doi: 10.1111/jop.12283
J Oral Pathol Med (2015) 44: 707–713 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
Scalp hair and blood cadmium levels in association with chewing gutkha, mainpuri, and snuff, among patients with oral cancer in Pakistan Sadaf Sadia Arain1, Tasneem Gul Kazi1, Hassan Imran Afridi1, Farah Naz Talpur1, Atif Gul Kazi2, Kapil Dev Brahman1, Abdul Haleem Panhwar1, Mariam Shahzadi Arain1 1
National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, Pakistan; 2Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan
BACKGROUND: It has been reported that smokeless tobacco (SLT) chewing can lead to caners of oral cavity. In this study, the relationship between cadmium exposure via consumption of different SLT products by patients with oral cancer with related to referents belongs to different cities of Pakistan are investigated. METHODS: The cadmium in different types of SLT products (gutkha, mainpuri and snuff) consumed by studied population and biological samples (scalp hair and blood) of cancerous and healthy subjects were analyzed. Both referents and patients with cancer have same age group (ranged 30–60 years), socioeconomic status, localities, and dietary habits. The concentrations of cadmium in SLT products and biological samples were measured by electrothermal atomic absorption spectrophotometer after microwave-assisted acid digestion. The validity and accuracy of the methodology were checked by certified reference materials. RESULTS: It was estimated that 10 g intake of different types of gutkha, mainpuri, and snuff could contribute, 18–40%, 15.7–33.6%, and 14–68% of the provisional maximum tolerable daily intake of cadmium, respectively for adults (60 kg). The results of present study showed that the mean value of cadmium was significantly higher in scalp hair and blood samples of patients with oral cancer as compared to those results obtained for referents (P < 0.001). CONCLUSIONS: The high exposure of cadmium due to consuming different SLT products may have synergistic effects with other risk factors associated for oral cancer. J Oral Pathol Med (2015) 44: 707–713
Correspondence: Hassan Imran Afridi, PhD, Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan. Tel: +92 0222 771379, Fax: +92 0221 771560, E-mail: hassanimranafridi@ yahoo.com Accepted for publication September 8, 2014
Keywords: atomic absorption spectrophotometer; biological samples; cadmium; oral cancer; smokeless tobacco products
Introduction The oral cancer is a common malignancy among people, who are smoker and chewer tobacco, with a worldwide incidence of over 300 000 new cases annually (1). The disease is characterized by a high rate of morbidity and mortality (2). The etiology of oral cancer is multifactorial, major risk factors are tobacco and alcohol consumption (3). Both tobacco smoking (cigarettes, cigars, and pipes) and chewing smokeless tobacco (SLT) products with and without other ingredients have been shown to increase the risk of developing oral cancer (3–5). Cancers caused by SLT use often begin as leukoplakia or erythroplakia; erythroplakia generally has a higher chance to becoming cancerous over time (6). Tobacco plant (Nicotiana tabacum) is well known for its capacity to concentrate toxic elements from its growing environment (7). Tobacco is known to contain numerous classes of carcinogenic substance such as tobacco-specific nitrosamines, which are often regarded as a major factor in SLT-related carcinogenesis. The combined exposure of nitrosamines and other classes of organic and inorganic substances, including toxic metals enhances the carcinogenetic effects (8). The cadmium (Cd) is a nonessential, potentially toxic, pollutant ion, which is accumulated by tobacco plants and diets and is transferred to human via smoking and chewing tobacco products (9–11). It was reported that Cd has adverse impact on human via genotoxic mechanisms including induction of single-strand DNA breaks, and also that it inhibits DNA repair by inactivation of the mismatch repair system (12). The Cd activates protooncogenes (13, 14) and inhibits normal apoptosis of cells (15). It was reported that Cd is an inhibitor of the enzymes with sulphydryl groups and disrupts the pathways for the oxidative metabolism (16). In vitro studies suggest that there
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is competition for transport mechanisms between Cd and some essential trace elements mainly Zn, Se, and Cu (17). Various regulatory agencies have reported that Cd is a human carcinogen (18–20). Several studies indicate a role for Cd in human prostatic, pancreatic, and renal cancers (21–24), while a few studies have associated Cd exposure with human cancer of the liver and urinary bladder (22, 25, 26). In non-occupationally exposed populations, Cd has also been associated with lung cancer incidence and mortality (27, 28). Prospective studies observed an association between dietary Cd and risk of breast cancer (29, 30) and endometrial cancer (31). Several other studies have investigated the role of Cd in cancer development in female reproductive organs (31, 32). The aim of present study was to evaluate and compare the concentration of Cd in different types of moist and dry snuff, mainpuri, and gutkha available and consumed in Pakistan, and to estimate the Cd levels in scalp hair and blood samples of patients diagnosed with oral cancer and healthy individuals.
Materials and methods Reagents and glassware Ultra-pure water obtained from ELGA labwater system (Bucks, UK). Concentrated nitric acid (65%) and hydrogen peroxide (30%) were obtained from Merck (Darmstadt, Germany). Working standard solutions of Cd were prepared prior to their use by stepwise dilution of certified standard solutions (1000 ppm) Fluka Kamica (Buchs, Switzerland), with 0.2 mol/l of HNO3. Moreover, matrix modifier was employed to analyze Cd [0.001 mg Pd + 0.0015 mg Mg (NO3)2], was prepared from Mg(NO3)2, and 99.999% Pd (Sigma, St. Louis, MO, USA). All solutions were stored in polyethylene bottles at 4°C. For the accuracy of methodology, certified reference materials (CRM), human hair BCR 397, Clincheckâ control-lyophilized human whole blood and Virginia tobacco leaves (ICHTJ-cta-VTL-2) were used. Glasswares and polyethylene containers were soaked in 10% (v/v) HNO3 for 24 h; washed with distilled water, finally with de-ionized water and dried in such a manner to ensure that no any contamination from glasswares occur. Instrumentation The determination of Cd was carried out by means of a double-beam Perkins-Elmer Atomic Absorption Spectrometer Model 700 (Perkin Elmer, Norwalk, CT, USA) equipped with the graphite furnace HGA-400, pyrocoated graphite tubes with integrated platform, an autosampler AS800 and deuterium lamp as background correction system. Hollow cathode lamp (Perkin Elmer) was used as radiation sources, and they were operating at a recommended current. All instrumental conditions were used according to the manufacturer’s recommendation. Portions of both standard or sample and modifier were transferred into autosampler cups, and 20 ll (standard or sample volume of 10 ll and 10 ll of modifier in each case) was injected to electrothermal graphite atomizer. A horizontal flask electrical shaker (220/60 Hz; Gallenkamp, England) was used for shaking the samples. The pH was measured by a pH meter (781-pH meter; Metrohm; Metrohm Ltd., Herisau, Switzerland). A J Oral Pathol Med
Pak Elektron Limited domestic microwave oven (Osaka, Japan), programmable for time and microwave power from 100 to 900 W, was used for digestion of samples. Study population A survey was carried out about the gutkha and mainpuri chewing, while snuff inhaling habits of both genders, age ranged 30–60 years, residing in the different cities of Pakistan. The data of hospital based case–control study population were collected from Nuclear Institute of Medicine and Radiotherapy (NIMRA) Jamshoro and Larkana institute of nuclear medicine and radiotherapy (LINAR), situated in different areas of Sindh, Pakistan, during 2010– 2012 years, by collecting files and extracting important information. During 1-year study period (2010), the information department of both hospitals recorded >5200 cases of cancers of all types, and mouth cancer comprised of 2.7% of the total. The patients with oral cancer were divided into subgroups according to the different over found locations of oral cancer, Lips, tongue, cheeks, floor of the mouth, hard and soft palate, sinuses, and pharynx (throat). Patients with oral cancer and referents were further grouped according to their SLT chewing habits, not consumed any SLT product (NU), gutkha (GU), snuff (SU), and mainpuri users (MPU). Complete demographic information is listed in Table 1. Physical examinations were performed to measure participant’s weight, height, blood pressure, and biochemical data. The biochemical tests of patients and referents were performed to estimate hemoglobin, red blood cells, packed cell volume, mean corpuscular hemoglobin concentration, mean corpuscular volume, and transferrin iron-binding capacity in the blood. The criteria for the selection of patients were of biopsy proved oral squamous cell carcinoma prior to any treatment and that they were not taking any mineral supplements during last 3 months. The criteria for selection of 1155 referent subjects were by matching to the same age group, socioeconomic status, and dietary habits, being free of any cancer diagnosis and not taking any mineral supplement. The biochemical results and histological information are not reported in present study. Prior to the biological samples collection, they have underwent a standard routine medical examination. This study was approved by ethical committee of Sindh University, working under the auspices of higher education commission of Pakistan. Sampling of SLT products A total of 23 brands of snuff (dry and moist), 11 brands of gutkha, and 12 brands of mainpuri were purchased from local markets of the different cities of Pakistan as per their availability over a 3-year period (January 2010–December 2013). The samples were packed in their original packing and placed in pre-washed dried plastic bags separately and stored at 4°C, until tested. Five composite samples of each brand of snuff, gutkha, and mainpuri were prepared by homogenizing the mixture after removing the wrappers. Care was taken to avoid any source of contamination, and this preparation was carried out in a clean environment. All samples were dried at 80°C. The dried samples were ground with agate mortar and pestle, sieved through nylon sieves with mesh sizes of 125 lm, and then stored in the labeled sample bottles.
Cadmium level in patients with oral cancer Arain et al.
Biological samples Venous blood samples (5 ml) were collected by 7 mm heparinized lithium Vacutainerâ tubes (Becton Dickinson, East Rutherford, NJ, USA). About 2 ml of venous blood samples was stored at 20°C until elemental analysis. The scalp hair samples were taken from five different parts of the scalp (frontal, cranial, occipital, right, and left lateral). The first 5 cm of hair from the root was used for analysis. Hair samples were put into separate plastic envelopes for each participant, tightly sealed and attached with identification number of the participant and questionnaire. In the laboratory, hair specimens were further cut into pieces, approximately 0.2–0.3 cm, and washed four times with a 1:200 v/v dilution of Triton X-100, then rinsed three times with ultra-pure water and two times with acetone (33). Then samples were then dried in an oven at 80–85°C.
Microwave-assisted acid digestion A microwave-assisted digestion (MAD) procedure was carried out, to achieve a shorter digestion time. Replicate six samples of each CRM (0.5 ml Clincheckâ controllyophilized human whole blood, 0.2 g of Virginia tobacco leaves and BCR 397 human hair) and duplicate samples of different types of SLT products (0.2 g), whole blood (0.5 ml), and scalp hair (0.2 g) were taken separately in polytetrafluoroethylene (PTFE) flasks (25 ml in capacity). Then added 3 ml of a freshly prepared mixture of concentrated HNO3–H2O2 (2:1, v/v), kept at room temperature for 10 min. Then placed the flasks in covered PTFE container and heated at 80% of total power (900 W) for 3–4 min. The digested samples were diluted up to 10 ml with 0.1 mol/l concentrated HNO3. A blank extraction (without sample) was carried out through the complete procedure.
pH determination of SLT products Weighed 1 g sample of each brand of gutkha, mainpuri, moist, and dry snuff, added 10 ml of ultrapure water in flask (100 ml capacity), and placed in an electrical shaker at 2 g for 30 min, then filtered the solution through Whatman No. 42 filter paper and extracts was taken to determine the pH.
Statistical analysis All statistical analyses were performed using computer program Excel X State (Microsoft Corp., Redmond, WA, USA) and Minitab 13.2 (Minitab Inc., State College, PA, USA). The data from triplicate samples of each composite samples were expressed as means SD. The Student’s ttest was used to assess the significant difference of Cd in certified and experimentally found values. The ANOVA was used to assess the significance of differences between the concentrations of Cd observed in the biological samples of patients and referent subjects, calculated by the unpaired two-sample t-test. A P < 0.05 was considered significant difference.
Table 1 Characteristics of study subjects (30–60) age groups Referents/Patients with oral cancer
Non-smokeless tobacco consumer
Gutkha users
Snuff users
Mainpuri users
192
158
135
209
31 23 18 21 20 22 18 153
54 51 37 27 34 26 23 252
47 29 26 24 21 25 30 202
55 34 32 38 27 31 26 243
136
120
107
98
23 18 20 14 18 17 19 129
31 31 15 19 19 28 15 158
28 23 22 15 25 15 19 147
24 27 19 26 23 20 26 165
Male Referents Patients with oral cancer Lips Tongue Cheeks Floor of the mouth Hard and soft palate Sinuses Pharynx (throat) Total Female Referents Oral cancer patients Lips Tongue Cheeks Floor of the mouth Hard and soft palate Sinuses Pharynx (throat) Total
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Analytical figures of merit The concentration range of Cd for calibration curve reached from the detection limit up to 10 lg/l. The detection and quantification limits, given by LOD = 3 9 s/m and LOQ = 10 9 s/m, respectively, where s is the standard deviation of ten measurements of a reagent blank and m is the slope of the calibration graph, were calculated for electrothermal atomic absorption spectrometer determination. The LOD and LOQ were calculated for Cd as 0.28 and 0.81 lg/l, respectively. The validity and efficiency of the MAD method were checked with certified values of human hair CRM 397, Clincheckâ control-lyophilized human whole blood, Virginia tobacco leaves (ICHTJ-cta-VTL-2) and with those obtained from conventional wet acid digestion method (Table 2). The MAD method was less time consuming, requiring DBK. The contents of Cd in different brands of mainpuri (n = 12) were found in the range of 0.954–2.35 lg/g, respectively. The contents of Cd in different brands of gutkha (n = 11) were found in the range of 0.657–1.09 lg/g.
Cadmium concentration in biological samples of referents and patients with oral cancer The levels of Cd in patients with oral cancer versus those values obtained in biological samples of healthy referents are presented in Table 4. The resulted data indicated that contents of Cd were significantly higher in scalp hair and blood samples among patients with cancer (Lips, tongue, cheeks, floor of the mouth, hard and soft palate, sinuses, and pharynx) than those of referents (P < 0.001). The ranges of Cd in the scalp hair samples of male referents NU, GU, SU, and MPU were found at 95% confidence intervals (CI: 1.35–1.50,
Demographic characteristic of study population The patients and referents have same age, socioeconomic status, residential areas, and food habits. The referents and patients informed that they consumed mainpuri, snuff, and
Table 3 Cadmium concentrations in Gutkha, Mainpuri, dry, and moist Snuff samples –intake of cadmium by consuming 10 g of each snuff products (in micrograms per gram) Gutkha (G) G1 G2 G3 G4 G5 G6 G7 G8 G9 G 10 G 11
p x ts=n a
lg/10 gb
S
8.6–9.8c 6.5–7.6 7.52–8.81 7.85–7.93 8.17–8.29 5.20–5.27 9.27–9.38 8.72–8.83 9.64–9.77 7.08–7.18 9.05–9.22
BM1d BM2 BM3 BM4 BM5 BM6 BM7 BM8 GM9e GM10 GM11 GM12 GM13 GM14 DB1f DB2 DB3 DB4 DBK5g DBK6 DBK7 DBK8 DBK9
0.921 0.711 0.823 0.789 0.823 0.524 0.932 0.878 0.971 0.712 0.913
0.06 0.05 0.07 0.04 0.06 0.03 0.06 0.06 0.07 0.05 0.08
Average value confidence interval (P = 0.05). Intake of toxic metals/10 g of each snuff products. c Range. d Brown moist. e Green moist. f Dry brown. g Dry black. a
b
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Snuff p x ts=n a
lg/10 gb
MP
13.5–18.5 8.72–11.9 16.9–20.3 8.65–11.6 10.1–15.8 9.83–13.1 9.78–13.9 7.46–8.01 7.09–10.1 6.58–10.2 7.23–12.1 7.09–8.80 6.89–9.46 6.99–9.64 9.34–13.2 8.51–11.4 8.69–14.3 7.41–12.1 4.26–8.51 5.11–9.97 7.69–9.87 5.49–8.41 5.19–7.56
MP1 MP2 MP3 MP4 MP5 MP6 MP7 MP8 MP9 MP10 MP11 MP12
1.61 1.04 1.85 1.03 1.32 1.05 1.23 0.77 0.874 0.825 0.925 0.746 0.815 0.824 1.13 1.01 1.15 0.991 0.632 0.741 0.880 0.693 0.645
0.23 0.25 0.14 0.16 0.21 0.26 0.24 0.12 0.16 0.24 0.18 0.14 0.15 0.13 0.19 0.17 0.28 0.25 0.24 0.28 0.11 0.14 0.12
Mainpuri p x ts=n a
lg/10 gb
10.6–1.39 10.8–12.4 15.8–17.7 21.5–23.9 15.9–18.2 16.2–19.5 15.3–18.1 17.9–21.4 17.2–19.4 13.2–15.5 14.8–17.3 13.9–15.8
1.24 1.11 1.67 2.27 1.71 1.79 1.72 1.95 1.83 1.43 1.60 1.49
0.17 0.13 0.10 0.12 0.12 0.17 0.2 0.16 0.11 0.12 0.13 0.10
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Table 4 Cadmium concentrations in scalp hair and blood samples of referents and patients with oral cancer Patients with oral cancer Biological samples Male Scalp hair (lg/g)
Blood (lg/l)
Female Scalp hair
Blood
Types of patients
Referents
Lips
Tongue
Cheeks
Floor of the mouth
Hard and soft palate
Sinuses
Pharynx (throat)
NU GU SU MPU NU GU SU MPU
1.42 2.95 4.69 3.57 2.86 5.42 6.16 6.85
0.15 0.62 0.52 0.38 0.38 0.56 0.85 0.73
5.43 7.82 9.79 8.63 8.09 12.5 14.3 13.6
1.21 1.36 1.15 0.73 0.75 0.92 0.72 0.38
6.24 8.67 9.62 9.47 9.28 13.4 14.9 14.2
0.75 0.42 0.63 0.38 0.72 0.58 0.39 0.27
5.56 7.93 9.25 8.84 8.95 13.7 15.2 14.8
1.24 0.55 0.81 0.67 0.74 0.62 0.84 0.57
7.48 10.3 12.3 11.6 10.5 14.2 16.4 15.9
1.39 0.76 0.82 0.37 0.89 0.48 0.25 0.32
8.33 12.2 13.4 12.8 9.96 13.8 15.4 15.2
0.92 0.64 0.57 0.38 0.65 0.74 0.43 0.55
8.17 11.6 12.9 12.5 9.69 13.8 14.4 14.2
1.03 0.86 0.64 0.47 0.83 0.61 0.36 0.28
7.45 10.6 13.8 12.4 9.62 14.3 16.6 15.8
0.72 0.55 0.65 0.44 0.72 0.57 0.92 0.66
NU GU SU MPU NU GU SU MPU
1.35 2.98 4.63 3.92 2.79 5.35 5.93 6.67
0.26 0.52 0.71 0.95 0.18 0.79 0.92 0.55
5.38 12.8 14.2 13.8 7.85 12.4 14.2 13.5
0.85 1.53 2.38 1.85 0.62 0.51 0.49 0.21
6.16 13.7 14.5 14.2 9.05 13.3 15.3 13.8
0.52 1.82 2.05 1.74 0.51 0.44 0.76 0.34
6.78 13.9 14.9 14.6 8.89 13.5 15.6 14.6
0.99 2.35 1.81 1.62 0.65 0.54 0.73 0.45
7.59 13.5 15.2 14.8 10.3 13.6 16.8 15.5
1.05 1.94 1.76 1.38 1.20 0.67 0.53 0.48
8.21 14.5 15.6 15.1 9.92 13.9 14.8 15.6
0.60 1.25 0.85 1.74 0.78 0.56 0.69 0.31
8.29 15.8 16.9 16.3 9.36 13.6 14.3 14.6
0.49 2.18 2.54 1.42 0.77 0.72 0.58 0.50
8.09 15.6 16.7 16.3 9.52 14.5 16.3 15.5
1.95 1.72 1.68 1.53 0.81 0.43 0.60 0.37
NU, non-smokeless tobacco users; GU, gutkha users; SU, snuff users; MPU, mainpuri users.
2.63–3.25, 4.43–4.95, and CI: 3.38–3.75) lg/g, respectively, were significantly lower as compared to patients have consumed different SLT products (P < 0.001). The same trend was observed in females. The Cd concentrations in blood samples of male referents NU, GU, SU, and MPU were found at 95% confidence intervals (CI: 2.65, 3.06), (CI: 5.15, 5.68), (CI: 5.75, 6.68), (CI: 6.49, 7.26) and female (CI: 2.57–2.86), (CI: 4.98–5.43), (CI: 5.47–6.39), (CI: 6.40– 6.92) lg/l, respectively (Table 4). The unpaired student t-test between patients with cancerous and referents for different degrees of freedom was calculated at different probabilities. Our calculated tvalue exceeds that of tcritical value at 95% confidence intervals, which indicated that the difference between mean values of Cd in referents and patients with cancerous (both gender) showed significant differences (P < 0.001).
Discussion The pH of all SLT products was highly basic, found in the range of 8.1–8.7, which favors the formation of tobaccospecific amines thus making the product potentially toxic. The production of nitrosamines is major contributors to the increased risk of chewing SLT products for cancer of upper digestive tract (8). This case–control study was conducted to evaluate the possible association between Cd exposure via consumption of different types of SLT products and its altered levels in blood and scalp hair of patients with oral cancer with related to referents of both gender. The Cd concentration in mainpuri, snuff, and gutkha samples consumed by patients with cancerous and referents was determined. It was observed that the levels of Cd vary in biological samples of referents and patients, according to the types of SLT products consumed but difference was not significant (P > 0.05). In all SLT products, significant variation in metal contents would be expected (34). Although SLT is
described as a group 1 carcinogen by the International Agency for Research on Cancer (IARC), little is known regarding bioavailability, absorption, and toxicological effects of toxic and carcinogenic inorganic substances from them. The present analysis, based on dataset available on oral cancer in population of both gender, confirms that chewing SLT products could be major risk factors for the oral disease. The resulted data indicated that in referents of both gender, who not consumed any SLT have two to threefold lower levels of Cd in their biological samples as compared to those results obtained from referents consumed SLT products. The significant high levels of Cd were observed in hard and soft palate, pharynx among patients with cancer as compared to referents consumed SLT products (P < 0.001) as shown in Table 4. The significant high levels of Cd were observed in scalp hair of hard and soft palate, sinuses, patients with pharynx cancer in the range of (11.9–13.7; 11.2–13.2; 10.3–14.1) lg/g and blood samples (13.4–15.6; 13.5–14.6; 14.0–17.1) lg/l, respectively as compared to other oral cancers (P < 0.05). The Cd concentration in blood at 95% confidence levels reported in the United States for healthy non-exposed, nonsmokers is 0.4 lg/l (35). It was reported in literature that reference value of Cd in blood of non-exposed and nonsmokers was indicated in the range of 0.5–1.0 lg/l (36, 37). The present resulted data (Table 4) indicate that Cd levels exceeded reference ranges of Cd in both biological samples of referents as well as patients with cancer (38). It was reported that the mean Cd level in hair of exposed and normal subjects was 1.42 3.78 and 0.32 0.21 lg/g, respectively (39). It was indicated in literature that elevated blood Cd levels confirm recent acute exposure (40). It was reported in previous studies that certain toxic metals could be found in SLT products (41) and thus are inhaled (snuff) or ingested (gutkha and mainpuri), can cause serious diseases, including oral cancer (42). The International Agency of Research on Cancer has classified the Cd J Oral Pathol Med
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as a carcinogen of first category (43). The Cd has multiple effects on cells (44). It was reported in vivo studies that Cd affects cell cycle progression, proliferation, differentiation, as well as apoptotic pathways (45, 46). It adversely affects all four major pathways of DNA repair: (i) base excision, (ii) nucleotide excision, (iii) double-strand-break, and (iv) mismatch repair (47). The resulted data of Cd in referent and patients with cancerous have habit of chewing SLT is consisted with reported study that the people chewing SLT products are over five times more likely to be at risk of oral cancer (3, 48). The IARC now regards the betel nut which is part of mainpuri and gutkha, itself known human as a carcinogen (49). Many epidemiological studies have reported that oral cancer is strongly associated with the consumption of SLT products and alcohol drinking (1, 3). It is demonstrating that reactive oxygen species, such as hydroxyl radical, are formed in the human oral cavity during SLT products chewing, and that the activity might cause oxidative DNA damage, which transformed into oral cancer (50). There were some limitations in the present study. The study exposure period of Cd levels in biological samples via chewing SLT products reflected by the current metal levels of study subjects might not be long enough to cover the latency for oral cancer development (51). The study sample was not selected randomly and cannot be generalized to the whole population. While other Cd exposures due to food, drinking water, and industries were not collected. We also did not gather information about the smoking habits of other family members, but we do not think that we missed any additional findings because we did not select those referents and patients who were smokers. Another limitation of our study was that we could not attain biomedical validation of the current chewing habit of different SLT products.
Conclusion The results of this study revealed the significant differences in the Cd concentration in biological samples of patients with oral cancer as compared to non-cancerous referents consumed or not consumed any type of SLT products. The imbalance in Cd level in patients with oral cancer could be due to change of cellular metabolism in the cancer process. It was also observed that the socioeconomic factors may also play a role in higher mortality rates for patients with oral cancer, such as poor nutrition, irregular screening, late diagnosis, and unequal access to health care due to poverty, because the cost of cancer treatment is very high. As the role of Cd in the mechanism of different types of oral cancer development is still unclear, further detailed and comprehensive investigations are necessary.
References 1. Khandekar SP, Bagdey PS, Tiwari RR. Oral cancer and some epidemiological factors: a hospital based study. Indian J Community Med 2006; 31: 157–9. 2. Ferlay J, Bray F, Pisani P, Parkin DM. GLOBOCAN 2002: Cancer Incidence, Mortality and Prevalence Worldwide IARC Cancer Base (2002 Estimates). Lyon: IARC Press, 2004. J Oral Pathol Med
3. Kazi TG, Wadhwa SK, Afridi HI, et al. Evaluation of cadmium and zinc in biological samples of tobacco and alcohol user male mouth cancer, patients. Hum Exp Toxicol 2010; 29: 221–30. 4. Rodu B, Cole P. Smokeless tobacco use and cancer of the upper respiratory tract. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002; 93: 511–5. 5. Accortt AN, Waterbor WJ, Beall C, Howard G. Cancer incidence among a cohort of smokeless tobacco users (United States). Cancer Causes Control 2005; 16: 1107–15. 6. Bouquot JE. Reviewing oral leukoplakia. Clinical concepts for the 1990s. J Am Dent Assoc 1991; 122: 80–2. 7. Golia EE, Dimirkou EA, Mitsios EIK. Accumulation of metals on tobacco leaves (primings) grown in an agricultural area in relation to soil. Bull Environ Contam Toxicol 2007; 79: 158–62. 8. Stepanov I, Hecht SS. Tobacco-specific nitrosamines and their pyridine-N-glucuronides in the urine of smokers and smokeless tobacco users. Cancer Epidemiol Biomarkers Prev 2005; 14: 885–91. 9. Elinder CG. Cadmium as an environmental hazard. IARC Sci Publ 1992; 118: 123–32. 10. Chen J, Teo KC. Determination of cadmium, copper, lead and zinc in water samples by flame atomic absorption spectrometry after cloud point extraction. Anal Chim Acta 2001; 450: 215– 22. 11. Sravrides JC. Lung carcinogenesis: pivotal role of metals in tobacco smoke. Free Radic Biol Med 2006; 41: 1017–30. 12. McMurray CT, Tainer JA. Cancer, cadmium and genome integrity. Nat Genet 2003; 34: 239–41. 13. Zheng H, Liu J, Choo KH, Michalska AE, Klaassen CD. Metallothionein-I and -II knock-out mice are sensitive to cadmium-induced liver mRNA expression of c-jun and p53. Toxicol Appl Pharmacol 1996; 136: 229–35. 14. Abshire MK, Buzard GS, Shiraishi N, Waalkes MP. Induction of c-myc and c-jun proto-oncogene expression in rat L6 myoblasts by cadmium is inhibited by zinc preinduction of the metallothionein gene. J Toxicol Environ Health 1996; 48: 359–77. 15. Shimada H, Shiao YH, Shibata M, Waalkes MP. Cadmium suppresses apoptosis induced by chromium. J Toxicol Environ Health 1998; 54: 159–68. 16. Miccadei S, Floridi A. Sites of inhibition of mitochondrial electron transport by cadmium. Chem Biol Interact 1993; 89: 156–67. 17. Drasch G, Schopfer J, Schrauzer GN. Selenium/cadmium ratios in human prostates Indicators of prostate cancer risk of smokers and nonsmokers, and relevance to the cancer protective effects of selenium. Biol Trace Elem Res 2005; 103: 103–7. 18. Waalkes MP. Cadmium carcinogenesis in review. J Inorg Biochem 2000; 79: 241–4. 19. National Toxicology Program, Tenth Report on Carcinogens. Research Triangle Park, NC: Department of Health and Human Services, 2000; 42–4. 20. Cho YA, Kim J, Woo HD, Kang M. Dietary cadmium intake and the risk of cancer: a meta-analysis. PLoS ONE 2013; 8: 1–8. 21. Adams SV, Passarelli MN, Newcomb PA. Cadmium exposure and cancer mortality in the Third National Health and Nutrition Examination Survey cohort. Occup Environ Med 2012; 69: 153–6. 22. Julin B, Wolk A, Johansson JE, Andersson SO, Andren O, Akesson A. Dietary cadmium exposure and prostate cancer incidence: a population-based prospective cohort study. Br J Cancer 2012; 107: 895–900. 23. Hu J, Mao Y, White K. Canadian Cancer Registries Epidemiology Research Group. Renal cell carcinoma and occupational exposure in Canada. Occup Med 2002; 52: 157–64.
Cadmium level in patients with oral cancer Arain et al.
24. Schwartz GG, Reis IM. Is cadmium a cause of human pancreatic cancer. Cancer Epidemiol Biomarkers Prev 2000; 9: 139–45. 25. Avti PK, Kumar S, Pathak CM, Vaiphei K, LAL K. Smokeless tobacco impairs the antioxidant defense in liver, lung, and kidney of rats. Toxicol Sci 2006; 89: 547–53. 26. Chen YC, Pu YS, Wu HC, et al. Cadmium burden and the risk and phenotype of prostate cancer. BMC Cancer 2009; 9: 429. 27. Verougstraete V, Lison D, Hotz P. Cadmium, lung and prostate cancer: a systematic review of recent epidemiological data. J Toxicol Environ Health 2003; 6: 227–55. 28. Kazi TG, Memon AR, Afridi HI, et al. Determination of cadmium in whole blood and scalp hair samples of Pakistani male lung cancer patients by electrothermal atomic absorption spectrometer. Sci Total Environ 2008; 389: 270–6. 29. Nagata C, Nagao Y, Nakamura K, et al. Cadmium exposure and the risk of breast cancer in Japanese women. Breast Cancer Res Treat 2013; 138: 235–9. 30. McElroy JA, Shafer MM, Trentham-Dietz A, Hampton JM, Newcomb PA. Cadmium exposure and breast cancer risk. J Natl Cancer Inst 2006; 98: 869–73. 31. Akesson A, Julin B, Wolk A. Long-term dietary cadmium intake and postmenopausal endometrial cancer incidence: a population-based prospective cohort study. Cancer Res 2008; 68: 6435–41. 32. Julin B, Wolk A, Akesson A. Dietary cadmium exposure and risk of epithelial ovarian cancer in a prospective cohort of Swedish women. Br J Cancer 2011; 105: 441–4. 33. Afridi HI, Kazi TG, , Kazi GH, Jamali MK, Shar GQ et al. Analysis of heavy metals in scalp hair samples of hypertensive patients by conventional and microwave digestion methods. Spectrosc Lett 2006; 39: 203–14. 34. Asta J, Guillard E, Tissut M, Gaude T, Ravanel P. Heavy metal transfer from atmosphere to plants. J Phys 2003; 107: 65–7. 35. Centers for Disease Control and Prevention. Third National Report on Human Exposure to Environmental Chemicals. Atlanta, GA: National Center for Environmental Health, 2005. 36. Herber RF, Christensen JM, Sabbioni E. Critical evaluation and review of cadmium concentrations in blood for use in occupational health according to the TRACY protocol. Int Arch Occup Environ Health 1997; 69: 372–8. 37. Wilhelm M, Schulz C, Schwenk M. Revised and new reference values for arsenic, cadmium, lead, and mercury in blood or urine of children: basis for validation of human
38. 39. 40.
41. 42.
43.
44. 45.
46. 47. 48. 49. 50. 51.
biomonitoring data in environmental medicine. Int J Hyg Environ Health 2006; 209: 301–5. Horn PS, Pesce AJ. Reference intervals: an update. Clin Chim Acta 2003; 334: 5–23. Mehra R, Juneja M. Elements in scalp hair and nails indicating metal body burden in polluted environment. J Sci Ind Res 2005; 67: 119–24. Agency for Toxic Substances and Disease Registry. Toxicological Profile for Cadmium. Atlanta, GA: US Department of Health and Human Services. NTIS Report No. PB/89/194476/ AS, 1999. Al-Rmalli SW, Jenkins RO, Haris PI. Betel quid chewing elevates human exposure to arsenic, cadmium and lead. J Hazard Mater 2011; 190: 69–74. Kazi TG, Wadhwa SK, Afridi HI, et al. Interaction of cadmium and zinc in biological samples of smokers and chewing tobacco female mouth cancer patients. J Hazard Mater 2010; 176: 985–91. IARC Cadmium and Cadmium Compounds. Beryllium, cadmium, mercury, and exposures in the glass manufacturing industry. IARC Monogr Eval Carcinog Risks Hum 1993; 58: 119–237. Qu Q, Melikian AA, Li G, et al. Validation of biomarkers in humans exposed to benzene: urine metabolites. Am J Ind Med 2000; 37: 522–31. Fang MZ, Mar WC, Cho MH. Cell cycle was disturbed in the MNNG-induced initiation stage during in vitro two-stage transformation of Balb/3T3 cells. Toxicology 2001; 163: 175–84. Dong S, Shen HM, Ong CN. Cadmium-induced apoptosis and phenotypic changes in mouse thymocytes. Mol Cell Biochem 2001; 222: 11–20. Hartwig A, Schwerdtle T. Interactions by carcinogenic metal compounds with DNA repair processes: toxicological implications. Toxicol Lett 2002; 127: 47–54. Ahluwalia KP. Assessing the oral cancer risk of South-Asian immigrants in New York City. Cancer 2005; 104: 2959–61. Warnakulasuriya S, Trivedy C, Peters TJ. Areca nut use: an independent risk factor for oral cancer. Br Med J 2002; 324: 799–800. Hecht SS. Tobacco carcinogens, their biomarkers and tobaccoinduced cancer. Nat Rev Cancer 2003; 3: 733–44. Nordberg GF, Fowler BA, Nordberg M, Friberg LT, editors. Handbook on the Toxicology of Metals, 3rd edn. Burlinton, MA: Academic Press, 2007.
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J Oral Pathol Med (2015) 44: 707–713 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
Scalp hair and blood cadmium levels in association with chewing gutkha, mainpuri, and snuff, among patients with oral cancer in Pakistan Sadaf Sadia Arain1, Tasneem Gul Kazi1, Hassan Imran Afridi1, Farah Naz Talpur1, Atif Gul Kazi2, Kapil Dev Brahman1, Abdul Haleem Panhwar1, Mariam Shahzadi Arain1 1
National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, Pakistan; 2Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan
BACKGROUND: It has been reported that smokeless tobacco (SLT) chewing can lead to caners of oral cavity. In this study, the relationship between cadmium exposure via consumption of different SLT products by patients with oral cancer with related to referents belongs to different cities of Pakistan are investigated. METHODS: The cadmium in different types of SLT products (gutkha, mainpuri and snuff) consumed by studied population and biological samples (scalp hair and blood) of cancerous and healthy subjects were analyzed. Both referents and patients with cancer have same age group (ranged 30–60 years), socioeconomic status, localities, and dietary habits. The concentrations of cadmium in SLT products and biological samples were measured by electrothermal atomic absorption spectrophotometer after microwave-assisted acid digestion. The validity and accuracy of the methodology were checked by certified reference materials. RESULTS: It was estimated that 10 g intake of different types of gutkha, mainpuri, and snuff could contribute, 18–40%, 15.7–33.6%, and 14–68% of the provisional maximum tolerable daily intake of cadmium, respectively for adults (60 kg). The results of present study showed that the mean value of cadmium was significantly higher in scalp hair and blood samples of patients with oral cancer as compared to those results obtained for referents (P < 0.001). CONCLUSIONS: The high exposure of cadmium due to consuming different SLT products may have synergistic effects with other risk factors associated for oral cancer. J Oral Pathol Med (2015) 44: 707–713
Correspondence: Hassan Imran Afridi, PhD, Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan. Tel: +92 0222 771379, Fax: +92 0221 771560, E-mail: hassanimranafridi@ yahoo.com Accepted for publication September 8, 2014
Keywords: atomic absorption spectrophotometer; biological samples; cadmium; oral cancer; smokeless tobacco products
Introduction The oral cancer is a common malignancy among people, who are smoker and chewer tobacco, with a worldwide incidence of over 300 000 new cases annually (1). The disease is characterized by a high rate of morbidity and mortality (2). The etiology of oral cancer is multifactorial, major risk factors are tobacco and alcohol consumption (3). Both tobacco smoking (cigarettes, cigars, and pipes) and chewing smokeless tobacco (SLT) products with and without other ingredients have been shown to increase the risk of developing oral cancer (3–5). Cancers caused by SLT use often begin as leukoplakia or erythroplakia; erythroplakia generally has a higher chance to becoming cancerous over time (6). Tobacco plant (Nicotiana tabacum) is well known for its capacity to concentrate toxic elements from its growing environment (7). Tobacco is known to contain numerous classes of carcinogenic substance such as tobacco-specific nitrosamines, which are often regarded as a major factor in SLT-related carcinogenesis. The combined exposure of nitrosamines and other classes of organic and inorganic substances, including toxic metals enhances the carcinogenetic effects (8). The cadmium (Cd) is a nonessential, potentially toxic, pollutant ion, which is accumulated by tobacco plants and diets and is transferred to human via smoking and chewing tobacco products (9–11). It was reported that Cd has adverse impact on human via genotoxic mechanisms including induction of single-strand DNA breaks, and also that it inhibits DNA repair by inactivation of the mismatch repair system (12). The Cd activates protooncogenes (13, 14) and inhibits normal apoptosis of cells (15). It was reported that Cd is an inhibitor of the enzymes with sulphydryl groups and disrupts the pathways for the oxidative metabolism (16). In vitro studies suggest that there
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is competition for transport mechanisms between Cd and some essential trace elements mainly Zn, Se, and Cu (17). Various regulatory agencies have reported that Cd is a human carcinogen (18–20). Several studies indicate a role for Cd in human prostatic, pancreatic, and renal cancers (21–24), while a few studies have associated Cd exposure with human cancer of the liver and urinary bladder (22, 25, 26). In non-occupationally exposed populations, Cd has also been associated with lung cancer incidence and mortality (27, 28). Prospective studies observed an association between dietary Cd and risk of breast cancer (29, 30) and endometrial cancer (31). Several other studies have investigated the role of Cd in cancer development in female reproductive organs (31, 32). The aim of present study was to evaluate and compare the concentration of Cd in different types of moist and dry snuff, mainpuri, and gutkha available and consumed in Pakistan, and to estimate the Cd levels in scalp hair and blood samples of patients diagnosed with oral cancer and healthy individuals.
Materials and methods Reagents and glassware Ultra-pure water obtained from ELGA labwater system (Bucks, UK). Concentrated nitric acid (65%) and hydrogen peroxide (30%) were obtained from Merck (Darmstadt, Germany). Working standard solutions of Cd were prepared prior to their use by stepwise dilution of certified standard solutions (1000 ppm) Fluka Kamica (Buchs, Switzerland), with 0.2 mol/l of HNO3. Moreover, matrix modifier was employed to analyze Cd [0.001 mg Pd + 0.0015 mg Mg (NO3)2], was prepared from Mg(NO3)2, and 99.999% Pd (Sigma, St. Louis, MO, USA). All solutions were stored in polyethylene bottles at 4°C. For the accuracy of methodology, certified reference materials (CRM), human hair BCR 397, Clincheckâ control-lyophilized human whole blood and Virginia tobacco leaves (ICHTJ-cta-VTL-2) were used. Glasswares and polyethylene containers were soaked in 10% (v/v) HNO3 for 24 h; washed with distilled water, finally with de-ionized water and dried in such a manner to ensure that no any contamination from glasswares occur. Instrumentation The determination of Cd was carried out by means of a double-beam Perkins-Elmer Atomic Absorption Spectrometer Model 700 (Perkin Elmer, Norwalk, CT, USA) equipped with the graphite furnace HGA-400, pyrocoated graphite tubes with integrated platform, an autosampler AS800 and deuterium lamp as background correction system. Hollow cathode lamp (Perkin Elmer) was used as radiation sources, and they were operating at a recommended current. All instrumental conditions were used according to the manufacturer’s recommendation. Portions of both standard or sample and modifier were transferred into autosampler cups, and 20 ll (standard or sample volume of 10 ll and 10 ll of modifier in each case) was injected to electrothermal graphite atomizer. A horizontal flask electrical shaker (220/60 Hz; Gallenkamp, England) was used for shaking the samples. The pH was measured by a pH meter (781-pH meter; Metrohm; Metrohm Ltd., Herisau, Switzerland). A J Oral Pathol Med
Pak Elektron Limited domestic microwave oven (Osaka, Japan), programmable for time and microwave power from 100 to 900 W, was used for digestion of samples. Study population A survey was carried out about the gutkha and mainpuri chewing, while snuff inhaling habits of both genders, age ranged 30–60 years, residing in the different cities of Pakistan. The data of hospital based case–control study population were collected from Nuclear Institute of Medicine and Radiotherapy (NIMRA) Jamshoro and Larkana institute of nuclear medicine and radiotherapy (LINAR), situated in different areas of Sindh, Pakistan, during 2010– 2012 years, by collecting files and extracting important information. During 1-year study period (2010), the information department of both hospitals recorded >5200 cases of cancers of all types, and mouth cancer comprised of 2.7% of the total. The patients with oral cancer were divided into subgroups according to the different over found locations of oral cancer, Lips, tongue, cheeks, floor of the mouth, hard and soft palate, sinuses, and pharynx (throat). Patients with oral cancer and referents were further grouped according to their SLT chewing habits, not consumed any SLT product (NU), gutkha (GU), snuff (SU), and mainpuri users (MPU). Complete demographic information is listed in Table 1. Physical examinations were performed to measure participant’s weight, height, blood pressure, and biochemical data. The biochemical tests of patients and referents were performed to estimate hemoglobin, red blood cells, packed cell volume, mean corpuscular hemoglobin concentration, mean corpuscular volume, and transferrin iron-binding capacity in the blood. The criteria for the selection of patients were of biopsy proved oral squamous cell carcinoma prior to any treatment and that they were not taking any mineral supplements during last 3 months. The criteria for selection of 1155 referent subjects were by matching to the same age group, socioeconomic status, and dietary habits, being free of any cancer diagnosis and not taking any mineral supplement. The biochemical results and histological information are not reported in present study. Prior to the biological samples collection, they have underwent a standard routine medical examination. This study was approved by ethical committee of Sindh University, working under the auspices of higher education commission of Pakistan. Sampling of SLT products A total of 23 brands of snuff (dry and moist), 11 brands of gutkha, and 12 brands of mainpuri were purchased from local markets of the different cities of Pakistan as per their availability over a 3-year period (January 2010–December 2013). The samples were packed in their original packing and placed in pre-washed dried plastic bags separately and stored at 4°C, until tested. Five composite samples of each brand of snuff, gutkha, and mainpuri were prepared by homogenizing the mixture after removing the wrappers. Care was taken to avoid any source of contamination, and this preparation was carried out in a clean environment. All samples were dried at 80°C. The dried samples were ground with agate mortar and pestle, sieved through nylon sieves with mesh sizes of 125 lm, and then stored in the labeled sample bottles.
Cadmium level in patients with oral cancer Arain et al.
Biological samples Venous blood samples (5 ml) were collected by 7 mm heparinized lithium Vacutainerâ tubes (Becton Dickinson, East Rutherford, NJ, USA). About 2 ml of venous blood samples was stored at 20°C until elemental analysis. The scalp hair samples were taken from five different parts of the scalp (frontal, cranial, occipital, right, and left lateral). The first 5 cm of hair from the root was used for analysis. Hair samples were put into separate plastic envelopes for each participant, tightly sealed and attached with identification number of the participant and questionnaire. In the laboratory, hair specimens were further cut into pieces, approximately 0.2–0.3 cm, and washed four times with a 1:200 v/v dilution of Triton X-100, then rinsed three times with ultra-pure water and two times with acetone (33). Then samples were then dried in an oven at 80–85°C.
Microwave-assisted acid digestion A microwave-assisted digestion (MAD) procedure was carried out, to achieve a shorter digestion time. Replicate six samples of each CRM (0.5 ml Clincheckâ controllyophilized human whole blood, 0.2 g of Virginia tobacco leaves and BCR 397 human hair) and duplicate samples of different types of SLT products (0.2 g), whole blood (0.5 ml), and scalp hair (0.2 g) were taken separately in polytetrafluoroethylene (PTFE) flasks (25 ml in capacity). Then added 3 ml of a freshly prepared mixture of concentrated HNO3–H2O2 (2:1, v/v), kept at room temperature for 10 min. Then placed the flasks in covered PTFE container and heated at 80% of total power (900 W) for 3–4 min. The digested samples were diluted up to 10 ml with 0.1 mol/l concentrated HNO3. A blank extraction (without sample) was carried out through the complete procedure.
pH determination of SLT products Weighed 1 g sample of each brand of gutkha, mainpuri, moist, and dry snuff, added 10 ml of ultrapure water in flask (100 ml capacity), and placed in an electrical shaker at 2 g for 30 min, then filtered the solution through Whatman No. 42 filter paper and extracts was taken to determine the pH.
Statistical analysis All statistical analyses were performed using computer program Excel X State (Microsoft Corp., Redmond, WA, USA) and Minitab 13.2 (Minitab Inc., State College, PA, USA). The data from triplicate samples of each composite samples were expressed as means SD. The Student’s ttest was used to assess the significant difference of Cd in certified and experimentally found values. The ANOVA was used to assess the significance of differences between the concentrations of Cd observed in the biological samples of patients and referent subjects, calculated by the unpaired two-sample t-test. A P < 0.05 was considered significant difference.
Table 1 Characteristics of study subjects (30–60) age groups Referents/Patients with oral cancer
Non-smokeless tobacco consumer
Gutkha users
Snuff users
Mainpuri users
192
158
135
209
31 23 18 21 20 22 18 153
54 51 37 27 34 26 23 252
47 29 26 24 21 25 30 202
55 34 32 38 27 31 26 243
136
120
107
98
23 18 20 14 18 17 19 129
31 31 15 19 19 28 15 158
28 23 22 15 25 15 19 147
24 27 19 26 23 20 26 165
Male Referents Patients with oral cancer Lips Tongue Cheeks Floor of the mouth Hard and soft palate Sinuses Pharynx (throat) Total Female Referents Oral cancer patients Lips Tongue Cheeks Floor of the mouth Hard and soft palate Sinuses Pharynx (throat) Total
709
Analytical figures of merit The concentration range of Cd for calibration curve reached from the detection limit up to 10 lg/l. The detection and quantification limits, given by LOD = 3 9 s/m and LOQ = 10 9 s/m, respectively, where s is the standard deviation of ten measurements of a reagent blank and m is the slope of the calibration graph, were calculated for electrothermal atomic absorption spectrometer determination. The LOD and LOQ were calculated for Cd as 0.28 and 0.81 lg/l, respectively. The validity and efficiency of the MAD method were checked with certified values of human hair CRM 397, Clincheckâ control-lyophilized human whole blood, Virginia tobacco leaves (ICHTJ-cta-VTL-2) and with those obtained from conventional wet acid digestion method (Table 2). The MAD method was less time consuming, requiring DBK. The contents of Cd in different brands of mainpuri (n = 12) were found in the range of 0.954–2.35 lg/g, respectively. The contents of Cd in different brands of gutkha (n = 11) were found in the range of 0.657–1.09 lg/g.
Cadmium concentration in biological samples of referents and patients with oral cancer The levels of Cd in patients with oral cancer versus those values obtained in biological samples of healthy referents are presented in Table 4. The resulted data indicated that contents of Cd were significantly higher in scalp hair and blood samples among patients with cancer (Lips, tongue, cheeks, floor of the mouth, hard and soft palate, sinuses, and pharynx) than those of referents (P < 0.001). The ranges of Cd in the scalp hair samples of male referents NU, GU, SU, and MPU were found at 95% confidence intervals (CI: 1.35–1.50,
Demographic characteristic of study population The patients and referents have same age, socioeconomic status, residential areas, and food habits. The referents and patients informed that they consumed mainpuri, snuff, and
Table 3 Cadmium concentrations in Gutkha, Mainpuri, dry, and moist Snuff samples –intake of cadmium by consuming 10 g of each snuff products (in micrograms per gram) Gutkha (G) G1 G2 G3 G4 G5 G6 G7 G8 G9 G 10 G 11
p x ts=n a
lg/10 gb
S
8.6–9.8c 6.5–7.6 7.52–8.81 7.85–7.93 8.17–8.29 5.20–5.27 9.27–9.38 8.72–8.83 9.64–9.77 7.08–7.18 9.05–9.22
BM1d BM2 BM3 BM4 BM5 BM6 BM7 BM8 GM9e GM10 GM11 GM12 GM13 GM14 DB1f DB2 DB3 DB4 DBK5g DBK6 DBK7 DBK8 DBK9
0.921 0.711 0.823 0.789 0.823 0.524 0.932 0.878 0.971 0.712 0.913
0.06 0.05 0.07 0.04 0.06 0.03 0.06 0.06 0.07 0.05 0.08
Average value confidence interval (P = 0.05). Intake of toxic metals/10 g of each snuff products. c Range. d Brown moist. e Green moist. f Dry brown. g Dry black. a
b
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Snuff p x ts=n a
lg/10 gb
MP
13.5–18.5 8.72–11.9 16.9–20.3 8.65–11.6 10.1–15.8 9.83–13.1 9.78–13.9 7.46–8.01 7.09–10.1 6.58–10.2 7.23–12.1 7.09–8.80 6.89–9.46 6.99–9.64 9.34–13.2 8.51–11.4 8.69–14.3 7.41–12.1 4.26–8.51 5.11–9.97 7.69–9.87 5.49–8.41 5.19–7.56
MP1 MP2 MP3 MP4 MP5 MP6 MP7 MP8 MP9 MP10 MP11 MP12
1.61 1.04 1.85 1.03 1.32 1.05 1.23 0.77 0.874 0.825 0.925 0.746 0.815 0.824 1.13 1.01 1.15 0.991 0.632 0.741 0.880 0.693 0.645
0.23 0.25 0.14 0.16 0.21 0.26 0.24 0.12 0.16 0.24 0.18 0.14 0.15 0.13 0.19 0.17 0.28 0.25 0.24 0.28 0.11 0.14 0.12
Mainpuri p x ts=n a
lg/10 gb
10.6–1.39 10.8–12.4 15.8–17.7 21.5–23.9 15.9–18.2 16.2–19.5 15.3–18.1 17.9–21.4 17.2–19.4 13.2–15.5 14.8–17.3 13.9–15.8
1.24 1.11 1.67 2.27 1.71 1.79 1.72 1.95 1.83 1.43 1.60 1.49
0.17 0.13 0.10 0.12 0.12 0.17 0.2 0.16 0.11 0.12 0.13 0.10
Cadmium level in patients with oral cancer Arain et al.
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Table 4 Cadmium concentrations in scalp hair and blood samples of referents and patients with oral cancer Patients with oral cancer Biological samples Male Scalp hair (lg/g)
Blood (lg/l)
Female Scalp hair
Blood
Types of patients
Referents
Lips
Tongue
Cheeks
Floor of the mouth
Hard and soft palate
Sinuses
Pharynx (throat)
NU GU SU MPU NU GU SU MPU
1.42 2.95 4.69 3.57 2.86 5.42 6.16 6.85
0.15 0.62 0.52 0.38 0.38 0.56 0.85 0.73
5.43 7.82 9.79 8.63 8.09 12.5 14.3 13.6
1.21 1.36 1.15 0.73 0.75 0.92 0.72 0.38
6.24 8.67 9.62 9.47 9.28 13.4 14.9 14.2
0.75 0.42 0.63 0.38 0.72 0.58 0.39 0.27
5.56 7.93 9.25 8.84 8.95 13.7 15.2 14.8
1.24 0.55 0.81 0.67 0.74 0.62 0.84 0.57
7.48 10.3 12.3 11.6 10.5 14.2 16.4 15.9
1.39 0.76 0.82 0.37 0.89 0.48 0.25 0.32
8.33 12.2 13.4 12.8 9.96 13.8 15.4 15.2
0.92 0.64 0.57 0.38 0.65 0.74 0.43 0.55
8.17 11.6 12.9 12.5 9.69 13.8 14.4 14.2
1.03 0.86 0.64 0.47 0.83 0.61 0.36 0.28
7.45 10.6 13.8 12.4 9.62 14.3 16.6 15.8
0.72 0.55 0.65 0.44 0.72 0.57 0.92 0.66
NU GU SU MPU NU GU SU MPU
1.35 2.98 4.63 3.92 2.79 5.35 5.93 6.67
0.26 0.52 0.71 0.95 0.18 0.79 0.92 0.55
5.38 12.8 14.2 13.8 7.85 12.4 14.2 13.5
0.85 1.53 2.38 1.85 0.62 0.51 0.49 0.21
6.16 13.7 14.5 14.2 9.05 13.3 15.3 13.8
0.52 1.82 2.05 1.74 0.51 0.44 0.76 0.34
6.78 13.9 14.9 14.6 8.89 13.5 15.6 14.6
0.99 2.35 1.81 1.62 0.65 0.54 0.73 0.45
7.59 13.5 15.2 14.8 10.3 13.6 16.8 15.5
1.05 1.94 1.76 1.38 1.20 0.67 0.53 0.48
8.21 14.5 15.6 15.1 9.92 13.9 14.8 15.6
0.60 1.25 0.85 1.74 0.78 0.56 0.69 0.31
8.29 15.8 16.9 16.3 9.36 13.6 14.3 14.6
0.49 2.18 2.54 1.42 0.77 0.72 0.58 0.50
8.09 15.6 16.7 16.3 9.52 14.5 16.3 15.5
1.95 1.72 1.68 1.53 0.81 0.43 0.60 0.37
NU, non-smokeless tobacco users; GU, gutkha users; SU, snuff users; MPU, mainpuri users.
2.63–3.25, 4.43–4.95, and CI: 3.38–3.75) lg/g, respectively, were significantly lower as compared to patients have consumed different SLT products (P < 0.001). The same trend was observed in females. The Cd concentrations in blood samples of male referents NU, GU, SU, and MPU were found at 95% confidence intervals (CI: 2.65, 3.06), (CI: 5.15, 5.68), (CI: 5.75, 6.68), (CI: 6.49, 7.26) and female (CI: 2.57–2.86), (CI: 4.98–5.43), (CI: 5.47–6.39), (CI: 6.40– 6.92) lg/l, respectively (Table 4). The unpaired student t-test between patients with cancerous and referents for different degrees of freedom was calculated at different probabilities. Our calculated tvalue exceeds that of tcritical value at 95% confidence intervals, which indicated that the difference between mean values of Cd in referents and patients with cancerous (both gender) showed significant differences (P < 0.001).
Discussion The pH of all SLT products was highly basic, found in the range of 8.1–8.7, which favors the formation of tobaccospecific amines thus making the product potentially toxic. The production of nitrosamines is major contributors to the increased risk of chewing SLT products for cancer of upper digestive tract (8). This case–control study was conducted to evaluate the possible association between Cd exposure via consumption of different types of SLT products and its altered levels in blood and scalp hair of patients with oral cancer with related to referents of both gender. The Cd concentration in mainpuri, snuff, and gutkha samples consumed by patients with cancerous and referents was determined. It was observed that the levels of Cd vary in biological samples of referents and patients, according to the types of SLT products consumed but difference was not significant (P > 0.05). In all SLT products, significant variation in metal contents would be expected (34). Although SLT is
described as a group 1 carcinogen by the International Agency for Research on Cancer (IARC), little is known regarding bioavailability, absorption, and toxicological effects of toxic and carcinogenic inorganic substances from them. The present analysis, based on dataset available on oral cancer in population of both gender, confirms that chewing SLT products could be major risk factors for the oral disease. The resulted data indicated that in referents of both gender, who not consumed any SLT have two to threefold lower levels of Cd in their biological samples as compared to those results obtained from referents consumed SLT products. The significant high levels of Cd were observed in hard and soft palate, pharynx among patients with cancer as compared to referents consumed SLT products (P < 0.001) as shown in Table 4. The significant high levels of Cd were observed in scalp hair of hard and soft palate, sinuses, patients with pharynx cancer in the range of (11.9–13.7; 11.2–13.2; 10.3–14.1) lg/g and blood samples (13.4–15.6; 13.5–14.6; 14.0–17.1) lg/l, respectively as compared to other oral cancers (P < 0.05). The Cd concentration in blood at 95% confidence levels reported in the United States for healthy non-exposed, nonsmokers is 0.4 lg/l (35). It was reported in literature that reference value of Cd in blood of non-exposed and nonsmokers was indicated in the range of 0.5–1.0 lg/l (36, 37). The present resulted data (Table 4) indicate that Cd levels exceeded reference ranges of Cd in both biological samples of referents as well as patients with cancer (38). It was reported that the mean Cd level in hair of exposed and normal subjects was 1.42 3.78 and 0.32 0.21 lg/g, respectively (39). It was indicated in literature that elevated blood Cd levels confirm recent acute exposure (40). It was reported in previous studies that certain toxic metals could be found in SLT products (41) and thus are inhaled (snuff) or ingested (gutkha and mainpuri), can cause serious diseases, including oral cancer (42). The International Agency of Research on Cancer has classified the Cd J Oral Pathol Med
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as a carcinogen of first category (43). The Cd has multiple effects on cells (44). It was reported in vivo studies that Cd affects cell cycle progression, proliferation, differentiation, as well as apoptotic pathways (45, 46). It adversely affects all four major pathways of DNA repair: (i) base excision, (ii) nucleotide excision, (iii) double-strand-break, and (iv) mismatch repair (47). The resulted data of Cd in referent and patients with cancerous have habit of chewing SLT is consisted with reported study that the people chewing SLT products are over five times more likely to be at risk of oral cancer (3, 48). The IARC now regards the betel nut which is part of mainpuri and gutkha, itself known human as a carcinogen (49). Many epidemiological studies have reported that oral cancer is strongly associated with the consumption of SLT products and alcohol drinking (1, 3). It is demonstrating that reactive oxygen species, such as hydroxyl radical, are formed in the human oral cavity during SLT products chewing, and that the activity might cause oxidative DNA damage, which transformed into oral cancer (50). There were some limitations in the present study. The study exposure period of Cd levels in biological samples via chewing SLT products reflected by the current metal levels of study subjects might not be long enough to cover the latency for oral cancer development (51). The study sample was not selected randomly and cannot be generalized to the whole population. While other Cd exposures due to food, drinking water, and industries were not collected. We also did not gather information about the smoking habits of other family members, but we do not think that we missed any additional findings because we did not select those referents and patients who were smokers. Another limitation of our study was that we could not attain biomedical validation of the current chewing habit of different SLT products.
Conclusion The results of this study revealed the significant differences in the Cd concentration in biological samples of patients with oral cancer as compared to non-cancerous referents consumed or not consumed any type of SLT products. The imbalance in Cd level in patients with oral cancer could be due to change of cellular metabolism in the cancer process. It was also observed that the socioeconomic factors may also play a role in higher mortality rates for patients with oral cancer, such as poor nutrition, irregular screening, late diagnosis, and unequal access to health care due to poverty, because the cost of cancer treatment is very high. As the role of Cd in the mechanism of different types of oral cancer development is still unclear, further detailed and comprehensive investigations are necessary.
References 1. Khandekar SP, Bagdey PS, Tiwari RR. Oral cancer and some epidemiological factors: a hospital based study. Indian J Community Med 2006; 31: 157–9. 2. Ferlay J, Bray F, Pisani P, Parkin DM. GLOBOCAN 2002: Cancer Incidence, Mortality and Prevalence Worldwide IARC Cancer Base (2002 Estimates). Lyon: IARC Press, 2004. J Oral Pathol Med
3. Kazi TG, Wadhwa SK, Afridi HI, et al. Evaluation of cadmium and zinc in biological samples of tobacco and alcohol user male mouth cancer, patients. Hum Exp Toxicol 2010; 29: 221–30. 4. Rodu B, Cole P. Smokeless tobacco use and cancer of the upper respiratory tract. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002; 93: 511–5. 5. Accortt AN, Waterbor WJ, Beall C, Howard G. Cancer incidence among a cohort of smokeless tobacco users (United States). Cancer Causes Control 2005; 16: 1107–15. 6. Bouquot JE. Reviewing oral leukoplakia. Clinical concepts for the 1990s. J Am Dent Assoc 1991; 122: 80–2. 7. Golia EE, Dimirkou EA, Mitsios EIK. Accumulation of metals on tobacco leaves (primings) grown in an agricultural area in relation to soil. Bull Environ Contam Toxicol 2007; 79: 158–62. 8. Stepanov I, Hecht SS. Tobacco-specific nitrosamines and their pyridine-N-glucuronides in the urine of smokers and smokeless tobacco users. Cancer Epidemiol Biomarkers Prev 2005; 14: 885–91. 9. Elinder CG. Cadmium as an environmental hazard. IARC Sci Publ 1992; 118: 123–32. 10. Chen J, Teo KC. Determination of cadmium, copper, lead and zinc in water samples by flame atomic absorption spectrometry after cloud point extraction. Anal Chim Acta 2001; 450: 215– 22. 11. Sravrides JC. Lung carcinogenesis: pivotal role of metals in tobacco smoke. Free Radic Biol Med 2006; 41: 1017–30. 12. McMurray CT, Tainer JA. Cancer, cadmium and genome integrity. Nat Genet 2003; 34: 239–41. 13. Zheng H, Liu J, Choo KH, Michalska AE, Klaassen CD. Metallothionein-I and -II knock-out mice are sensitive to cadmium-induced liver mRNA expression of c-jun and p53. Toxicol Appl Pharmacol 1996; 136: 229–35. 14. Abshire MK, Buzard GS, Shiraishi N, Waalkes MP. Induction of c-myc and c-jun proto-oncogene expression in rat L6 myoblasts by cadmium is inhibited by zinc preinduction of the metallothionein gene. J Toxicol Environ Health 1996; 48: 359–77. 15. Shimada H, Shiao YH, Shibata M, Waalkes MP. Cadmium suppresses apoptosis induced by chromium. J Toxicol Environ Health 1998; 54: 159–68. 16. Miccadei S, Floridi A. Sites of inhibition of mitochondrial electron transport by cadmium. Chem Biol Interact 1993; 89: 156–67. 17. Drasch G, Schopfer J, Schrauzer GN. Selenium/cadmium ratios in human prostates Indicators of prostate cancer risk of smokers and nonsmokers, and relevance to the cancer protective effects of selenium. Biol Trace Elem Res 2005; 103: 103–7. 18. Waalkes MP. Cadmium carcinogenesis in review. J Inorg Biochem 2000; 79: 241–4. 19. National Toxicology Program, Tenth Report on Carcinogens. Research Triangle Park, NC: Department of Health and Human Services, 2000; 42–4. 20. Cho YA, Kim J, Woo HD, Kang M. Dietary cadmium intake and the risk of cancer: a meta-analysis. PLoS ONE 2013; 8: 1–8. 21. Adams SV, Passarelli MN, Newcomb PA. Cadmium exposure and cancer mortality in the Third National Health and Nutrition Examination Survey cohort. Occup Environ Med 2012; 69: 153–6. 22. Julin B, Wolk A, Johansson JE, Andersson SO, Andren O, Akesson A. Dietary cadmium exposure and prostate cancer incidence: a population-based prospective cohort study. Br J Cancer 2012; 107: 895–900. 23. Hu J, Mao Y, White K. Canadian Cancer Registries Epidemiology Research Group. Renal cell carcinoma and occupational exposure in Canada. Occup Med 2002; 52: 157–64.
Cadmium level in patients with oral cancer Arain et al.
24. Schwartz GG, Reis IM. Is cadmium a cause of human pancreatic cancer. Cancer Epidemiol Biomarkers Prev 2000; 9: 139–45. 25. Avti PK, Kumar S, Pathak CM, Vaiphei K, LAL K. Smokeless tobacco impairs the antioxidant defense in liver, lung, and kidney of rats. Toxicol Sci 2006; 89: 547–53. 26. Chen YC, Pu YS, Wu HC, et al. Cadmium burden and the risk and phenotype of prostate cancer. BMC Cancer 2009; 9: 429. 27. Verougstraete V, Lison D, Hotz P. Cadmium, lung and prostate cancer: a systematic review of recent epidemiological data. J Toxicol Environ Health 2003; 6: 227–55. 28. Kazi TG, Memon AR, Afridi HI, et al. Determination of cadmium in whole blood and scalp hair samples of Pakistani male lung cancer patients by electrothermal atomic absorption spectrometer. Sci Total Environ 2008; 389: 270–6. 29. Nagata C, Nagao Y, Nakamura K, et al. Cadmium exposure and the risk of breast cancer in Japanese women. Breast Cancer Res Treat 2013; 138: 235–9. 30. McElroy JA, Shafer MM, Trentham-Dietz A, Hampton JM, Newcomb PA. Cadmium exposure and breast cancer risk. J Natl Cancer Inst 2006; 98: 869–73. 31. Akesson A, Julin B, Wolk A. Long-term dietary cadmium intake and postmenopausal endometrial cancer incidence: a population-based prospective cohort study. Cancer Res 2008; 68: 6435–41. 32. Julin B, Wolk A, Akesson A. Dietary cadmium exposure and risk of epithelial ovarian cancer in a prospective cohort of Swedish women. Br J Cancer 2011; 105: 441–4. 33. Afridi HI, Kazi TG, , Kazi GH, Jamali MK, Shar GQ et al. Analysis of heavy metals in scalp hair samples of hypertensive patients by conventional and microwave digestion methods. Spectrosc Lett 2006; 39: 203–14. 34. Asta J, Guillard E, Tissut M, Gaude T, Ravanel P. Heavy metal transfer from atmosphere to plants. J Phys 2003; 107: 65–7. 35. Centers for Disease Control and Prevention. Third National Report on Human Exposure to Environmental Chemicals. Atlanta, GA: National Center for Environmental Health, 2005. 36. Herber RF, Christensen JM, Sabbioni E. Critical evaluation and review of cadmium concentrations in blood for use in occupational health according to the TRACY protocol. Int Arch Occup Environ Health 1997; 69: 372–8. 37. Wilhelm M, Schulz C, Schwenk M. Revised and new reference values for arsenic, cadmium, lead, and mercury in blood or urine of children: basis for validation of human
38. 39. 40.
41. 42.
43.
44. 45.
46. 47. 48. 49. 50. 51.
biomonitoring data in environmental medicine. Int J Hyg Environ Health 2006; 209: 301–5. Horn PS, Pesce AJ. Reference intervals: an update. Clin Chim Acta 2003; 334: 5–23. Mehra R, Juneja M. Elements in scalp hair and nails indicating metal body burden in polluted environment. J Sci Ind Res 2005; 67: 119–24. Agency for Toxic Substances and Disease Registry. Toxicological Profile for Cadmium. Atlanta, GA: US Department of Health and Human Services. NTIS Report No. PB/89/194476/ AS, 1999. Al-Rmalli SW, Jenkins RO, Haris PI. Betel quid chewing elevates human exposure to arsenic, cadmium and lead. J Hazard Mater 2011; 190: 69–74. Kazi TG, Wadhwa SK, Afridi HI, et al. Interaction of cadmium and zinc in biological samples of smokers and chewing tobacco female mouth cancer patients. J Hazard Mater 2010; 176: 985–91. IARC Cadmium and Cadmium Compounds. Beryllium, cadmium, mercury, and exposures in the glass manufacturing industry. IARC Monogr Eval Carcinog Risks Hum 1993; 58: 119–237. Qu Q, Melikian AA, Li G, et al. Validation of biomarkers in humans exposed to benzene: urine metabolites. Am J Ind Med 2000; 37: 522–31. Fang MZ, Mar WC, Cho MH. Cell cycle was disturbed in the MNNG-induced initiation stage during in vitro two-stage transformation of Balb/3T3 cells. Toxicology 2001; 163: 175–84. Dong S, Shen HM, Ong CN. Cadmium-induced apoptosis and phenotypic changes in mouse thymocytes. Mol Cell Biochem 2001; 222: 11–20. Hartwig A, Schwerdtle T. Interactions by carcinogenic metal compounds with DNA repair processes: toxicological implications. Toxicol Lett 2002; 127: 47–54. Ahluwalia KP. Assessing the oral cancer risk of South-Asian immigrants in New York City. Cancer 2005; 104: 2959–61. Warnakulasuriya S, Trivedy C, Peters TJ. Areca nut use: an independent risk factor for oral cancer. Br Med J 2002; 324: 799–800. Hecht SS. Tobacco carcinogens, their biomarkers and tobaccoinduced cancer. Nat Rev Cancer 2003; 3: 733–44. Nordberg GF, Fowler BA, Nordberg M, Friberg LT, editors. Handbook on the Toxicology of Metals, 3rd edn. Burlinton, MA: Academic Press, 2007.
713
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