| Abstract|| |
Context: Recently, non-communicable diseases have snatched the lead from infectious diseases in causing mortality. Of these, oral cancer accounts for a significant proportion of deaths. Every year in India significant percentage of newly diagnosed malignancy is oral cancer attributed to various reasons.
Aims: The aim of this study was to assess the extent of oxidative stress and its effect on modification of DNA by urinary nucleoside 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in oral cancer subjects. To see the relationship between the nucleoside 8-OHdG and antioxidant capacity ferric reducing ability plasma (FRAP) in oral cancer subjects.
Settings and Design: Case–control study included three groups with 60 volunteers, who were divided into 30 controls, and equal number of clinically diagnosed oral cancer male patients: (Subdivided into newly diagnosed [n = 15] and 1-year treatment follow-up oral cancer subjects [n = 15]).
Materials and Methods: A random urine sample was used for analysis of 8-OHdG concentration. Serum triglycerides, lipid peroxidation, protein thiols, and FRAP assay were performed by spectrophotometric technique.
Statistical Analysis Used: Student's t-test and one-way analysis of variance were performed for group comparison and Pearson's correlation analysis were used. A P < 0.05 was considered the optimum level of significance.
Results: The urinary 8-OHdG and serum malondialdehyde levels were significantly elevated in newly diagnosed oral cancer subjects in their 1-year treatment compared to the control group (P < 0.05). A significant correlation was observed between urinary 8-OHdG and FRAP in both groups of oral cancer subjects.
Conclusions: Urinary 8-OHdG can be a useful diagnostic marker of oxidative DNA damage in oral cancer subjects.
Keywords: 8-hydroxy-2'-deoxyguanosine, ferric reducing ability plasma, marker, malondialdehyde, oral cancer
|How to cite this article:|
Murugaiyan SB, Ramasamy R, Nakkeeran M, Rangdhol V, Srinivasan A R, Niranjan G. Urinary 8-hydroxydeoxyguanosine as a marker of oxidative stress induced genetic toxicity in oral cancer patients. Indian J Dent Res 2015;26:226-30
Oral cancer is one of the most common malignancies in both developed and developing nations like India. Oral cancer is responsible for considerable morbidity and mortality due to various reasons worldwide. There are emerging experimental evidences to implicate oxidative stress and dyslipidemia in a large number of pathological states including cancer.
|How to cite this URL:|
Murugaiyan SB, Ramasamy R, Nakkeeran M, Rangdhol V, Srinivasan A R, Niranjan G. Urinary 8-hydroxydeoxyguanosine as a marker of oxidative stress induced genetic toxicity in oral cancer patients. Indian J Dent Res [serial online] 2015 [cited 2019 Aug 18];26:226-30. Available from: http://www.ijdr.in/text.asp?2015/26/3/226/162880
The 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most sensitive biomarkers for oxidative stress to DNA that can be detected in plasma and it is reasonably soluble in water and excreted in urine., Till date, very few studies have been documented from this region, to evaluate the extent of oxidative stress, as assessed by 8-OHdG, lipid peroxidation product malondialdehyde (MDA), protein thiols, and ferric reducing ability plasma (FRAP) levels in oral cancer subjects of south Indian population. Hence, aim and objectives were to study the role of urinary 8-OHdG (noninvasive) marker to predict the extent of cellular oxidative stress on genetic material DNA, and its relationship with the antioxidant status in oral cancer patients.
| Materials and Methods|| |
Following clearance by the Institute Ethical Committee, the study was undertaken in 60 subjects divided into two groups: Thirty healthy controls (Group I) and 30 clinically diagnosed oral cancer subjects (Group II) further subdivided into newly diagnosed oral cancer patients without anticancer medication (Group IIA) (n = 15) and oral cancer patients who were on 1-year treatment (Group IIB) (n = 15). Subjects with a history of diabetes mellitus, hypertension, coronary artery disease, myocardial infarction, peripheral vascular disease, renal disease, and those on vitamin supplements were excluded from the study. All volunteers enrolled in this study were males between ages 20 and 65 years. Random urine sample was used for estimation of 8-OHdG concentration. Fasting venous blood sample obtained was centrifuged at 3000 rpm for 10 min in refrigerated remi table top centrifuge, and the separated serum sample was used for estimation of glucose, lipid profile by International Federation of Clinical Chemistry approved assay procedure on clinical chemistry analyzer using reagent kit supply by Agappe Diagnostics (Kerala, India). Serum MDA was estimated by the thiobarbituric acid-reactive substances method, total antioxidant status were estimated by FRAP assay method, protein thiols was estimated by Ellman's method using a spectrophotometer.
The sample size was calculated using "n" master sample size calculator provided by CMC, Vellore with 85% power of the study. All values were entered in Micro Soft Excel 2010, and data were expressed in mean ± standard deviation (SD). Student's t-test and one-way analysis of variance and post-hoc analysis were performed for group comparison. Pearson's correlations analysis was performed using commercial statistical software graph pad InStat (San Diego, CA 92130, USA). A P < 0.05 was considered as significant.
| Results|| |
In the present study, oral cancer subjects were males between the age group of 25 and 45 years adjoining Pondicherry. The mean age of affected patients was 36.9 ± 6 SD. Eighty-five percent of the oralcancer subject had revealed the history of using tobacco usage for many years. Glycemic status in the study subjects showed no significant difference (P = 0.7) as depicted in [Table 1]. When compared to healthy controls, MDA level was significantly higher in oral cancer subjects (P < 0.00), but between subgroups the levels of MDA was significantly low in subjects on treatment follow-up [Table 2]. Antioxidant capacity in plasma as assessed by FRAP assay significantly low in newly diagnosed subjects when compared to control (P < 0.01) in [Table 1]. There is a significant increase in the levels of FRAP assay in subjects with 1-year of treatment follow-up in comparison to the newly diagnosed subjects (P < 0.001) [Table 2]. There was no significant alteration noted in the levels of serum protein thiols.
|Table 1: Characteristic of study parameters between control and oral cancer patients|
Click here to view
|Table 2: Comparison of oxidant, antioxidant status, and 8-.OHdG levels in the different study groups|
Click here to view
When compared to the control, the urinary 8-OHdG level in oral cancer subjects was significantly higher [Table 1]. However, the levels of urinary 8-OHdG in newly diagnosed subjects of oral cancer is significantly high (P < 0.001) as depicted in [Table 2]. However, when compared to controls, there are no significant differences in the patients who were on 1-year of treatment follow-up (P > 0.05).
When compared between the subgroups of oral cancer subjects, there was a significantly lowered level of 8-OHdG in the patient on the treatment of 1-year follow-up.
When compared to control, serum total cholesterol, triacylglycerol (TAG), and low-density lipoprotein (LDL) levels were the significantly higher; and high-density lipoprotein (HDL) level was lower in oral cancer subjects as depicted in [Table 1]. Similarly, both the newly diagnosed and subjects who were on treatment follow-up have showed the significantly higher level of total cholesterol levels, TAG and LDL cholesterol (P < 0.001). HDL cholesterol level was significantly lowered in the treatment follow-up subjects when compared to control (P < 0.01) as shown in [Table 3]. There was a significant correlation noted between urinary 8-OHdG and FRAP assay in controls (r = −0.7; P = 0.03), newly diagnosed (r = −0.8; P = 0.04), and treatment follow-up subjects (r = −0.65; P = 0.01) as shown in [Table 4].
|Table 4: Relationship between FRAP and 8-.OHdG in newly diagnosed oral cancer subjects|
Click here to view
| Discussion|| |
The present study showed a high level of oxidative stress as assessed by MDA levels in oral cancer subjects. MDA levels were much higher in newly diagnosed cancer subjects than subjects on treatment and follow-up [Table 1] and [Table 2]. In concordance with our study, studies have documented increased level of oxidative stress (lipid peroxide levels) in oral cancer subjects. Moreover, 85% oral cancer subjects revealed that they had a habit of tobacco chewing and smoking for long duration justifying the relationship between tobaccos in the development of oxidative stress; its complications. Oxidative stress can induce a deleterious effect on lipids, proteins, carbohydrates, and nucleic acids. As a result, the product of lipid peroxidation such as MDA, proteinthiols, sialic acid, and 8-OHdG are formed in increased amount. These modifications due to oxidative stress can hamper the cell signal transduction, membrane transport, and other cellular metabolic functions., A study by Marnett, 1999 documents MDA reacts readily with proteins and other biomolecules to form a variety of adducts including cross-linked products. MDA also forms adducts with DNA bases that are mutagenic and possibly leading to cancer.
Free radical sources can be endogenous or exogenous in our body, and it is neutralized by the antioxidant system. The level of oxidative stress is determined by the balance between the rate at which oxidative damage is induced and the rate at which it is efficiently cleared, and this is regulated by an individual's hereditary factors, as well as environment and lifestyle pattern. In our study, the antioxidant status, as assessed by FRAP is comparatively low in oral cancer subject, emphasizing a cumulative evidence to support the role of oxidative stress in the pathogenesis oral cancer. Though in our study, there is no significant change in protein thiols, a decrease in FRAP level is relatively pronounced in newly diagnosed untreated cases. Similarly, studies also have documented decrease levels of total antioxidants in thyroid cancer, colon cancer, and periodontitis. Studies have documented that depleted or low antioxidant capacity is involved in both the initiation and the promotion of multistage carcinogenesis including oral cancer. Antioxidant defenses of the body have been proposed to protect the cells against oxidative DNA damage at initiation, promotion, and transformational stages of carcinogenesis.,
In our study, urinary 8-OHdG an endogenous noninvasive marker of oxidative DNA damage is significantly higher. In addition, there is an association between the urinary 8-OHdG level and antioxidant status as assessed by FRAP in both subgroups of oral cancer patients. Ergun et al. 2011 have documented increased oxidative stress and decrease in antioxidants and inverse correlation between them. Another study by Shariff et al. 2009 have documented the beneficial effect of supplementation of antioxidant during radiotherapy for head and neck cancer in order to prevent of oxidative stress. In our study, antioxidant capacity assessed by FRAP is significantly low in newly diagnosed cases, and in 1-year treated subject there was an increase in FRAP suggesting an improvement in antioxidant defense against intensified oxidative DNA stress., Reactive oxygen species (ROS) induced modification of cellular DNA to produce about 30 modified nucleobases this collective modification due to oxidative stress is involved in cancer and aging. Till date origin of the modified base not identified, but recent cell culture study suggests nudix hydrolase on oxidative modification of nucleotide pool.
Various nucleobases of DNA react with HO• (free radical) in a similar manner and this biochemical alteration acts as a pro-mutagen, and, therefore, can be a potential endogenous biomarker of carcinogenesis by decreasing cellular anti-oxidant defense mechanisms. Similar to this other studies have documented oxidative DNA modification 8-OHdG as a potent stress marker in cardiovascular diseases. It is known that cellular DNA and mitochondrial DNA in aerobic organisms are constantly damaged even in the absence of any prior exposure to genotoxic carcinogens. Like damaging processes include endogenous sources such as DNA instability (e.g., depurination), errors of DNA replication, repair, and by other physicochemical means of generating ROS. Sezer et al. 2012 suggest that the correlation between 8-OHdG levels in saliva on periodontal diseases. Patients with periodontitis had an increased 8-OHdG concentration compared to controls and following periodontal treatment, these levels declined and approached the levels as observed in controls. Urinary 8-OHdG exhibits favorable properties such as noninvasiveness, stability, most sensitive, as marker of repair rate, and level tend to decrease following treatment water soluble hence excretion of the repair products in urine represents the average rate of damage in the total body.
Study by Weaver et al. 2013 documents cancer survivor's life end due to cardiac disease. In our study, subjects of newly diagnosed oral cancer, and subjects who were on treatment showed altered lipid profile with significant enhancements in LDL level and decreased HDL level in both newly diagnosed and subjects who were on treatment, when compared to controls. This makes us to conclude that a patient with oral cancer is susceptible to cardiovascular risk. In addition, oxidative stress and decreased antioxidant status can thus play a synergistic role in increasing cardiovascular mortality of oral cancer patients.
Some of the limitation of our study includes sample size, gender difference, and assessment of other antioxidant enzymes might have encouraged the present report futures subdivisions based on histological grading or precancerous lesions continue to act as a major drawback of our study.
| Conclusion|| |
A significant increase in the urinary 8-OHdG and other oxidative stress markers suggest firmly that increased rate of oxidative DNA modifications in oral cancer subjects especially in patients who are in needs of treatment. The association between urinary 8-OHdG and FRAP in oral cancer patients suggest the preventive role of antioxidant on DNA modifications as induced by oxidative stress. In future, more research has to be planned to study gender variation, histological type, different grades of tumor, survival rate (in oral cancer patients), and beneficial effect of antioxidants supplementation in oral cancer subjects.
| Acknowledgments|| |
Dr. Sethuraman KR, Vice-Chancellor, Sri Balaji Vidyapeeth University and Dr. Anathakrishnan N, Dean Research, Mahatma Gandhi Medical College and Research Institute.
| References|| |
Bansal H, Sandhu VS, Bhandari R, Sharma D. Evaluation of micronuclei in tobacco users: A study in Punjabi population. Contemp Clin Dent 2012;3:184-7.
Matsuda M, Shimomura I. Increased oxidative stress in obesity: Implications for metabolic syndrome, diabetes, hypertension, dyslipidemia, atherosclerosis, and cancer. Obes Res Clin Pract 2013;7:330-41.
Cooke MS, Olinski R, Loft S, European Standards Committee on Urinary (DNA) Lesion Analysis. Measurement and meaning of oxidatively modified DNA lesions in urine. Cancer Epidemiol Biomarkers Prev 2008;17:3-14.
Dröge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82:47-95.
Vinayagamoorthi R, Bobby Z, Sridhar MG. Antioxidants preserve redox balance and inhibit c-Jun-N-terminal kinase pathway while improving insulin signaling in fat-fed rats: Evidence for the role of oxidative stress on IRS-1 serine phosphorylation and insulin resistance. J Endocrinol 2008;197:287-96.
Espinosa O, Jiménez-Almazán J, Chaves FJ, Tormos MC, Clapes S, Iradi A, et al.
Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a reliable oxidative stress marker in hypertension. Free Radic Res 2007;41:546-54.
Wu LL, Chiou CC, Chang PY, Wu JT. Urinary 8-OHdG: A marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin Chim Acta 2004;339:1-9.
Richards D. Clinical recommendations for oral cancer screening. Evid Based Dent 2010;11:101-2.
Saito S, Yamauchi H, Hasui Y, Kurashige J, Ochi H, Yoshida K. Quantitative determination of urinary 8-hydroxydeoxyguanosine (8-OH-dg) by using ELISA. Res Commun Mol Pathol Pharmacol 2000;107:39-44.
Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta 1978;90:37-43.
Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": The FRAP assay. Anal Biochem 1996;239:70-6.
Ellman G, Lysko H. A precise method for the determination of whole blood and plasma sulfhydryl groups. Anal Biochem 1979;93:98-102.
Carnevali S, Petruzzelli S, Longoni B, Vanacore R, Barale R, Cipollini M, et al.
Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 2003;284:L955-63.
van der Vaart H, Postma DS, Timens W, ten Hacken NH. Acute effects of cigarette smoke on inflammation and oxidative stress: A review. Thorax 2004;59:713-21.
Cha HJ, Yim H. The accumulation of DNA repair defects is the molecular origin of carcinogenesis. Tumour Biol 2013;34:3293-302.
Patel BP, Rawal UM, Dave TK, Rawal RM, Shukla SN, Shah PM, et al.
Lipid peroxidation, total antioxidant status, and total thiol levels predict overall survival in patients with oral squamous cell carcinoma. Integr Cancer Ther 2007;6:365-72.
Beevi SS, Rasheed AM, Geetha A. Evaluation of oxidative stress and nitric oxide levels in patients with oral cavity cancer. Jpn J Clin Oncol 2004;34:379-85.
Marnett LJ. Lipid peroxidation-DNA damage by malondialdehyde. Mutat Res 1999;424:83-95.
Wang CH, Wu SB, Wu YT, Wei YH. Oxidative stress response elicited by mitochondrial dysfunction: Implication in the pathophysiology of aging. Exp Biol Med (Maywood) 2013;238:450-60.
Suma HR, Prabhu K, Shenoy RP, Annaswamy R, Rao S, Rao A. Estimation of salivary protein thiols and total antioxidant power of saliva in brain tumor patients. J Cancer Res Ther 2010;6:278-81.
Pathak HR, Singh BP. Defining the role of antioxidants in the prevention of prostate cancer. Indian J Urol 2009;25:417-8.
Korde SD, Basak A, Chaudhary M, Goyal M, Vagga A. Enhanced nitrosative and oxidative stress with decreased total antioxidant capacity in patients with oral precancer and oral squamous cell carcinoma. Oncology 2011;80:382-9.
Shariff AK, Patil SR, Shukla PS, Sontakke AV, Hendre AS, Gudur AK. Effect of oral antioxidant supplementation on lipid peroxidation during radiotherapy in head and neck malignancies. Indian J Clin Biochem 2009;24:307-11.
Barut O, Vural P, Sirin S, Aydin S, Dizdar Y. The oxidant/antioxidant status and cell death mode in oral squamous cell carcinoma. Acta Odontol Scand 2012;70:303-8.
Cooke MS, Evans MD. 8-Oxo-deoxyguanosine: Reduce, reuse, recycle? Proc Natl Acad Sci U S A 2007;104:13535-6.
Masugata H, Senda S, Murao K, Inukai M, Himoto T, Hosomi N, et al.
Association between urinary 8-hydroxydeoxyguanosine, an indicator of oxidative stress, and the cardio-ankle vascular index in hypertensive patients. J Atheroscler Thromb 2012;19:747-55.
Valavanidis A, Vlachogianni T, Fiotakis C 8-hydroxy-2' -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 2009;27:120-39.
Sezer U, Ciçek Y, Canakçi CF. Increased salivary levels of 8-hydroxydeoxyguanosine may be a marker for disease activity for periodontitis. Dis Markers 2012;32:165-72.
Weaver KE, Foraker RE, Alfano CM, Rowland JH, Arora NK, Bellizzi KM, et al.
Cardiovascular risk factors among long-term survivors of breast, prostate, colorectal, and gynecologic cancers: A gap in survivorship care? J Cancer Surviv 2013;7:253-61.
Department of Biochemistry, Mahatma Gandhi Medical College and Research Institute, Pondicherry
Source of Support: Sri Balaji Vidyapeeth Charitable Trust, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]