|Year : 2012 | Volume
| Issue : 3 | Page : 309-315
Determinants of oxidative stress and DNA damage (8-OhdG) in squamous cell carcinoma of head and neck
A Kumar1, MC Pant2, HS Singh3, S Khandelwal1
1 CSIR-Indian Institute of Toxicology Research, Lucknow, India
2 C.S.M. Medical University, Lucknow, India
3 Ch. Charan Singh University, Meerut, India
|Date of Web Publication||12-Dec-2012|
CSIR-Indian Institute of Toxicology Research, Lucknow
Source of Support: None, Conflict of Interest: None
Background: Squamous cell carcinoma of head and neck (SCCHN) is a major concern of health risk in developing countries, such as India. Apart from genetic configuration, environmental and lifestyle factors, as well as poor oral hygiene, provide free radical-generating environment, which may contribute to the development of cancer through DNA damage. Materials and Methods: Here we ascertained the various oxidative stress determinants in diagnosed SCCHN patients with health risk addictions. This study further evaluated the incremental effects inflicted by these lifestyle factors on redox status. The study included 100 consenting SCCHN patients and 90 matched healthy controls. Salivary total antioxidant capacity (TAC), glutathione (GSH), free radicals: such as reactive nitrogen species (RNS) and reactive oxygen species (ROS) along with oxidative DNA adduct (8-OHdG) were monitored. Results: Our findings indicated altered salivary oxidant-antioxidant status in SCCHN. A substantial rise in ROS (~2.0 folds) and RNS (~1.4 folds), together with significant lowering in TAC (~1.2 folds) and GSH (~1.7 folds) was observed. The 8-OHdG levels were also found to be considerably higher (P < 0.001) in salivary cell's DNA of these patients. Conclusions: Our results demonstrate significant redox imbalance in cancer patients suggesting their paramount importance in the development of SCCHN.
Keywords: 8-OHdG, redox status, saliva, squamous cell carcinoma of head and neck
|How to cite this article:|
Kumar A, Pant M C, Singh H S, Khandelwal S. Determinants of oxidative stress and DNA damage (8-OhdG) in squamous cell carcinoma of head and neck. Indian J Cancer 2012;49:309-15
|How to cite this URL:|
Kumar A, Pant M C, Singh H S, Khandelwal S. Determinants of oxidative stress and DNA damage (8-OhdG) in squamous cell carcinoma of head and neck. Indian J Cancer [serial online] 2012 [cited 2020 Jul 10];49:309-15. Available from: http://www.indianjcancer.com/text.asp?2012/49/3/309/104499
| » Introduction|| |
Squamous cell carcinoma of head and neck (SCCHN) including cancer of oral cavity, larynx and pharynx contributes majorly towards total cancer burden worldwide  and accounts for one fourth of male cancers in India.  In developing countries, such as India, SCCHN is of large concern due to its associated morbidity and mortality. It is mainly a disease of middle age and elderly, but early addiction to alcohol, tobacco chewing and smoking has tilted the risk pendulum to the left. Another threat that can also contribute to head and neck cancer is the human papilloma virus (HPV), which gets transmitted sexually or from mother to child. It is recognized as the major risk factor in about 70% of cervical cancer cases and in about 60% of head and neck cancers, particularly among the younger subjects with no history of tobacco or alcohol use. ,
Addiction of tobacco and tobacco products, poor oral hygiene and inadequate nutrition leads to a more venerable population. Apart from genetic configuration, environmental and lifestyle factors play a crucial role, particularly, in the progression of head and neck cancer.  Eighty-five percent of head and neck cancers are linked to tobacco use. People who consume both tobacco and alcohol are at a higher risk of developing SCCHN than those addicted to either of them. Differences in the habit of tobacco usage in India and western countries, could lead to variation in the incidence of oral cancer. 
Oxidative stress, a prominent feature of various diseases and their progression, is well established. Free radicals, which induce oxidative and nitrative stress, are the chief inducers of oral cancer and Ma et al demonstrated that oxidative and nitrative stress contribute to the development of oral carcinogenesis through DNA damage. In another study, Patel et al reported oxidized proteins and DNA in saliva to be the possible link between salivary free radicals, antioxidants and oral cancer. Recently, alterations in antioxidant activities and their association with nitric oxide production in oral cancer was shown by Nair et al.
Pan masala and gutkha have clastogenic and carcinogenic effects.  They are capable of generating free radicals during auto-oxidation of polyphenols in saliva.  Free radicals including reactive oxygen species (ROS), such as superoxide anions (O 2 ), hydroxyl radicals (OH) and hydrogen peroxide (H 2 O 2 ), malondialdehyde; and reactive nitrogen species (RNS) in the form of nitrosamines (NO 3 and NO 2 ), play a crucial role in human cancer development as they can cause DNA damage, leading to mutations in oncogenesis and tumor suppressor genes.  Overproduction of ROS might stimulate malignant transformation.  An important free radical, that is, hydroxyl radical when added to purines, gives rise to C4-OH- C5-OH-, and C8-OH- adducts. , The 8-OHdG lesions which are easily formed are highly mutagenic.
Dietary nitrates after intestinal absorption, reach saliva through salivary glands by an active transport system.  In the saliva, they are partially converted into nitrite by nitrate reductase derived from commensal bacteria. The nitrites are of special importance as carcinogenic promoters because they can react with amines and amides to form carcinogenic nitrosamines.  NO can be converted into strong nitrosating agent N 2 O 3 in the presence of oxygen, which can deaminate various DNA bases. It can react with secondary amines to form carcinogenic N-nitrosamines, that can damage DNA by alkylation.  DNA strand breaks caused by peroxynitrite and NO can activate poly-ADP ribosylase.  This rapid activation of poly-ADP ribosylase results in the depletion of NAD + , followed by prevention of ATP synthesis, leading to acute cell dysfunction and cell death. 
RNS can modify proteins through oxidation, nitrosation and nitration. Modified proteins with altered structure and function accumulate during aging, oxidative stress and carcinogenesis. Nitration of tyrosine has shown to affect the signal transduction pathway. 
Oral cavity is well equipped with an advanced salivary antioxidant system that also contains antinitrosamine inhibitory agents.  The antioxidant system is composed of both enzymatic and non-enzymatic machinery. The enzymatic component includes catalase, superoxide dismutase, glutathione peroxidase and nonenzymatic part such as uric acid and glutathione (GSH).  GSH is a critical factor in protecting organisms against toxicity and disease, since it provides reducing capacity for several reactions and also plays an important role in the detoxification of hydrogen peroxide and other free radicals.  It is a lifestyle indicator of oxidative stress but not of age and gender. 
The pivotal role of salivary free radicals and antioxidant system in prognosis of oral cancer has gained prominence in the recent past. ,,, Direct involvement of oxidized proteins and DNA in saliva of oral cancer patients was demonstrated by Patel et al. Alterations in antioxidant activities in blood with higher RNS levels in oral cancer suggest their importance in cancer development. 
The use of oral fluids is broadening perspectives in clinical diagnosis, disease monitoring and decision making for patient care. We therefore, aimed to evaluate oxidant-antioxidant-related biologic indexes (ROS, RNS, GSH), total antioxidant capacity (TAC), and oxidative DNA adduct (8-OHdG) in saliva of SCCHN patients for better understanding of the pathogenesis of this disease.
| » Materials and Methods|| |
The study included 100 consenting SCCHN patients and 90 matched healthy controls and was approved by the Ethical Committees for Clinical Research. All cases were newly diagnosed, biopsy proven squamous cell carcinoma of head and neck (SCCHN) and received no chemo-/radiotherapy. The mean age of SCCHN patients was 53 years (range 25-75 years) and that of controls was 51 years (range 25-60 years). Subjects having regular smoking habits and smoking index (cigarettes/ day × 365) of more than 730 and regular smokeless tobacco chewers with chewing index more than 365 (CY = frequency of tobacco chewed/kept/day × 365), were considered in the category of smokers  and tobacco chewers,  respectively. Approximately, 4 mL of saliva was collected under resting condition in a quiet room between 10 am to 12.30 pm, at least 1 h after food intake. Subjects were asked to generate saliva in their mouth and spit into a wide mouth collection tube. The saliva samples were immediately kept in ice till further use.
The saliva samples were filtered through 100 μm nylon mesh and were centrifuged at 800 × g, for 10 min at 4°C. The resulting supernatant was used for the analysis of TAC, ROS, RNS and GSH in saliva supernatant of all the study subjects.
Reactive oxygen species
The ROS was measured with DCF-DA, a fluorescent probe, which reacts with ROS to yield highly fluorescent DCF.  DCF-DA diffuses through cell membranes and is subsequently deacetylated by intracellular esterases to nonfluorescent DCF-H. In a cell-free system, DCF-DA is treated with 0.1 NaOH for 30 min at room temperature to convert DCF-DA to DCF-H, which can readily react with free radicals. , Briefly, 50 μL of saliva supernatant was added in a microplate and the volume was made up to 200 μL with phosphate-buffered saline (PBS). Twenty-five microliters of freshly prepared DCF-H (500 μM final concentration) was then added to each well and after 1 h incubation at 37°C, the florescence was measured at 485 nm excitation and 528 nm emission on a plate reader (BMG FLUOstar Omega). 2′,7′ Dichlorofluorescein was used as the standard, for quantification of total ROS.
Reactive nitrogen species
The RNS was quantified by Griess Reagent. The nitrite present in saliva reacts with sulfanilamide and N-(naphthyl)ethylenediamine to produce a red color.  Briefly, 20 μL of saliva supernatant was added in a microplate and the volume was made up to 100 μL with PBS. Fifty microliters each of Griess Reagent I and II were then added to each well and after 10 min incubation at 37°C, the absorbance was measured at 550 nm on a plate reader (BMG FLUOstar Omega). Sodium nitrite solution was used as the standard, for determining total nitrite levels.
GSH in saliva was evaluated using o-phthaldialdehyde (OPT),  which reacts with both GSH amino- and sulfhydryl groups, yielding a highly fluorescent cyclic product. Briefly, 25 μL of saliva supernatant was added in a microplate and the volume was made up to 200 μL with HEPES buffer (0.1 M, pH 7.4). Ten microliters of OPT (100 mM)) was then added to each well and after 10 min incubation at 37°C, the fluorescence was measured at 360 nm excitation and 460 nm emission on a plate reader (BMG FLUOstar Omega). GSH reduced was used as the standard, for determining GSH levels.
Total antioxidant capacity
TAC was measured by using Antioxidant Assay Kit (Cayman Chemical Company, USA). The assay relies on the ability of antioxidants in the sample to inhibit oxidation of ABTS (2,2′-Azino-di[3-ethybenzthiazoline sulfonate]) by metmyoglobin, which can be monitored by reading the absorbance at 750 nm. The capacity of antioxidants in the sample to prevent ABTS oxidation is compared with that of Trolox and quantified as millimolar Trolox equivalents.
Ten microliters of saliva (2 times diluted) were taken in a microplate and the procedure followed according to the manual. The absorbance of ABTS oxidation was measured at 750 nm on a plate reader (BMG FLUOstar Omega).
Oxidative DNA damage
DNA adduct (8-OHdG) was quantitated by using DNA Damage Quantification Kit (BioVision, USA). After treating DNA-containing abasic sites (AP) with aldehyde reactive probe reagent (ARP), AP sites were tagged with biotin residues, which can be determined using avidin-biotin assay followed by colorimetric detection.
For extraction of DNA from salivary cells, the saliva samples were centrifuged at 10,000×g for 10 min and the pellet was used for DNA isolation following the method of Miller et al with some modifications. The pellet was suspended in 3 mL of lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 100 mM Na 2 EDTA, 0.5% SDS adjusted to pH 8.0) and 100 μL of 10% SDS and 500 μL of proteinase K (1 mg/mL in 2mM Na 2 EDTA), and further incubated overnight at 37°C. One milliliter of 6 M sodium chloride was then added and after vigorous shaking for 15 sec, the samples were centrifuged at 2500 rpm for 15 min. The pellet was discarded and the supernatant was taken in a separate tube and double volume of cold ethanol (100%) was added, inverting the tube 7-8 times to precipitate DNA. The precipitated DNA was resuspended in 200-400 μL of Tris-HCl, pH 8.5 and was left at 37°C to dissolve. The amount and purity of DNA was measured by NanoDrop Spectrophotometer (ND 1000 V3.3.1).
Five microliters of highly purified DNA sample (0.1 μg/μL), isolated from salivary cells (as given above) was taken in a microcentrifuge tube, mixed with 5 μL of ARP solution and incubated for 60 min at 37°C to tag AP sites of DNA. Assay was carried out according to the manual provided. 40ARP-DNA Standard (40ARP sites per 10 5 bp) was used for determination of AP sites in the samples to ascertain oxidative DNA damage.
The data were statistically analyzed using SPSS Statistical software (version 12.0 for windows; SPSS Chicago, IL, USA). Student's t test was performed to compare the levels between controls and patients. Differences between groups and variables were analyzed for significance using one-way analysis of variance test using GraphPad PRISM 5 software (CA, USA) and were considered significant when P values were 0.05 or less.
| » Results|| |
General profile of TAC, GSH, RNS and ROS of controls and SCCHN patients is given in [Figure 1]. TAC of the patients evaluated in saliva was found to be substantially suppressed when compared with healthy controls. The control TAC value in saliva was 0.95 mM, whereas in SCCHN patients, the levels dropped to 0.74 mM. A ~1.3-fold reduction (P < 0.001) in TAC indicates a compromised redox status.
|Figure 1: Salivary TAC, GSH, RNS, and ROS in controls (n = 90) and SCCHN patients (n = 100) 8-OHdG in salivary cells of controls (n = 50) and SCCHN patients (n = 50). The level of TAC is expressed as mM, GSH, RNS, and ROS as μM, 8-OHdG as number of apurinic sites/105 bp. All values are expressed as mean ± S.E. ***P < 0.001, **P < 0.01 vs control, by Student's t test. SCCHN, squamous cell carcinoma of head and neck; TAC, total antioxidant capacity; GSH, glutathione; RNS, reactive nitrogen species; ROS, reactive oxygen species; 8-OHdG, oxidative DNA adduct.|
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Likewise, GSH levels of SCCHN patients indicated a lowering pattern. A ~1.7-fold reduction in GSH, suggests an oxidant/antioxidant imbalance in the cancer patients. GSH values in saliva were 2.65 μM as compared with 1.59 μM in controls.
ROS and RNS, on the other hand, were significantly elevated in SCCHN patients. The nitrite levels in saliva showed a 1.4-fold rise (P < 0.01), that is, from 145.04 μM in controls to 205.75 μM in patients. The ROS levels were also raised by ~2.2-folds (control 11.20 μM, patients 24.60 μM). Similarly, DNA adduct (8-OHdG) levels in saliva, also registered a significant rise, that is, 1.6-folds (P < 0.001) [Figure 1].
These salivary determinants when compared in groups habituated to tobacco, smoking, and chewing provide a clear representation of the lifestyle effects.
[Table 1] demonstrates the salivary TAC values in nonhabituates, smokers, chewers and smokers + chewers. Case smokers showed a significant reduction in TAC, from 0.94 to 0.73 mM (P < 0.01) when compared with control smokers. In control and case smokers + chewers, a further decline of TAC was observed, that is, from 0.90 to 0.70 mM (P < 0.001).
GSH levels in smokers + chewers control and case failed to show any statistical significance. However, a significance of P < 0.05 (lowering from 2.42 to 1.43 μM) was observed in control smokers versus case smokers + chewers.
As shown in [Table 2], a ~2-fold increase in ROS levels was evident in all the study groups when compared with their respective controls, but statistical significance was observed only between the control and case smokers and control and case smokers + chewers groups. A marked increase was also seen between control smokers and case smokers + chewers [Table 2].
Regarding the RNS values in saliva, control smokers + chewers versus respective case SCCHN exhibited no statistical significance. However, a mild significance of P < 0.05 was observed in case of smokers + chewers when compared with control smokers [Table 2]. The data suggests that the patients with smoking and chewing habits exhibit a higher ROS and RNS, suppressed GSH and TAC values.
Mean 8-OHdG levels representing oxidized DNA in saliva [Table 2] increased by ~2-folds in case with smoking habits (P < 0.05) and also in smoking + chewing habituates (P < 0.05). The overall data of salivary analysis clearly demonstrates oxidative stress determinants, including 8-OHdG levels, to be highly altered in SCCHN patients addicted to both tobacco chewing and smoking.
| » Discussion|| |
In this study we have determined the levels of antioxidants (TAC, GSH), free radicals (ROS, RNS) and oxidative DNA damage to understand the involvement of these biologic indexes in head and neck squamous cell carcinoma.
Interplay of oxidant and antioxidant systems is one of the deciding factors for health of an individual and is a continuous process. Under adverse conditions when there is an inadequate antioxidant pool, oxidants such as free radicals tend to damage various body structures. They may lead to oxidation of macromolecules such as proteins, DNA and lipids, which in altered configuration for long, may lead to deleterious outcomes. Here we ascertain the levels of oxidants as well as antioxidants in human saliva since it forms the frontline defense to encounter various oxidants present in tobacco smoke, tobacco chewing, alcohol and food. Several studies have demonstrated an association of these free radicals with diseases, such as diabetes, neurodegenerative diseases and cancer. , Although RNS are produced by auto-oxidation of nitric oxide (NO), dietary nitrate affects concentrations of both nitrite and nitrate in saliva. In the oral cavity, nitrate is reduced to nitrite and the latter is reduced to NO by bacterial population in oral cavity. Takahama et al reported that nitrate is salivary concentration of nitrite as a function of age and an increase of 6.6% in serum ROS every 10 years has also been demonstrated by Hayashi et al. In our study, a difference of 2 years (average age) is not likely to affect the free radical change observed in cancer subjects.
Cancer progression is dependent on several factors, such as genetic makeup, lifestyle habits and nutritional status. In this study, a significant lowering of TAC in the saliva of case smokers and smokers + chewers, indicates compromised salivary antioxidant defenses in habituates. Reduced TAC in saliva of oral cancer ,,, and in breast cancer  has been reported. However, no association has been shown by Mekary et al in colorectal cancer.
GSH, another important antioxidant studied in this investigation is also found to be reduced in case smokers + chewers group when compared with control smokers. This finding suggests that smoking along with chewing has a greater impact on GSH leading to its decline. Modulation of GSH has been determined under various disease-associated conditions, such as viral diseases, cigarette smoke, chemical pollutants and occupational exposures. Lowering of salivary GSH in head and neck cancer was also shown by Singh et al.  In contrast, elevated salivary GSH was observed by Arioz et al. and in brain tumor patients by Suma et al. Raised GSH levels were also reported in laryngeal and breast tumor tissues.  Yet, there are some other studies suggesting no significant difference in GSH in the case of cancer patients. ,
In this study, out of 100 cancer patients, 93 were addicted to tobacco chewing and/or smoking. Increased free radicals, that is, ROS and NO 2 levels in tobacco-habituated group appear to have originated from exposure to cigarette smoke and tobacco chewing. In oral cancer patients, higher production of blood NO 2 + NO 3 levels  and elevated levels in blood and oral tissue  indicate oxidative stress, which appears to be one of the prime factors responsible for oral pathogenesis. In numerous other diseases, increase in ROS has also been extensively reported, for example, in oral, , lung,  and gastric cancers. 
Enhanced levels of ROS and RNS along with DNA oxidation has been reported by many groups. ,, In this case-control study, increased salivary DNA adduct (8-OHdG) in the habituate group (smokers + chewers) along with other oxidative stress markers, suggest a strong contribution toward increased DNA oxidation by free radicals. Similar findings of 8-OHdG production in tumor-associated lymphocytes,  oral cancer,  breast cancer,  colorectal tumors,  and in bladder cancer  are available.
To sum up, these results indicate an oxidant/antioxidant imbalance, resulting in reduced TAC and GSH and enhanced production of ROS and NO 2 in head and neck cancer patients.
This redox imbalance appears to have a marked effect on DNA oxidation (DNA adduct) one of the causative factors for oral cancer development. The latter can, therefore, be considered as a biomarker for head and neck cancer. Since these biochemical alterations are of a higher magnitude in those patients with both smoking and chewing habits, it is imperative to believe that lifestyle habits do play a central role in the onset of SCCHN.
Our results clearly demonstrate a significant role of oxidative stress in squamous cell carcinoma of head and neck. Saliva being noninvasive and easy to collect, can be used as an alternate to blood for mass screening. To determine the true impact of Redox imbalance on the progression of SCCHN, more correlation studies are warranted.
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[Table 1], [Table 2]
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