Mutation Research 770 (2016) 12–25
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Mutation Research/Reviews in Mutation Research
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Community address: www.elsevier.com/locate/mutres
Review
Molecular mechanisms by which in vivo exposure to exogenous
chemical genotoxic agents can lead to micronucleus formation in
lymphocytes in vivo and ex vivo in humans
Michael Fenecha,* , Siegfried Knasmuellerb , Claudia Bolognesic , Stefano Bonassid ,
Nina Hollande, Lucia Miglioref , Fabrizio Palittig , Adayapalam T. Natarajang,
Micheline Kirsch-Voldersh
a
Commonwealth and Scientific Industrial Research Organization, Genome Health and Personalised Nutrition Laboratory, Food and Nutrition Flagship,
Adelaide, South Australia 5000, Australia
b
Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna,
Austria
c
Environmental Carcinogenesis Unit, IRCCS Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale Ricerca sul Cancro, Largo Rosanna Benzi 10,
Genova 16132, Italy
d
Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome, Italy
e
School of Public Health, University of California, Berkeley, CA, USA
f
Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy
g
Department of Ecological and Biological Sciences, University of Tuscia, Via San Camillo de Lellis snc, 01100 Viterbo, Italy
h
Laboratory of Cell Genetics, Faculty of Science and Bio-Engineering, Free University of Brussels (VUB), Pleinlaan 2, 1050 Brussel, Belgium
A R T I C L E I N F O
Article history:
Received 24 February 2016
Accepted 18 April 2016
Available online 7 June 2016
Keywords:
Molecular
Mechanisms
Micronucleus
Lymphocytes
Exogenous
Chemical
Genotoxins
Exposure
In vivo
Ex vivo
Humans
A B S T R A C T
The purpose of this review is to summarise current knowledge on the molecular mechanisms by which in
vivo exposure to exogenous chemical genotoxins in humans induces micronuclei (MNi) and other nuclear
anomalies in lymphocytes in vivo and ex vivo after nuclear division in vitro. MNi originate from acentric
chromosome fragments and/or whole chromosomes that are unable to engage with the mitotic spindle
and/or fail to segregate properly to the daughter nuclei during anaphase. The lagging fragments or whole
chromosomes are surrounded by membrane and become MNi. Acentric fragments are caused by failure
of repair or mis-repair of DNA strand breaks which may be induced by chemicals that (i) damage the
phosphodiester backbone of DNA, and/or (ii) inhibit the DNA damage response mechanisms or repair of
DNA strand breaks and/or (iii) cause DNA replication stress due to DNA adduct or cross-link formation.
MNi originating from lagging whole chromosomes may be induced by chemicals that cause defects in
centromeres or the mitotic machinery. Mis-repair of chemically-induced DNA breaks may also cause
formation of dicentric chromosomes and nucleoplasmic bridges (NPBs) between daughter nuclei in
mitosis. NPBs may break and initiate recurring breakage-fusion-bridge cycles and chromosomal
instability. The review also explores knowledge on (i) the routes by which lymphocytes in the human
body may be exposed to genotoxic chemicals, (ii) kinetics of MNi expression in lymphocytes in vivo and ex
vivo in the lymphocyte cytokinesis-block micronucleus (L-CBMN) assay and (iii) current evidence on the
efficiency of the L-CBMN assay in detecting in vivo exposure to chemical genotoxins and its concordance
with MNi expression in epithelial tissues. The review also identifies important knowledge gaps (e.g. effect
of nanomaterials; interactions with nutritional deficiencies etc.) regarding mechanisms by which in vivo
chemical genotoxin exposure may cause MNi formation in lymphocytes in vivo and ex vivo in
lymphocytes.
ã 2016 Published by Elsevier B.V.
* Corresponding author.
E-mail address: [email protected] (M. Fenech).
http://dx.doi.org/10.1016/j.mrrev.2016.04.008
1383-5742/ã 2016 Published by Elsevier B.V.
M. Fenech et al. / Mutation Research 770 (2016) 12–25
13
Contents
1.
2.
3.
4.
5.
6.
7.
8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Routes by which lymphocytes in the body can be exposed to exogenous genotoxic chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Molecular mechanisms by which genotoxic chemicals can induce MNi and other nuclear anomalies in lymphocytes in vivo . . . . . . . . . . . . 14
Molecular mechanisms by which DNA and protein lesions induced by chemicals in circulating lymphocytes in vivo can be expressed as MNi and
other nuclear anomalies ex vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Kinetics of MNi expression in lymphocytes in vivo and ex vivo in the L-CBMN assay after acute or chronic genotoxin exposure . . . . . . . . . 20
Current evidence that the L-CBMN assay is efficient in detecting in vivo exposure to chemical genotoxins and its concordance with other DNA
damage biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Important knowledge gaps regarding mechanisms by which genotoxic chemicals induce MN in lymphocytes in vivo and ex vivo . . . . . . . . 23
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
1. Introduction
The lymphocyte cytokinesis-block micronucleus cytome
(L-CBMN) assay is one of the most widely used methods to
measure genome damage in humans which may be induced by
endogenous and environmental genotoxins [1]. It detects both
structural and numerical chromosome aberrations [1–4]. The
L-CBMN assay has the distinct feature that it allows (i) micronuclei
(MNi) expressed in vivo to be observed in lymphocytes without the
need of completing nuclear division ex vivo as well as (ii)
micronucleus (MN) expression ex vivo in lymphocytes that have
been stimulated to divide in in vitro culture [2]. The most
commonly used mitogen in this assay is phytohaemagglutinin
(PHA) which predominantly stimulates T-lymphocytes to divide,
however other mitogens can be used that stimulate other
lymphocyte subsets to divide. The use of lymphocytes is
particularly important because it allows the L-CBMN assay to be
performed both in vivo and ex vivo for biomonitoring studies and
also for the purpose of in vitro genetic toxicology testing of
chemicals and pharmaceuticals [5].
The ex vivo step in the assay is important because it allows
persistent DNA or protein damage accumulated during maturation
in and after exit from the bone-marrow to be converted to
structural chromosome aberrations and malsegregated chromosomes during mitosis from which MN and other associated nuclear
anomalies (nucleoplasmic bridges, NPB, and nuclear buds, NBUD)
may ultimately arise [1–4]. For example a DNA strand break may
lead to an acentric chromosome fragment that cannot engage the
spindle, and protein damage to kinetochore or spindle proteins
could lead to a whole chromosome lagging during anaphase. The
exclusion of the acentric fragment and/or whole chromosome from
the daughter nuclei during anaphase/telophase ultimately results
in their conversion to MNi (Fig. 1). Misrepair of DNA strand breaks
or impairment of chromatid separation due to defects in cohesins
Fig. 1. Schematic diagram describing the origin of micronuclei and nucleoplasmic bridges and their detection using the cytokinesis-block micronucleus assay in lymphocyte
cultures. Nucleoplasmic bridges may be accompanied with a micronucleus if the dicentric chromosome is a result of mis-repair of double stranded DNA breaks which also
yields acentric chromosome fragments from which micronuclei may arise. The figure was adapted from (A) in Fenech M, The in vitro micronucleus technique. Mutat Res 200; 455:
81–95, with permission from Elsevier.
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M. Fenech et al. / Mutation Research 770 (2016) 12–25
and separases can lead to dicentric chromosome formation or
sister chromatids being terminally attached to each other,
respectively. If the centromeres of these abnormal chromosomes
are pulled to opposite poles of the cell it will cause the formation of
anaphase bridges and NPBs during the anaphase/telophase stages
of mitosis (Fig. 1) [3,4,6–8]. The resulting physical tension often
leads to breakage of the chromosomes in the NPB which initiates
breakage-fusion-bridge cycles, causing gene amplification and
elimination of amplified DNA as nuclear buds (NBUDs) which are
also scored in the L-CBMN assay together with MNi and NPBs
(Fig. 2a and b).
The purpose of this review is to provide a summary of current
knowledge on:
(i) Routes by which lymphocytes in the human body can be
exposed to genotoxic chemicals;
(ii) Molecular mechanisms by which genotoxic chemicals may
induce MNi and other nuclear anomalies in lymphocytes in
vivo;
(iii) Molecular mechanisms by which DNA and protein lesions
induced by chemicals in circulating lymphocytes in vivo can be
expressed as MNi and other nuclear anomalies ex vivo;
(iv) Kinetics of MNi expression in lymphocytes in vivo and ex vivo
in the L-CBMN assay
(v) Current evidence of the efficiency of the L-CBMN assay in
detecting in vivo exposure to chemical genotoxins and its
concordance with other DNA damage biomarkers.
Another objective is to identify important knowledge gaps
regarding such mechanisms to stimulate future research in this
important area of environmental mutagenesis and MN biology.
2. Routes by which lymphocytes in the body can be exposed to
exogenous genotoxic chemicals
Exogenous chemical genotoxins may enter the body via the oral
route, the gastro-intestinal tract, the respiratory system and the
skin. These chemicals or their metabolites may be absorbed into
the body fluids and organs in which lymphocytes reside such as the
circulatory system, the lymphatic system, bone marrow, lymph
nodes, spleen and thymus. As chemical genotoxins are absorbed
and circulate in the body they may become activated or detoxified
by different organs and thus influence the degree to which
lymphocytes are exposed to the risk of chemically-induced DNA
damage.
The bone marrow is the site where haematopoietic stem cells
are located. In humans hematopoiesis ceases in the long bones of
humans between 5 and 7 years of age and in adulthood the major
sites of hematopoiesis are bones of the axial skeleton (the cranium,
sternum, ribs and vertebrae) in addition to the ilium [9]. The spleen
does not support hematopoiesis after birth in humans, although
extramedullary splenic hematopoiesis can occur as a consequence
of hematopoietic stress [10].
Chemical genotoxins reaching haematopoiesis sites, such as the
bone marrow, may cause genome damage in the stem cells and
precursor cells from which lymphocytes and other leukocytes are
derived [11]. These regenerative proliferating cells are particularly
sensitive to chemical genotoxins that cause DNA-damage-induced
replication stress during S-phase of the cell cycle [12,13]. After
maturation in the bone marrow the majority of lymphocytes may
remain in the naive stage and are short-lived (half-life of weeks to
months) whilst others that have been primed to recognise a foreign
antigen become long-lived (half-life of months to years) [14,15]. A
significant fraction of lymphocytes are further matured as
T-lymphocytes in the thymus. Both naive and committed
lymphocytes circulate in the blood, lymphatic system and
interstitial fluid and they may also become located temporarily
in lymph nodes and spleen. Theoretically, long-lived lymphocytes
are likely to accumulate much more DNA damage than naive shortlived lymphocytes before they die because of their longer duration
of exposure, however, this has not been tested yet. There is
evidence indicating that the rates of MN formation may differ
substantially between lymphocyte subsets such as B cells versus T
cells and CD4+ T cells versus CD8+ T cells depending on the
chemical genotoxin [16–18]. These differences may reflect intrinsic
cell susceptibility or difference of residence and life-span relative
to chemical genotoxin exposure.
As chemical genotoxins circulate in the body, they may
become activated or de-toxified by different organs and thus
influence the degree to which lymphocytes are exposed to the
risk of chemically-induced DNA damage. Chemicals may also
exert their genotoxic effects in lymphocytes indirectly via
induction of inflammation-generated reactive oxygen species
or activation of procarcinogens. For example, it was shown that
phorbol ester activation of neutrophils caused conversion of
mixantrone to genotoxic metabolites causing DNA adduct
formation [19]. MN frequency in the WIL2-NS lymphoblastoid
cell line was increased 30-fold when co-cultured in a transwell
with neutrophils activated using phorbol 12-myristate acetate
[20]. Both of these examples indicate the plausibility of
inflammation, via neutrophil activation, causing DNA damage
to bystander lymphocytes. One of the most likely sites where such
indirect effects might occur is the lung for which evidence of
indirect chemical-induced genotoxicity via inflammation is
accumulating including examples such as crystalline silica,
asbestos, nanoparticles, formaldehyde and smoking [21–25].
3. Molecular mechanisms by which genotoxic chemicals can
induce MNi and other nuclear anomalies in lymphocytes in vivo
The mechanisms by which ionising radiation leads to acentric
chromosome fragments and dicentric chromosomes in lymphocytes and thus to MNi and NPBs are well known and have been
described in detail in several publications [3,6,7,8]. These mechanisms are also relevant to so-called “radiomimetic chemicals” such
as bleomycin that predominantly induce DNA strand breaks.
However, much less is known about the multitude of mechanisms
by which other types of genotoxic chemicals can potentially cause
the type of numerical and chromosome or chromatid aberrations
that can induce the formation of MNi, NPBs and NBUDs.
MNi mainly originate from lagging whole chromosomes or
lagging chromosome fragments and NPB are caused by dicentric
chromosomes or inseparable chromatids at anaphase/telophase.
For these reasons, the expression of these nuclear anomalies in
lymphocytes can be expected to occur during the various stages of
their production from stem cell and precursor cell divisions in vivo.
Because NPBs ultimately break during telophase they are not
observed in post-mitotic cells unless the cell division cycle is
stalled at the binucleate stage. When a NPB breaks it can lead to the
formation of a MN or nuclear bud (NBUD) [3,26,27]. Therefore MNi
and NBUDs are the nuclear anomalies that are mainly observed in
post-mitotic lymphocytes in vivo in the peripheral blood.
The bone marrow is the site where haematopoietic stem cells
reside. Therefore in vivo induction of MNi by genotoxic chemicals
in lymphocyte precursors can occur at this early stage and such
MNi may become present in nascent lymphocytes in the bone
marrow and retained after exiting into the blood circulation
system. After leaving the bone marrow lymphocytes may also be
exposed to genotoxic chemicals in the blood stream and interstitial
fluids as well as in organs such as lymph nodes, spleen and thymus
where they may mature into T cells or B cells or further divide in
response to pathogenic insults. It is possible that further nuclear
M. Fenech et al. / Mutation Research 770 (2016) 12–25
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Fig. 2. (a) Schematic diagram showing expression of micronuclei (MN) in vivo and expression of MN, nucleoplasmic bridges (NPB) and nuclear buds (NBUD) ex vivo/in vitro in
the L-CBMN assay in relationship to exposure to genotoxic factors, DNA damage and DNA repair at the various cell cycle stages. Measurements in cells prior to completion of
mitosis and after mitosis in binucleated cells allows discrimination between MN expressed in vivo and those expressed ex vivo respectively. CYT-B, cytochalasin-B; PHA,
phytohaemagglutinin. This figure is a copy of Fig. 3 in Kirsch-Volders M, Bonassi S, Knasmueller S, Holland N, Bolognesi C, Fenech MF. Commentary: critical questions,
misconceptions and a road map for improving the use of the lymphocyte cytokinesis-block micronucleus assay for in vivo biomonitoring of human exposure to genotoxic chemicals-a
HUMN project perspective. Mutat Res Rev Mutat Res. 2014 Jan-Mar;759:49-58. Permission to re-use this figure was obtained from the publisher Elsevier. (b) Photomicrographs of
the various cell types and biomarkers of DNA damage, cytostasis and cell death measured in the L-CBMN cytome assay. (A) binucleated cell with nucleoplasmic bridge (NPB)
and micronucleus (MN); (B) binucleated cell with MN; (C) binucleated cell with nuclear buds (NBUD); (D) mononucleated cell with MN, (E) mononucleated cell with one
NBUD; (F) apoptotic cell; (G) necrotic cell; (H) mononucleated cell; (I) binucleated cell; (J) multinucleated cell. The frequency of MN, NPB and NBUD in binucleated cells
provides a measure of DNA damage expressed ex vivo. The frequency of MN and NBUD in mononucleated cells provides a measure of DNA damage expressed in vivo.
Measurement of apoptotic and necrotic cells gives information on the frequency of dead/dying cells and the mechanism by which cell death occurred. The relative frequency
of mononucleated, binucleated and multinucleated cells is an index of immune responsiveness of lymphocytes and their regenerative capacity. This figure is adapted from
Fig. 1B in Fenech M. Cytokinesis-block micronucleus assay evolves into a “cytome” assay of chromosomal instability, mitotic dysfunction and cell death. Mutat Res. 2006 Aug 30; 600
(1–2):58-66. Permission was obtained from the publisher Elsevier.
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M. Fenech et al. / Mutation Research 770 (2016) 12–25
divisions in organs other than the bone marrow may also lead to
MN expression in vivo in lymphocytes.
There are multiple mechanisms by which genotoxic chemicals
in the body can cause lesions in DNA that lead to MNi expression in
vivo or ex vivo. These mechanisms are summarised in Fig. 3 and
listed below:
(i) Chemicals that directly or indirectly generate reactive oxygen
or reactive nitrogen species that break the phosphodiester
backbone of DNA leading to DNA strand breaks (e.g. hydrogen
peroxide, bleomycin, phorbol esters).
(ii) Chemicals that inhibit DNA damage response (DDR) by
interfering with the catalytic site or displacing important
DNA synthesis or DNA repair enzyme cofactors (e.g.
3-aminobenzamide, cytosine arabinoside, nickel, cadmium).
Such inhibitors may result in mis-repair or mis-replication of
DNA strand breaks or persistence of DNA lesions such as DNA
strand breaks or adducts.
(iii) Chemicals that induce small or bulky DNA adducts such as
8-hydroxy-20 -deoxyguanosine or n7-methylguanine and
benzo-[a]-pyrene diolepoxide adducts respectively.
(iv) Chemicals that induce DNA strand cross-links such as
mitomycin-C or DNA-protein cross-links such as formaldehyde.
(v) Chemicals that cause numerical chromosome abnormalities
by inhibiting the polymerisation of proteins (e.g. tubulin,
actin) required to form cytoskeletal structures (e.g. microtubules, microfilaments) that are essential for the mitotic
process or cause damage to kinetochores or cohesins required
for chromatid segregation during anaphase. Examples include
taxol (inhibitor of tubulin polymerisation), tryprostatin-A
(inhibitor of microfilament formation), Sepin-1 (inhibitor of
separase).
(vi) Chemicals interfering with epigenetic mechanisms such as
inhibitors of DNA methylation including impairment of
methyl donor metabolism or direct inhibition of DNA
methyltransferases or histone modifications of centromeric
heterochromatin. Examples include 5-azacytidine (inhibitor
of DNA methyl transferase) and Vorinostat (inhibitor of
histone deacetylases).
The DNA lesions (i)-(iv) above, if left unrepaired will cause
replication stress and stalling of the DNA replication fork, leading
to formation of transient or permanent DNA breaks from which
acentric chromosome fragments and MNi originate or induce
asymmetric chromatid exchanges from which NPB and MNi may
arise. In addition, simultaneous excision repair of DNA adducts on
opposite strands of DNA within a few bases of each other may also
Fig. 3. The various mechanisms by which chemical genotoxins cause micronuclei (MN), nucleoplasmic bridges (NPB) and nuclear buds (NBUD). MN containing whole
chromosomes are centromore positive (Cen +ve) as detected by molecular probes for centromeric DNA, while MN containing acentric chromosome fragments are centromere
negative (Cen ve). ROS = reactive oxygen species; RNS = reactive nitrogen species.? = plausible but insufficient data. This figure was adapted from Fig. 1 in Kirsch-Volders M,
Bonassi S, Knasmueller S, Holland N, Bolognesi C, Fenech MF. Commentary: critical questions, misconceptions and a road map for improving the use of the lymphocyte cytokinesisblock micronucleus assay for in vivo biomonitoring of human exposure to genotoxic chemicals-a HUMN project perspective. Mutat Res Rev Mutat Res. 2014 Jan-Mar;759:49-58.
Permission was obtained from the publisher Elsevier.
M. Fenech et al. / Mutation Research 770 (2016) 12–25
lead to DNA double strand break formation leading to MNi
expression.
The action of genotoxic chemicals on formation of chromatid
and chromosome aberrations has been shown to vary depending
on the cell cycle stage (Table 1) [28–31]. For example, inhibitors of
DNA synthesis (e.g. hydroxyurea, methotrexate) or repair (e.g.
cytosine arabinoside, PARP inhibitors, such as 3-aminobenzamide)
induce chromatid gaps and deletions during the S and G2 phases of
the cell cycle, whilst other compounds (such as bleomycin)
produce chromatid aberrations of all types in late S and G2. Most of
the other genotoxic chemicals may cause such aberrations due to
their actions at all stages of the cell cycle [28–32]. Genotoxic
chemicals have also been classified as being S-independent or
S-dependent in their mode of action. In the former case the DNA
lesions are repaired or mis-repaired independently of DNA
synthesis while in the case of S-dependent agents the induced
DNA lesion is not repaired before the DNA synthesis leading to misreplications or replication stress that may result in the formation of
chromatid-type or chromosome-type aberrations [28–32]. Fig. 4 is
an adaptation of a detailed schematic diagram designed by John
Savage [33] which depicts the various types of chromatid and
chromosome type aberrations that may be generated by genotoxic
chemicals and which lead to formation of MNi and NPBs.
Several types of chemicals have been identified as being capable
of inducing the expression of phosphorylated gH2AX foci
(a quantitative indicator of DNA double strand breaks) in
mammalian cells [34] such as DNA damaging agents (bleomycin,
tirapazamine, 4-nitro-quinoline-N-oxide, methyl methanesulfonate,
N-methyl-N0-nitro-N-nitrosoguanidine,
adozelesin,
calicheamicin), DNA intercalating agents (cisplatin), DNA synthesis
inhibitors (hydroxyurea, aphidicolin), RNA synthesis inhibitors
(actinomycin D), DNA topoisomerase I inhibitors (camptothecin,
topotecan), DNA topoisomerase II inhibitors (etoposide, teniposide, doxorubicin, mitoxantrone) DNA topoisomerase I and II
inhibitors (genistein, quercetin), Poly(ADP-ribose)polymerase
inhibitors (NU1025), reactive oxygen species (hydrogen peroxide),
heavy metals (selenium, arsenic, chromium compounds), complex
mixtures (tobacco smoke) [34,35]. This list indicates that a large
number of chemicals with diverse mechanisms may induce double
strand breaks (DSBs) in DNA from which acentric chromosome
fragments and dicentric chromosomes originate and which lead to
the formation of MNi and NPBs respectively.
Other mechanisms by which genotoxic chemicals may induce
MNi in vivo are those that lead to malsegregation of whole
chromosomes. The types of chemicals that could cause such events
include agents that disrupt the function of centrosomes (e.g.,
estrogens, the antiretroviral drug zidovudine, 30 -azido-30 -deoxythymidine), mitotic spindle (e.g. colchicine, colcemid, nocodazole,
taxol, benomyl, and its metabolite carbendazim), kinetochores
(estrogenic chemicals, farnesyl transferase inhibitors) that are
required for chromosome segregation during mitosis [3,4,36,37].
There is growing evidence that mechanisms involving methylation pattern alterations or histone tails modifications can lead to
MNi expression [38]. It is likely that the epigenetic mechanisms of
MNi formation, identified until now mainly in vitro, could also act
in vivo and ex vivo. A reduced availability of methyl groups because
of a lower concentration of S-adenosyl-L-methionine (SAM) (due to
enzyme polymorphisms, nutritional deficiencies, and exposure to
chemical agents) is related to a greater chromosome damage
vulnerability [39]. There are increasing examples of in vitro
treatments with many chemicals which have been shown to be
able to interfere with methyl groups availability, among which is
arsenic; the frequency of cells with MNi containing whole
chromosomes in cultures treated with sodium arsenite is counteracted by the presence of increased concentrations of SAM [40]. It is
known that the hypomethylation of repetitive DNA sequences in
the centromeric and pericentromeric regions of chromosomes is
highly correlated with chromosomal instability. For example
hypomethylation of centromeric DNA induced by the DNA
methyltransferase inhibitor 5-azacytidine causes malsegregation
of chromosomes 1, 9 and 16 possibly by affecting the binding of the
kinetochore to the centromere or spindle fibers [41–43]. Another
example is the nucleoside analogue 5-fluorouracil (5FU), used in
cancer chemotherapy, which is able to induce MNi in vitro, in the
Chinese hamster V79 cell line [44], and possibly, ex vivo, in
lymphocytes of nurses handling cytostatic drugs [45]. Moreover in
vitro (in H4IIE rat hepatoma cells) 5FU was shown to affect
multiple changes in DNA methylation [46] and to interfere with the
transcriptional state and the epigenetic histone modifications of
centromeric heterochromatin, possibly contributing to the defects
detected in chromosome segregation [47].
4. Molecular mechanisms by which DNA and protein lesions
induced by chemicals in circulating lymphocytes in vivo can be
expressed as MNi and other nuclear anomalies ex vivo
All lymphocytes in the circulating blood in healthy individuals
are non-dividing post-mitotic cells in G0 phase. These cells may
have already acquired unrepaired lesions to the genome such as
abasic sites, DNA strand breaks or unrepaired adducts from their
parent cells due to chemical genotoxin exposure. However, as they
travel around the body they may experience further of these insults
to the genome.
Specific sites in the genome such as the telomeres accumulate
DNA strand breaks and base lesions because they are resistant to
repair [48–50]. Damage to the telomere sequence can lead to
Table 1
Different chemical types cause different chromatid aberrations depending on cell cycle stage treated.a
Compounds producing gaps and deletions in late S and G2 cells
Inhibitors of DNA synthesis (e.g. hydroxyurea, methotrexate)
Inhibitors of DNA repair (e.g. cytosine arabinoside, 3-aminobenzamide)
Compounds producing chromatid-type aberrations in late S and G2 cells
May also induce chromosome aberrations in G0 or G1
Induce DNA strand breaks as well as base lesions directly
Radiomimetic (e.g. bleomycin) or oxidative radical generators (e.g. H2O2)
Compounds producing chromatid-type aberrations, but only in cells treated in G1 and early S phase
Most are known to react with DNA or its precursors directly
Cause excision-repairable DNA lesions
If unrepaired may cause replication stress leading to gaps in daughter strand
Most compounds that cause chromatid/chromosome aberrations are in this class
Compounds that cause all types of chromatid aberrations at all stages of the cell cycle
Probably many compounds because of indirect effects on DNA metabolism
a
17
The table is an adaptation of information in the following publications: Bender et al. [28] and Natarajan [31].
18
M. Fenech et al. / Mutation Research 770 (2016) 12–25
Fig. 4. The principal chromatid and chromosome aberrations that contribute to micronucleus (MN) and nucleoplasmic bridge (NPB) formation in the L-CBMN cytome assay.
Adapted from Savage JRK (2000) Micronuclei: pitfalls and problems. Atlas Genet Cytogenet Oncol Haematol. (http://atlasgeneticsoncology.org/Deep/MicronucleiID20016.
html). Permission was obtained from Atlas of Genetics and Cytogenetics in Oncology and Haematology. The figure is also a copy of Fig. 2 in Kirsch-Volders M, Bonassi S,
Knasmueller S, Holland N, Bolognesi C, Fenech MF. Commentary: critical questions, misconceptions and a road map for improving the use of the lymphocyte cytokinesis-block
micronucleus assay for in vivo biomonitoring of human exposure to genotoxic chemicals-a HUMN project perspective. Mutat Res Rev Mutat Res. 2014 Jan-Mar;759:49-58. Permission
to re-use the figure was obtained from the publisher Elsevier.
telomere dysfunction due to inefficient binding of telosome
proteins that regulate telomere length and stability and may lead
to telomere end fusions or deletions [51,52]. In addition damage in
telomeric DNA triggers the pro-inflammatory senescence associated secretory phenotype (SASP) which can lead to excessive
oxidative stress if senescence mechanisms malfunction [53]. When
lymphocytes are stimulated to divide ex vivo, as in the L-CBMN
assay, these persistent telomere lesions may result in the
formation of NPBs. When the NPBs break, MNi and NBUDs may
be formed and become evident during late telophase in the
binucleated cell stage or after nuclear division is completed
[27,54,55].
Chemically-induced unrepaired base lesions persisting in
lymphocytes in vivo may cause replication stress during nuclear
division ex vivo leading to a single strand break (SSB) formation
during S phase and an achromatic gap in the chromosome that,
however, may not result in MN formation in the first division cycle
[12,13,28,29]. Nevertheless, as Bender et al. [28,29] suggested in
the early 19700 s, single strand nucleases (SSN) may be involved
together with glycosylases in base excision repair [56–59] in
excising the chemically-induced adduct and nicking the resulting
single strand leading to a SSB during G1 phase. The SSB is then
converted to a DSB during S phase causing the formation of an
acentric chromatid fragment which could result in MN formation
during anaphase/telophase. In addition, single strand exchange
events may also result in the conversion of the resulting DSBs on
one chromatid to the formation of a recombined isochromatid
deletion which will also lag behind at anaphase and result in MN
formation. These mechanisms, explained diagrammatically in
Fig. 5a, also indicate how MN may arise from the resulting
chromatid breaks. Chemically-induced SSBs caused by replication
stress in vivo can also convert to DSBs during S-phase ex vivo and
form acentric chromatid fragments and isochromatid deletions
which result in MN formation in cytokinesis-blocked BN cells in
the ex vivo L-CBMN assay (Fig. 5b). Furthermore, it was shown that
simultaneous excision of adducts on opposite strands of DNA
within 12 bases of each other can also generate DSBs [60] from
which MN may originate. Conversion of SSBs to DSBs can be
M. Fenech et al. / Mutation Research 770 (2016) 12–25
19
Fig. 5. (a) Mechanisms by which unrepaired base lesions induced in vivo in G0 can be converted to acentric chromatid fragments from which micronuclei and other nuclear
anomalies may originate ex vivo after one mitosis. SSN = single strand nuclease; SSE = single strand exchange;? = plausible but insufficient evidence. Diagram adapted from
original Fig. 1 in Bender et al. [29]. Permission to re-use and adapt the figure was obtained from publisher Elsevier. (b) Mechanisms by which unrepaired base lesions induced
in vivo in G2 can be converted to acentric chromatid fragments from which micronuclei and other nuclear anomalies may originate ex vivo after one mitosis. SSN = single
strand nuclease; SSE = single strand exchange;? = plausible but insufficient evidence. Diagram adapted from original Fig. 3 in Bender et al. [29]. Permission to re-use and adapt
the figure was obtained from publisher Elsevier.
induced to occur ex vivo/in vitro in all stages of the cell cycle, during
the long patch repair of SSBs, by using single strand specific
endonucleases such as Neurospora endonuclease [61,62].
Conversion of excision-repairable DNA adducts induced in G0
phase to MNi can be achieved within one cell cycle. This was shown
by using a modification of the L-CBMN assay in which mitogenstimulated lymphocytes were treated with cytosine arabinoside
(ARA-C) which inhibits the gap-filling step of the excision repair
process during G1 [63,64] (Fig. 6). It is essential to wash out ARA-C,
an analogue of cytidine, prior to the onset of S-phase before
proceeding with the L-CBMN assay. Using this procedure excision
repaired sites are efficiently converted to SSBs prior to S-phase and
into DSBs and MN after S-phase. In vitro studies showed between a
1.8 fold and up to 10-fold or more increase in the slope of the MN
dose-response effect for different genotoxic agents that induce
excision-repairable DNA lesions in binucleated cells; the ARA-C/LCBMN assay also enhanced the capacity to detect spontaneous
DNA damage ex vivo although with reduced efficacy in older
subjects possibly due to a decline of excision repair capacity with
age [63–65].
In the absence of ARA-C treatment during G1 phase ex vivo it
will only possible for an unrepaired DNA adduct to be converted
into an MN after two or more divisions ex vivo because a first
mitosis is needed to cause replication stress and a SSB and a second
mitosis is required to convert the SSB to a DSB and chromatid
acentric fragment from which an MN may arise. For these reasons a
protocol allowing more than one division ex vivo may enable the
full complement of chemically induced DNA damage to be
expressed as MN (Fig. 7). However, this approach, remains
untested.
In addition, it is already well-established that certain chemicals
may cause chromosome malsegregations and numerical chromosome aberrations by disrupting the formation of the mitotic
spindle, damage to centrosomes, centromeres and kinetochores or
by disabling the processes by which chromatids align on the
20
M. Fenech et al. / Mutation Research 770 (2016) 12–25
Fig. 6. Mechanism by which an excision-repairable DNA adduct induced in vivo in a peripheral blood lymphocyte can be converted to a single strand DNA break (SSB) ex vivo
using cytosine arabinoside (ARA-C) and the conversion of the SSB to a double stranded DNA break (DSB) leading to the formation of an acentric chromosome fragment from
which a micronucleus is formed.
spindle or separate from each other during anaphase [4,36,37]. The
encapsulation of a mal-segregated whole chromosome within a
MN can itself lead to massive numbers of breaks within that
chromosome because of incomplete DNA synthesis and inefficient
joining of Okazaki DNA fragments due to either (i) lack of
synchrony between the DNA replication cycle in the main nucleus
and MNi and/or (ii) dysfunctional uptake of DNA replication
enzymes and cofactors into MNi, both of which might be affected
by the extent of expression of lamin A and lamin B in the
micronucleus membrane [66–68]. This means that MNi containing
whole chromosomes, that are already present within lymphocytes
in vivo, may lead to further chromatid and chromosome aberrations when the cell divides ex vivo. This is known as the process of
chromoanagenesis and chromothripsis and has been identified as
one of the most rapid and devastating mutational mechanism
within single chromosomes [66,67,69].
5. Kinetics of MNi expression in lymphocytes in vivo and ex vivo
in the L-CBMN assay after acute or chronic genotoxin exposure
The kinetics of MNi expression in lymphocytes in the bonemarrow and lymphocytes in blood in vivo and ex vivo is expected to
be different between acute and chronic in vivo exposures. In the
case of acute exposure to chemicals that rapidly reach the bonemarrow one can expect induction and expression of MNi in the
dividing population of stem cells and other precursor cells within
24 h of exposure because this will allow one nuclear division and
MNi expression to occur. However this period will not allow
sufficient time for maturation of the daughter lymphocytes and
their emergence into the blood stream. Mature lymphocytes in the
blood stream will experience DNA damage from acute chemical
genotoxin exposure and such damage may persist depending on
the time frame between exposure and blood collection and the
extent to which the DNA lesions are repaired or mis-repaired while
the cell is in G0 phase. Persistent DNA lesions may convert to MNi
ex vivo in the L-CBMN assay via mechanisms described above or
possibly be eliminated by apoptosis. The final result will depend on
the type of induced DNA damage profile. This is explained in
Fig. 8a.
In the case of chronic (continuous) exposure the situation is less
complex because one can expect, a steady-state situation to occur
with regards to the rate of MNi being induced in the bone-marrow,
the emergence of lymphocytes with MNi into the bloodstream and
the propensity of persistent DNA damage in peripheral blood
lymphocytes to be retained and ultimately expressed ex vivo in the
L-CBMN assay [70] (Fig. 8b). Results from studies in children
exposed to radioactive isotopes from the Chernobyl disaster
showed a good correlation between lymphocyte MNi formed in
vivo in mononucleated cells and MNi expressed ex vivo in
binucleated cells in the L-CBMN assay with similar efficacy of
detecting the genotoxic effects of chronic exposure to radioisotopes [71]. Furthermore, concordance for association with lung
cancer in smokers or exposure to asbestos was also observed for
MNi in mononucleated and binucleated cells in the L-CBMN assay
[72,73]. Discordance between MNi expressed in vivo in mononucleated cells and MNi expressed ex vivo in binucleated cells in the
L-CBMN assay might be expected if the chemical genotoxin is
highly reactive and as a consequence only affects peripheral blood
lymphocytes but does not reach the dividing precursor cells in the
bone marrow.
In vitro modelling experiments of long-term lymphocyte
cultures with either chronic folate deficiency (a model of
replication stress) or chronic exposure to alcohol indicate that it
takes at least 7–14 days of culture before a steady state level of MNi
expression becomes apparent [74–77]. If the same situation
applies in vivo it has important implications with regards to the
timing of blood sample collection if one is measuring MNi already
expressed in vivo in lymphocytes. The time-frame required to
achieve a steady state rate of expression of MNi would be
additional to that required for nascent mature lymphocytes in the
M. Fenech et al. / Mutation Research 770 (2016) 12–25
21
Fig. 7. Mechanism by which a unrepaired DNA adduct occurring in vivo in a lymphocyte may cause replication stress induction of a single strand break (SSB) in DNA after the
first mitosis ex vivo and the conversion of the SSB to a double strand break during a second mitosis ex vivo that results in the formation of an acentric chromosome fragment
that lags at anaphase and becomes a micronucleus.
bone-marrow to appear in the blood. It is evident that in the event
of acute exposures there will be a specific time-frame delay when
lymphocytes bearing MNi in vivo will appear and then disappear
depending on the proportion of dividing precursor cells in the bone
marrow and the rate of exit into the blood stream.
6. Current evidence that the L-CBMN assay is efficient in
detecting in vivo exposure to chemical genotoxins and its
concordance with other DNA damage biomarkers
A recurring question is whether the ex vivo component of the LCBMN assay correctly reflects the genome damaging effects of in
vivo exposure to genotoxic chemicals.
Occupational exposure studies (asbestos, smoking, styrene,
radioisotopes) in which MN were scored in both mononucleated
(in vivo induced) and binucleated lymphocytes (ex vivo induced)
showed good agreement with respect to increases in the exposed
group [71–73,78].
In other studies MN were scored both in dividing epithelial cells
in vivo (e.g. buccal cells, urothelial cells) and ex vivo in the L-CBMN
assay binucleated cells. Results form 19 occupational exposure
studies showed high concordance in MN frequency ratios in buccal
epithelial cells and in binucleated lymphocytes (correlation R
squared = 0.74, P < 0.0001) [79]. Another 11 studies showed high
concordance in MN frequency ratios in bladder epithelial
(urothelial) cells and in binucleated lymphocytes in the L-CBMN
assay (correlation R squared = 0.89, P < 0.0001) [80].
There are a number of plausible mechanisms that explain why
genome damage measured using the L-CBMN assay correlates well
with MN frequency in other tissues such as buccal or urothelial
epithelial cells as follows: (i) chemically-induced genome damage
in lymphocytes and epithelial cells may occur when chemical
genotoxins are transported via the peripheral blood stream to the
basal regenerative layers of epithelial tissues and/or (ii) chemical
genotoxins may also exert their genome–damaging effect by
penetrating the epithelial layers from the exterior (e.g. gut lumen,
oral cavity, bladder cavity) down to the basal layer, where nuclear
division and MN expression occurs, and ultimately may enter the
blood stream and damage DNA in lymphocytes given the close
proximity of the basal layers and the microvasculature. Lymphocytes in the peripheral blood may, therefore, experience the
genome damaging effects of chemical genotoxins both as the
chemicals are being transported in the blood to epithelial tissues as
well as via their absorption into the blood stream through the
epithelial tissues. An important difference which can modify
susceptibility to genome damage is that lymphocytes in the
peripheral blood are mainly in the Go phase of the cell cycle but the
epithelial basal cells may be at any stage of the cell cycle,
depending on the proliferative rate of the tissue. The relative
22
M. Fenech et al. / Mutation Research 770 (2016) 12–25
a
b
Fig. 8. (a) Kinetics of micronucleus formation in lymphocytes in vivo (in bone marrow, spleen or thymus), their migration to blood and ex vivo expression of DNA damage in
the L-CBMN assay. The example shown is that of an in vivo acute exposure to a genotoxin with sampling occurring in a time-frame shorter than the time it takes for the
damaged cells from the bone marrow, spleen and thymus to enter the blood stream. In this case MN induced in dividing lymphocytes in vivo will not be evident in the L-CBMN
assay which, therefore, in this case would only detect the conversion of in vivo DNA lesions in peripheral blood lymphocytes that lead to structural and numerical chromosome
aberrations in culture ex vivo and are expressed as micronuclei and other nuclear anomalies. (b) Kinetics of micronucleus formation in lymphocytes in vivo (in bone marrow,
spleen or thymus), their migration to peripheral blood and ex vivo expression of DNA damage in the L-CBMN assay. The example shown is that of an in vivo chronic exposure to
a genotoxin (over several days, weeks or months) which allows ample time for the damaged cells from the bone marrow, spleen and thymus to enter the blood stream. In this
case MN induced in dividing lymphocytes in vivo will be evident in the L-CBMN assay in mononucleated cells. The L-CBMN assay will also detect the conversion of in vivo DNA
lesions in peripheral blood lymphocytes that lead to structural and numerical chromosome aberrations in culture ex vivo and are expressed as micronuclei and other nuclear
anomalies.
efficiency with which MNi are expressed may also differ between
tissues depending on (i) the extent to which a chemical genotoxin
can induce defects in DNA or the mitotic machinery that cause MN
formation in lymphocytes (in vivo or ex vivo) and in the dividing
epithelial cells in vivo, (ii) the metabolic form and concentration of
the chemical genotoxin within the blood stream and the epithelial
M. Fenech et al. / Mutation Research 770 (2016) 12–25
tissue and (iii) differences between tissues with regards to their
intrinsic detoxification and DNA repair capacities.
These results together with a large body of other studies using
ex vivo cytogenetic assays in lymphocytes in which chromosome
aberrations, from which MN and NPB originate, were measured
provide robust evidence that the nuclear anomalies scored in the
L-CBMN assay in lymphocytes are sensitive enough to detect in
vivo exposure to genotoxic chemicals that also induce structural
and numerical chromosome aberrations. Comparison of the
L-CBMN assay and chromosome aberration assay methods, their
relative advantages, disadvantages, specificity and confounding
factors can be found in an earlier published review [81].
Other papers in this special issue provide further evidence of the
agreement of the L-CBMN assay with a wider range of DNA damage
biomarkers used to measure the genomic effects of exposure to
different classes of exogenous genotoxic chemicals in humans.
7. Important knowledge gaps regarding mechanisms by which
genotoxic chemicals induce MN in lymphocytes in vivo and ex
vivo
Despite several studies showing increases in chromosome
aberrations and MN in relation to in vivo exposure to chemical
genotoxins the vast array of man-made chemicals and their
interactions in the body raises the question whether current
knowledge is sufficient to explain all the mechanisms underlying
in vivo and ex vivo results with the L-CBMN assay. Furthermore
there are some important knowledge gaps as follows:
(i) Very little is known about the persistence of chemicallyinduced damage to proteins involved in the chromatid and
chromosome segregation machinery of G0 lymphocytes. The
possibility of differences between affect of mitotic machinery
poisons is suggested by the example of nocodazole and taxol,
both of which bind tubulins, but at different sites and with
different affinity and kinetics [82,83]. Studies are needed with
model aneugens to verify whether such chemicals can adduct
persistently to mitotic machinery proteins within lymphocytes in vivo and exert an effect ex vivo when the L-CBMN
assay is performed.
(ii) A key question relates to whether so-called S-phase dependent chemicals can induce chromosome aberrations ex vivo in
the first cell division cycle that would be expressed as MN or
other nuclear anomalies such as NPBs. In those cases where
DNA adducts persist through S-phase one can expect the
formation of SSBs due to stalling of the replication fork in the
case of bulky adducts but this may only lead to chromatid gap
formation which may not be converted into a MN in the first
division cycle ex vivo unless an ARA-C protocol is used. This
therefore raises the question whether an extension of the
L-CBMN assay protocol may be required to allow two S phases
to occur prior to cytokinesis-block and thus enable the
conversion of an SSB to a DSB and ultimately an acentric
fragment from which an MN may be formed.
(iii) Several studies have shown that the susceptibility to chemical
genotoxins is increased under conditions of deficiency of
micronutrients required for DNA synthesis and repair (e.g.
folate or zinc deficiency). However, the ex vivo L-CBMN assay
is performed with culture medium that is optimised for cell
growth and contains supra-physiological amounts of folate. It
is therefore important to explore whether sensitivity of the ex
vivo L-CBMN assay to in vivo chemical genotoxin exposure
may be increased if culture medium with physiological
concentration of micronutrients is used.
(iv) Because some agents have to be present during a specific
mitotic phase of the cell cycle (e.g. Camptothecin during
23
S-phase, Colcemid during metaphase) to exert their genotoxic
effects, their impact ex vivo in peripheral blood lymphocyte
cultures may be diminished because the blood is diluted 1:10
in the culture medium in the ex vivo L-CBMN assay. The extent
of this possibility leading to false negative results remains
unclear.
(v) In the large majority of biomonitoring studies applying the
L-CBMN assay an unexplained inter- and intra-individual
variability was observed in control and exposed groups
indicating that other confounding factors, apart from those
already known, still need to be identified and controlled in
order to be considered in the study design and/or accounted
for in the analysis of the results.
(vi) The current low level exposure to genotoxic agents in
occupational settings makes it even more difficult to trace
back the occurrence of chromosome damage and MNi
formation to a specific exposure. The simultaneous effects
of different low level exposures, the presence of interaction
with other agents, nutritional factors, and genetic susceptibility requires a more detailed assessment of exposure using
the new tools of exposomics. In this special issue a quality
score taking into account known variables was introduced to
start addressing whether, at least, the known variables (e.g.
age, gender, smoking, alcohol, chemical genotoxin exposure,
number of binucleated cells scored in the L-CBMN assay) were
properly controlled or measured.
(vii) Most of the data on chemicals are collected after exposure to
soluble chemicals, except asbestos. The exponential use and
release in the environment of nanomaterials which are often
poorly soluble and have specific physico-chemical characteristics and biokinetics address a new challenge, including
measuring the extent of exposure of the target cells, such as
lymphocytes. Adaptation of the ex vivo L-CBMN assay might
therefore be necessary to ascertain whether genotoxic effects
of nanoparticles were caused directly or indirectly. For
example, the feasibility of including measurements of nanoparticles within the same lymphocytes used for the CBMN
assay should be explored.
8. Conclusion
There are several plausible generic mechanisms by which
exposure to genotoxic chemicals in vivo may, directly or indirectly,
induce MNi in vivo or ex vivo in the L-CBMN assay. However, more
knowledge is required to unravel the unique mechanisms involved
for each type of chemical and chemical class. The use of molecular
probes to define the type and origin of MNi in the L-CBMN assay
(e.g. centromere positive or negative MNi) and associated gene
expression patterns should help to distinguish between the effects
of endogenous and exogenous chemical genotoxins. Increasingly
more rigorous study designs in population monitoring are
recommended to control for all known variables that affect MNi
expression and to make more certain the attribution of an increase
of MNi frequency to specific exogenous chemical genotoxin
exposures.
Acknowledgement
This review was written in our personal time and was not
supported by funding from government or private institutions.
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