0.51 vs.
1.42  0.57). Interestingly, the decrease in arsenic exposure
146 B. Annangi et al. / Mutation Research 770 (2016) 140–161

resulted in a significant reduction in the number of individuals
experiencing dermatological disorders. However, an increase in
the incidence of non-dermatological diseases, such as peripheral
neuropathy, conjunctivitis and respiratory distress, were equally
observed over the study period.
4.4. Meta-analysis
A regression model with all studies was initially fitted to
estimate a summary effect of exposure to arsenic. The presence of a
significant heterogeneity (P < 0.029) and the large proportion of
variability due to heterogeneity (I2 = 51.7%) suggested to evaluate
separately studies according to the source of exposure, i.e.,
occupational exposure (3 studies) and environmental exposure
(7 studies). This stratification reduced sensibly heterogeneity and
the results of the meta-analysis are shown in the forest plot (Fig. 2).
A clear difference between occupational (n.s.) and environmental
exposures (SMD = 0.30 P < 0.001) is evident. The random effect
estimate of effect after transformation from SMD to log Odds Ratio
showed an overall increase of 72% (95% Confidence Interval 24–
138%) in the mean frequency of micronuclei in subjects drinking
water contaminated by arsenic as compared to unexposed. The
model with fixed effects showed an effect smaller but still highly
significant (P < 0.01).
The results of this meta-analysis clearly showed the presence of
heterogeneity, with studies showing highly significant increases
[56,57,59] and other which did not show any differences between
exposed and unexposed. Even after stratification by the source of
exposure some heterogeneity remained in the group of studies
investigating environmental exposures. The absolute value of the
increase of MN frequency, i.e. 72%, supports the evidence that
prolonged exposure to arsenic induces genomic instability at a
level which may imply long term risks in exposed populations.
4.5. Summing up and potential gaps
People are exposed to arsenic compounds mainly by environ-
mental and occupational exposure. Although workers in the
copper and other industries are exposed to arsenic, as to other
metals, the majority of people are exposed to arsenic that has made
its way into the food and water supply, either through the
contamination of soils used to grow vegetables or the water used in
agriculture and for human consumption. The importance of this
environmental exposure is evident from the studies reviewed
herein, where 6 studies out of 10 (Table 1) were carried out in
people who had been exposed environmentally. This is in
agreement with the high number of individuals exposed to
contaminated water, mainly in the area of West Bengal,

Fig. 2. Meta-analysis of population studies investigating MN frequency in individuals exposed to arsenic. Bars indicate Standardized Mean Difference (SMD) of MN frequency
between exposed and unexposed groups with the corresponding 95% CI.; Grey squares are proportional to the size of the study; Diamonds represent random meta effects and
for environment exposures also fixed effect estimate.%weight indicates the proportion of variance explained by each study.
 B. Annangi et al. / Mutation Research 770 (2016) 140–161
Bangladesh, and part of China, where millions of people are
drinking water with a high content of arsenic, exceeding the
amount recommended by the World Health Organization (WHO).
As an example if we take into account the WHO guideline on
arsenic content in drinking water (10 mg/L), the number of people
living in Bangladesh who are exposed to values above this
threshold is approximately 45 million people. From the six
biomonitoring studies carried out with people who had been
environmentally exposed, two were carried out in South America
(Chile and Argentina) and four in West Bengal. Significant
differences existed between both sets of data, mainly because
the observed effects were more drastic in the West Bengal studies,
as indicated by the fold changes in the MN frequency (Fig. 3). Other
differences between the two sets of exposed groups include a
higher incidence of dermatological problems and vascular
pathologies (black-foots) in West Bengal. Since high interindivid-
ual variability is observed among people exposed to equivalent
doses of arsenic, genetic susceptibility factors have been postulat-
ed to be involved in the incidence of health problems, as previously
reviewed [60]. In this context, it has been shown that variants in
the AS3MT gene are associated with higher MMAIII values in urine
and higher levels of genetic damage [45], which would suggest an
increased risk of cancer or other pathologies related to arsenic
exposure (skin lesions, diabetes and vascular lesions, among
others). Polymorphisms in other genes, such as GST genes, have
also been mentioned in studies showing that those individuals
carrying the null variant of GSTM1 present an increased risk of skin
lesions [58]. In spite of the well-known interfering role of arsenic in
Fig. 3. Graphic presentation of the data presented in Table 1. The fold changes in MN frequency in PBL, as well as in other biomarkers, of groups exposed to arsenic relative to
controls are indicated.
147
DNA repair processes, neither of the biomonitoring studies has
determined the potential role of genetic variation in these genes on
the levels of BNMN. Confirming the important role of this set of
genes, a recent study has demonstrated that polymorphisms in
XRCC genes may modify the associations between skin cancer risk
and exposure to sunlight or arsenic [61]. This means that there is an
important lack of association studies to determine potential
genetic factors modulating the genotoxic risk associated with
arsenic exposure.
5. Chromium
Chromium is naturally present in the earth’s crust with
oxidation states ranging from chromiumII to chromiumIV. In
addition, chromium ores in the environment are stable in their
trivalent and hexavalent states [7,62].
5.1. Human exposure to chromium
Similar to what occurs with other metals, chromium can be
found in air, water and soil from natural or industrial sources.
Industries such as metal processing, tannery facilities, chromate
production, stainless steel welding, and ferrochrome and chrome
pigment production contribute to chromium release into the
environment. Metallurgical, refractory and chemical industries are
mainly responsible for the increase in environmental chromium
concentrations, and released chromium mainly occurs in its
hexavalent state [63]. ChromiumVI is regarded as a possible human
148 B. Annangi et al. / Mutation Research 770 (2016) 140–161
carcinogen by several regulatory and non-regulatory agencies
[63–65]. The toxicity of chromium depends upon its valence
state; its metal form is considered the least toxic and the
hexavalent state is regarded as the most toxic form. ChromiumVI
can be found on ground and water surfaces at concentrations
exceeding the prescribed World Health Organization limit for
drinking water (50 mg/L) [66]. Industrial and commercial appli-
cations of chromium compounds, such as welding, chrome plating,
dyes and pigments, leather tanning, wood preservation and as an
anticorrosive in cooking systems and boilers are the main sources
of human exposure [67,68]. It is estimated that occupational
exposure to chromium and chromium compounds may affect
300,000 workers annually and there is a major concern with
regards to chromium-related diseases in industrial workers
exposed to chromiumVI [69].
5.2. Health effects of chromium
Chromium can enter the human body by inhalation, thus the
lungs may be considered the primary target organ for its exposure.
In addition, significant human exposure may also take place via
skin penetration [70,71]. Among the health risks posed by
chromium compounds, genotoxicity/carcinogenicity in humans
has been widely studied [72,73]. Fig. 4 show intracellular
mechanisms explaining how chromium acts as a genotoxic
compound. A number of epidemiological studies were carried
out in chromium-exposed workers suggesting that occupational
exposure to chromium increased carcinogenic risk in the
respiratory tract [65,74]. With regard to the genotoxic potential
of chromiumVI, different types of DNA damage have been observed
Fig. 4. Intracellular mechanisms explaining how chromium acts as a genotoxic compound inducing micronuclei.
both in vivo and in vitro. DNA strand breaks, inter-strand cross-links
and chromium-DNA adducts have been identified as potential
mechanisms of chromiumVI genotoxicity [75,76]. Although chro-
miumIII has been largely considered non-genotoxic, experiments
carried out using cell-free systems demonstrated that it binds to
DNA, interfering with base-pair stacking and consequently, leading
to mutations [77].
5.3. Biomonitoring of human populations using the MN assay
Some biomonitoring studies have been carried out in people
exposed to chromium using the frequency of BNMN in PBL as a
biomarker of genotoxicity (Table 2). Workers exposed to chromium
at worksites have been the subjects of these studies and,
surprisingly, no studies have been carried out using the MN assay
in PBL in people environmentally exposed to chromium.
5.3.1. Studies on people occupationally exposed
The first study [78] evaluated the genetic risk associated with
occupational exposure of chromium in electroplater workers
(n = 40) in Bulgaria. Workers were classified as highly exposed
(n = 24, 83  10 mg/m3 chromium in air) and less exposed (n = 16,
43  10 mg/m3 chromium in air). A significant increase in the total
number of MN (53.08 and 29.06) and in binucleated cells carrying
MN (42.13 and 25.4) was reported in exposed workers as compared
with the control group (n = 18, 21.05 and 19.8, respectively;
0.3  0.1 mg/m3 chromium in air). The authors suggested a direct
correlation between chromium levels in air, erythrocytes, or urine
and the frequency of BNMN. Further, the study also showed a lower
induction of MN in the lymphocytes of chromium-exposed
 B. Annangi et al. / Mutation Research 770 (2016) 140–161 149

Table 2
Results of L-CBMN and other genotoxicity assay studies in various cell types of chromium exposed persons.

Exposure subjects, country Number of Exposure Exposure Number of binucleated cells (BNCs) TQS Results and Ref.
participants sex measurement (type period evaluated in L-CBMN assay and stain comments
(age) and value)

.Electroplater workers, E1: 16 m Cr in erythrocytes: 12 years 1000 BNCs 18/27 "(2.25) [78]
Bulgaria (40.81). E1: 8.4 mg/L (E group) Giemsa medium E1: 42.13
E2: 24 m E2:4.3 mg/L E2: 22.5
(40.25). C: 0.5 mg/L C: 18.7
C: 18 m (41.2) Cr in urine:
E1: 5.0 mg/L
E2: 4.3 mg/L
C: 0.5 mg/L
Chromium platers in Yambol, E: 15 Total urinary Cr n.s. 2000 BNCs 15/27 L- " (1.2) [79]
Bulgaria (14m + 1f) (44.8) E: 18.6 3.1 mg/L Giemsa low CBMN 23.4 vs 18.5
C: 18 (44.7) C: 1.1 0.2 mg/L 1000 B-cells B-MN " (2.08)
24.0 vs 11.5
CA 1.38 vs 1.28
SCE 7.07 vs 6.52
Tannery workers (CrIII) and E1:33 (41) Cr in plasma (mg/L) n.s. 1000 BNCs 17/27 " (1.8) [80]
welders (CrVI) in Portugal (tannery) E1: 2.43 Giemsa medium E1: 6.5 vs 3.58
E2: 5 (40) E2:1.55 $
(welders) C: 0.41 E2: 5.4 vs 3.58
C: 30 (43) Cr in urine (mg/g)
E1: 2.63
E2:1.9
C: 0.7
Tannery workers exposed with Cr E:20 n.s. E:8.5 500 BNCs 11/27 L-CBMN " (11.5) [81]
in Morocco (15m + 5f) (32.4) years Giemsa low woman
C: 7 m (34.3) 61.2 vs 5.3
" (7.9) men
42.0 vs 5.3
CA "
woman
10.4 vs 0.0
"
men 6.4 vs
0.0
Tannery workers and surrounding E1: 36 Total urinary Cr (mg/ E1: 11–15 1000 BNCs 19/27 L-CBMN " (2.4) [82]
co-inhabitants in Tamil Nadu, (9m + 27f) (33.1) g) y Giemsa medium E1: 13.6 vs
India E2:36(9m + 27f) E1: 2.1 E2: 71–80 5.6
(46.0) E2: 1.81 y E2: 10.2 vs
C: 36 C:0.54 5.6
(9m + 27f) (ns) CA " (5.6)
E1: 5.6 vs
1.0
E2:3.3 vs
1.0
Comet $
E1: 4.80 vs
3.20
E2: 4.01 vs
3.20
Welders with Cr(VI) exposure, E: 93 (35.5) n.s. E: 8.6 y nd BNCs 15/27 L-CBMN " (2.2) [83]
India C: 60 (35.3) Acridine orange low 9.09 vs
4.05
Comet " (1.3)
15.1 vs 11.4

E  exposed; DE  directly exposed, IE  indirectly exposed, C  control, B  buccal cells; L  lymphocytes; MN  micronuclei; L-CBMN lymphocyte cytokinesis-block
micronucleus assay; CA  chromosomal aberrations assay; B-MN  buccal micronucleus assay; n.s.  not specified, m  male, f  female, y  years; h  hours, TQS  total
quality study score " significant increase, number following arrow in brackets indicates fold increase, $ no effect

workers after in vitro gamma irradiation of their lymphocytes,
suggesting an adaptive response.
A second study was also carried out in Bulgaria, this time with
chromium platers (n = 15) using the cytokinesis block MN assay in
peripheral lymphocytes [79]. The authors found a significant
increase in the number of cells with micronuclei (MN) in
peripheral lymphocytes (23.4  1.3; 15.70 mg/m3 chromium in
air) with respect to the values obtained from controls (n = 18,
18.5  1.2; 1.0 mg/m3 chromium in air). In this study, other
cytogenetic parameters were also evaluated. Thus, when exfoliated
buccal cells of the exposed and control groups were used to
determine the frequency of micronuclei, more pronounced effects
in the buccal cells of chromium-exposed workers were observed
(24.0  4.3) as compared to the controls (11.5  2.3). Interestingly,
when the FISH technique was applied to both cell types, the results
revealed the presence of both centromere positive (C+) as well as
centromere negative (C) MN in both groups, with no significant
differences reported between them. When CA and SCE analyses
were carried out in PBL, no significant differences were observed.
CA and SCE values for exposed and controls were 1.38 vs. 1.28 and
7.97 vs. 6.52, respectively.
The incidence of MN in peripheral lymphocytes was also
evaluated in a group of tannery workers (n = 33) exposed to
trivalent chromium and in a small group of manual metal arc
150 B. Annangi et al. / Mutation Research 770 (2016) 140–161
stainless steel welders (n = 5) exposed to hexavalent chromium.
The authors observed a significant increase in the frequency of
BNMN in tannery workers (6.35  2.94) relative to controls
(n = 30; 3.58  1.69); however, in the group of welders, no
significant increase in the BNMN frequency was observed
(5.40  1.67) relative to the controls. When urinary chromium
levels were determined as a measure of exposure, increased
values were observed in both groups, with a greater increase in
tanners compared with controls (2.63  1.62 mg/g creatinine)
than in welders (1.90  0.37) in comparison with controls
(0.70  0.38 mg/g creatinine). This study determined that the
formation of DNA-protein crosslinks, a biomarker of the biologi-
cally active dose in the tannery workers exposed to trivalent
chromium, was higher in welders (2.22) than in tannery workers
(0.88) but significantly higher than the values observed in controls
(0.57). From these data, the authors concluded that chronic
exposure to chromium (trivalent) increases DNA damage in
lymphocytes and this effect correlates with exposure to this metal
[80].
A small study was carried out in tannery workers in Morocco
[81]. This study comprised 20 exposed persons (15 men and 5
women) and 7 unexposed control men. Significant increases in the
BNMN frequency in blood lymphocytes were observed in exposed
men (42.0) and women (61.2) compared with the unexposed
controls (5.3). In addition, the frequency of aberrant cells was also
significantly increased in exposed women (19.2) and exposed men
(6.4) when compared with that of healthy controls (0.0). The
incidence of micronuclei in tannery workers from India was also
determined [82]. The study included 36 tannery workers and 36
surrounding inhabitants chronically exposed to hexavalent chro-
mium. The frequency of BNMN in PBL was higher in tannery
Fig. 5. Graphic presentation of the data presented in Table 2. The fold changes in MN frequency in PBL, as well as in other biomarkers, of groups exposed to chromium relative
to controls are indicated.
workers (10.29  5.75 and 13.13  8.89 for men and women) and in
persons indirectly exposed (13.62  4.94; 18.4  8.36) than in
controls (5.62  3.58; 7.27 4.38). This increased damage corre-
lated with the levels of exposure, measured as chromium content
in urine, which were 2.11 1.01, 1.81  0.88 and 0.54  0.39 for the
three groups, respectively. Two other biomarkers of genotoxicity
namely, chromosome aberrations and the comet assay, were also
evaluated in this study. The total number of chromosomal
aberrations in tannery workers (5.62  4.48 and 8  4.78 for
men and women), and in persons indirectly exposed
(3.38  2.85; 3.87  2.07) were significantly higher than in control
subjects (1.05  1.49; 2.08  1.83). When the comet assay was used,
slight but significant increases in the percentages of DNA in tail
were observed in tannery workers (4.80) relative to people living in
the surrounding area (4.01) and controls (3.20). Another study was
carried out in India with 93 welders of similar age, smoking habits
and alcohol consumption using the BNMN assay [83]. The results
indicated a significant increase in the frequency of binucleated
cells with MN (9.09) relative to controls (4.05). In addition, the
extent of genomic damage was also determined using the comet
assay, which revealed significant increases in the percentages of
DNA in the tail (15.1 vs. 11.4). Although authors determined the
potential role of the XPD gene polymorphism, claiming that a
deficient DNA repair would explain the observed increases in MN
and comet results, the reported data did not support this view.
5.4. Summing up and potential gaps
Chromium and its compounds are considered to be possible
human carcinogens, mainly under conditions of occupational
exposure [9]. All six of the studies reviewed in this article revealed
 B. Annangi et al. / Mutation Research 770 (2016) 140–161 151

significant increases in the MN frequency in PBL relative to the
values observed in controls. Interestingly, all of these studies were
carried out in occupational scenarios, which would confirm the
potential occupational risk of chromium exposure [64]. However,
some of the studies also reported negative data when other genetic
end-points, such as CA, SCE and the comet assay, were utilized
[79,82], rendering it difficult to determine the underlying
mechanism explaining the effects of chromium. It is important
to point out that, as also occurs with other metals, the effects of
chromium depend on its valence. Thus, most of the studies
indicated that chromiumVI had a higher genotoxic potential than
chromiumIII, likely due to fact that chromiumVI could enter the cell
more easily than chromiumIII. Chromium genotoxicity is based on
the intracellular reduction of chromiumVI by cellular metabolism
to its lower oxidation states: V, IV and III. This reduction suggests
that chromium-mediated reactive oxygen species generation may
cause persistent oxidative stress, which could play a key role in the
mechanism of chromiumVI-induced genotoxicity [78,84,85]. As-
suming that oxidative stress is the underlying mechanism of
chromium-induced genotoxicity, the lack of concordance with
other assays is surprising, and particularly in the case of the comet
assay, which is especially sensitive to oxidative damage.
In most of the studies, there was a positive correlation between
chromium levels in the urine and erythrocytes as markers of
exposure and the MN frequency in PBL, corroborating both the
sensitivity of the assay and the genotoxic potential of chromium
exposure. Although there was clear evidence of a higher incidence
of MN formation among studies, differences were observed among
them when fold increases of MN were considered (Fig. 5). The
variability observed among the various studies of chromium
exposure could be due to the influence of different variables, such
as level, duration and route of exposure and other confounding
factors, such as gender, smoking habits, alcohol consumption and
age, among others [86]. Nevertheless, it is not clear from the
analysis of these factors what could modulate chromium-induced
genotoxic response. It should be noted that the relatively low
quality scores we assigned to the chromium studies in our analysis
support this inability to further explain the underlying mecha-
nisms that generated the differences we observed between studies.
No studies including the genetic characterization of the exposed
individuals have been conducted. This is an important gap in the
existing literature.
6. Nickel
Nickel is a metallic compound found abundantly in the Earth’s
crust, the primary source being the Earth’s molten core, which
renders it unusable. Nickel is also seen in volcanic eruptions, soils,
oceanic floors and water [11]. Because of its resistance to corrosion,
nickel is pre-eminently an alloy metal used in combination with
many other metals.
6.1. Human exposure to nickel
The uses of nickel include stainless steel manufacturing,
electroplating, various foundry applications and printing inks
[87]. Nickel exists in various forms, including elemental nickel (Ni),
nickel oxide (NiO), nickel chloride (NiCl2), nickel sulfate (NiSO4),
nickel carbonate (NiCO3), nickel monosulfide (NiS) and nickel
subsulfide (Ni3S2). The release of nickel into the environment
occurs during nickel mining, industrial production of stainless
steel and other nickel alloys, or by industries that use nickel and its
compounds [88]. This environmental presence contributes to
human exposure through air, drinking water, food consumption or
tobacco smoking. Generally, populations are environmentally
exposed to high levels of nickel by dietary and drinking water
intake [89]. In addition, humans are in direct contact with nickel-
containing products, such as jewellery, stainless steel and coins. In
addition, nickel is used in the manufacture of prostheses, resulting
in human exposure to nickel-containing alloys [90]. Nickel-
producing industries increase the likelihood of nickel contamina-
tion of drinking water and soil with significantly high concen-
trations (up to 9000 ppm) [91]. Among several routes of exposures
to humans, total nickel is significantly higher in occupationally

Table 3
Results of L-CBMN and other genotoxicity assay studies in various cell types of nickel and vanadium exposed persons.

Exposure subjects, country Number of Exposure Exposure Number of binucleated TQS Results and comments Ref.
participants sex measurement (type period cells (BNCs) evaluated in
(age) and value) L-CBMN assay and stain

Smelters in a copper-nickel E: 9 (n.s.) Ni in hair (mg/g) E: 18 y 2000 BNCs 14/27 L-CBMN $ [100]
sulfide plant in Russia. C: 6 (n.s.) E: 18.1 Giemsa low 9.9 vs
C: 7.6 10.3
SCE $
68
Dental technicians exposed to E: 27 m (29.18) Total urinary Ni E: 13 2000 BNCs 18/27 L-CBMN " [86]
Cr (20–30%), in Ankara, C:15 m (28.40) E: 4.43 mg/g years May-Grünwald and medium (2.8)
Turkey Giemsa 4.0 vs
3000 N-cells 1.4
Fast Green N-MN " (1.9)
3.5 vs
1.8
Workers exposed to vanadium E: 23 m (43) Vanadium in plasma n.s. 2000 BNCs 18/27 L-CBMN " (2.5) [111]
in Austria C: 24 m (38) E: 5.0 mg/g Diff Quick medium 5.0 vs
C: 2.0 mg/g 2.0
Comet $
TM: 2.3
vs 2.4
Comet + FPF + endoIII $
TM: 2.7
vs 2.5
TM: 3.8
vs 3.5

E—exposed; DE—directly exposed, IE—indirectly exposed, C—control, N—nasal cells; L—lymphocytes; MN—micronuclei; L-CBMN lymphocyte cytokinesis-block micronucleus
assay; SCE lymphocyte sister chromatid exchange assay; N-MN nasal micronucleus assay, n.s.—not specified, m—male, f  female, y—years; h—hours, TQS—total quality study
score "—significant increase, number following arrow in brackets indicates fold increase, $—no effect.
152 B. Annangi et al. / Mutation Research 770 (2016) 140–161
exposed individuals than in the general population [92]. In the
general population, 0.1–0.25 mg nickel per day is likely to be
inhaled, whereas workers in nickel refining operations, such as
matte handling or grinding, could inhale 0.3–0.8 mg nickel per day.
Furthermore, it has been reported that the nickel exposure of
cigarette smokers was increased by 0.0004 mg per day relative to
non-smokers [93].
6.2. Health effects of nickel
Among the most known health related effects of nickel
exposure are skin allergies, lung fibrosis, variable degrees of
kidney and cardiovascular system poisoning and neoplastic
transformation [94]. Inhalation exposure by nickel industrial
workers was implicated in lung cancer incidence and different
epidemiologic studies reported a significant increase in nasal and
lung cancers in such occupational workers as reviewed by several
authors [95–98]. In addition, nickel compounds induce tumors at
virtually any site of administration in different animal models, and
insoluble nickel compounds like nickel subsulfide efficiently
transform rodent and human cells in vitro. According to that the
International Agency for Research on Cancer (IARC) classified all
nickel compounds (except metallic nickel) as carcinogenic to
humans [11]. The genotoxic potential of nickel compounds has
been demonstrated by various studies, showing that nickel can
produce DNA strand breaks and DNA cross-links at a time that
interferes with DNA repair mechanisms [86]. Nickel has been
shown to interfere with DNA repair mechanisms by enhancing the
genotoxicity of various agents, such as UV light, X-rays and
cytostatic agents. This effect is presumably due to alterations at the
incision step of nucleotide excision repair [99].
6.3. Biomonitoring of human populations using the MN assay
Several studies have been carried out in exposed groups to
demonstrate potential associations between exposure to nickel
and increases in the levels of genetic damage. Smelters and dental
technicians have been the subjects of these studies, which use
various biomarkers of genotoxicity, mainly the micronucleus in
buccal cells. However, only two studies have reported data using
PBL and the MN assay (Table 3).
Fig. 6. Graphic presentation of the data presented in Table 3. The fold changes in MN frequency in PBL, as well as in other biomarkers, of groups exposed to nickel and
vanadium relative to controls are indicated.
The first study was carried out in workers in the smelting
shop of a copper-nickel sulfide processing plant in Russia [100].
This study addressed a small number (n = 9) of workers exposed
to nickel as compared with another small group (n = 6) of
unexposed workers. The content of nickel in the hair of smelting
workers was significantly higher than of workers in other
professions (18.1 and 7.6 mg/g, respectively). In addition, high
levels of nickel in smelters’ hair (27–31 mg/g) correlated with the
length of service in smelting shops. When the frequency of
BNMN was determined, no differences were observed between
exposed workers (9.9) and controls (10.3). Although SCE
detection was also carried out in both sets of workers, no
specific data were reported; however, it was indicated that the
range was 6–8 SCE/cell. Workers received ascorbic acid to
determine its potential antimutagenic effect. Results indicated
that, in spite of the high variability observed, those workers
showing initial high levels of DNA damage responded better to
the effects of ascorbic acid.
Dental technicians constitute a group characterized by nickel
exposure through the use of metal alloys. A study was conducted in
27 male workers involved in the production of skeletal prostheses
together with 15 male controls [86]. Exposure levels were
measured according to the content of nickel in urine and the
reported values were 7.65  2.50 mg/g creatinine and
2.42  1.15 mg/g creatinine in the exposed and control groups,
respectively. When the frequency of BNMN was determined in
peripheral lymphocytes, the values in the exposed workers
(4.00  2.98) were significantly higher than those obtained from
the controls (1.40  1.30). In addition, in this study, the frequency of
MN in exfoliated nasal cells was also determined. In this case the
observed values in the exposed workers (3.50  1.80) were also
significantly higher than the control values (1.19  0.53). In spite of
this positive response, the fact that alloys contained other metals
did not permit the authors to attribute the observed genotoxic
effects exclusively to nickel. The genotoxic effects associated with
nickel exposure would be consistent with the results of those using
chromosome aberrations as a biomarker [101], but not with the
study carried out in Finland, where the frequency of micro-
nucleated epithelial cells in the buccal mucosa of nickel refinery
workers was not significantly elevated relative to control values
 B. Annangi et al. / Mutation Research 770 (2016) 140–161
[102]. In this work, no relationship was observed between
micronucleus frequencies and levels of nickel in air, urine or blood.
6.4. Summing up and potential gaps
Few biomonitoring studies have been conducted aiming to
determine the genotoxic risk of nickel exposure, as indicated by
increases in the levels of MN in PBL (Fig. 6). This renders it difficult
to obtain sound conclusions. From the two reported studies
involving nickel, one gave negative results, but it was conducted
only in a very small group of 9 smelters. The study reporting
positive results was conducted in dental technicians, but the alloys
they used only contained 20%-30% nickel, which makes it difficult
to attribute the observed effects to nickel itself. This is a clear
example of the poor quality of many of the reported studies. Few
subjects and a lack of accurate characterization of the different
potential confounding factors leads to poor-quality data that gives
a poor estimation of the genotoxic/carcinogenic risk associated
with this exposure. In addition, it must be remembered that nickel
compounds may interfere with DNA repair mechanisms [88]
particularly the repair of DNA lesions, in addition to their potential
enhancement of the genotoxic effects of different genotoxic agents
[99]. Again, the lack of well-characterized routes of exposure and
of information on confounding factors makes it difficult to extract
fruitful conclusions from these studies.
7. Vanadium
Vanadium is a non-essential metal that is becoming a serious
matter of discussion due to the adverse effects on human beings
caused by its mobility from soil to plants [103]. The main source of
vanadium in the environment is its presence in parental rocks.
7.1. Human exposure to vanadium
In addition to its presence in parental rocks, anthropogenic
activities such as mining, industries, burning of fossil fuels,
fertilizer and pesticide application and recycling of domestic
wastes, contribute to the environmental presence of vanadium
[103,104]. Vanadium is used for the production of metal alloys, the
manufacturing of lithium batteries and high-pressure lamps and
the synthesis of chemicals. Thus, human exposure to vanadium
occurs via inhalation on worksites and in their surroundings or
through the consumption of contaminated foods. Nevertheless the
general population is mainly exposed to vanadium by ingestion of
contaminated food [105].
7.2. Health effects of vanadium
Vanadium complexes act as cofactors for several enzymes. They
exhibit insulin-mimetic properties and, consequently, they are
involved in the regulation of glucose metabolism, including in
patients with diabetes. In addition, vanadium salts may also
normalize blood pressure and play a key role in the metabolism of
the thyroid and of iron as well as in the regulation of total
cholesterol, cholesterol HDL and triglyceride (TG) levels in blood
[106]. Regarding to their carcinogenic potential, the IARC has
classified vanadium into Group 2B, as possibly carcinogenic to
humans, due to the lack of available information from human
studies and sufficient data in experimental animals [105]. This
means that there is serious need for epidemiological data on the
potential carcinogenic risk of vanadium exposure. The genotoxicity
of vanadium compounds has been reviewed by the IARC [105] and
also by Beyersmann and Hartwig [12]. According to these reviews
in animal experiments vanadium compounds induced micro-
nuclei, chromosomal aberrations and aneuploidy in bone marrow
153
cells. In in vitro studies using human cells, vanadium compounds
induced DNA strand breaks causing the formation of chromosome
aberrations and aneuploidy [107]. Similar to most of the
carcinogenic metal compounds, no consistent results were
observed in bacterial mutagenicity assays. The genotoxic mecha-
nism of action of vanadium compounds includes induction of
oxidative stress, inhibition of DNA repair and interference with the
activity of protein phosphatases and kinases [12]. The induction of
aneuploidy is attributed to the inhibition of spindle formation and
the disruption of microtubule assembly [108]. Nevertheless, recent
studies have demonstrated that vanadium compounds can exert
antigenotoxic effects, potentially reducing the genotoxic effects
induced by cisplatin in mice [109].
7.3. Biomonitoring of human populations using the MN assay
Only two biomonitoring studies have been carried out to
determine the potential genotoxic risk associated with vanadium
exposure (Table 3). The first study used the comet assay and the
SCE assay in the PBL of 49 exposed workers but no changes in the
levels of DNA breaks, SCE frequencies or levels of 8-hydroxy-20 -
deoxyguanosine were found relative to those obtained from a
control group. Nevertheless, the exposed workers showed signifi-
cant vanadium uptake [110]. The only study that used the MN
frequency in lymphocytes was carried out in Austria with 23
workers in the vanadium industry. The exposure levels were
clearly increased in the exposed group, showing plasma vanadium
concentrations that were 7-fold higher than those observed in the
controls (0.31 mg/L). When the BNMN frequency was evaluated,
the workers showed a 2.5-fold higher MN frequency (5.0 vs. 2.0). In
addition, other parameters, such as nucleoplasmic bridges and
nuclear buds, were also significantly increased. Genomic damage
was also determined using the comet assay, but no differences in
DNA migration under standard conditions were observed.
Nevertheless, when the comet assay was complemented with
the use of formamidopyrimidine glycosylase and endonuclease III
to detect oxidized DNA bases, significant increases were obtained
[111], which would indicate the potential induction of oxidative
damage.
7.4. Summing up and potential gaps
The only biomonitoring study carried out in people who had
been occupationally exposed to vanadium showed significant
increases in the BNMN frequency that correlated well with the
content of vanadium in plasma. This was one of the few studies
that included nucleoplasmic bridges and buds as complementary
markers of MN. In this case, both markers were also significantly
increased in the exposed workers. As occurs with other studies,
although exposure levels were determined and showed significant
increases relative to controls, no correlation studies were carried
out to demonstrate the relationship between the internal level of
the evaluated metal and the levels of BNMN. From this study, only
few data can be supplied to the studies characterizing the potential
carcinogenic risk of vanadium exposure to humans, which is
classified as a “possible carcinogen” due to the inadequate
information available for humans and sufficient data in experi-
mental animals [105].
8. Complex mixtures of metals
Metals are ubiquitous elements present in different combina-
tions in both the environment and working places. Since mineral
ores are complex and contain different metals, they are present
with variable concentrations at smelting sites and, consequently,
spread into the environment.
154 B. Annangi et al. / Mutation Research 770 (2016) 140–161
8.1. Human exposure to metal mixtures
Human exposure to metals is complex and associated to
different sources. In addition to natural sources, anthropogenic
activities mainly coal power plants and waste incinerators have
increased tremendously its presence in the environment. Due to
these activities metals become important components of ambient
air particulate matter (PM), and especially some of those that are
within the fine PM fraction, have been cited as PM components
that are most likely to be toxic [112]. Different studies showed
evidences that the inhalation of some metals in ambient air PM is
associated with adverse health effects at concentrations near or
not much higher than current ambient levels. Nickel, vanadium
and lead are included among the most harmful metals, although
suggestive evidences exist for others, such as zinc [113]. In addition
to polluted air, contamination of dietary substances by metals is
another important source of exposure in the general population
[114]. This dietary intake of metal contaminated food can have a
series of adverse effects on the body of humans and animals
[115,116].
8.2. Health effects of metals exposure
Overall health status of people exposed to metal mixtures is
significantly affected, and this exposure is also associated to all
causes of mortality. At this point it is interesting to indicate the
studies carried out in the Utah valley using extracts of PM collected
in a steel mill plant before and after a strike. Samples obtained
during the strike had the lowest metal content, specifically soluble
iron, copper, lead and zinc. When these samples were adminis-
tered to rats no cytotoxicity, minimal induction of cytokines and
lowest oxidant generation ability was obtained in the strike
samples as compared to extracts from PM obtained before the
strike with higher metal content. In addition, exposures of human
lungs in vivo to aqueous extracts of PM collected before closure and
after reopening of the steel mill provoked a greater inflammatory
response than PM extracts from filters taken during the plant
shutdown [reviewed in 113]. This view is also supported by the US
Environmental Protection Agency (EPA) review of national
ambient air quality standards. From both epidemiological and
experimental studies the conclusion is that although many
components contribute to the health effects of PM, not sufficient
evidence exist to differentiate those constituents more closely
related to specific health outcomes [117].
Although the mechanisms of hazardous effects of metals have
been wide studied [7] few studies have focused on the potential
interactions in co-exposures to metal/metalloid mixtures. In this
context there are interesting studies showing that combinations of
arsenic, lead and cadmium produced more severe effects than their
components alone [118]. In addition, human co-exposure to
cadmium and inorganic arsenic resulted in a more pronounced
renal damage than exposure to each of the elements alone [119].
8.3. Biomonitoring of people exposed to mixtures of metals using the
MN assay
In most of the activities related to the metal industry, the
presence of metal mixtures on worksites or in the environment of
the surrounding areas can be detected. As indicated in Table 4 all
biomonitoring studies that use the MN assay have been carried
with people occupationally exposed, with the exception of one
study. In such type of exposures it is difficult to assign the observed
effects to one particular source of exposure. This was the case in a
study set up to determine the genotoxic damage sustained by
workers from different cobalt and hard metal plants in Belgium,
Norway, Finland, Sweden and England. The levels of exposure in
these workers were evaluated according to the levels of cobalt in
the urine, which were significantly high in both the cobalt (21.5 m/g
creatinine) and hard metal industry workers (19.9 m/g creatinine)
with respect to those reported in the controls (1.7 m/g creatinine).
No significant increases in the levels of BNMN were observed,
neither in the cobalt (5.3) nor in the hard metal workers (3.8),
when compared with control values (3.9). No differences were
observed when the comet assay was used, neither in the standard
version or when FPG enzyme was used. Additionally, the levels of
urinary concentration of 8-OHdG did not differ between the
exposed groups and controls [120]. Interestingly, the same authors
carried out a retrospective study in these workers, examining the
role of polymorphisms in genes involved in base-excision (OGG1,
XRCC1) and DNA double-strand break (XRCC3) repair. A signifi-
cantly higher frequency of micronuclei in mononucleated cells was
observed in workers with a genotype that included the variant
OGG1(326) [121]. Although the toxic effects of metals can depend
on both the forms and routes of exposure, this studies support the
view that oxidative and DNA damage are considered the main
mechanisms involved in their hazardous effects. In this context,
the antioxidant status of exposed individuals is an important factor
modulating such effects [116].
In workers involved in a primary aluminium production plant
located in Sardinia, Italy nonsignificant differences in the
frequency of BNMN between workers and controls (8.5  5.4%
vs. 9.7 4.9%, respectively) were observed. Unfortunately, no
biomarkers of metal exposure were evaluated in these workers.
In addition, when the comet assay was used, no differences
between groups (0.53  0.53 vs. 0.49  0.45) were observed in the
tail moment, used as parameter of DNA damage. However, when
lymphocytes were cultured in the presence of cytosine arabinoside
(ara-C), significant differences in tail moment values between
aluminium workers and controls (1.73  1.05 vs. 0.93  0.88) were
observed. This would indicate the presence of relatively stable DNA
lesions in workers, detected only when gap refilling is inhibited by
ara-C [122].
Workers from different workshops exposed to metal mixtures
were also evaluated in France. In this study, exposure was
determined according to the presence in blood and urine of
different metals (lead, cadmium, chromium, cobalt, aluminium,
manganese, nickel and zinc). The concentration of all metals was
increased in the studied workers with the exception of aluminium
and zinc. When the levels of BNMN were determined, welders
presented significantly higher values (6.3  2.9%) than controls
(4.7  1.8%). Similar positive effects were observed when the comet
assay was used; tail moment values in welders were significantly
higher at the end of the week (4.54  1.68) than at the beginning of
the week (2.84  0.75). Variations in XRCC1 and XRCC3 genotypes
were included in the study and indicated that the XRCC1 variant
allele (Arg399Gln) was associated with a higher number of DNA
breaks, enumerated by the comet assay [123].
A study was carried out to evaluate the genotoxic damage
caused by environmental and occupational exposures to metal
mixtures (arsenic, chromium, lead, manganese, molybdenum and
zinc) in populations residing in the Panasqueira mine area of
central Portugal [124]. Micronuclei frequency, together with other
genotoxicity biomarkers (TCR mutations, CA and comet), were
determined. In addition, the modulating roles of genetic poly-
morphisms of metabolism and DNA repair genes were included in
the study. The MN test was performed in a total of 122 subjects
who were occupationally (n = 41) and environmentally (n = 41)
exposed and in 40 unexposed controls. The authors revealed a
nonsignificant increase in MN frequency (4.98  3.06 vs.
6.45  4.47) in the occupationally exposed group; nevertheless, a
higher incidence of genetic damage was reported in environmen-
tally exposed subjects (8.46  5.27 vs. 6.45  4.47). All of the other
 B. Annangi et al. / Mutation Research 770 (2016) 140–161 155

Table 4
Results of L-CBMN and other genotoxicity assay studies in various cell types of persons exposed to mixtures of metals.

Exposure subjects, country Number of Exposure measurement Exposure Number of binucleated cells TQS Results and Ref.
participants (type and value) period (BNCs) evaluated in L-CBMN comments
sex (age) assay and stain
Workers from cobalt and hard metal E1: 35 m Cobalt in urine E1: 9.9 2000 BNCs 23/27 L- CBMN $ [120]
plants in Belgium, Norway, Finland, (38.5) (mg/g creatinine) E2: 10.3 Giemsa high E1:5.3
Sweden and England E2: 29 m E1: 21.5 E2: 3.8
(40.70) E2: 19.9 C: 3.9
C: 35 m (38) C: 1.7 Comet (% $
DNA T) E1:
0.50
E2:
0.57
C: 0.51
Comet + fpg $
(%DNA T) E1:
0.20
E2:
0.06
C:
0.069
8-OHdG $
E1:
1.52
E2:
1.63
C: 1.46
Workers aluminium industry in E: 42 m (46) n.s. n.s. 1000 BNCs 17/27 L- CBMN $ [122]
Sardinia, Italy C: 16 m (44) Giemsa medium E: 8.5
C: 9.7
Comet $
(TM) E: 0.43
C: 0.47
Welders from different workshops in E1: 27 m Blood levels of Al, Cd, Cr, 0.5 to 45 y 1000 BNCs 23/27 L- CBMN " (1.3) [123]
France (43.9) Co, Pb, Mn, Ni, Zn Giemsa high E: 6.3
E2: 33 m C: 4.7
(44.4) Comet "
C: 30 m (43.1) (TM) E1:
4.54
$
E2: 2.9
C: 2.9
Panasqueira mine exposed to E1: 41 m As, Cr, Mn, Mo, Pb and Zn E: 25 1000 BNCs 13/27 L- CBMN " (1.31) [124]
metalloids in the area of central (62.05) levels in blood, urine, years Giemsa low E2:
Portugal E2:41 nails and hair 8.46 vs
(16m + 25 f) 6.45
(61.71) $
C: 40 E1:
(17m + 23f) 4.98 vs
(56.6) 6.45
Comet " (1.9)
%DNAT E2:
24.6 vs
12.4
" (1.5)
E1:
18.7 vs
12.4
CA " (2.1)
E2:
5.56 vs
2.65
$
E1:
3.24 vs
2.65
TCR " (1.3)
mutation E2: 4.9
vs 3.8
" (1.5)
E1: 5.8
vs 3.8
People living in 5 Bosnian regions E: 41f + 43m n.s. n.s. 1000 BNCs 19/27 L- CBMN $ [126]
C: 10f + 10m Giemsa medium E: 8.33
vs C:
8.80
NPB " (2.3)
E: 3.87
156 B. Annangi et al. / Mutation Research 770 (2016) 140–161

Table 4 (Continued)
Exposure subjects, country Number of Exposure measurement Exposure Number of binucleated cells TQS Results and Ref.
participants (type and value) period (BNCs) evaluated in L-CBMN comments
sex (age) assay and stain
vs C:
1.65
NBUD $
E: 2.59
vs C:
2.10
Workers of solid-waste incinerators in E: 23 m Metals in urine n.s. 1000 BNCs 21/27 L- CBMN $ [128]
Austria (43.7) Cr, Mn, Ni, As Giemsa high E: 13.4
C: 19 m (43.4) vs
C: 14.5
Comet $
(visual E: 6.5
scoring) vs C:
7.1
Workers with occupational exposure to E: 71 m Metals in blood 12.67 y 2000 BNCs 21/27 L- CBMN " (2.4) [129]
coal in Candiota, Brazil (42.75) Al, Mg, Cu, Zn Giemsa high E: 7.46
C: 57 m vs
(41.51) C: 3.12
NPB " (2.2)
E: 12.3
vs C:
5.6
NBUD $
E: 6.10
vs C:
5.17
Comet " (2.2)
(visual E: 33.7
scoring) vs C:
15.5
Dental technicians in Japan E: 54 m Metals in hair 19.0 y 1000 BNCs 21/27 L- CBMN " [132]
(40.7) Al, Cd, Pb, Mg, Co, Zn, Mo Giemsa high (2.07)
C: 38 m E: 8.5
(46.4) vs
C: 4.1

E—exposed; DE—directly exposed, IE—indirectly exposed, C—control, IC—internal control, L—lymphocytes; MN—micronuclei; L-CBMN—lymphocyte cytokinesis-block
micronucleus assay; TCR—T-cell receptor, NPB—nucleoplasmic bridges, NBUD—nuclear buds, n.s.—not specified, m—male, f—female, y—years; h—hours, TQS—total quality
study score, "—significant increase, number following arrow in brackets indicates fold increase, $—no effect.

genotoxicity biomarkers (TCR mutations, CA and comet) were
significantly increased in the exposed groups, mainly in those who
had been exposed environmentally. The levels of exposure
detected by the metal levels in blood, urine, nails and hair were
clearly increased in the exposed group [125]. When the modulat-
ing effects of polymorphisms in the GSTA2, GSTM1, GSTP1, GSTT1,
XRCC1, APEX1, MPG, MUTYH, OGG1, PARP1, PARP4, ERCC1, ERCC4 and
ERCC5 genes were determined, significant associations were
observed for GSTM1 and OGG1 variants with CA frequencies, the
APEX1 variant with comet, the ERCC1 variant with comet and CA,
and the ERCC4 variant with MN and CA values.
A recent study has been carried out in five Bosnian regions that
were highly contaminated with metals [126]. In this study the
frequency of BNMN was studied in a group of 84 persons living in
five contaminated areas and 20 control individuals living in a non-
contaminated area. Results indicated that no differences were
observed between exposed subjects (8.33) and controls (8.80).
Additional nucleoplasmic bridges and nuclear bud parameters
were recorded. In this case, significant differences were observed
when nucleoplasmic bridges were taken into account (3.87 vs.
1.65) [127].
Exposed workers at solid waste incinerators in Austria were
evaluated for genotoxic risk. Exposure levels were determined
according the content of metals in urine. Results indicated
nonsignificant increases in the levels of chromium (0.79 vs.
0.19), manganese (0.87 vs. 0.94), nickel (9.66 vs. 8.10) and arsenic
(9.56 vs. 9.46) expressed as mg metal per gram of creatinine. No
effects were observed when the levels of BNMN were determined
(13.4 vs. 14.5). In the same way, the results of the comet assay did
not show any significant differences between exposed subjects and
controls (6.5 vs. 7.1) [128].
Coal mine workers also constitute a group that is occupationally
exposed to the different metals contained in extracted minerals. A
biomonitoring study of coal workers was carried out in Brazil [129]
to determine the genotoxic risk using BNMN and comet biomark-
ers. A previous study using samples from the same mine workers
detected increased levels of magnesium, aluminium, copper and
zinc in the blood cells [130] indicating that this group had been
exposed to high levels of metals. When MN analysis was carried on
the lymphocytes of these workers, a significant increase was
observed in workers (7.46  4.64) relative to control subjects
(3.12  2.93). Significant effects were also observed when nucleo-
plasmic bridges were taken into account (12.33  7.48 vs.
5.59  4.06). No effects were detected when the presence of
nuclear buds was evaluated (6.10  4.48 vs. 5.17  3.31). The
presence of DNA strand breaks was determined using the comet
assay and a damage index determined by visual scoring. In this
case, the damage observed in workers (33.69  28.70) was
significantly higher than in control subjects (15.53  8.80). The
fact that high levels of superoxide dismutase were observed in
workers suggested that some kind of oxidative stress associated
with exposure take place in these workers.
Orthodontic patients are exposed to a noticeable amount of
metal alloys in the mouth, resulting in a potential release of metal
 B. Annangi et al. / Mutation Research 770 (2016) 140–161
ions and the induction of DNA damage in their buccal cells [131].
Although different studies have used the micronucleus test as a
biomarker of genotoxic damage in these patients, the assay has
been carried out on buccal cells but not in lymphocytes.
Nevertheless, technicians preparing orthodontic appliances have
been the subject of one study in Japanese technicians where the
levels of various metals in hair (Al, Cd, Pb, Mg, Co, Zn and Mo) were
evaluated as biomarkers of exposure [132]. Results showed that
cobalt and zinc levels were significantly increased in the group of
54 technicians when compared with a matched control group
(n = 38). The frequency of BNMN was also significantly higher in
technicians (8.5  6.6) than in controls (4.1  3.3). The levels of
aluminium in hair as well as the period of time they had spent
working were contributing factors to the levels of BNMN.
8.4. Summing up and potential gaps
With the exception of environmental exposure to arsenic, in all
the other cases (mainly at workplaces or industrial sites) exposure
is a complex parameter with many metals at variable concen-
trations being present in the exposure scenario. This is the reason
why the section on exposure to metal mixtures contains an
important number of studies (8/27). As it seems obvious under
such conditions, it is difficult to assign the observed effects to one
defined metal. In addition, only half of the studies reported positive
results that would reflect the complexity of the exposure scenarios
(Fig. 7). In general, these studies fail to meet several of the criteria
Fig. 7. Graphic presentation of the data presented in Table 4. The fold changes in MN frequency in PBL, as well as in other biomarkers, of groups exposed to metal mixtures
relative to controls are indicated.
157
that a correct biomonitoring study must satisfy. Thus, the number
of exposed individuals was low, ranging from 23 to 84 and,
although most of the studies included a correct characterization of
exposure by measuring metal content in different biological
matrices, two of them did not present any characterization of the
exposure levels [122,127]. Interestingly, three of the studies
included a genetic characterization of the participants, showing
the relevance of the OGG1 gene [120], which would emphasize the
potential role of oxidative stress as an effect related to metal
exposure [133], as well as XRCC1 and ERCC4 genes [123,124], which
would support the induction of DNA damage repaired by NER
mechanisms.
In studies related to metal pollution, chronic low dose exposure
to multiple elements represents a major public health concern. In
this scenario elucidating the mechanistic basis of heavy metal
interactions is essential for health risk assessment and manage-
ment of chemical mixtures. Thus, more studies are required to fil in
this gap. In addition, other shortage of the biomonitoring studies
carried out with complex metal exposures is the lack of
information related to dietary habits that are not taken into
consideration in these studies.
9. Discussion
Anthropogenic or geological releases are considered the main
sources of toxic metal and metalloid contaminant distribution in
air, water, soil and other environmental compartments. Toxic
158 B. Annangi et al. / Mutation Research 770 (2016) 140–161
metals and metalloid contamination due to anthropogenic sources
generally follows a cyclic order: industry, atmosphere, soil, water,
food and humans [134].
Metal exposure is of great environmental concern due to its
potential long-term effects on human health. Many studies, as
described in different IARC Monographs, have demonstrated the
potential carcinogenic risk of metal exposure. Among the major
health effects on humans caused by heavy metals, genotoxic risk or
DNA damaging potential is considered the most important aspect,
because the genotoxicity/mutagenicity of metal and metalloids
have been linked to carcinogenic processes [135].
To meet the challenges inherent in complex epidemiological
studies aiming to detect carcinogenic exposure, the biomonitoring
of human populations using biomarkers of genotoxic effects is
considered a useful tool for the identification of at-risk popula-
tions. In this way, biomonitoring studies can be used as surrogate
biomarkers of carcinogenic risk, by identifying at-risk groups in the
early stages of the process. The association between increases in
biomarkers of genetic damage, such as chromosomal aberrations
and micronuclei, and increases in carcinogenic risk has already
been demonstrated [8,136,137]. In the face of the complexity of CA
assays, the MN assay using PBL has become a gold standard in the
biomonitoring of populations to demonstrate potential exposure
to genotoxicants. The use of cytochalasin-B to identify divided
(binucleated) cells, together with the ability of discriminate
clastogenic and aneugenic mechanisms using FISH methodologies,
has popularized this assay and has increased our knowledge of the
underlying mechanisms linking specific exposures and cancer
outcome. In addition, the possibility of extending the target from
PBL to epithelial cells (buccal, nasal, urothelial) has added new
value to biomonitoring studies using the MN assay. In fact, a lot of
biomonitoring studies in human populations exposed to metals
use the incidence of MN in epithelial cells as a biomarker of
genotoxic damage and indeed as a potential indicator of future
increased risk of cancer [138].
In spite of the well demonstrated carcinogenic potential of
exposure to different metals, the low number of biomonitoring
studies carried out in these populations using the MN assay in PBL
is surprising. As indicated in this review, only 10 studies have been
carried out in people exposed to arsenic, 6 to chromium, 2 to nickel,
1 to vanadium and 8 with people exposed to mixtures of metals.
Another surprising finding is the low number of exposed
individuals included in the studies. Only in the biomonitoring
studies on people exposed to arsenic were more than 100 exposed
individuals included. In addition, few studies determined the
potential sources of confounding factors – specifically nutritional
factors – that could have reduced the importance of the reported
values. In fact, we have tried to emphasize the relevance of the
reported values by including in the supporting tables a quality
score parameter that takes into consideration the sample size of
the exposed and control groups as well as the different
confounding factors as modulating factors. The quality score used
to measure the relevance of the data contained in the various
studies included in this review indicates that only the studies
carried out with people exposed to complex mixtures reach a high
level.
Our conclusion is that, of the biomonitoring studies carried out
in people who have been environmentally exposed to arsenic
where the exposure is easily well characterized, the remaining
studies lack important components that are necessary for a strong
characterization of the exposure scenario. This means that
reported exposure conditions differ significantly between studies,
rendering difficult any extrapolation exercise. The quality score
included in this study clearly reflects the different aspects that
were not completely covered by the reported studies. To avoid
weaknesses in data obtained in biomonitoring studies, they should
be conducted according to the criteria we have outlined in our
quality scoring scheme. Perhaps it is difficult for most research
groups involved in biomonitoring studies to fulfil all of the
requirements; in this case, a close collaboration between groups
would be required to generate a wide set of data that give a
complete view of the genotoxic risk of defined exposures as well as
the importance of the parameters that modulate such risk.
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgements
The authors thank Dr. Aliaksei Kisialou, IRCCS San Raffaele
Pisana, Rome, Italy for his kind assistance with meta-analysis.
References
[1] J.O. Nriagu, A global assessment of natural sources of atmospheric trace
metals, Nature 338 (1989) 47–49.
[2] Z.L. He, X.E. Yang, P.J. Stoffella, Trace elements in agroecosystems and impacts
on the environment, J. Trace Elem. Med. Biol. 19 (2005) 125–140.
[3] M.P. Taylor, A.K. Mackay, K.A. Hudson-Edwards, E. Holz, Soil Cd, Cu, Pb and Zn
contaminants around Mount Isa City, Queensland, Australia : Potential
sources and risks to human health, Appl. Geochem. 25 (2010) 841–855.
[4] J. Csavina, J. Field, M.P. Taylor, S. Gao, A. Landázuri, E.A. Betterton, A.E. Sáez, A
review on the importance of metals and metalloids in atmospheric dust and
aerosol from mining operations, Sci. Total Environ. 433 (2012) 58–73.
[5] WHO/FAO/IAEA, World Health Organization Switzerland, Geneva. Trace
Elements in Human Nutrition and Health, 1996.
[6] C.G. Fraga, Relevance, essentiality and toxicity of trace elements in human
health, Mol. Aspects Med. 26 (2005) 235–244.
[7] P.B. Tchounwou, C.G. Yedjou, A.K. Patlolla, D.J. Sutton, Heavy metal toxicity
and the environment, EXS 101 (2012) 133–164.
[8] IARC (International Agency for Research in Cancer), Some Metals and
Metallic Compounds, IARC Monographs on the Evaluation of Carcinogenic
Risks to Humans, 23, International Agency for Research in Cancer, Lyon,
France, 1980.
[9] IARC (International Agency for Research in Cancer), Chromium, Nickel and
Welding, IARC Monographs on the Evaluation of Carcinogenic Risks to
Humans, 49, International Agency for Research in Cancer, Lyon, France, 1990.
[10] IARC(International Agency for Research in Cancer), Cobalt in Hard Metals and
Cobalt Sulfate, Gallium Arsenide, Indium Phosphide and Vanadium
Pentoxide, IARC Monographs on the Evaluation of Carcinogenic Risks to
Humans, 86, International Agency for Research in Cancer, Lyon, France, 2006.
[11] IARC (International Agency for Research in Cancer), Arsenic, Metals, Fibres,
and Dusts, IARC Monographs on the Evaluation of Carcinogenic Risks to
Humans, 100C, International Agency for Research in Cancer, Lyon, France,
2012.
[12] D. Beyersmann, A. Hartwig, Carcinogenic metal compounds: recent insight
into molecular and cellular mechanisms, Arch. Toxicol. 82 (2008) 493–512.
[13] P. Koedrith, Y.R. Seo, Advances in carcinogenic metal toxicity and potential
molecular markers, Int. J. Mol. Sci. 12 (2011) 9576–9595.
[14] P. Møller, P.H. Danielsen, D.G. Karottki, K. Jantzen, M. Roursgaard, H.
Klingberg, et al., Oxidative stress and inflammation generated DNA damage
by exposure to air pollution particles, Mutat. Res. Rev. 762 (2014) 133–166.
[15] H. Ha, J.R. Olson, L. Bian, P.A. Rogerson, Analysis of heavy metal sources in soil
using kriging interpolation on principal components, Environ. Sci. Technol.
48 (2014) 4999–5007.
[16] P. Li, C. Lin, H. Cheng, X. Duan, K. Lei, Contamination and health risks of soil
heavy metals around a lead/zinc smelter in south western China, Ecotoxicol.
Environ. Saf. 113 (2015) 391–399.
[17] I. Silins, J. Högberg, Combined toxic exposures and human health: biomarkers
of exposure and effect, Int. J. Environ. Res. Public Health 8 (2011) 629–647.
[18] S. Bonassi, A. Znaor, M. Ceppi, C. Lando, W.P. Chang, N. Holland, et al., An
increased micronucleus frequency in peripheral blood lymphocytes predicts
the risk of cancer in humans, Carcinogenesis 28 (2007) 625–631.
[19] J.T. MacGregor, Dietary factors affecting spontaneous chromosomal damage
in man, Prog. Clin. Biol. Res. 347 (1990) 139–153.
[20] J.W. Crott, S.T. Mashiyama, B.N. Ames, M. Fenech, MTHFR C677T
polymorphism does not alter folic acid deficiency-induced uracil
incorporation into primary lymphocyte DNA in vitro, Carcinogenesis 22
(2001) 1019–1025.
[21] K. Umegaki, M. Fenech, Cytokinesis-block micronucleus assay in WIL2-NS
cells: a sensitive system to detect chromosomal damage induced by reactive
oxygen species and activated human neutrophils, Mutagenesis 15 (2000)
261–269.
[22] M. Kimura, K. Umegaki, M. Higuchi, P. Thomas, M. Fenech,
Methylenetetrahydrofolate reductase C677T polymorphism, folic acid and
 B. Annangi et al. / Mutation Research 770 (2016) 140–161
riboflavin are important determinants of genome stability in cultured human
lymphocytes, J. Nutr. 134 (2004) 48–56.
[23] H. Rajagopalan, P.V. Jallepalli, C. Rago, V.E. Velculescu, K.W. Kinzler, B.
Vogelstein, C. Lengauer, Inactivation of hCDC4 can cause chromosomal
instability, Nature 428 (2004) 77–81.
[24] M. Fenech, P. Baghurst, W. Luderer, J. Turner, S. Record, M. Ceppi, S. Bonassi,
Low intake of calcium folate, nicotinic acid, vitamin E, retinol, b-carotene and
high intake of pantothenic acid, biotin and riboflavin are significantly
associated with increased genome instability—results from a dietary intake
and micronucleus index survey in South Australia, Carcinogenesis 26 (2005)
991–999.
[25] M. Fenech, N. Holland, W.P. Chang, E. Zeiger, S. Bonassi, The HUman
MicroNucleus project—an international collaborative study on the use of the
micronucleus technique for measuring DNA damage in humans, Mutat. Res.
428 (1999) 271–283.
[26] M. Fenech, Cytokinesis-block micronucleus assay evolves into a cytome assay
of chromosomal instability, mitotic dysfunction and cell death, Mutat. Res.
600 (2006) 58–66.
[27] M. Fenech, Cytokinesis-block micronucleus cytome assay, Nat. Protoc. 2
(2007) 1084–1104.
[28] S. Bonassi, U. Ugolini, M. Kirsch-Volders, U. Strömberg, R. Vermeulen, J.D.
Tucker, Human population studies with cytogenetic biomarkers: review of
the literature and future prospectives, Environ. Mol. Mutagen. 45 (2005)
258–270.
[29] Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0
[updated March 2011], in: J.P.T. Higgins, S. Green (Eds.), The Cochrane
Collaboration, 2011.
[30] J. Cohen, Statistical Power Analysis for the Behavioral Sciences, second ed.,
Lawrence Erlbaum Associates, 1988.
[31] B.K. Mandal, K.T. Suzuki, Arsenic round the world: a review, Talanta 58 (2002)
201–235.
[32] V. Bencko, A. Dobisová, M. Mácaj, Arsenic in the hair of a non-occupationally
exposed population, Atmos. Environ. 5 (1971) 275–279.
[33] C.O. Abernathy, Y.P. Liu, D. Longfellow, H.V. Aposhian, B. Beck, B. Fowler, et al.,
Arsenic: health effects, mechanisms of actions, and research issues, Environ.
Health Perspect. 107 (1999) 593–597.
[34] J.A. Centeno, F.G. Mullick, L. Martinez, N.P. Page, H. Gibb, D. Longfellow, C.
Thompson, E.R. Ladich, Pathology related to chronic arsenic exposure,
Environ. Health Perspect. 110 (2002) 883–886.
[35] M.E. Sears, K.J. Kerr, R.I. Bray, Arsenic, cadmium, lead, and mercury in sweat: a
systematic review, J. Environ. Public Health 2012 (2012) 184745.
[36] M. Vahter, Health effects of early life exposure to arsenic, Basic Clin.
Pharmacol. Toxicol. 102 (2008) 204–211.
[37] K.S. Mohammed Abdul, S.S. Jayasinghe, E.P. Chandana, C. Jayasumana, P.M. De
Silva, Arsenic and human health effects: a review, Environ. Toxicol.
Pharmacol. 40 (2015) 828–846.
[38] M.F. Hughes, Arsenic toxicity and potential mechanisms of action, Toxicol.
Lett. 133 (2002) 1–16.
[39] K. Salnikow, A. Zhitkovich, Genetic and epigenetic mechanisms in metal
carcinogenesis and cocarcinogenesis: nickel, arsenic, and chromium, Chem.
Res. Toxicol. 21 (2008) 28–44.
[40] D. Beyersmann, A. Hartwig, Carcinogenic metal compounds: recent insight
into molecular and cellular mechanisms, Arch. Toxicol. 82 (2008) 493–512.
[41] S.J. Flora, Arsenic-induced oxidative stress and its reversibility, Free Radic.
Biol. Med. 51 (2011) 257–281.
[42] F. Faita, L. Cori, F. Bianchi, M.G. Andreassi, Arsenic-induced genotoxicity and
genetic susceptibility to arsenic-related pathologies, Int. J. Environ. Res.
Public Health 10 (2013) 1527–1546.
[43] N. Marchiset-Ferlay, C. Savanovitch, M.P. Sauvant-Rochat, What is the best
biomarker to assess arsenic exposure via drinking water? Environ. Int. 39
(2012) 150–171.
[44] L. Paiva, V. Martínez, A. Creus, D. Quinteros, R. Marcos, Evaluation of
micronucleus frequencies in blood lymphocytes from smelting plant workers
exposed to arsenic, Environ. Mol. Mutagen. 49 (2008) 200–205.
[45] A. Hernández, L. Paiva, A. Creus, D. Quinteros, R. Marcos, Micronucleus
frequency in copper-mine workers exposed to arsenic is modulated by the
AS3MT Met287Thr polymorphism, Mutat. Res. Genet. Toxicol. Environ.
Mutagen. 759 (2014) 51–55.
[46] D. Lewin  ska, J. Palus, M. Stepnik, E. Dziubałtowska, J. Beck, K. Rydzyn
 ski, A.T.
Natarajan, R. Nilsson, Micronucleus frequency in peripheral blood
lymphocytes and buccal mucosa cells of copper smelter workers, with special
regard to arsenic exposure, Int. Arch. Occup. Environ. Health 80 (2007) 371–
380.
[47] R. Nilsson, A.N. Ja, Z. Zaprianov, A.T. Natarajan, Chromosomal aberrations in
humans exposed to arsenic in the Srednogorie area, Bulgaria, Fresenius
Environ. Bull. 2 (1993) 59–64.
[48] D. Sumi, S. Himeno, Role of arsenic (+3 oxidation state) methyltransferase in
arsenic metabolism and toxicity, Biol. Pharm. Bull. 35 (2012) 1870–1875.
[49] T. Watanabe, S. Hirano, Metabolism of arsenic and its toxicological relevance,
Arch. Toxicol. 87 (2013) 969–979.
[50] A. Hernández, N. Xamena, C. Sekaran, H. Tokunaga, A. Sampayo-Reyes, D.
Quinteros, A. Creus, R. Marcos, High arsenic metabolic efficiency in
AS3MT287Thr allele carriers, Pharmacogenet. Genomics 18 (2008) 349–355.
159
[51] F.N. Dulout, C.A. Grillo, A.I. Seoane, C.R. Maderna, R. Nilsson, M. Vahter, F.
Darroudi, A.T. Natarajan, Chromosomal aberrations in peripheral blood
lymphocytes from native Andean women and children from northwestern
Argentina exposed to arsenic in drinking water, Mutat. Res. 370 (1996) 151–
158.
[52] C.H. Tseng, A review on environmental factors regulating arsenic methylation
in humans, Toxicol. Appl. Pharmacol. 235 (2009) 338–350.
[53] J. Mahata, A. Basu, S. Ghoshal, J.N. Sarkar, A.K. Roy, G. Poddar, et al.,
Chromosomal aberrations and sister chromatid exchanges in individuals
exposed to arsenic through drinking water in West Bengal, India, Mutat. Res.
534 (2003) 133–143.
[54] L. Paiva, V. Martínez, A. Creus, D. Quinteros, R. Marcos, Sister chromatid
exchange analysis in smelting plant workers exposed to arsenic, Environ.
Mol. Mutagen. 47 (2006) 230–235.
[55] V. Martínez, A. Creus, W. Venegas, A. Arroyo, J.P. Beck, T.W. Gebel, J. Surrallés,
R. Marcos, Evaluation of micronucleus induction in a Chilean population
environmentally exposed to arsenic, Mutat. Res. 564 (2004) 65–74.
[56] A. Basu, J. Mahata, A.K. Roy, J.N. Sarkar, G. Poddar, A.K. Nandy, et al., Enhanced
frequency of micronuclei in individuals exposed to arsenic through drinking
water in West Bengal, India, Mutat. Res. 516 (2002) 29–40.
[57] A. Basu, P. Ghosh, J.K. Das, A. Banerjee, K. Ray, A.K. Giri, Micronuclei as
biomarkers of carcinogen exposure in populations exposed to arsenic
through drinking water in West Bengal, India: a comparative study in three
cell types, Cancer Epidemiol. Biomarkers Prev. 14 (2004) 820–827.
[58] P. Ghosh, A. Basu, J. Mahata, S. Basu, M. Sengupta, J.K. Das, et al., Cytogenetic
damage and genetic variants in the individuals susceptible to arsenic-
induced cancer through drinking water, Int. J. Cancer 118 (2006) 2470–2478.
[59] S. Paul, N. Das, P. Bhattacharjee, M. Banerjee, J.K. Das, N. Sarma, et al., Arsenic-
induced toxicity and carcinogenicity: a two-wave cross-sectional study in
arsenicosis individuals in West Bengal, India, J. Expo. Sci. Environ. Epidemiol.
23 (2013) 156–162.
[60] A. Hernández, R. Marcos, Genetic variations associated with interindividual
sensitivity in the response to arsenic exposure, Pharmacogenomics 9 (2008)
1113–1132.
[61] S. Surdu, E.F. Fitzgerald, M.S. Bloom, F.P. Boscoe, D.O. Carpenter, R.F. Haase, E.
Gurzau, P. Rudnai, K. Koppova, M. Vahter, G. Leonardi, W. Goessler, R. Kumar,
T. Fletcher, Polymorphisms in DNA repair genes XRCC1 and XRCC3
occupational exposure to arsenic and sunlight, and the risk of non-melanoma
skin cancer in a European case-control study, Environ. Res. 134 (2014) 382–
389.
[62] J.A. Jacobs, S.M. Testa, Overview of chromium(VI) in the environment:
background and history, in: J. Guertin, J.A. Jacobs, C.P. Avakian (Eds.),
Chromium(VI) Handbook, CRC Press, Boca Raton, FL, 2005, pp. 1–22.
[63] ATSDR(Agency for Toxic Substances and Disease Registry), Toxicological
Profile for Chromium, U.S. Department of Public Health and Human Services,
Public Health Service, Atlanta, GA, 2012.
[64] IARC (International Agency for Research in Cancer), Chromium, nickel and
welding, IARC Monographs on the Evaluation of Carcinogenic Risks to
Humans, International Agency for Research in Cancer, Lyon, France, 1990, pp.
49.
[65] U.S. EPA (United States Environmental Protection Agency), Environmental
Criteria and Assessment Office, Integrated Risk Information System (IRIS),
Cincinnati, OH, 1992.
[66] V. Velma, S.S. Vutukuru, P.B. Tchounwou, Ecotoxicology of hexavalent
chromium in freshwater fish: a critical review, Rev. Environ. Health 24 (2009)
129–145.
[67] T. Norseth, The carcinogenicity of chromium, Environ. Health Perspect 40
(1981) 121–130.
[68] X.F. Wang, M.L. Xing, Y. Shen, X. Zhu, L.H. Xu, Oral administration of Cr(VI)
induced oxidative stress, DNA damage and apoptotic cell death in mice,
Toxicology 228 (2006) 16–23.
[69] J. Guertin, Toxicity and health effects of chromium (all oxidation states), in: J.
Guertin, J.A. Jacobs, C.P. Avakian (Eds.), Chromium(VI) Handbook, CRC Press,
Boca Raton, FL, 2005, pp. 216–234.
[70] M. Costa, Toxicity and carcinogenicity of Cr(VI) in animal models and
humans, Crit. Rev. Toxicol. 27 (1997) 431–442.
[71] S.R. Shelnutt, P. Goad, D.V. Belsito, Dermatological toxicity of hexavalent
chromium, Crit. Rev. Toxicol. 37 (2007) 375–387.
[72] T.L. Chen, S.S. Wise, S. Kraus, F. Shaffiey, K. Levine, D.W. Thompson, T. Romano,
T. O’Hara, J.P. Wise, Particulate hexavalent chromium is cytotoxic and
genotoxic to the North Atlantic right whale (Eubalaena glacialis) lung and skin
fibroblasts, Environ. Mol. Mutagen. 50 (2009) 387–393.
[73] W.V. Christian, L.D. Oliver, D.J. Paustenbach, M.L. Kreider, B.L. Finley,
Toxicology-based cancer causation analysis of CoCr-containing hip implants:
a quantitative assessment of genotoxicity and tumorigenicity studies, J. Appl.
Toxicol. 34 (2014) 939–967.
[74] M. Cohen, B. Kargacin, C.B. Klein, M. Costa, Mechanisms of chromium
carcinogenicity and toxicity, Crit. Rev. Toxicol. 23 (1993) 255–281.
[75] K. Salnikow, A. Zhitkovich, Genetic and epigenetic mechanisms in metal
carcinogenesis and co-carcinogenesis: nickel, arsenic and chromium, Chem.
Res. Toxicol. 21 (2008) 28–44.
[76] K. Khorsandi, A. Rabbani-Chadegani, Studies on the genotoxic effect of
chromium oxide (Cr VI): Interaction with deoxyribonucleic acid in solution,
Mutat. Res. Gen. Tox. Env. Mutagen. 750 (2013) 105–110.
160 B. Annangi et al. / Mutation Research 770 (2016) 140–161
[77] Z. Fang, M. Zhao, H. Zhen, L. Chen, P. Shi, Z. Huang, Genotoxicity of tri- and
hexavalent chromium compounds in vivo and their modes of action on DNA
damage in vitro, PLoS One 11 (2014) e103194.
[78] A. Vaglenov, M. Nosko, R. Georgieva, E. Carbonell, A. Creus, R. Marcos,
Genotoxicity and radioresistance in electroplating workers exposed to
chromium, Mutat. Res. 446 (1999) 23–34.
[79] D. Benova, V. Hadjidekova, R. Hristova, T. Nikolova, M. Boulanova, I.
Georgieva, et al., Cytogenetic effects of hexavalent chromium in Bulgarian
chromium platers, Mutat. Res. 514 (2002) 29–38.
[80] M.G. Medeiros, A.S. Rodrigues, M.C. Batoréu, A. Laires, J. Rueff, A. Zhitkovich,
Elevated levels of DNA-protein crosslinks and micronuclei in peripheral
lymphocytes of tannery workers exposed to trivalent chromium,
Mutagenesis 18 (2003) 19–24.
[81] A. Hilali, R. Anane, N. Jaaouani, E.E. Creppy, L. Verschaeve, Cytogenetic
analysis of tannery workers in Morocco, J. Appl. Toxicol. 28 (2008) 439–442.
[82] V. Balachandar, M. Arun, S. Mohana Devi, P. Velmurugan, P. Manikantan, A.
Karthick Kumar, et al., Evaluation of the genetic alterations in direct and
indirect exposures of hexavalent chromium [Cr(VI)] in leather tanning
industry workers North Arcot District, South India, Int. Arch. Occup. Environ.
Health 83 (2010) 791–801.
[83] S. Sellappa, K.S. Prathyumnan, S. Joseph, B.S. Vasudevan, K. Sasikala,
Evaluation of DNA damage induction and repair inhibition in welders
exposed to hexavalent chromium, Asian Pac. J. Cancer Prev. 11 (2010) 95–100.
[84] K.S. Kasprzak, Possible role of oxidative damage in metal-induced
carcinogenesis, Cancer Invest. 13 (1995) 411–430.
[85] X. Shi, A. Chiu, C.T. Chen, B. Halliwell, V. Castranova, V. Vallyathan, Reduction
of chromium VI and its relationship to carcinogenesis, J. Toxicol. Environ.
Health 2 (1999) 87–104.
[86] G.C. Burgaz, M. Yilmazer, N. Ertaş, Y. Kemaloglu, Y. Burgaz, Assessment of
cytogenetic damage in lymphocytes and in exfoliated nasal cells of dental
laboratory technicians exposed to chromium, cobalt, and nickel, Mutat. Res.
521 (2002) 47–56.
[87] Nickel Institute, Nickel and Its Uses, (2006) . www.nickelinstitute.org.
[88] K.S. Cameron, V. Buchner, P.B. Tchounwou, Exploring the molecular
mechanisms of nickel-induced genotoxicity and carcinogenicity: a literature
review, Rev. Environ. Health 26 (2011) 81–92.
[89] T.P. Coogan, D.M. Latta, E.T. Snow, M. Costa, Toxicity and carcinogenicity of
nickel compounds, Crit. Rev. Toxicol. 19 (1989) 341–384.
[90] A. Hartmann, F. Hannemann, J. Lützner, A. Seidler, H. Drexler, K.P. Günther, J.
Schmitt, Metal ion concentrations in body fluids after implantation of hip
replacements with metal-on-metal bearing–systematic review of clinical
and epidemiological studies, PLoS One 8 (2013) e70359.
[91] ATSDR (Agency for Toxic Substances and Disease Registry), Toxicological
Profile for Nickel (Update) Atlanta, GA, U.S. Department of Public Health and
Human Services, Public Health Service, 2005. http:/www.atsdr.cdc.gov.
[92] EPA (Environmental Protection Agency), Nickel and Nickel Compounds,
(2002) . http://www.epa.state.oh.us/ocapp/p2/mercury_pbt.
[93] P. Grandjean, G.D. Nielson, O. Anderson, Human nickel exposure and
chemobiokinetics, in: H.E. Maibach, T. Menné (Eds.), Nickel and the Skin:
Immunology and Toxicology, CRC Press, Boca Raton, FL, 1989, pp. 9–34.
[94] E. Denkhaus, K. Salnikow, Nickel essentiality, toxicity, and carcinogenicity,
Crit. Rev. Oncol. Hematol. 42 (2002) 35–56.
[95] J. Zhao, X. Shi, V. Castranova, M. Ding, Occupational toxicology of nickel and
nickel compounds, J. Environ. Pathol. Toxicol. Oncol. 28 (2009) 177–208.
[96] J.E. Goodman, R.L. Prueitt, D.G. Dodge, S. Thakali, Carcinogenicity assessment
of water-soluble nickel compounds, Crit. Rev. Toxicol. 39 (2009) 365–417.
[97] B. Zambelli, S. Ciurli, Nickel and human health, Met. Ions Life Sci. 13 (2013)
321–357.
[98] A. Binazzi, P. Ferrante, A. Marinaccio, Occupational exposure and sinonasal
cancer: a systematic review and meta-analysis, BMC Cancer 15 (2015) e49.
[99] A. Hartwig, I. Krüger, D. Beyersmann, Mechanisms in nickel genotoxicity: the
significance of interactions with DNA repair, Toxicol. Lett. 72 (1994) 353–358.
[100] I.N. Perminova, T.A. Sinel’shchikova, N.I. Alekhina, E.V. Perminova, G.D.
Zasukhina, Individual sensitivity to genotoxic effects of nickel and
antimutagenic activity of ascorbic acid, Bull. Exp. Biol. Med. 131 (2001) 367–
370.
[101] V. Senft, F. Losan, M. Tucek, Cytogenetic analysis of chromosomal aberrations
of peripheral lymphocytes in workers occupationally exposed to nickel,
Mutat. Res. 279 (1992) 171–179.
[102] M. Kiilunen, J. Utela, T. Rantanen, H. Norppa, A. Tossavainen, M. Koponen,
et al., Exposure to soluble nickel in electrolytic nickel refining, Ann. Occup.
Hyg. 41 (1997) 167–188.
[103] M. Imtiaz, M.S. Rizwan, S. Xiong, H. Li, M. Ashraf, S.M. Shahzad, M. Shahzad,
M. Rizwan, S. Tu, Vanadium, recent advancements and research prospects: a
review, Environ. Int. 80 (2015) 79–88.
[104] J.O. Nriagu, Vanadium in the Environment, John Wiley and Sons, Inc., New
York, 1998.
[105] IARC (International Agency for Research on Cancer), Vanadium pentoxide.
IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 86,
International Agency for Research in Cancer, Lyon, France, 2006.
[106] K. Gruzewska, A. Michno, T. Pawelczyk, H. Bielarczyk, Essentiality and
toxicity of vanadium supplements in health and pathology, J. Physiol.
Pharmacol. 65 (2014) 603–611.
[107] J. Rodríguez-Mercado, R.A. Mateos-Nava, M.A. Altamirano-Lozano, DNA
damage induction in human cells exposed to vanadium oxides in vitro,
Toxicol. In Vitro 25 (2011) 1996–2002.
[108] J.B. Mailhes, C. Hilliard, J.W. Fuseler, S.N. London, Vanadate, an inhibitor of
tyrosine phosphatases, induced premature anaphase in oocytes and
aneuploidy and polyploidy in mouse bone marrow cells, Mutat. Res. 538
(2003) 101–107.
[109] A. Basu, P. Ghosh, A. Bhattacharjee, A.R. Patra, S. Bhattacharya, Prevention of
myelosuppression and genotoxicity induced by cisplatin in murine bone
marrow cells: effect of an organovanadium compound vanadium(III)-l-
cysteine, Mutagenesis 30 (2015) 509–517.
[110] S. Ivancsits, A. Pilger, E. Diem, A. Schaffer, H.W. Rüdiger, Vanadate induces
DNA strand breaks in cultured human fibroblasts at doses relevant to
occupational exposure, Mutat. Res. 519 (2002) 25–35.
[111] V.A. Ehrlich, A.K. Nersesyan, C. Hoelzl, F. Ferk, J. Bichler, E. Valic, et al.,
Inhaltive exposure to vanadium pentoxide causes DNA damage in workers:
results of a multiple end point study, Environ. Health Perspect. 116 (2008)
1689–1693.
[112] J.L. Mauderly, J.C. Chow, Health effects of organic aerosols, Inhal. Toxicol. 20
(2008) 257–288.
[113] L.C. Chen, M. Lippmann, Effects of metals within ambient air particulate
matter (PM) on human health, Inhal. Toxicol. 21 (2009) 1–31.
[114] J. Zukowska, M. Biziuk, Methodological evaluation of method for dietary
heavy metal intake, J. Food Sci. 73 (2008) 21–29.
[115] S.J.S. Flora, M. Mittal, A. Mehta, Heavy metal induced oxidative stress and its
reversal by chelation therapy, Ind. J. Med. Res. 128 (2008) 501–523.
[116] A.T. Jan, M. Azam, K. Siddiqui, A. Ali, I. Choi, Q.M. Haq, Heavy metals and
human health: mechanistic insight into toxicity and counter defense system
of antioxidants, Int. J. Mol. Sci. 16 (2015) 29592–29630.
[117] EPA, Integrated Science Assessment for Particulate Matter (final Report),
United States Environmental Protection Agency, Washington, DC, 2009.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=216546#Download.
[118] G. Wang, B.A. Fowler, Roles of biomarkers in evaluating interactions among
mixtures of lead: cadmium and arsenic, Toxicol. Appl. Pharmacol. 233 (2008)
92–99.
[119] G.F. Nordberg, T. Jin, F. Hong, A. Zhang, J.P. Buchet, A. Bernard, Biomarkers of
cadmium and arsenic interactions, Toxicol. Appl. Pharmacol. 206 (2005) 191–
197.
[120] M. De Boeck, S. Lardau, J.P. Buchet, M. Kirsch-Volders, D. Lison, Absence of
significant genotoxicity in lymphocytes and urine from workers exposed to
moderate levels of cobalt-containing dust: a cross-sectional study, Environ.
Mol. Mutagen. 36 (2000) 151–160.
[121] R. Mateuca, P.V. Aka, M. De Boeck, R. Hauspie, M. Kirsch-Volders, D. Lison,
Influence of hOGG1, XRCC1 and XRCC3 genotypes on biomarkers of
genotoxicity in workers exposed to cobalt or hard metal dusts, Toxicol. Lett.
156 (2005) 277–288.
[122] R. Crebelli, P. Carta, C. Andreoli, G. Aru, G. Dobrowolny, S. Rossi, A. Zijno,
Biomonitoring of primary aluminium industry workers: detection of
micronuclei and repairable DNA lesions by alkaline SCGE, Mutat. Res. 516
(2002) 63–70.
[123] G. Iarmarcovai, I. Sari-Minodier, I.F. Chaspoul, C. Botta, M. De Méo, T. Orsière,
J.L. Bergé-Lefranc, P. Gallice, A. Botta, Risk assessment of welders using
analysis of eight metals by ICP-MS in blood and urine and DNA damage
evaluation by the comet and micronucleus assays; influence of XRCC1 and
XRCC3 polymorphisms, Mutagenesis 20 (2005) 425–432.
[124] P. Coelho, J. García-Lestón, S. Costa, C. Costa, S. Silva, S.V. Dall’Armi, et al.,
Genotoxic effect of exposure to metal(loid)s. A molecular epidemiology
survey of populations living and working in Panasqueira mine area Portugal,
Environ. Int. 60 (2013) 163–170.
[125] P. Coelho, S. Costa, S. Silva, A. Walter, J. Ranville, A.C. Sousa, et al., Metal(loid)
levels in biological matrices from human populations exposed to mining
contamination—Panasqueira Mine (Portugal), J. Toxicol. Environ. Health A 75
(2012) 893–908.
[126] M. Manojlovic’, B.R. Singh, Trace elements in soils and food chains of the
Balkan region, Acta Agric. Scand. Sect. B Soil Plant Sci. 62 (2012) 673–695.
[127] A. Mesic, H. Nefic, Assessment of the genotoxicity and cytotoxicity in
environmentally exposed human populations to heavy metals using the
cytokinesis-block micronucleus cytome assay, Environ. Toxicol. 30 (2015)
1331–1342.
[128] G. Wultsch, M. Mišík, A. Nersesyan, S. Knasmueller, Genotoxic effects of
occupational exposure measured in lymphocytes of waste-incinerator
workers, Mutat. Res. 720 (2011) 3–7.
[129] P. Rohr, K. Kvitko, F.R. da Silva, A.P. Menezes, C. Porto, M. Sarmento, et al.,
Genetic and oxidative damage of peripheral blood lymphocytes in workers
with occupational exposure to coal, Mutat. Res. 758 (2013) 23–28.
[130] P. Rohr, J. da Silva, F.R. da Silva, M. Sarmento, C. Porto, R. Debastiani, C.E. Dos
Santos, J.F. Dias, K. Kvitko, Evaluation of genetic damage in open-cast coal
mine workers using the buccal micronucleus cytome assay, Environ. Mol.
Mutagen. 54 (2013) 65–71.
[131] E. Fernandez-Minano, C. Ortiz, A. Vicente, J.L. Calvo, A.J. Ortiz, Metallic ion
content and damage to the DNA in oral mucosa cells of children with fixed
orthodontic appliances, Biometals 5 (2011) 935–941.
[132] S. Ishikawa, H. Ishikawa, T. Shindo, T. Yoshida, Y. Shimoyama, T. Satomi, et al.,
Effects of occupational environmental controls and work management on
chromosomal damage in dental technicians in Japan, Int. J. Hyg. Environ.
Health 216 (2013) 100–107.
[133] C. Angelé-Martínez, C. Goodman, J. Brumaghim, Metal-mediated DNA
damage and cell death: mechanisms, detection methods, and cellular
consequences, Metallomics 6 (2014) 1358–1381.
 B. Annangi et al. / Mutation Research 770 (2016) 140–161
[134] F. Gil, A.F. Hernández, Toxicological importance of human biomonitoring of
metallic and metalloid elements in different biological samples, Food Chem.
Toxicol. 80 (2015) 287–297.
[135] A.I. Seoane, F.N. Dulout, Genotoxic ability of cadmium, chromium and nickel
salts studied by kinetochore staining in the cytokinesis-blocked
micronucleus assay, Mutat. Res. (GTEM) 490 (2001) 99–106.
[136] S. Bonassi, R. El-Zein, C. Bolognesi, M. Fenech, Micronuclei frequency in
peripheral blood lymphocytes and cancer risk: evidence from human studies,
Mutagenesis 26 (2011) 93–100.
[137] S. Bonassi, H. Norppa, M. Ceppi, U. Strömberg, R. Vermeulen, A. Znaor, A.
Cebulska-Wasilewska, E. Fabianova, A. Fucic, S. Gundy, I.L. Hansteen, L.E.
161
Knudsen, J. Lazutka, P. Rossner, R.J. Sram, P. Boffetta, Chromosomal aberration
frequency in lymphocytes predicts the risk of cancer: results from a pooled
cohort study of 22 358 subjects in 11 countries, Carcinogenesis 29 (2008)
1178–1183.
[138] M. Fenech, N. Holland, E. Zeiger, W.P. Chang, S. Burgaz, P. Thomas, C.
Bolognesi, S. Knasmueller, M. Kirsch-Volders, S. Bonassi, The HUMN and
HUMNxL international collaboration projects on human micronucleus assays
in lymphocytes and buccal cells—past, present and future, Mutagenesis 26
(2011) 239–245.