|Year : 2018 | Volume
| Issue : 3 | Page : 125-130
Oxidative DNA damage and RUNX1-RUNX1T1 translocation induced by cigarette smoking as a potential risk factor for leukemogenesis
Mohammed M El-Khawanky1, Ahmed M Solaiman1, Basel A Abdel-Wahab2
1 Department of Pathology, College of Medicine, Najran University, Najran; Department of Clinical Pathology, College of Medicine, Al-Azhar University, Assuit, Saudi Arabia
2 Department of Pathology, College of Medicine, Najran University, Najran; Department of Pharmacology, College of Medicine, Assiut University, Assiut, Saudi Arabia
|Date of Submission||04-Apr-2018|
|Date of Acceptance||27-Jun-2018|
|Date of Web Publication||3-Dec-2018|
Mohammed M El-Khawanky
7 El-Nady Street, Belbies, Sharkia, Egypt. 44622
Source of Support: None, Conflict of Interest: None
Background Cigarette smoking, one of the main causes of avertible morbidity and mortality, has a multitude of well-known side effects. Cigarette smoking contains large amounts of reactive oxygen species that induce oxidative DNA damage and increase the incidence of chromosomal aberrations and thus increases the incidence of oncogenesis.
Aim The aim of this work is to study the hematological effects of smoking and its ability to induce DNA damage and specific RUNX1-RUNX1T1 gene translocation.
Patients and methods The hematological studies and measurement of plasma concentration of 8-hydroxy-2’-deoxyguanosine (8-OHDG) (using enzyme-linked immunosorbent assay) and RUNX1-RUNX1T1 translocation t(8;21) (using reverse transcription-PCR) were conducted on two groups of participants one smoking and the other is a nonsmoking control group.
Results Smokers group showed a highly significant (P≤0.001) increase in plasma 8-OHDG and with a significant correlation between 8-OHDG and hemoglobin concentrations. In addition, the incidence of translocation(8;21) was 8.3% in the smokers’ group with obvious myelodysplasia of peripheral white blood cells was detected in 29.2% of smoking persons.
Keywords: 8-hydroxy-2’-deoxyguanosine, leukemogenesis, RUNX1-RUNX1T1, smoking, translocation(8, 21)
|How to cite this article:|
El-Khawanky MM, Solaiman AM, Abdel-Wahab BA. Oxidative DNA damage and RUNX1-RUNX1T1 translocation induced by cigarette smoking as a potential risk factor for leukemogenesis. Egypt J Haematol 2018;43:125-30
|How to cite this URL:|
El-Khawanky MM, Solaiman AM, Abdel-Wahab BA. Oxidative DNA damage and RUNX1-RUNX1T1 translocation induced by cigarette smoking as a potential risk factor for leukemogenesis. Egypt J Haematol [serial online] 2018 [cited 2019 Aug 23];43:125-30. Available from: http://www.ehj.eg.net/text.asp?2018/43/3/125/246776
| Introduction|| |
Cigarette smoking (CS) is a major public health problem, globally associated with extensive preventable morbidity . About 30% of the world’s population over the age of 15 years is smoking cigarettes;  most of them (94.9%) are men . Up to half of the current smokers will eventually die of a tobacco-related disease. Tobacco is an established factor that can cause carcinogenicity, in particular, acute myeloid leukemia (AML) .
Cigarette combustion produces a smoke with more than 4000 noxious components ; nevertheless, the underlying mechanisms for carcinogenicity are still indistinctly understood. One proposed mechanism is through oxidative DNA damage and gene alteration as cigarette smoke induces the formation of reactive oxygen species that can induce direct damage of the DNA, resulting in mutagenic and carcinogenic effects .
8-Hydroxy-2’-deoxyguanosine (8-OHDG) is one of the predominant forms of free radical-inducing oxidative DNA lesions. 8-OHDG is a resultant of DNA damage due to the hydroxyl radical attack at the C8 position of the nucleobase guanine. Such damage is usually successfully repaired by competent cells, but if unrepaired may contribute to mutagenicity and cancer promotion . According to previously conducted studies, it can be suggested that the concentration of 8-OHDG may be considered as a biomarker to assess the extent of oxidative DNA damage and an indicator to the liability of oncogenesis .
Many reports have shown that increased levels of chromosome aberrations in peripheral blood cells were associated with subsequent increased cancer risk, especially for hematological malignancies , particularly if chromosome 21 is involved . One of the reported gene translocations among cigarette smokers was RUNX1-RUNX1T1 (AML1-ETO) in the translocation (8;21) [t(8;21)] (q22;q22) translocation, which is considered a smoking-associated risk factor for AML . Moreover, it was suggested that prenatal parental smoking is a possible risk factor for childhood leukemia, mainly AML .
In this study, we investigated the oxidative stress marker ‘8-OHDG’ in the plasma, t(8;21) in peripheral mononuclear cells, and the presence of myelodysplastic features in the peripheral blood cells resultant of CS as a risk factor for leukemia incidence.
| Patients and methods|| |
This study was carried out on 43 patients who were classified into two groups: group I included 19 apparently healthy individuals non-CS, whose ages ranged from 23 to 60 years, and group II that included 24 cigarette smokers, whose ages ranged from 23 to 63 years. The study was approved by the medical ethics committee, and informed consents were obtained from the patients to participate in the study.
The study groups were subjected to full history taking filled in a questionnaire with a signed consent, complete blood count using automatic cell counter Sysmex KX-21 (Sysmex KX-21, Hematology Analyzer, Kobe, Japan), examination of peripheral blood smears, measurement of plasma 8-OHDG concentration, and detection of t(8;21).
Specimen collection and preparation
Four milliliters of blood was collected aseptically by venipuncture from all the studied groups. Two milliliters was added to a sterile tube containing EDTA (1.5 mg EDTA/1 ml blood) for complete blood count and for blood smear examination. Two milliliters of blood was divided into two separate sterile EDTA-containing tubes, 1 ml in each tube. One tube for measuring the plasma concentration of 8-OHDG and the other to detect t(8;21).
Specific laboratory investigations
Plasma 8-OHDG was detected by the enzyme-linked immunosorbent assay system TecanSpectra (TecanSpectra II Microplate Reader, Austria) using StressXpress 8-OHDG-enzyme immunoassay (EIA) kit (SKT120). In this assay, 8-OHDG (EIA) was a competitive assay. The EIA utilized an anti-mouse IgG-coated plate and a tracer consisting of an 8-OHDG-enzyme conjugate. This format has the advantage of providing low variability and increased sensitivity.
RUNX1-RUNX1T1 ‘t(8;21)’ was detected by qualitative reverse transcription-PCR using the HemaVision-8;21 kit Catalog# KS-2010050, innuPREP Blood RNA Kit (Catalog# KS-2010050, innuPREP Blood RNA Kit, DNA Diagnostic, Risskov, Denmark). The principle of this procedure had four phases: (a) RNA extraction from the whole blood providing the template for the synthesis of cDNA. (b) cDNA synthesis by using the cDNA primer mix and reverse transcriptase. (c) PCR amplification of the resultant cDNA using the PCR primer mix. (d) Detection of PCR products by using horizontal agarose gel electrophoresis that was visualized by staining with ethidium bromide and was detected under ultraviolet transilluminator.
The collected data were revised, coded, tabulated, and analyzed statistically using the SPSS statistical software (version 20; IBM SPSS Inc., Chicago, Illinois, USA). Data were presented, and suitable analyses were carried out according to the type of data obtained for each parameter. Statistical difference was considered significant if the P value was less than 0.05 and highly significant if the P value was less than 0.001.
| Results|| |
This study encompassed two groups: group I, which included 19 apparently healthy individuals nonsmokers, their ages ranged from 23 to 60 years with a mean±SD of 36.84±10.34 years; they comprised 63% (12/19) men and 27% (7/19) women. The other group (group II) included 24 cigarette smokers, their ages ranged from 23 to 63 years with mean±SD of 42.25±11.46 years; they comprised 75% (18/24) men and 25% (6/24) women ([Table 1]).
|Table 1 Descriptive analysis of the demographic and laboratory characteristics of normal nonsmoking (group I) and smoking (group II) patients|
Click here to view
The oxidative DNA stress marker, 8-OHDG showed highly significant (P≤0.001) increased levels in the smokers’ group when compared with the control group. The mean±SD levels of 8-OHDG were 27.8±7.7, 12.5±3.1 pg/ml, respectively ([Figure 1]a, [Table 2]).
|Figure 1 These graphs show plasma 8-OHDG and SI levels among different groups: (a) There is a highly significant increase of 8-OHDG in the smokers’ group ((P≤0.001). (b) 8-OHDG in the control group shows more increased levels in men than women. (c) According to the sex, 8-OHDG levels in the smokers’ group shows more increased 8-OHDG levels in men. (d) Smokers with positive t(8;21) have higher levels of SI. 8-OHDG, 8-hydroxy-2’-deoxyguanosine; SI, smoking index; t(8;21), translocation (8;21).|
Click here to view
|Table 2 Changes in plasma 8-hydroxy-2’-deoxyguanosine level and percentage of translocation (8:21) in normal nonsmoking (group I) and smoking (group II) individuals|
Click here to view
Statistically, there was no significant difference in the 8-OHDG plasma level between men and women of neither the control group nor the smokers’ group. Nevertheless, the mean level of 8-OHDG in the control group was higher in men (13.18±3.5 pg/ml) than in women (11.414±2.1 pg/ml); moreover, in the smokers’ group, the mean levels of 8-OHDG were 29.072±8.5, 23.867±2.4 pg/ml, respectively ([Figure 1]b and c).
In the smokers’ group, the smoking index (SI) ranged between 180 and 1760 with a mean±SD of 717.1±469.3. SI was calculated as the number of smoked cigarettes/day×number of smoking years. The SI did not show any significant correlation with 8-OHDG levels (P=0.175) nor age or any of the blood parameters except with the red blood cell count, which showed a highly significant positive correlation (P=0.003, r=0.573) ([Figure 2]a). In addition, there was a highly significant positive correlation (P=0.009, r=0.521) between 8-OHDG levels and hemoglobin concentrations ([Figure 2]b).
|Figure 2 Positive correlations in the smokers’ group. (a) There is a highly positive correlation between SI and RBC count (P=0.003, r=0.573). (b) There is a positive correlation between 8-OHDG levels and hemoglobin concentrations in the smokers’ group (r=0.521, P=.009). 8-OHDG, 8-hydroxyl-2-deoxyguanosine; HGB, hemoglobin; RBC, red blood cell; SI, smoking index.|
Click here to view
In the control group, 8-OHDG did not show any significant correlation with age or any of the blood parameters.
The incidence of RUNX1-RUNX1T1 ‘t(8;21) (q22;q22)’ in the cigarette smokers’ group comprised 8.3% (2/24) ([Figure 3]), while in the nonsmokers’ group all individuals were negative for this translocation ([Table 2]).
|Figure 3 Agarose gel electrophoresis plate showing a positive case for t(8;21). t(8;21), translocation (8;21).|
Click here to view
The positive cases for RUNX1-RUNX1T1 translocation were of heavy smoking persons as there was a highly significant (P=0.005) increase in SI among smokers positive to t(8;21). The smoking indices among positive and negative t(8;21) persons were 922.5±3.536, 374.76±498.67, respectively ([Figure 1]d). Furthermore, 8-OHDG levels in smokers positive to t(8;21) tend to be higher in comparison with negative cases in the smokers’ group. The 8-OHDG among positive and negative cases were 17.35±0.07and 21.2±9.97 pg/ml, respectively.
Peripheral blood cells of the smokers’ group exhibited some morphological and structural alterations identified as myelodysplastic features. These altered features appeared in the form of hypogranular and/or hypolobulated neutrophil, giant platelets, vacuolated, and/or hypogranular monocytes ([Figure 4]). These myelodysplastic features were encountered in 29.2% (7/24) of smoking persons.
|Figure 4 Dysplastic features of peripheral white blood cells: (a) neutrophil with a bilobed nucleus, (b) monocytes with vacuoles in the cytoplasm, and (c) giant platelet.|
Click here to view
| Discussion|| |
CS is known for its deleterious effects on many systems and organs . In comparison with the research on the relationship of smoking and other organ systems, relatively little research has been performed with the aim of studying the effects of smoking on the possibility of DNA damage and translocation as possible changes that can contribute to leukemogenicity. The present work studied the harmful effects of CS through the measurement of 8-OHDG levels in the plasma and detection of t(8;21) in mononuclear white blood cells, considering the effect of CS on the morphology of peripheral blood cells.
Normally, each human cell metabolizes ∼1012 molecules of oxygen per day, resulting in the generation of radical oxygen species causing an oxidative DNA damage in the form of 8-OHDG . There is a balance maintained between endogenous oxidants and antioxidant defenses. When an imbalance occurs, extensive oxidative damage to the DNA occurs . It is established that CS increases DNA damage and reduces its repair .
We have reported highly significant (P=0.000) increased levels of oxidative DNA damage marker (8-OHDG) in the smokers’ group when compared with the nonsmokers’ (control) group. The mean±SD levels of 8-OHDG were 27.8±7.7, 12.5±3.1 pg/ml, respectively.
It has been established that ‘8-OHDG’ is an important biomarker to assess the risk to cancer after exposure to various carcinogenic substances and environmental pollutants, by this CS could be implied .
Radical oxygen species attack not only the DNA bases but also other cellular components such as lipids that in turn can couple to DNA bases and leave behind gene alterations, which represents the first step of carcinogenesis . Different mechanisms are thought to play a role in the development of carcinogenesis, through modulation of gene expression, chromosomal rearrangements , stimulation of protein kinase, and signal transduction pathways, moreover, and their ability to induce DNA base changes in certain oncogenes and tumor suppressor genes .
In the smokers’ group; the SI ‘the result of multiplying the number of cigarettes that smoked daily by the number of smoking years’ showed a significant positive correlation with red blood cells count and tended to be positively correlated with 8-OHDG levels. Several studies have confirmed that plasma 8-OHDG levels were or tended to be positively correlated with the number of cigarettes smoked per day when adjusted for the confounding factors . In contrast, other studies have shown negative correlations between passive smoking and 8-OHDG levels . Although the reason for this discrepancy is unknown, the grade of the cigarette filter or differences in the quality of cigarettes among countries might be a factor.
Our study identified the presence of RUNX1-RUNX1T1 ‘t(8;21) (q22;q22) ’ in smokers’ peripheral mononuclear white blood cells, the fact that was not present in the nonsmoking persons ‘control group.’ Structural chromosome aberrations have long been known to be widely present in tumor cells . Gene alterations in peripheral blood lymphocytes of healthy individuals are significantly associated with increased cancer risks. In our results, the incidence of t(8;21) was 8.3% (2/24) among the smokers’ group, which has been implicated in leukemogenesis .
Translocation frequencies have been shown to be elevated in smokers compared with nonsmokers, although other studies have not observed such an association . t(8;21) is frequently observed in AML, acute lymphoblastic leukemia, chronic myelomonocytic leukemia, and in myelodysplastic syndrome .
The mechanism by which tobacco smoking exposure leads to increased levels of translocations between chromosomes 8 and 21 is not clear. One possible mechanism may be related to topoisomerase II inhibition, as it was observed that t(8;21) exhibited a clonal abnormality associated with AML caused by topoisomerase-inhibiting drugs . This rearrangement ‘t(8;21)’ generates the transcriptional active fusion gene 5’RUNX1/3’RUNX1T1. The leukemogenic potential of this fusion gene is being established .
Normally, during embryogenesis, hematopoietic development consists of two distinct waves of discrete cellular components. The first wave is primitive hematopoiesis, which is followed by definitive hematopoiesis as the second wave, at which ‘second wave’ RUNX1 has an essential role .
t(8;21) encoding the chimeric RUNX1/RUNX1T1 transcription factor represses the expression of RUNX1 target genes, such as the nuclear receptor corepressor (NCOR1), histone deacetylase (HDAC1), and SIN3A/HDAC, which block hematopoietic differentiation . Moreover, this chimeric RUNX1/RUNX1T1 transcription factor inhibits transcriptional activation by dysregulation of E proteins which are crucial for the commitment of blood cell progenitors and considered an important step in t(8;21) leukemogenesis . RUNX1/RUNX1T1 expression upregulated NTRK1, which in normal cells regulate cell differentiation and silences AML1and target hematopoietic genes through directly recruiting the HDAC complex to their promoters disrupting gene transcription impeding cell differentiation, and hence leukemia occurs .Dysregulation of tumor suppressors has been observed in RUNX1-RUNX1T1 leukemia. The pro-apoptotic p53-stabilizing p14ARF (CDKN2A), NF1, and silencing of miR-9-170 expression were transcriptionally repressed through dominant inhibition of RUNX1 function ,. Several studies supported our results and reported that smoking was significantly associated with cytogenetic abnormalities t(8;21) as a risk factor for AML occurrence ,.
Myelodysplasia of the peripheral white blood cells was one of the most prominent features associated with CS in 29.2% (7/24) of cases in the form of hypogranular and or hypolobulated neutrophil, giant platelet, vacuolated, and/or hypogranular monocytes. Cigarette smokers were shown to have a 45% higher risk of developing myelodysplastic syndrome than never smokers , as tobacco products can induce chromosomal aberrations in primitive bone marrow cells to differentiate into morphologically and functionally abnormal myeloid lineages .
| Conclusion|| |
CS has deleterious effects on blood cells and increases both the oxidative stress biomarker (8-OHDG) level and the incidence of t(8;21), consequently increasing the risk for oncogenesis particularly t(8;21)-positive leukemia.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Mathers CD, Loncar D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med
Kulikowska-Karpińska E, Czerw K. Estimation of 8-hydroxy-2’-deoxyguanosine (8-OHDG) concentration in the urine of cigarette smokers. Wiad Lek
Kovac JR, Khanna A, Lipshultz LI. The effects of cigarette smoking on male fertility. Postgrad Med
Wang P, Liu H, Jiang T, Yang J. Cigarette smoking and the risk of adult myeloid disease: a meta-analysis. PLoS ONE
EPA. EPA Report/600/6-90/006F: respiratory health effects of passive smoking: lung cancer and other disorders
. Washington, DC; EPA; 1992.
El-khawanky MM, Abdel-Wahab BA, Metwally ME, Hashem AM. Benzene induced hematotoxicity and DNA damage in rats. Nat Sci
Hasselbalch HC. Smoking as a contributing factor for development of polycythemia vera and related neoplasms. Leuk Res
:1137–1145. pii: S0145-2126(15)30373-8.
Bonassi S, El-Zein R, Bolognesi C, Fenech M. Micronuclei frequency in peripheral blood lymphocytes and cancer risk: evidence from human studies, Mutagenesis
Yu HZ, Cheng Z, Xie SS, Yi YF, Peng HL. Analysis of clinical characteristics in 31 acute myeloid leukemia patients with chromosome 21 aberrations. Zhongguo Shi Yan Xue Ye Xue Za Zhi
Metayer C, Zhang L, Wiemels JL, Bartley K, Schiffman J, Ma X et al.
Tobacco smoke exposure and the risk of childhood acute lymphoblastic and myeloid leukemias by cytogenetic subtype. Cancer Epidemiol Biomarkers Prev
Metayer C, Petridou E, Aranguré JM, Roman E, Schüz J, Magnani C et al.
Parental tobacco smoking and acute myeloid leukemia: the childhood leukemia international consortium. Am J Epidemiol
Li W, Zhou J, Chen Y, Zhang G, Jiang P, Hong L et al.
Cigarette smoke enhances initiation and progression of lung cancer by mutating Notch1/2 and dysregulating downstream signaling molecules. Oncotarget
Beckman B, Ames N. Oxidative decay of DNA. J Biol Chem
Lee HW, Park S-H, Weng M-W, Wang H-T, Huang WC, Lepor H et al.
E-cig damages DNA in lung, heart, and bladder. Proceedings of the National Academy of Sciences USA
; January 2018, 201718185
Nilsson R, Nordlinder R, Moen E, Øvrebø S, Bleie K, Skorve A et al.
Increased urinary excretion of 8-hydroxydeoxyguanosine in engine room personnel exposed to polycyclic aromatic hydrocarbons. Occup Environ Med
Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem
Crawford DR, Edbauer-Nechamen CA, Schools GP, Salmon SL, Davies JMS, Davies KJA. Oxidant-modulated gene expression. In: Davies KJA, Ursini F, editors. The Oxygen Paradox
. Padova, Italy: Cleup University Press; 1995. 327–335.
Chen H, Liou S, Ho S, Wu K, Sun C, Chen MF et al.
Oxidative DNA damage estimated by plasma 8-hydroxydeoxyguanosine (8-OHDG): influence of 4,4-methylenebis (2-chloroaniline) exposure and smoking. J Occup Health
Smith J, Fischer H, Heavner L, Rumple A, Bowman DL, Brown BG et al.
Urinary thromboxane, prostacyclin, cortisol, and 8-hydroxy-2’-deoxyguanosine in nonsmokers exposed and not exposed to environmental tobacco smoke. Toxicol Sci
Rossner P, Boffetta P, Ceppi M, Bonassi S, Smerhovsky Z, Landa K et al.
Chromosomal aberrations in lymphocytes of healthy subjects and risk of cancer. Environ Health Perspect
Whitehouse CA, Edwards AA, Tawn EJ, Stephan G, Oestreicher U, Moquet JE et al.
Translocation yields in peripheral blood lymphocytes from control populations. Int J Radiat Biol
Sood R, Kamikubo Y, Liu P. Role of RUNX1 in hematological malignancies. Blood
Mondrala S, Eastmond DA. Topoisomerase II inhibition by the bioactivated benzene metabolite hydroquinone involves multiple mechanisms. Chem Biol Interact
Byrd JC, Mrozek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC et al.
Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood
Yamagata T, Maki K, Mitani K. Runx1/AML1 in normal and abnormal hematopoiesis. Int J Hematol
Elagib KE, Goldfarb AN. Oncogenic pathways of AML1-ETO in acute myeloid leukemia: multifaceted manipulation of marrow maturation. Cancer Lett
Zhang J, Kalkum M, Yamamura S, Chait BT, Roeder RG. E protein silencing by the leukemogenic AML1-ETO fusion protein. Science
Mulloy JC, Jankovic V, Wunderlich M, Delwel R, Cammenga J, Krejci O et al.
AML1-ETO fusion protein up-regulates TRKA mRNA expression in human CD34(+) cells, allowing nerve growth factor-induced expansion. Proc Nat Acad Sci
Fu L, Shi J, Liu A, Zhou L, Jiang M, Fu H et al.
A minicircuitry of microRNA-9-1 and RUNX1-RUNX1T1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia. Int J Cancer
Strom SS, Oum R, Elhor Gbito KY, Garcia-Manero G, Yamamura Y. De novo acute myeloid leukemia risk factors: a Texas case-control study. Cancer
Tong H, Hu C, Yin X, Yu M, Yang J, Jin J. A meta-analysis of the relationship between cigarette smoking and incidence of myelodysplastic syndromes. PLoS ONE
Bjork J, Albin M, Mauritzson N, Stromberg U, Johansson B, Hagmar L. Smoking and myelodysplastic syndromes. Epidemiology
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]