10.14639/0392-100X-N0490
The aim of the study is to determine and compare micronucleus (MN), nucleoplasmic bridge (NPB) and nuclear bud (NBUD) frequencies in patients with laryngeal carcinoma and healthy controls. The study was conducted in the School of Medicine of Onsekiz Mart University. A total of 102 volunteers, 51 of whom had laryngeal carcinoma and 51 of whom were healthy control subjects, participated in this study. The Cytokinesis- Block Micronucleus Assay (CBMN) was applied to peripheral blood lymphocytes taken from patients and controls. We evaluated MN, NPBs and NBUDs frequencies in patients with laryngeal carcinoma and compared the results with those in the control group. The frequencies of MN, NPBs and NBUDs of patients with laryngeal carcinoma were found significantly higher than those in the control group (P = 0.01, P = 0.004, P = 0.01, respectively). MN, NPB and NBUD frequencies were also compared in the patients with and without pesticide exposure, and the means of all frequencies was higher in patients with pesticide exposure (P = 0.001, P = 0.02 respectively). The MN, NPBs and NBUDs frequencies of the patients with laryngeal cancer were significantly higher than those of the control group, and pesticide exposure might be a risk factor that increases genomic instability and risk of laryngeal cancer.
application/xml
1827-675X
490
6
en
Sefa Dereköy
https://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
frequency
Micronucleus, nucleoplasmic bridge and nuclear bud frequencies in patients with laryngeal carcinoma
Text.Serial.Journal
https://www.actaitalica.it/
40
Laryngology
2021-01-21
2019-09-30
2021-01-21
2021-01-21
1
Sefa Dereköy
2020
10.14639/0392-100X-N0490
Deparment of Otorhinolaryngology, Çanakkale Onsekiz Mart University, Faculty of Medicine, Çanakkale, Turkey
1827-675X
6
frequency
Micronucleus, nucleoplasmic bridge and nuclear bud frequencies in patients with laryngeal carcinoma
https://www.actaitalica.it/article/download/490/492
40
ACTA OTORHINOLARYNGOLOGICA ITALICA 2020;40:410-414; doi: 10.14639/0392-100X-N0490




Laryngology


Micronucleus, nucleoplasmic bridge and nuclear bud
frequencies in patients with laryngeal carcinoma
Micronucleo, ponte nucleoplasmatico e frequenze di gemme nucleari in pazienti
con carcinoma laringeo
Ibrahim Yazici1, Ozge Caglar2, Oguz Guclu2, Hayal Cobanoglu3, Mahmut Coskun4, Münevver Coskun3, Aytac Kilic2,
Fevzi Sefa Dereköy2
1
 Deparment of Otorhinolaryngology, Ezine State Hospital Çanakkale, Turkey; 2 Deparment of Otorhinolaryngology, Çanakkale Onsekiz
Mart University, Faculty of Medicine, Çanakkale, Turkey; 3 Çanakkale Onsekiz Mart University, Health Services Vocational College,
Çanakkale, Turkey; 4 Çanakkale Onsekiz Mart University, Faculty of Medicine, Department of Medical Biology, Çanakkale, Turkey


SUMMARY
The aim of the study is to determine and compare micronucleus (MN), nucleoplasmic
bridge (NPB) and nuclear bud (NBUD) frequencies in patients with laryngeal carcinoma
and healthy controls. The study was conducted in the School of Medicine of Onsekiz
Mart University. A total of 102 volunteers, 51 of whom had laryngeal carcinoma and
51 of whom were healthy control subjects, participated in this study. The Cytokinesis-
Block Micronucleus Assay (CBMN) was applied to peripheral blood lymphocytes taken
from patients and controls. We evaluated MN, NPBs and NBUDs frequencies in patients                Received: September 30, 2019
with laryngeal carcinoma and compared the results with those in the control group. The             Accepted: May 5, 2020
frequencies of MN, NPBs and NBUDs of patients with laryngeal carcinoma were found
significantly higher than those in the control group (P = 0.01, P = 0.004, P = 0.01, re-           Correspondence
spectively). MN, NPB and NBUD frequencies were also compared in the patients with                  Ozge Caglar
and without pesticide exposure, and the means of all frequencies was higher in patients            Onsekiz Mart University, Faculty of Medicine,
with pesticide exposure (P = 0.001, P = 0.02 respectively). The MN, NPBs and NBUDs                 Department of Otorhinolaryngology, Canakkale-
frequencies of the patients with laryngeal cancer were significantly higher than those of          Turkey 09000
the control group, and pesticide exposure might be a risk factor that increases genomic            Tel. 05326364459
instability and risk of laryngeal cancer.                                                          E-mail: ozgecaglar@comu.edu.tr

KEY WORDS: micronucleus, nucleoplasmic bridge, nuclear bud, larynx cancer, frequency               Funding
                                                                                                   This work was supported by Çanakkale Onsekiz
RIASSUNTO                                                                                          Mart University The Scientific Research
                                                                                                   Coordination Unit, Project number: TTU-2014-197.
Lo scopo dello studio è quello di valutare e confrontare le frequenze relative alla presenza
di micronucleo (MN), ponte nucleoplasmatico (NPB) e gemma nucleare (NBUD) in pazien-
                                                                                                   Conflict of interest
ti con carcinoma laringeo e popolazione sana. Lo studio è stato condotto presso un istituto
                                                                                                   The Authors declare no conflict of interest.
universitario di medicina. Sono stati arruolati 102 volontari, 51 dei quali con carcinoma
laringeo e 51 controlli sani. Il metodo di Cytokinesis-Block Micronucleus Assay (CBMN) è
stato applicato ai linfociti del sangue periferico dei casi e dei controlli. Abbiamo valutato le     How to cite this article: Yazici I, Caglar O,
frequenze di MN, NPB e NBUD nel paziente con carcinoma laringeo e confrontato i risul-               Guclu O, et al. Micronucleus, nucleoplasmic
tati con il gruppo dei controlli. Le frequenze MN, NPB e NBUD dei pazienti con carcinoma             bridge and nuclear bud frequencies in pa-
laringeo sono risultate significativamente più alte rispetto al gruppo di controllo (P = 0,01,       tients with laryngeal carcinoma. Acta Otorhi-
                                                                                                     nolaryngol Ital 2020;40:410-414. https://doi.
P = 0,004, P = 0,01, rispettivamente). Nello studio, le frequenze MN, NPB e NBUD sono
                                                                                                     org/10.14639/0392-100X-N0490
state confrontate in pazienti con e senza esposizione ai pesticidi. I risultati hanno mostrato
che la media delle frequenze di NBP, MN e NBUD era significativamente più alta nei pa-
                                                                                                   © Società Italiana di Otorinolaringoiatria
zienti con esposizione ai pesticidi (P = 0,001, P = 0,02 rispettivamente). Il presente studio      e Chirurgia Cervico-Facciale
ha indicato che un aumento delle frequenze di MN, NPB e NBUD nei linfociti periferici
umani potrebbe essere utilizzato per prevedere il rischio individuale di sviluppare un car-                       OPEN ACCESS
cinoma laringeo. Inoltre, i nostri risultati indicano che l’esposizione ai pesticidi potrebbe
aumentare il rischio di carcinoma laringeo.                                                        This is an open access article distributed in accordance with
                                                                                                   the CC-BY-NC-ND (Creative Commons Attribution-Non-
PAROLE CHIAVE: micronucleo, ponte nucleoplasmatico, gemma nucleare, cancro alla                    Commercial-NoDerivatives 4.0 International) license. The
                                                                                                   article can be used by giving appropriate credit and mentio-
laringe, frequenza                                                                                 ning the license, but only for non-commercial purposes and
                                                                                                   only in the original version. For further information: https://
                                                                                                   creativecommons.org/licenses/by-nc-nd/4.0/deed.en




410
                                                                                                           DNA damage in laryngeal cancer




Introduction                                                     Methods
Laryngeal carcinoma (LC) comprises 30% of all head and           Population
neck malignancies 1. The incidence of LC is 5 times higher       A total of 102 volunteers, 51 with LC and 51 healthy control
in males than in females, although the incidence of LC in        subjects, participated in this study. LC patient files were re-
female patients has increased in recent years. This is re-       viewed. Patients’ age, gender, occupation, pesticide exposure,
lated to the increasing smoking rates in females and to the      clinical history, anatomical location of disease, smoking his-
increases in participation in work life which also elevates      tory, radiotherapy history, physical examination, and TNM
toxic exposure 2. Cancer rates increase after the fifth dec-     classifications were recorded (Tab. I). Cancers were staged ac-
ade of life and reache the highest rates in the seventh or       cording to the 2002 AJCC guidelines 17. All patients had under-
eighth decade of life. The incidence is 1% before the third      gone video laryngoscope examination. Lymph node positivity
decade 3. Squamous cell carcinoma comprises 85-90% of            was evaluated with both physical examination and ultrasonog-
laryngeal malignancies 4. To increase the disease-free life      raphy. The type of the treatment, recurrence and metastasis fre-
span, survival and organ protection rates in laryngeal carci-    quency were noted. Lymph node invasion and tumour grade
noma, better understanding of behaviours is needed.              were recorded as well. Pathology reports were evaluated. LC
Tumour stage, anatomical localisation, histopathological dif-    patients were divided based on anatomical location of disease
ferentiation and neck metastasis are general prognostic fac-     (supraglottic, glottic, subglottic), surgery type (partial, total lar-
tors: neck metastasis is accepted as a particularly poor prog-   yngectomy, microlaryngoscopic resection), radiotherapy his-
nostic sign 5. However, different tumour behaviours can be       tory and lymph node positivity of the neck. The control group
observed even in tumours with similar histopathological di-      was selected from healthy individuals who had no cancer cases
agnosis, grade and stage, leading investigators to study addi-   in their family and who were over 40 years of age. This re-
tional prognostic factors and conduct new molecular studies.     search was approved by the Ethics Committee of the Faculty
The accumulation of genetic damage leading to genomic            of Medicine. Informed consent was obtained from each pa-
instability is a recognised cause of cancer 6,7. Therefore, it   tient and control.
is important to measure the genetic damage 8. In human pe-
ripheral lymphocytes (PBL), the CBMN assay is one of the         Table I. Size and characteristics of the studied population.
standard cytogenetic tests for evaluation of genetic damage                                                    Case (%)       Control (%)
because of its sensitivity, reliablity and low cost. CBMN is      N                                             51               51
a comprehensive method that allows determination of mul-          Age
tiple markers such as MN, NPB and NBUD arising from               Mean ± sd                                  64 ± 8.3          51 ± 7
different genetic mechanisms 9,10. The mechanisms of MN           Range                                       38-81            41-67
are chromosome and/or chromatid breaks and mal-segre-             Gender
                                                                  Male                                       51 (100)         24 (47.1)
gated chromosomes. Dicentric chromosomes and gene                 Female                                        0             27 (52.9)
amplifications are the mechanisms of NPB and NBUD,                Smoking
respectively. MN and NBUD may sometimes occur via                 Yes                                        51 (100)         39 (76.0)
breakage of NPBs 9,11. These nuclear anomalies, which are         No                                            0             12 (24.0)
markers of genomic instability, are frequently seen in can-       Tumour stage                                                    -
cer 6. Numerous studies have validated that there is a posi-      T1                                         18 (35.3)
                                                                  T2                                         22 (43.1)
tive correlation between increased MN, NPB and NBUD               T3                                         9 (17.6)
frequencies in PBL and cancer risk 6,8,12-15.                     T4                                          2 (3.9)
The CBMN assay is a useful method to monitor genomic              Tumour Location                                                 -
instability, and many recent events reveal that the increased     Glottic                                    46 (90.2)
genomic instability in PBL might be an early event in car-        Supraglottic                                5 (9.8)
cinogenesis 6,16. To the best of the authors’ knowledge,          Lymph node metastasis                                           -
                                                                  Yes                                        11 (21.6)
there is no data about MN, NPB and NBUD frequencies in            No                                         40 (78.4)
patients with LC.
                                                                  Radiotherapy                                                    -
In the present study, we aimed to determine the frequencies of    Yes                                        24 (47.1)
MN, NPB and NBUD in PBL collected from individuals with           No                                         27 (52.9)
LC, who were diagnosed by the ear, nose and throat (ENT)          Occupational exposure to pesticide                              -
department, and healthy individuals who lived in Çanakkale,       Yes                                        24 (47.1)
Turkey. For this purpose, we carried out the CBMN assay.          No                                         27 (52.9)




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I. Yazici et al.




Chemicals                                                        Table II. Descriptive statistics of MN, NPB. and NBUDS values in cancer and
Chemicals used in the study were purchased from the fol-         control groups.
lowing suppliers; cytochalasin B, culture medium RPMI                                                  N      Mean ± SD (‰)           p
1640, foetal calf serum from Sigma (Germany), phytohae-           MN              Cancer group          51         28.1 ± 15.4           0.01
magglutinin from Biological Industries (Israel), Giemsa,                          Control group         51         13.9 ± 4.07
methanol, glacial acetic acid, potassium chloride from            NPB             Cancer group          51         1.31 ± 2.28           0.004
Merck (Germany).                                                                  Control group         51         0.29 ± 0.75
                                                                  NBUD            Cancer group          51         3.33 ± 3.60           0.01
Cytokinesis-block micronucleus assay                                              Control group         51         0.02 ± 0.14
Fenech’s method was used with minor modifications 10. A 5        MN: micronucleus; NPB: nucleoplasmic bridge; NBUD: nuclear bud; SD: standard
mL peripheral blood sample obtained from each donor tak-         deviation.
en in sterile heparinised tubes. A culture medium mixture,
including 4 mL medium (RPMI 1640), 1 mL serum (foetal            tively. In the control group, the MN, NPB and NBUD values
calf serum), 0.2 mL phytohemagglutinin (as a mitogen),           were 13.9, 0.29 and 0.02, respectively. The analyses showed
was prepared for each donor. 0.5 mL whole blood, taken           that there were significant differences between the control and
from each donor, was added to the medium. All cultures           the cancer groups in terms of MN, NPB and NBUDS values
were incubated at 37°C for 72 h. At hour 44, cytochalasin B      (p = 0.01, p = 0.004, p = 0.01, respectively).
(6 µg/L) was added to each culture to stop cytokinesis. Af-      The MN, NPB and NBUDS values were compared in the
ter 72 h, the cultures were harvested. At the harvest stage,     patients with and without radiotherapy treatment (Tab. III).
the cells were firstly treated with cold potassium chloride      The average MN, NBP and NBUD frequencies in the group
(0.075 mol/L) as a hypotonic solution and culture tubes          treated with radiotherapy were 32.8, 1.88 and 4.13, respec-
were centrifuged at 1000 rpm for 5 min; the supernatant          tively. In the group treated without radiotherapy, the aver-
was discarded. Secondly, the pellet was resuspended and          age MN, NBP and NBUD frequencies were 24.0, 0.81 and
fixed in methanol-acetic acid (7:1, v/v) three times. 5%         2.63, respectively. There were no significant differences
Giemsa was used to stain slides. We evaluated 1000 binu-         between the two groups in terms of MN, NPB and NBUD
cleated cells under a light microscope for each individual.      frequencies (p > 0.05).
The microscopic evaluation of the slides was performed at        The MN, NPB and NBUD frequencies were compared in
x1000 magnification according to Fenech’s criteria 18.           patients with and without pesticide exposure (Tab. IV). The
                                                                 results showed that the mean of MN, NBP and NBUD fre-
Statistical evaluation                                           quencies in the patients with pesticide exposure were high-
We used frequency distribution, one sample t test, one-way       er than in the patients without pesticide exposure. However,
analysis of variance (ANOVA) and correlation analysis. We        the differences in the median values of the MN and NBUD
used the SPSS (Statistical Package for the Social Sciences)      frequencies between the two groups were statistically sig-
10.0 statistical program to evaluate the data. The Kolmogorow-   nificant (p = 0.01, p = 0.02 respectively).
Smirnov goodness-of-fit test was used to check the normal dis-
tribution of data. The MN, NPB and NBUDS rates were com-
                                                                 Discussion
pared between LC patients and the control group. Results with
p < 0.05 were considered statistically significant.              LC comprises 25-30% of all head and neck malignancies,
                                                                 and is the most common cancer of the upper respiratory
Results
                                                                 Table III. Descriptive statistics of MN, NPB, NBUD in LC with and without
A total of 102 volunteers, 51 with LC diagnosis by the           radiotherapy treatment.
ENT department and 51 control subjects, participated in                               RT              N      Mean ± SD (‰)         p
the study. In the cancer group, all patients (n = 51) with LC     MN                 With RT            24         32.7 ± 20.6           0.06
were male, smokers, and the mean age was 64 (38-81). The                            Without RT          27         23.9 ± 6.5
tumour locations were 90.2% glottic and 9.8% supraglot-           NPB                With RT            24          1.8 ± 2.8            0.11
tic. In the control group, 47.1% were male, 52.9% were                              Without RT          27          0.8 ± 1.4
female and 76% were smokers.                                      NBUDS              With RT            24         4.13 ± 4.52           0.14
Table II shows the MN, NPB and NBUD values of the con-                              Without RT          27         2.63 ± 2.41
trol group and the cancer group. In the cancer group, the        MN: micronucleus; NPB: nucleoplasmic bridge; NBUD: nuclear bud; SD: standard
MN, NPB and NBUD values were 28.1, 1.31 and 3.33, respec-        deviation; RT: radiotherapy.




412
                                                                                                                    DNA damage in laryngeal cancer




Table IV. Descriptive statistics of MN, NPB and NBUD values according to         more common in men 2. In the present study, all the patients
pesticide use status of patients in the cancer group.                            with LC were men and smokers. The result is consistent
               Pesticide exposure          N        Mean ± SD      p             with previous findings investigating the relationship be-
 MN                     Yes               24          36.0 ± 17.7         0.01   tween smoking/gender and LC.
                        No                27           21.1 ± 8.4                Since the two groups did not match in terms of gender and
 NPB                    Yes               24          1.92 ± 2.82         0.09   smoking habit, we compared only males and smokers in
                        No                27          0.78 ± 1.52                both groups. The mean of the MN, NPB, and NBUD fre-
 NBUD                   Yes               24          4.63 ± 4.61         0.02   quencies for smokers were compared between the patient
                        No                27          2.19 ± 1.81                group (n = 51) and the control group (n = 39). The analyses
MN: micronucleus; NPB: nucleoplasmic bridge; NBUD: nuclear bud; SD: standard
                                                                                 demonstrated that the MN, NPB, and NBUD frequencies
deviation.                                                                       were significantly higher in the cancer group than the con-
                                                                                 trol group (p < 0.001, p < 0.001, p < 0.001 respectively).
tract 19,20. In the current study, we evaluated genomic dam-                     Similarly, the male controls (n = 24) and the male patients
age in patients with LC. For this purpose, the CBMN assay                        (n = 51) were compared in terms of MN, NPB, and NBUD
was used in PBL. CBMN is sensitive, reliable and has low                         values and a significant difference was found between the
cost. Moreover, it is a comprehensive and preferred method                       two groups (p < 0.001, p < 0.01, p < 0.001 respectively).
allowing assessment of chromosomal damage 9,10,18. In ge-                        As a result, gender and smoking habit were not found to be
netic toxicology, genetic damage at chromosome level is                          the confounding factors for the increase in MN, NPB, and
important because the mutation of a chromosome is a nota-                        NBUD frequencies.
ble event for carcinogenesis 18.                                                 There are many pesticides in the 2A (probably carcinogen-
The MN, NPB and NBUD frequencies were determined                                 ic to humans) and 2B (possibly carcinogenic to humans)
in the patient group, and compared with those of the con-                        lists of the IARC 25. Moreover, there have been relation-
trol group. The analyses demonstrated that there were sig-                       ships between many types of cancer and pesticide exposure
nificant differences between the control and cancer groups                       such as brain, leukaemia, kidney, lung and prostate can-
in terms of MN, NPB and NBUD frequencies (p = 0.01,                              cers 25. There are some studies on cancer risk in farmers
p = 0.004, p = 0.01, respectively). To our knowledge, there                      exposed to pesticides, which indicated a link between pes-
is no data related to genomic damage assessed in PBL in                          ticide exposure and increased risk of cancer 26,27. However,
patients having LC. However, there are many studies on                           only a few studies examined the relationship between pes-
genomic damage in different cancer types. The result on                          ticide exposure and head and neck cancers. These studies
the MN frequency obtained in the present study is consist-                       reported that pesticide exposure might be a risk factor for
ent with the results of other studies 8,12-15. There are only a                  head and neck cancers 28. In our study, approximately half
few studies evaluating the frequency of NPB, NBUD and                            of the patients with LC had previous pesticide exposure.
MN 21. Pardini 21 reported that MN and NBUD frequencies                          Therefore, we compared MN, NPB and NBUD frequencies
were significantly elevated in bladder cancer patients com-                      in patients with and without pesticide exposure. The results
pared to controls. Similarly, a significant relationship was                     showed that the mean of the NBP frequencies was higher
found in the present study. However, contrary to Pardini 21,                     and that the mean of the MN and NBUD frequencies were
NPB, MN and NBUD were all significantly increased in                             significantly higher in patients with pesticide exposure
the present study. NPB is an indicator of dicentric chro-                        (p = 0.001, p = 0.02 respectively). Therefore, our findings
mosomes in the anaphase stage of mitosis. It occurrs be-                         suggest that pesticide exposure might be a risk factor that
cause of misrepair of DNA breaks and/or telomere end fu-                         increases genomic instability and risk of LC.
sions. NBUDs caused by gene amplification and MN arise                           In the cancer group, the frequencies of MN, NPB, and
from chromosome fragments and/or whole chromosome,                               NBUD were compared between patients with and without
but MN and NBUD may sometimes occur via breakage of                              radiotherapy treatment. All biomarkers were found to be
NPBs. Our findings showed that MN, NPB, and NBUDs                                higher in patients with radiotherapy treatment, but none of
frequencies representing the above-mentioned genetic                             the increases was statistically significant (p > 0.05). To our
mechanisms were increased in the cancer group, which was                         knowledge, there is no data comparing genomic damage in
statistically significant.                                                       patients with and without radiotherapy treatment. However,
Many epidemiological and experimental studies 2,22,23 and                        in the literature, there are some studies evaluating genomic
the 2004 and 2010 monographs of the International Agency                         damage in cancer patients undergoing radiotherapy. These
for Research on Cancer (IARC) concluded that smoking                             studies showed that genomic damage increased significant-
habit is an important risk factor in cancer 24 and LC was                        ly in cancer patients undergoing radiotherapy 29,30.



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I. Yazici et al.




The main limitation of the present study was that it had a                     13
                                                                                    Çelik DA, Koşar PA, Özçelik N, et al. Cytogenetic finding of breast
small number of patients. For this reason, future studies                           cancer cases and in their first-degree relatives. J Breast Cancer
                                                                                    2013;16:285-90. https://doi.org/10.4048/jbc.2013.16.3.285.
should be designed with larger cohorts. Another limitation                     14
                                                                                    Devi SM, Balachandar V, Arun M, et al. Analysis of genetic damage
of the study was lack of information on pesticide exposure of                       and gene polymorphism in hepatocellular carcinoma (HCC) patients
patients with LC, such as what types of pesticides were used.                       in a South Indian population. Dig Dis Sci 2013;58:759-67. https://doi.
                                                                                    org/10.1007/s10620-012-2409-8
                                                                               15
                                                                                    Alfaisal AH, Al-Ramahi IJ, Abdul-Hassan IA, et al. Micronucleus
Conclusions                                                                         frequency among Iraqi thyroid disorder patients. Comp Clin Path
                                                                                    2014;23:683-8. https://doi.org/10.1007/s00580-012-1671-7
The MN, NPBs and NBUDs frequencies of patients with
                                                                               16
                                                                                    Bolognesi C, Filiberti R, Neri M, et al. High frequency of micronuclei
LC were significantly higher than those in the control group.
                                                                                    in peripheral blood lymphocytes as index of susceptibility to pleural
When we compared the genetic damage in the pesticide-                               malignant mesothelioma. Cancer Res 2002;62:5418-9
exposed and nonexposed patients with LC, genetic damage                        17
                                                                                    Greene FL, Page DL, Fleming ID, et al. AJCC cancer staging manual.
was significantly higher in the patients with LC who were                           USA: Springer; 2002.
exposed to pesticides. Our findings suggest that pesticide ex-                 18
                                                                                    Fenech M. The in vitro micronucleus technique. Mutat Res
posure might be a risk factor that increases genomic instabil-                      2000;455:81-95.
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                                                                                    Lu B, Li J, Gao Q, et al. Laryngeal cancer risk and common sin-
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future studies with a larger number of cases.                                       genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5 and XPA. Gene
                                                                                    2014;542:64-8. https://doi.org/10.1016/j.gene.2014.02.043
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     doi.org/10.1158/1055-9965                                                      org/10.1016/j.toxlet.2012.04.020




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