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Analysis of blood and tissue in gallbladder cancer
Nuclear Instruments and Methods in Physics Research B 267 (2009) 2878–2883
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Analysis of blood and tissue in gallbladder cancer T.R. Rautray a,*, V. Vijayan b, M. Sudarshan c, S. Panigrahi d a
Department of Dental Biomaterials, School of Dentistry, Kyungpook National University, 2-188-1 Samduk-dong, Jung-gu, Daegu, Republic of Korea Dept. of Physics, Valliammai Engineering College, SRM Nagar, Chennai, India UGC-DAE Consortium for Scientific Research, Kolkata Centre, 3/LB-8 Bidhan Nagar, Kolkata 700 098, West Bengal, India d Dept. of Physics, National Institute of Technology, Rourkela 769 008, Orissa, India b c
a r t i c l e
i n f o
Article history: Received 16 March 2009 Received in revised form 30 May 2009 Available online 18 June 2009 PACS: 87.64.Ni 87.68.+z 87.64.Gb 78.30.Er 65.60.+a
a b s t r a c t Particle induced X-ray emission, particle induced c-ray emission studies has been carried out to analyse normal and carcinoma tissues and blood samples of gallbladder of both sexes and seventeen trace elements namely Na, Mg, Al, K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br and Pb were estimated in the tissue and blood samples. In the present study, concentration of Zn in the carcinoma gallbladder tissue is less than that of the normal gallbladder tissue. Tobacco habit could be one of the important factors to decrease the elemental concentrations in blood and tissue samples. Ó 2009 Elsevier B.V. All rights reserved.
Keywords: PIXE PIGE Trace elements Gallbladder
1. Introduction Carcinoma of the gallbladder is the most common malignant disease of the biliary tract. It is a highly fatal disease with dismal prognosis. It is the most common malignant lesion of the biliary tract and one of the most common among malignant neoplasms of the digestive tract. There have been some attempts to understand the role played by trace elements in either initiating or promoting or inhibiting the growth of cancer [1–3]. In such investigations, the concentrations of different elements in the tissues of the cancer-afflicted organ as well as the normal tissue of the same organ are measured employing a high precision technique like external proton induced X-ray emission. Trace elements are an important and emerging class of carcinogen [4]. Many trace elements are potent carcinogen in laboratory animals [5]. A few are potent carcinogen and several are suspected human carcinogen [6]. Trace elements are ubiquitous in both natural environment and work place. Beyond this, trace elements are typically persistent within the natural and man made environment with growing concentration in biosphere [7]. The trace elements absorbed by the * Corresponding author. Tel.: +82 536606897; fax: +82 534229631. E-mail address: [email protected] (T.R. Rautray). 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.06.084
body enter the digestive system, pass through gastrointestinal tract and are deposited in the liver by blood stream. From the liver they are carried to different organs for participating in biochemical reactions. Usually, the ions of trace elements act as coordination centres to build up the structure of enzymes or proteins [8]. Thus, when the concentrations of the trace elements in the body differ from the normal values, many clinical and pathological disorders arise. The excess or deficiency of these elements in the tissues of the cancer-afflicted organ when compared with that in the normal organ, is sought to be correlated with the pathology of cancer of that organ. Toxic elements like cadmium and nickel are promoters in the cancer process [4]. It was reported that the presence of specific elements in human subjects is an indicator of cancer. The serum copper level and serum zinc level were reported to correlate with various cancers [9–12]. Zinc is a ubiquitous trace metal required for the activity of over 300 metalloenzymes, including many involved in nucleic acid synthesis and cellular replication. Additionally, there are over 1400 zinc-finger proteins that participate in the genetic expression of many proteins. Deficiency of zinc may result in increased wound complications and cell-mediated immune dysfunctions in humans and increased rates of tumor development in experimental animals [13].
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2. Materials and methods 2.1. Sample preparation The normal and carcinoma tissues of gallbladder of both sexes were collected in the age group of 38 to 71 years. The tissue samples were washed with deionised distilled water 5–6 times and freeze dried in a lypholiser for 8 h till the samples got fully dried. After drying, the tissue samples were crushed to make pellets. For external PIXE and PIGE irradiation, 500 mg of powdered samples were mixed with 500 mg of high pure cellulose powder in 1:1 ratio by mass to make pellets of size 25 mm diameter in a hydraulic pressure and taken for analysis. Similarly, the blood samples were pressed into pellets of size 13 mm diameter after lypholisation for vacuum PIXE irradiation. 2.2. External PIXE set-up The external PIXE set-up at Institute of Physics (Fig. 1), which is the unique of its kind in India, was installed by us in 2003 [14]. The proton beam of 3 MeV energy was obtained from the 3 MV tandem type horizontal pelletron accelerator (Model: 9SDH-2, make: National Electrostatics Corporation, Madison, USA) and collimated by a graphite collimator to a beam size of 2 mm diameter. The beam was extracted into air using a KaptonTM foil (8 lm thick) at the exit point of a vacuum scattering chamber [14]. The scattering chamber has an inner diameter of 80 cm and was designed to cater to the requirements of the external beam as well as to serve for the charged particle reaction studies for nuclear physics experiments. The beam was first focused and centered at the target location inside the scattering chamber and then let through the thin Kapton foil placed at the exit port. The Kapton foil is used as exit window due to its several special characteristics like low beam-induced background emission, minimal energy loss and resistance to radiation damage. The beam was allowed to travel a few cm in air after which it irradiates the samples. Beam charge measurements were carried out by using a rotating vane chopper designed by us [15]. For the measurements to be described below, the samples were kept in air over a sample stand (of 5 kg capacity) making an angle of 45° to the beam direction. The samples were irradiated with maximum beam current of 30 nA. A Si (Li) detector (active area 30 mm2) having energy resolution of 170 eV at 5.9 KeV placed at 90° with respect to the beam direction was used to detect characteristic X-rays emitted from the target [14,16]. The detector has an entrance beryllium window of 8 lm thickness. A 25 lm thick aluminium absorber (with 6% hole) was kept in front of the detector to attenuate the bremsstrahlung background and the dominant low energy X-ray peaks [17]. Spectra were recorded by using a PC based multi channel analyser. The PIXE spectral analyses were per-
Fig. 1. External PIXE set-up at Institute of Physics.
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formed using GUPIX-2000 software. This provides a non-linear least square fitting of the spectrum, together with subsequent conversion of the fitted X-ray peak intensities into elemental concentrations, utilizing the fundamental parameter method (FPM) for quantitative analysis. The PIGE spectral analyses were done using the ANGES (IAEA, Vienna) software [18–20]. 3. Results and discussion The external PIXE spectrum of a normal gallbladder tissue and carcinoma gallbladder tissue are shown in Figs. 2 and 3 respectively. Similarly, the PIGE spectrum of a normal gallbladder tissue and carcinoma gallbladder tissue are shown in Figs. 4 and 5 respectively. The analysis of the tissue samples were carried out by simultaneous external PIXE and PIGE technique whereas the analysis of the blood samples of the healthy persons and the patients affected by gallbladder cancer were carried out by vacuum PIXE technique [21]. In the present study, seventeen trace elements namely Na, Mg, Al, K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br and Pb were estimated in both normal tissue and carcinoma tissue, normal blood and carcinoma blood samples of gallbladder which are provided in Table 1. From PIGE technique, the concentration of Na, Mg and Al have been established in the tissue samples. In the gallbladder carcinoma tissues, concentration of elements namely Na, Mg, Al, Ca, Zn and Se are less than that in the normal gallbladder tissues, whereas the concentration of K and Ca has been decreased in case of the blood samples from the patients affected with gallbladder cancer. The concentration of elements namely Ti, Fe, Co, Ni and Cu have increased in the carcinoma tissue samples. But average concentration of Cu has been increased in carcinoma blood samples. The ten major elements in living bodies are H, C, N, O, Na, Mg, P, S, K and Ca. The total amount of these elements reaches 99% of all elements in the living bodies. The concentration of elements of N, Na, P, S and Ca are higher in animals, and concentration of elements like O, Mg and K are lower than in plants. High contents of these elements in animals are originating from proteins for N and S, from bones for P and Ca, and from electrolytes for Na. Low contents of O, Mg and K in animals are due to lack of celluloses as a major constituent to make cell wall membranes. Together with these major elements, such minor elements as F, Si, Cr, Mn, Fe, Co, Ni. Cu, Zn, Se, Mo, Sn, I etc. are indispensable for animals. These elements are distributed in each organ in a characteristic way. If the
Fig. 2. External PIXE spectrum of a normal gallbladder tissue.
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Fig. 3. External PIXE spectrum of a carcinoma gallbladder tissue.
Fig. 4. PIGE spectrum of a normal gallbladder tissue.
Fig. 5. PIGE spectrum of a carcinoma gallbladder tissue.
balance of these elements is broken, living bodies have to experience ‘‘diseases” [22]. The concentration of Na in the carcinoma tissues is about 0.7 times less than that in the normal tissues. But, the concentration of Mg in the carcinoma gallbladder tissues is about 0.4 times less than that in the normal tissues. The relative concentration of elements in normal versus carcinoma gallbladder tissue and blood are depicted in Figs. 6 and 7 respectively. High intakes of dietary fibre lower magnesium absorption. In contrast, high intakes of zinc decrease magnesium absorption and contribute to a shift toward negative balance in adult males [23]. It was observed in the present work that K levels in the carcinoma blood of gallbladder is lower than those in the normal blood samples. K is a major intracellular cation which is also excreted in gastrointestinal tract, saliva, gastric juice, bile, pancreatic and intestinal juices. Prolonged hypokalemia (potassium deficiency) causes injury to myocardium and kidneys. The glycolytic enzyme ‘‘pyruvate kinase” requires potassium for its maximum activity [24]. K deficiency causes acidosis, renal damage and cardiac arrest. Shonk and his associates [25–27] studied the activities of different glycolytic enzymes in carcinoma of rectum and colon and in the corresponding non-malignant counterparts. They observed that glycolytic enzyme activities of the neoplastic tissues of rectum and colon are much higher than the activities in the corresponding normal rectum and colon. They reported the ratio of neoplastic to normal ‘‘pyruvate kinase” enzyme activity is 2.1 for colon and 2.4 for rectum. Hence, it is expected that the levels of potassium which may be a cofactor for ‘‘pyruvate kinase” enzyme should be higher in the carcinoma tissue [28]. But the observation of low K levels in the blood of carcinoma gallbladder in the present work does not support the above statement. This may possibly be due to different organs. Ca is a major constituent of bone and teeth. It is required for coagulation of blood, regulation of neuromuscular irritability and muscular contractility. Concentration of Ca in both the gallbladder carcinoma tissue and blood samples is about 0.6 times less than that in the gallbladder normal tissue and blood. In a strictly operational sense, Ca balance is determined by the relationship between Ca intake and Ca absorption and excretion. A striking feature of the system is that relatively small changes in Ca absorption and excretion can neutralise a high intake or compensate for a low one. The concentration of Se in the carcinoma tissue samples is slightly less than that of the normal. Se has long been marked as a carcinogen. Nevertheless, the Committee on Medical and Biological effects of Environmental pollutants (1976) have reviewed the confounding data and concluded that selenium may be an anti-carcinogen. The inorganic Se at lower concentration acts as an anticarcinogen and it is deficient in cancerous patients. Se can be a good metabolic risk modifier against cancer. Several elements like Ni, Se, Cu and Cr are considered essential at certain concentrations, but they are toxic when intake is excess [29]. The main source of Se is plant extracts and the cereals are the likely major dietary sources. The variation depends on the differences of Se content of the soil of that particular place. Areas of high Se have higher prevalence of dental caries [30]. Selenium is a relatively toxic element. Intakes averaging 1.2 mg/day can induce changes in nail structure. Chronic selenium intakes over 3.2 mg/day can result in the loss of hair and nails, mottling of the teeth, lesions in the skin and nervous system, nausea, weakness and diarrhoea. Selenium, which is biologically important as an anion, is homeostatically regulated by excretion, primarily in the urine but some also is excreted in the breath. Selenate, selenite and selenomethionine are all highly absorbed by the gastrointestinal tract; absorption percentages for these forms of selenium are commonly found to be
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T.R. Rautray et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2878–2883 Table 1 Average concentration (in ppm by weight) of elements in normal gallbladder and carcinoma gallbladder tissue and blood. Tissue
By PIGE Na Mg Al By external PIXE K Ca Ti Cr Mn Fe Co Ni Cu Zn As Se Br Pb
Blood
Normal (n = 10)
Carcinoma (n = 9)
Normal (n = 10)
Carcinoma (n = 9)
717.2 ± 61.8 8621.7 ± 551.2 559.2 ± 50.4
512.6 ± 47.2 3807.5 ± 227.5 262.3 ± 32.5
– – –
– – –
408.2 ± 38.9 616.3 ± 54.5 23.7 ± 3.8 11.1 ± 1.9 27.6 ± 3.6 1066.9 ± 93.8 8.25 ± 1.3 7.53 ± 1.3 11.1 ± 2.0 101.3 ± 16.9 3.8 ± 0.6 3.14 ± 0.6 12.6 ± 2.1 13.5 ± 2.1
359.4 ± 41.3 366.2 ± 40.8 37.2 ± 5.2 9.0 ± 1.7 23.4 ± 3.4 1579.7 ± 113.4 13.1 ± 2.3 12.3 ± 2.1 27.8 ± 4.3 75.6 ± 13.3 4.3 ± 0.7 2.06 ± 0.4 10.5 ± 1.8 11.8 ± 1.9
2082.7 ± 136.1 321.7 ± 38.9 1.97 ± 0.4 1.07 ± 0.2 4.13 ± 0.8 597.3 ± 52.5 0.74 ± 0.2 1.13 ± 0.3 1.10 ± 0.3 2.54 ± 0.5 0.11 ± 0.03 0.16 ± 0.04 1.04 ± 0.3 0.09 ± 0.03
1834.4 ± 122.7 201.2 ± 31.2 2.01 ± 0.4 1.02 ± 0.2 3.21 ± 0.6 657.1 ± 48.7 0.87 ± 0.2 1.74 ± 0.4 2.11 ± 0.4 2.06 ± 0.4 0.13 ± 0.03 0.14 ± 0.04 1.0 ± 0.3 0.16 ± 0.04
Fig. 6. Concentration of elements in normal versus carcinoma gallbladder tissue.
Fig. 7. Concentration of elements in gallbladder normal versus carcinoma blood.
in the 80–90% range. Lead is very useful, but very harmful as well when intake is more. The concentration of Fe in carcinoma tissue samples have been increased as compared to the Fe level in normal tissue. But the concentration of Cu in the carcinoma blood and tissue samples has increased as compared to the normal blood and tissue samples. Deficiency of Fe results in anaemia. It is responsible for oxygen transport and cellular respiration. Signs of Cu deficiency are anaemia, blood vessel necrosis, dyspigmentation, neuropathy, emphysema and diarrhoea. But excess intake of Cu causes stomach-ache, diarrhoea, vomiting, liver dysfunction and hemolysis. Zinc contents in every organ, except for the cancer affected organ where Zn is enriched, are reported to become low if a human is attacked by cancer. This is caused by a high transfer rate of Zn to wastes [22]. But, to contrary, in the present study, average concentration of Zn in the carcinoma gallbladder tissue is less than that of the normal gallbladder tissue. Zinc is present in all body tissues and fluids. The total body zinc content has been estimated to be 30 mmol (2 g). Plasma zinc has a rapid turnover rate and it represents only about 0.l% of total body zinc content. This level appears to be under close homeostatic control [31]. Deficiency of Zn causes poor growth, sexual infantilism in adolescents, loss of taste and delayed wound healing. The utilisation of zinc depends on the overall composition of the diet. Experimental studies have identified a number of dietary factors as potential promoters or antagonists of zinc absorption. Soluble low molecular weight organic substances, such as amino and hydroxy acids facilitate zinc absorption. In contrast, organic compounds forming stable and poorly soluble complexes with zinc can impair absorption. In addition, competitive interactions between zinc and other ions with similar physicochemical properties can affect the uptake and intestinal absorption of zinc. The risk for competitive interactions seems mainly to be related to high doses in the form of supplements or in aqueous solutions. However, at levels present in food and at realistic fortification levels, zinc absorption appears not to be affected by iron and copper [32]. The lack of specific and sensitive indexes for zinc status limits the possibilities for evaluating zinc requirements from epidemiologic observations. Zinc requirements were estimated by using the factorial technique (i.e. by adding the requirements for tissue growth, maintenance, metabolism and endogenous losses). Experimental zinc repletion studies with low zinc intakes have clearly
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shown that the body has a pronounced ability to adapt to different levels of zinc intakes by changing the endogenous zinc losses through the kidneys, intestine and skin [33–37]. The concentration of toxic elements like As and Pb did not show much variation between the normal and carcinoma gallbladder tissue and blood. Moreover, As and Pb concentration may be attributed to be contamination during sample lypholisation, pelletization or handling. The profound negative influence of malnutrition on cancer treatment is well recognised, with nutritional deficiencies impacting on all modalities of treatment. It therefore becomes critical to assess the nutritional status of patients before therapy and identify those at greater risk for morbidity. Given the presence of carcinoma effects coupled with years of tobacco and alcohol abuse, the high incidence of nutritional deficiency in the gallbladder is not surprising. Diet may influence human carcinogenesis in six general ways [38]: (i) Diet provides the carcinogens or their immediate precursors, (ii) Diet facilitates or inhibits the endogenous production of carcinogens, (iii) The modification of carcinogens by metabolic activation or inactivation could be affected by other dietary components, (iv) Increasing or impeding the delivery of carcinogens to their site of action may be influenced by dietary changes, (v) Diet may alter the susceptibility of tissues to cancer induction or growth by dietary effects or tissue metabolism, (vi) Diet may alter the body’s capacity to eliminate transformed cells. In the field of nutrition and cancer, turmeric is emphasised as an anti-cancer agent. It prevents the formation of preneoplastic lesions induced in the initiation as well as post initiative phase. The As concentrations are found relatively high in spices, whereas lead and iron are found more in tobacco-products. Clinical studies have shown that iron promotes cancer cell growth. It acts more like a co-carcinogen than a primary one and in case of tobacco user’s local absorption of concentrated iron in oral region could be high. Tobacco addicts who normally require more antioxidants as compared to a normal person tend to consume a less adequate diet with subnormal antioxidant levels than do others [39]. From the epidemiological evidences and different animal models, it is established that Mn and Se are non-carcinogens. But, excess exposure to Cr, As, Cd, Ni, Be, Co, Pb, Si and Sn may lead to cancer. The carcinogenicity of Al, Cu, Fe, Mo, Sc, Ti, V and Zn are yet to be established [40]. Daily nutritional intake of elements of different individuals from different areas and studies on trace element imbalances in different environment and different biomedi-
cal samples may lead to better diagnosis of diseases like cancer which will definitely guide the clinicians to supplement or withdraw the elements and minerals to the human body for cure and maintenance of better health. It was observed from Table 2 that all the tobacco-products were very highly enriched with multi-elements. The present results for tobacco-shoots when compared with those reported by Oliveira et al. [41], there is an exception in the strontium and copper levels. Low Sr-levels and high Cu-levels were observed in the tobaccosamples. These elemental variations in the tobacco-chemistry could be due to some agricultural and industrial factors like preharvest and post-harvest operations, pre-blend operations, drying, storing, processing, flavor addition and casings. Among the common smoking tobaccos, it was found that the Indian bidi, which contains raw tobacco leaves, have higher elemental levels than that of the cigarettes. Clinical studies have shown that Fe promotes cancer cell growth and it acts more like a co-carcinogen [42]. All the tobacco-products were found to be very highly enriched with iron. The branded tobacco-products like gutkha and zarda, whose other ingredients are betel nuts, lime, cardamom catechu, silver leaves, aromatic spices and saffron, have comparatively lower iron level than that of the raw tobacco-products such as gudakhu. Exposure to arsenic is associated with the on set of cancers of skin and lung. Table 3 shows the elemental variations in human blood with respect to their tobacco habits. From the results, it was observed that all the elemental levels in human blood decrease with respect to increase in the tobacco habits. The selenium and copper contents in the blood decreases significantly in case of heavy users of tobacco in comparison with that of the control group, who have no tobacco habits [43]. The blood iron and copper values in case of moderate and heavy users of tobacco has become lower than the reported normal ranges [44]. Of course, these lower elemental values could be due to the inadequate and poor dietary habits of the tobacco users [45]. The copper and zinc ratio, which is an important index in disease activity [46], decreases significantly with respect to increasing habits of tobacco. 4. Conclusion Decreasing levels of Zn and Se in the tissue samples have a definite importance in the oxidative processes in the human body.
Table 2 Concentration (in ppm by weight) of some tobacco-products by PIXE analysis.
Cigarette (n = 8) Bidi (n = 8) Gutkha (n = 8) Zarda (n = 8) Khaini (n = 7) Gudakhu (n = 7)
K%
Ca%
Mn
Fe
Cu
Zn
As
Se
Sr
Pb
0.63 ± 0.04 0.76 ± 0.06 1.03 ± 0.06 0.69 ± 0.05 0.87 ± 0.04 0.81 ± 0.04
2.03 ± 0.08 4.21 ± 0.04 2.01 ± 0.07 1.76 ± 0.07 4.01 ± 0.04 1.63 ± 0.07
151.6 ± 23.6 97.8 ± 19.7 73.6 ± 17.8 49.5 ± 10.3 157.4 ± 28.5 121.4 ± 17.0
687.6 ± 68.7 1397.2 ± 159.5 472.8 ± 72.4 484.9 ± 83.7 615.8 ± 117.3 992.3 ± 147.3
15.4 ± 3.1 12.5 ± 2.0 9.6 ± 1.6 13.6 ± 2.7 20.3 ± 3.1 14.8 ± 2.2
29.6 ± 5.7 61.8 ± 9.3 71.1 ± 12.4 43.4 ± 12.7 18.2 ± 3.5 72.5 ± 16.2
0.17 ± 0.03 0.31 ± 0.05 0.23 ± 0.05 0.20 ± 0.04 0.19 ± 0.04 0.30 ± 0.06
0.10 ± 0.02 0.15 ± 0.03 0.14 ± 0.03 0.11 ± 0.02 0.15 ± 0.03 0.19 ± 0.04
14.8 ± 3.1 19.6 ± 4.0 15.3 ± 2.9 21.5 ± 3.8 19.3 ± 4.1 15.8 ± 2.8
0.47 ± 0.10 1.25 ± 0.22 0.68 ± 0.16 0.71 ± 0.15 0.69 ± 0.14 0.97 ± 0.18
Table 3 Average concentration (in lg/ml) of various elements of human blood with respect to tobacco habits by PIXE technique. Groups
K Ca Fe Cu Zn Se Pb
Average elemental concentrations of whole blood I (No habit)
II (Light habits)
III (Moderate habits)
IV (Heavy habits)
2132.5 ± 127.3 272.4 ± 45.2 532.7 ± 82.6 1.06 ± 0.22 2.41 ± 0.50 0.16 ± 0.03 0.07 ± 0.01
1982.6 ± 120.4 263.4 ± 46.4 504.0 ± 80.6 0.91 ± 0.20 2.32 ± 0.49 0.13 ± 0.02 0.07 ± 0.01
1971.8 ± 121.0 251.1 ± 44.9 372.8 ± 69.8 0.83 ± 0.17 2.21 ± 0.46 0.10 ± 0.02 0.07 ± 0.01
1711.3 ± 198.2 239.3 ± 45.2 359.3 ± 71.8 0.61 ± 0.17 1.68 ± 0.47 0.07 ± 0.02 0.06 ± 0.01
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But, to contrary, in the present study, concentration of Zn in the carcinoma gallbladder tissue is less than that of the normal gallbladder tissue. Tobacco habit could be one of the important factors to decrease the elemental concentrations in blood and tissue samples. Gradual increase in tobacco habits decreases the dietary intake of a person and hence trace element levels in blood. Therefore, proportionately adequate supplementation of trace elements to tobacco users is very much essential to scavenge the free radicals. The local absorption of highly concentrated iron and calcium from the tobacco-products could be dangerous. Tobacco products are rich in multi-elements. The more the local absorption of multi-elements, the more is the local irritation and subsequent development of cancer. Acknowledgements The help extended by the staff members of Institute of Physics (IOP) is acknowledged and the authors would like to thank the authorities of IOP for providing beam time to carry out the current research. References [1] V. Valkovic, Proton-induced X-ray emission: applications in medicine, Nucl. Instr. and Meth. 142 (1977) 151. [2] W. Maenhaut, J. Vandenhaute, H. Duflou, Applications of PIXE to biological and biomedical samples at the university of Kent, Nucl. Instr. and Meth. B 22 (1987) 138. [3] M. Tanaka, E. Mataugi, K. Miyasaki, T. Yamagata, M. Inoue, H. Ogata, S. Shimoura, PIXE measurement applied to trace elemental analysis of human tissues, Nucl. Instr. and Meth. B 22 (1987) 152. [4] P. Boffetta, Involuntary smoking and lung cancer, Scand. J. Work Environ. Health. 28 (2002) 30. [5] E. Nogueira, A. Cardesa, U. Mohr, Experimental models of kidney tumors, J. Cancer Res. Clin. Oncol. 119 (4) (1993) 190. [6] B. Delahunt, J.N. Nacey Bethwaite, Occupational risk for renal cell carcinoma, Br. J. Urol. 75 (1997) 578. [7] N.S. Mandel, J.K. McLaughlin, B. Schlehofer, A. Mellemgaard, International renal-cell cancer study IV, Occupat. Int. J. Cancer 61 (5) (1995) 601. [8] U. Lindh, Cell biology, trace elements and nuclear microscopy, Nucl. Instr. and Meth. B 104 (1995) 285. [9] F. Martin-Lagos, M. Navarro-Alarcon, C. Terres-Martos, S. Lopez-G de la Serrana, M.C. Lopez-Martinez, Serum copper and zinc concentrations in serum from patients with cancer and cardiovascular disease, Sci. Total Environ. 204 (1997) 27. [10] J.L. Poo, R.R. Romero, J.A. Robles, A.C. Montemayor, F. Isoard, A. Estanes, M. Uribe, Diagnostic value of the copper/zinc ratio in digestive cancer: a case control study, Arch. Med. Res. 28 (1997) 259. [11] T. Magalova, V. Bella, A. Brtkova, I. Beno, M. Kudlackova, K. Volovova, Copper, zinc and superoxide dismutase in precancerous, benign diseases and gastric, colorectal and breast cancer, Neoplasma 46 (1999) 100. [12] D. Ferrigno, G. Buccheri, T. Camilla, Serum copper and zinc content in nonsmall cell lung cancer: abnormalities and clinical correlates, Monaldi Arch. Chest Dis. 54 (1999) 204. [13] A.S. Prasad, Zinc: an overview, Nutrition 11 (1) (1995) 93. [14] V. Vijayan, R.K. Choudhury, B. Mallick, S. Sahu, S.K. Choudhury, H.P. Lenka, T.R. Rautray, P.K. Nayak, External particle induced X-ray emission, Current Sci. 85 (2003) 772. [15] S. Sahu, S.K. Choudhury, B. Mallick, T.R. Rautray, V. Vijayan, R.K. Choudhury, Design of a rotating vane chopper for external PIXE analysis, in: Proceedings of Indian Particle Accelerator Conference, Indore, India, 2003, p. 695. [16] R.K. Choudhury, V. Vijayan, N.C. Mohanty, Scientific study of metallic compositions of Orissa state museum specimens, Orissa Rev. 59 (2003) 48.
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[17] T.R. Rautray, V. Vijayan, S. Panigrahi, Analysis of Indian pigment gallstones, Nucl. Instr. and Meth. B 255 (2007) 409. [18] J.L. Campbell, T.L. Hopmann, J.A. Maxwell, Z. Nezedly, The Guelph PIXE software package III: alternative proton database, Nucl. Instr. and Meth. B 170 (2000) 193. [19] ANGES software package, IAEA, Vienna. Available from. [20] P.K. Hota, S.N. Senapati, S.K. Giri, U.R. Parija, R.K. Jena, V. Vijayan, PIXE analysis of human blood in cancer, Int. J. PIXE 12 (1–2) (2002) 47. [21] T.R. Rautray, V. Vijayan, S. Panigrahi, Analysis of cholesterol gallstones by PIXE and TG-DTG, Euro. J. Gastroenterol. Hepatol. 18 (2006) 999. [22] M. Uda, K. Maeda, Y. Sasa, H. Kusuyama, Y. Yokode, An attempt to diagnose cancer by PIXE, Nucl. Instr. and Meth. B 22 (1987) 184. [23] H. Spencer, C. Norris, D. Williams, Inhibitory effect of zinc on magnesium balance and absorption in man, J. Am. Coll. Nutr. 13 (1994) 479. [24] A.V.S.S. Rama Rao, A Text Book of Biochemistry, L.K. and S Publishers, 2002, p. 438. [25] C.E. Shonk, B.J. Koven, H. Majima, G.E. Boxer, Enzyme patterns in human tissues II: glycolytic enzyme patterns in non-malignant human tissues, Cancer. Res. 24 (1964) 722. [26] C.E. Shonk, R.N. Arison, B.J. Koven, H. Majima, G.E. Boxer, Enzyme patterns in human tissues III: glycolytic enzymes in normal and malignant tissues of the colon and rectum, Cancer. Res. 25 (1965) 206. [27] C.E. Shonk, H. Majima, B.J. Koven, G.E. Boxer, Enzyme patterns in human tissues IV: comparison of glycolytic enzymes in surgical biopsies and autopsy specimens, Cancer. Res. 26 (1966) 607. [28] S.B. Reddy, M.J. Charles, G.J.N. Raju, V. Vijayan, B.S. Reddy, M.R. Kumar, B. Sundareswar, Trace elemental analysis of carcinoma kidney and stomach by PIXE method, Nucl. Instr. and Meth. B 207 (2003) 345. [29] M.J. Sadler, J.J. Strain, B. Caballero, Encyclopedia of Human Nutrition, Vol. 1, Academic Press, New York, 1999. [30] J.E. Park, K. Park, Textbook of Preventive and Social Medicine, 12th ed., 1989, p. 334. [31] K.M. Hambidge, Zinc, in: W. Mertz (Ed.), Trace Elements in Human and Animal Nutrition, Academic Press Inc., Orlando, Florida, 1987, p. 1. [32] B. Sandström, B. Lönnerdal, Promoters and antagonists of zinc absorption, in: C.F. Mills (Ed.), Zinc in Human Biology, Springer–Verlag, U.K., 1989, p. 57. [33] H.C. Lukaski, W.W. Bolonchuk, L.M. Klevay, D.B. Milne, H.H. Sandstead, Changes in plasma zinc content after exercise in men fed a low-zinc diet, Am. J. Physiol. 247 (1984) E88. [34] D.B. Milne, W.K. Canfield, S.K. Gallagher, J.R. Hunt, L.M. Klevay, Ethanol metabolism in postmenopausal women fed a diet marginal in zinc, Am. J. Clin. Nutr. 46 (1987) 688. [35] M.J. Baer, J.C. King, Tissue zinc levels and zinc excretion during experimental zinc depletion in young men, Am. J. Clin. Nutr. 39 (1984) 556. [36] F.M. Hess, J.C. King, S. Margen, Zinc excretion in young women on low zinc intakes and oral contraceptive agents, J. Nutr. 107 (1977) 1610. [37] D.B. Milne, W.K. Canfield, J.R. Mahalko, H.H. Sandstead, Effect of dietary zinc on whole body surface loss of zinc: impact on estimation of zinc retention by balance method, Am. J. Clin. Nutr. 38 (1983) 181. [38] B.K. Armstrong, A.J. McMichael, R. McLennan, Diet, in: D. Schottenfeld, J. Fraumeni (Eds.), Cancer epidemiology and prevention, WB Saunders, Philadelphia, 1982 [39] S.H. Jeffrey, M.B. Nancy, Cigarette use during adolescence: effects on nutritional status, Nutrition Rev. 57 (7) (1999) 215. [40] W.C. Louis, Toxicology of Metals, CRC Press Inc., Boca Raton, Florida, 1996. [41] H. Oliveira, E.A.N. Fernandes, M.A. Bacchi, G.A. Sarries, F.S. Tagliaferro, Tobacco element composition determined by INAA, J. Radioanal. Nucl. Chem. 244 (2) (2000) 299. [42] M.D. Cohen, D.H. Bowser, M. Costa, Carcinogenicity and genotoxicity of Pb, Be, and other metals, in: Louis W. Chang (Ed.), Toxicology of Metals, CRC Press Inc., Boca Raton, 1996, p. 253. [43] T.R. Rautray, V. Vijayan, P.K. Hota, Elemental analysis of blood in oral cancer, Int. J. PIXE 12 (1–2) (2002) 41–45. [44] G.V. Iyengar, Elemental Analysis of Biological Systems, Vol. 1, CRC Press Inc., USA, 1989. [45] J.S. Hampl, N.M. Betts, Cigarette use during adolescence: effects on nutritional status, Nutrition Reviews 57 (7) (1999) 215. [46] Y. Beguin, V. Bours, J.M. Delbrouck, G. Robaye, I. Roelandts, G. Fillet, G. Weber, Use of PIXE to measure serum Cu, Zn, Se and Br in patients with hematological malignancies, Nucl. Instr. and Meth. B 49 (1990) 202.
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Analysis of blood and tissue in gallbladder cancer T.R. Rautray a,*, V. Vijayan b, M. Sudarshan c, S. Panigrahi d a
Department of Dental Biomaterials, School of Dentistry, Kyungpook National University, 2-188-1 Samduk-dong, Jung-gu, Daegu, Republic of Korea Dept. of Physics, Valliammai Engineering College, SRM Nagar, Chennai, India UGC-DAE Consortium for Scientific Research, Kolkata Centre, 3/LB-8 Bidhan Nagar, Kolkata 700 098, West Bengal, India d Dept. of Physics, National Institute of Technology, Rourkela 769 008, Orissa, India b c
a r t i c l e
i n f o
Article history: Received 16 March 2009 Received in revised form 30 May 2009 Available online 18 June 2009 PACS: 87.64.Ni 87.68.+z 87.64.Gb 78.30.Er 65.60.+a
a b s t r a c t Particle induced X-ray emission, particle induced c-ray emission studies has been carried out to analyse normal and carcinoma tissues and blood samples of gallbladder of both sexes and seventeen trace elements namely Na, Mg, Al, K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br and Pb were estimated in the tissue and blood samples. In the present study, concentration of Zn in the carcinoma gallbladder tissue is less than that of the normal gallbladder tissue. Tobacco habit could be one of the important factors to decrease the elemental concentrations in blood and tissue samples. Ó 2009 Elsevier B.V. All rights reserved.
Keywords: PIXE PIGE Trace elements Gallbladder
1. Introduction Carcinoma of the gallbladder is the most common malignant disease of the biliary tract. It is a highly fatal disease with dismal prognosis. It is the most common malignant lesion of the biliary tract and one of the most common among malignant neoplasms of the digestive tract. There have been some attempts to understand the role played by trace elements in either initiating or promoting or inhibiting the growth of cancer [1–3]. In such investigations, the concentrations of different elements in the tissues of the cancer-afflicted organ as well as the normal tissue of the same organ are measured employing a high precision technique like external proton induced X-ray emission. Trace elements are an important and emerging class of carcinogen [4]. Many trace elements are potent carcinogen in laboratory animals [5]. A few are potent carcinogen and several are suspected human carcinogen [6]. Trace elements are ubiquitous in both natural environment and work place. Beyond this, trace elements are typically persistent within the natural and man made environment with growing concentration in biosphere [7]. The trace elements absorbed by the * Corresponding author. Tel.: +82 536606897; fax: +82 534229631. E-mail address: [email protected] (T.R. Rautray). 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.06.084
body enter the digestive system, pass through gastrointestinal tract and are deposited in the liver by blood stream. From the liver they are carried to different organs for participating in biochemical reactions. Usually, the ions of trace elements act as coordination centres to build up the structure of enzymes or proteins [8]. Thus, when the concentrations of the trace elements in the body differ from the normal values, many clinical and pathological disorders arise. The excess or deficiency of these elements in the tissues of the cancer-afflicted organ when compared with that in the normal organ, is sought to be correlated with the pathology of cancer of that organ. Toxic elements like cadmium and nickel are promoters in the cancer process [4]. It was reported that the presence of specific elements in human subjects is an indicator of cancer. The serum copper level and serum zinc level were reported to correlate with various cancers [9–12]. Zinc is a ubiquitous trace metal required for the activity of over 300 metalloenzymes, including many involved in nucleic acid synthesis and cellular replication. Additionally, there are over 1400 zinc-finger proteins that participate in the genetic expression of many proteins. Deficiency of zinc may result in increased wound complications and cell-mediated immune dysfunctions in humans and increased rates of tumor development in experimental animals [13].
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2. Materials and methods 2.1. Sample preparation The normal and carcinoma tissues of gallbladder of both sexes were collected in the age group of 38 to 71 years. The tissue samples were washed with deionised distilled water 5–6 times and freeze dried in a lypholiser for 8 h till the samples got fully dried. After drying, the tissue samples were crushed to make pellets. For external PIXE and PIGE irradiation, 500 mg of powdered samples were mixed with 500 mg of high pure cellulose powder in 1:1 ratio by mass to make pellets of size 25 mm diameter in a hydraulic pressure and taken for analysis. Similarly, the blood samples were pressed into pellets of size 13 mm diameter after lypholisation for vacuum PIXE irradiation. 2.2. External PIXE set-up The external PIXE set-up at Institute of Physics (Fig. 1), which is the unique of its kind in India, was installed by us in 2003 [14]. The proton beam of 3 MeV energy was obtained from the 3 MV tandem type horizontal pelletron accelerator (Model: 9SDH-2, make: National Electrostatics Corporation, Madison, USA) and collimated by a graphite collimator to a beam size of 2 mm diameter. The beam was extracted into air using a KaptonTM foil (8 lm thick) at the exit point of a vacuum scattering chamber [14]. The scattering chamber has an inner diameter of 80 cm and was designed to cater to the requirements of the external beam as well as to serve for the charged particle reaction studies for nuclear physics experiments. The beam was first focused and centered at the target location inside the scattering chamber and then let through the thin Kapton foil placed at the exit port. The Kapton foil is used as exit window due to its several special characteristics like low beam-induced background emission, minimal energy loss and resistance to radiation damage. The beam was allowed to travel a few cm in air after which it irradiates the samples. Beam charge measurements were carried out by using a rotating vane chopper designed by us [15]. For the measurements to be described below, the samples were kept in air over a sample stand (of 5 kg capacity) making an angle of 45° to the beam direction. The samples were irradiated with maximum beam current of 30 nA. A Si (Li) detector (active area 30 mm2) having energy resolution of 170 eV at 5.9 KeV placed at 90° with respect to the beam direction was used to detect characteristic X-rays emitted from the target [14,16]. The detector has an entrance beryllium window of 8 lm thickness. A 25 lm thick aluminium absorber (with 6% hole) was kept in front of the detector to attenuate the bremsstrahlung background and the dominant low energy X-ray peaks [17]. Spectra were recorded by using a PC based multi channel analyser. The PIXE spectral analyses were per-
Fig. 1. External PIXE set-up at Institute of Physics.
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formed using GUPIX-2000 software. This provides a non-linear least square fitting of the spectrum, together with subsequent conversion of the fitted X-ray peak intensities into elemental concentrations, utilizing the fundamental parameter method (FPM) for quantitative analysis. The PIGE spectral analyses were done using the ANGES (IAEA, Vienna) software [18–20]. 3. Results and discussion The external PIXE spectrum of a normal gallbladder tissue and carcinoma gallbladder tissue are shown in Figs. 2 and 3 respectively. Similarly, the PIGE spectrum of a normal gallbladder tissue and carcinoma gallbladder tissue are shown in Figs. 4 and 5 respectively. The analysis of the tissue samples were carried out by simultaneous external PIXE and PIGE technique whereas the analysis of the blood samples of the healthy persons and the patients affected by gallbladder cancer were carried out by vacuum PIXE technique [21]. In the present study, seventeen trace elements namely Na, Mg, Al, K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br and Pb were estimated in both normal tissue and carcinoma tissue, normal blood and carcinoma blood samples of gallbladder which are provided in Table 1. From PIGE technique, the concentration of Na, Mg and Al have been established in the tissue samples. In the gallbladder carcinoma tissues, concentration of elements namely Na, Mg, Al, Ca, Zn and Se are less than that in the normal gallbladder tissues, whereas the concentration of K and Ca has been decreased in case of the blood samples from the patients affected with gallbladder cancer. The concentration of elements namely Ti, Fe, Co, Ni and Cu have increased in the carcinoma tissue samples. But average concentration of Cu has been increased in carcinoma blood samples. The ten major elements in living bodies are H, C, N, O, Na, Mg, P, S, K and Ca. The total amount of these elements reaches 99% of all elements in the living bodies. The concentration of elements of N, Na, P, S and Ca are higher in animals, and concentration of elements like O, Mg and K are lower than in plants. High contents of these elements in animals are originating from proteins for N and S, from bones for P and Ca, and from electrolytes for Na. Low contents of O, Mg and K in animals are due to lack of celluloses as a major constituent to make cell wall membranes. Together with these major elements, such minor elements as F, Si, Cr, Mn, Fe, Co, Ni. Cu, Zn, Se, Mo, Sn, I etc. are indispensable for animals. These elements are distributed in each organ in a characteristic way. If the
Fig. 2. External PIXE spectrum of a normal gallbladder tissue.
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Fig. 3. External PIXE spectrum of a carcinoma gallbladder tissue.
Fig. 4. PIGE spectrum of a normal gallbladder tissue.
Fig. 5. PIGE spectrum of a carcinoma gallbladder tissue.
balance of these elements is broken, living bodies have to experience ‘‘diseases” [22]. The concentration of Na in the carcinoma tissues is about 0.7 times less than that in the normal tissues. But, the concentration of Mg in the carcinoma gallbladder tissues is about 0.4 times less than that in the normal tissues. The relative concentration of elements in normal versus carcinoma gallbladder tissue and blood are depicted in Figs. 6 and 7 respectively. High intakes of dietary fibre lower magnesium absorption. In contrast, high intakes of zinc decrease magnesium absorption and contribute to a shift toward negative balance in adult males [23]. It was observed in the present work that K levels in the carcinoma blood of gallbladder is lower than those in the normal blood samples. K is a major intracellular cation which is also excreted in gastrointestinal tract, saliva, gastric juice, bile, pancreatic and intestinal juices. Prolonged hypokalemia (potassium deficiency) causes injury to myocardium and kidneys. The glycolytic enzyme ‘‘pyruvate kinase” requires potassium for its maximum activity [24]. K deficiency causes acidosis, renal damage and cardiac arrest. Shonk and his associates [25–27] studied the activities of different glycolytic enzymes in carcinoma of rectum and colon and in the corresponding non-malignant counterparts. They observed that glycolytic enzyme activities of the neoplastic tissues of rectum and colon are much higher than the activities in the corresponding normal rectum and colon. They reported the ratio of neoplastic to normal ‘‘pyruvate kinase” enzyme activity is 2.1 for colon and 2.4 for rectum. Hence, it is expected that the levels of potassium which may be a cofactor for ‘‘pyruvate kinase” enzyme should be higher in the carcinoma tissue [28]. But the observation of low K levels in the blood of carcinoma gallbladder in the present work does not support the above statement. This may possibly be due to different organs. Ca is a major constituent of bone and teeth. It is required for coagulation of blood, regulation of neuromuscular irritability and muscular contractility. Concentration of Ca in both the gallbladder carcinoma tissue and blood samples is about 0.6 times less than that in the gallbladder normal tissue and blood. In a strictly operational sense, Ca balance is determined by the relationship between Ca intake and Ca absorption and excretion. A striking feature of the system is that relatively small changes in Ca absorption and excretion can neutralise a high intake or compensate for a low one. The concentration of Se in the carcinoma tissue samples is slightly less than that of the normal. Se has long been marked as a carcinogen. Nevertheless, the Committee on Medical and Biological effects of Environmental pollutants (1976) have reviewed the confounding data and concluded that selenium may be an anti-carcinogen. The inorganic Se at lower concentration acts as an anticarcinogen and it is deficient in cancerous patients. Se can be a good metabolic risk modifier against cancer. Several elements like Ni, Se, Cu and Cr are considered essential at certain concentrations, but they are toxic when intake is excess [29]. The main source of Se is plant extracts and the cereals are the likely major dietary sources. The variation depends on the differences of Se content of the soil of that particular place. Areas of high Se have higher prevalence of dental caries [30]. Selenium is a relatively toxic element. Intakes averaging 1.2 mg/day can induce changes in nail structure. Chronic selenium intakes over 3.2 mg/day can result in the loss of hair and nails, mottling of the teeth, lesions in the skin and nervous system, nausea, weakness and diarrhoea. Selenium, which is biologically important as an anion, is homeostatically regulated by excretion, primarily in the urine but some also is excreted in the breath. Selenate, selenite and selenomethionine are all highly absorbed by the gastrointestinal tract; absorption percentages for these forms of selenium are commonly found to be
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By PIGE Na Mg Al By external PIXE K Ca Ti Cr Mn Fe Co Ni Cu Zn As Se Br Pb
Blood
Normal (n = 10)
Carcinoma (n = 9)
Normal (n = 10)
Carcinoma (n = 9)
717.2 ± 61.8 8621.7 ± 551.2 559.2 ± 50.4
512.6 ± 47.2 3807.5 ± 227.5 262.3 ± 32.5
– – –
– – –
408.2 ± 38.9 616.3 ± 54.5 23.7 ± 3.8 11.1 ± 1.9 27.6 ± 3.6 1066.9 ± 93.8 8.25 ± 1.3 7.53 ± 1.3 11.1 ± 2.0 101.3 ± 16.9 3.8 ± 0.6 3.14 ± 0.6 12.6 ± 2.1 13.5 ± 2.1
359.4 ± 41.3 366.2 ± 40.8 37.2 ± 5.2 9.0 ± 1.7 23.4 ± 3.4 1579.7 ± 113.4 13.1 ± 2.3 12.3 ± 2.1 27.8 ± 4.3 75.6 ± 13.3 4.3 ± 0.7 2.06 ± 0.4 10.5 ± 1.8 11.8 ± 1.9
2082.7 ± 136.1 321.7 ± 38.9 1.97 ± 0.4 1.07 ± 0.2 4.13 ± 0.8 597.3 ± 52.5 0.74 ± 0.2 1.13 ± 0.3 1.10 ± 0.3 2.54 ± 0.5 0.11 ± 0.03 0.16 ± 0.04 1.04 ± 0.3 0.09 ± 0.03
1834.4 ± 122.7 201.2 ± 31.2 2.01 ± 0.4 1.02 ± 0.2 3.21 ± 0.6 657.1 ± 48.7 0.87 ± 0.2 1.74 ± 0.4 2.11 ± 0.4 2.06 ± 0.4 0.13 ± 0.03 0.14 ± 0.04 1.0 ± 0.3 0.16 ± 0.04
Fig. 6. Concentration of elements in normal versus carcinoma gallbladder tissue.
Fig. 7. Concentration of elements in gallbladder normal versus carcinoma blood.
in the 80–90% range. Lead is very useful, but very harmful as well when intake is more. The concentration of Fe in carcinoma tissue samples have been increased as compared to the Fe level in normal tissue. But the concentration of Cu in the carcinoma blood and tissue samples has increased as compared to the normal blood and tissue samples. Deficiency of Fe results in anaemia. It is responsible for oxygen transport and cellular respiration. Signs of Cu deficiency are anaemia, blood vessel necrosis, dyspigmentation, neuropathy, emphysema and diarrhoea. But excess intake of Cu causes stomach-ache, diarrhoea, vomiting, liver dysfunction and hemolysis. Zinc contents in every organ, except for the cancer affected organ where Zn is enriched, are reported to become low if a human is attacked by cancer. This is caused by a high transfer rate of Zn to wastes [22]. But, to contrary, in the present study, average concentration of Zn in the carcinoma gallbladder tissue is less than that of the normal gallbladder tissue. Zinc is present in all body tissues and fluids. The total body zinc content has been estimated to be 30 mmol (2 g). Plasma zinc has a rapid turnover rate and it represents only about 0.l% of total body zinc content. This level appears to be under close homeostatic control [31]. Deficiency of Zn causes poor growth, sexual infantilism in adolescents, loss of taste and delayed wound healing. The utilisation of zinc depends on the overall composition of the diet. Experimental studies have identified a number of dietary factors as potential promoters or antagonists of zinc absorption. Soluble low molecular weight organic substances, such as amino and hydroxy acids facilitate zinc absorption. In contrast, organic compounds forming stable and poorly soluble complexes with zinc can impair absorption. In addition, competitive interactions between zinc and other ions with similar physicochemical properties can affect the uptake and intestinal absorption of zinc. The risk for competitive interactions seems mainly to be related to high doses in the form of supplements or in aqueous solutions. However, at levels present in food and at realistic fortification levels, zinc absorption appears not to be affected by iron and copper [32]. The lack of specific and sensitive indexes for zinc status limits the possibilities for evaluating zinc requirements from epidemiologic observations. Zinc requirements were estimated by using the factorial technique (i.e. by adding the requirements for tissue growth, maintenance, metabolism and endogenous losses). Experimental zinc repletion studies with low zinc intakes have clearly
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shown that the body has a pronounced ability to adapt to different levels of zinc intakes by changing the endogenous zinc losses through the kidneys, intestine and skin [33–37]. The concentration of toxic elements like As and Pb did not show much variation between the normal and carcinoma gallbladder tissue and blood. Moreover, As and Pb concentration may be attributed to be contamination during sample lypholisation, pelletization or handling. The profound negative influence of malnutrition on cancer treatment is well recognised, with nutritional deficiencies impacting on all modalities of treatment. It therefore becomes critical to assess the nutritional status of patients before therapy and identify those at greater risk for morbidity. Given the presence of carcinoma effects coupled with years of tobacco and alcohol abuse, the high incidence of nutritional deficiency in the gallbladder is not surprising. Diet may influence human carcinogenesis in six general ways [38]: (i) Diet provides the carcinogens or their immediate precursors, (ii) Diet facilitates or inhibits the endogenous production of carcinogens, (iii) The modification of carcinogens by metabolic activation or inactivation could be affected by other dietary components, (iv) Increasing or impeding the delivery of carcinogens to their site of action may be influenced by dietary changes, (v) Diet may alter the susceptibility of tissues to cancer induction or growth by dietary effects or tissue metabolism, (vi) Diet may alter the body’s capacity to eliminate transformed cells. In the field of nutrition and cancer, turmeric is emphasised as an anti-cancer agent. It prevents the formation of preneoplastic lesions induced in the initiation as well as post initiative phase. The As concentrations are found relatively high in spices, whereas lead and iron are found more in tobacco-products. Clinical studies have shown that iron promotes cancer cell growth. It acts more like a co-carcinogen than a primary one and in case of tobacco user’s local absorption of concentrated iron in oral region could be high. Tobacco addicts who normally require more antioxidants as compared to a normal person tend to consume a less adequate diet with subnormal antioxidant levels than do others [39]. From the epidemiological evidences and different animal models, it is established that Mn and Se are non-carcinogens. But, excess exposure to Cr, As, Cd, Ni, Be, Co, Pb, Si and Sn may lead to cancer. The carcinogenicity of Al, Cu, Fe, Mo, Sc, Ti, V and Zn are yet to be established [40]. Daily nutritional intake of elements of different individuals from different areas and studies on trace element imbalances in different environment and different biomedi-
cal samples may lead to better diagnosis of diseases like cancer which will definitely guide the clinicians to supplement or withdraw the elements and minerals to the human body for cure and maintenance of better health. It was observed from Table 2 that all the tobacco-products were very highly enriched with multi-elements. The present results for tobacco-shoots when compared with those reported by Oliveira et al. [41], there is an exception in the strontium and copper levels. Low Sr-levels and high Cu-levels were observed in the tobaccosamples. These elemental variations in the tobacco-chemistry could be due to some agricultural and industrial factors like preharvest and post-harvest operations, pre-blend operations, drying, storing, processing, flavor addition and casings. Among the common smoking tobaccos, it was found that the Indian bidi, which contains raw tobacco leaves, have higher elemental levels than that of the cigarettes. Clinical studies have shown that Fe promotes cancer cell growth and it acts more like a co-carcinogen [42]. All the tobacco-products were found to be very highly enriched with iron. The branded tobacco-products like gutkha and zarda, whose other ingredients are betel nuts, lime, cardamom catechu, silver leaves, aromatic spices and saffron, have comparatively lower iron level than that of the raw tobacco-products such as gudakhu. Exposure to arsenic is associated with the on set of cancers of skin and lung. Table 3 shows the elemental variations in human blood with respect to their tobacco habits. From the results, it was observed that all the elemental levels in human blood decrease with respect to increase in the tobacco habits. The selenium and copper contents in the blood decreases significantly in case of heavy users of tobacco in comparison with that of the control group, who have no tobacco habits [43]. The blood iron and copper values in case of moderate and heavy users of tobacco has become lower than the reported normal ranges [44]. Of course, these lower elemental values could be due to the inadequate and poor dietary habits of the tobacco users [45]. The copper and zinc ratio, which is an important index in disease activity [46], decreases significantly with respect to increasing habits of tobacco. 4. Conclusion Decreasing levels of Zn and Se in the tissue samples have a definite importance in the oxidative processes in the human body.
Table 2 Concentration (in ppm by weight) of some tobacco-products by PIXE analysis.
Cigarette (n = 8) Bidi (n = 8) Gutkha (n = 8) Zarda (n = 8) Khaini (n = 7) Gudakhu (n = 7)
K%
Ca%
Mn
Fe
Cu
Zn
As
Se
Sr
Pb
0.63 ± 0.04 0.76 ± 0.06 1.03 ± 0.06 0.69 ± 0.05 0.87 ± 0.04 0.81 ± 0.04
2.03 ± 0.08 4.21 ± 0.04 2.01 ± 0.07 1.76 ± 0.07 4.01 ± 0.04 1.63 ± 0.07
151.6 ± 23.6 97.8 ± 19.7 73.6 ± 17.8 49.5 ± 10.3 157.4 ± 28.5 121.4 ± 17.0
687.6 ± 68.7 1397.2 ± 159.5 472.8 ± 72.4 484.9 ± 83.7 615.8 ± 117.3 992.3 ± 147.3
15.4 ± 3.1 12.5 ± 2.0 9.6 ± 1.6 13.6 ± 2.7 20.3 ± 3.1 14.8 ± 2.2
29.6 ± 5.7 61.8 ± 9.3 71.1 ± 12.4 43.4 ± 12.7 18.2 ± 3.5 72.5 ± 16.2
0.17 ± 0.03 0.31 ± 0.05 0.23 ± 0.05 0.20 ± 0.04 0.19 ± 0.04 0.30 ± 0.06
0.10 ± 0.02 0.15 ± 0.03 0.14 ± 0.03 0.11 ± 0.02 0.15 ± 0.03 0.19 ± 0.04
14.8 ± 3.1 19.6 ± 4.0 15.3 ± 2.9 21.5 ± 3.8 19.3 ± 4.1 15.8 ± 2.8
0.47 ± 0.10 1.25 ± 0.22 0.68 ± 0.16 0.71 ± 0.15 0.69 ± 0.14 0.97 ± 0.18
Table 3 Average concentration (in lg/ml) of various elements of human blood with respect to tobacco habits by PIXE technique. Groups
K Ca Fe Cu Zn Se Pb
Average elemental concentrations of whole blood I (No habit)
II (Light habits)
III (Moderate habits)
IV (Heavy habits)
2132.5 ± 127.3 272.4 ± 45.2 532.7 ± 82.6 1.06 ± 0.22 2.41 ± 0.50 0.16 ± 0.03 0.07 ± 0.01
1982.6 ± 120.4 263.4 ± 46.4 504.0 ± 80.6 0.91 ± 0.20 2.32 ± 0.49 0.13 ± 0.02 0.07 ± 0.01
1971.8 ± 121.0 251.1 ± 44.9 372.8 ± 69.8 0.83 ± 0.17 2.21 ± 0.46 0.10 ± 0.02 0.07 ± 0.01
1711.3 ± 198.2 239.3 ± 45.2 359.3 ± 71.8 0.61 ± 0.17 1.68 ± 0.47 0.07 ± 0.02 0.06 ± 0.01
T.R. Rautray et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2878–2883
But, to contrary, in the present study, concentration of Zn in the carcinoma gallbladder tissue is less than that of the normal gallbladder tissue. Tobacco habit could be one of the important factors to decrease the elemental concentrations in blood and tissue samples. Gradual increase in tobacco habits decreases the dietary intake of a person and hence trace element levels in blood. Therefore, proportionately adequate supplementation of trace elements to tobacco users is very much essential to scavenge the free radicals. The local absorption of highly concentrated iron and calcium from the tobacco-products could be dangerous. Tobacco products are rich in multi-elements. The more the local absorption of multi-elements, the more is the local irritation and subsequent development of cancer. Acknowledgements The help extended by the staff members of Institute of Physics (IOP) is acknowledged and the authors would like to thank the authorities of IOP for providing beam time to carry out the current research. References [1] V. Valkovic, Proton-induced X-ray emission: applications in medicine, Nucl. Instr. and Meth. 142 (1977) 151. [2] W. Maenhaut, J. Vandenhaute, H. Duflou, Applications of PIXE to biological and biomedical samples at the university of Kent, Nucl. Instr. and Meth. B 22 (1987) 138. [3] M. Tanaka, E. Mataugi, K. Miyasaki, T. Yamagata, M. Inoue, H. Ogata, S. Shimoura, PIXE measurement applied to trace elemental analysis of human tissues, Nucl. Instr. and Meth. B 22 (1987) 152. [4] P. Boffetta, Involuntary smoking and lung cancer, Scand. J. Work Environ. Health. 28 (2002) 30. [5] E. Nogueira, A. Cardesa, U. Mohr, Experimental models of kidney tumors, J. Cancer Res. Clin. Oncol. 119 (4) (1993) 190. [6] B. Delahunt, J.N. Nacey Bethwaite, Occupational risk for renal cell carcinoma, Br. J. Urol. 75 (1997) 578. [7] N.S. Mandel, J.K. McLaughlin, B. Schlehofer, A. Mellemgaard, International renal-cell cancer study IV, Occupat. Int. J. Cancer 61 (5) (1995) 601. [8] U. Lindh, Cell biology, trace elements and nuclear microscopy, Nucl. Instr. and Meth. B 104 (1995) 285. [9] F. Martin-Lagos, M. Navarro-Alarcon, C. Terres-Martos, S. Lopez-G de la Serrana, M.C. Lopez-Martinez, Serum copper and zinc concentrations in serum from patients with cancer and cardiovascular disease, Sci. Total Environ. 204 (1997) 27. [10] J.L. Poo, R.R. Romero, J.A. Robles, A.C. Montemayor, F. Isoard, A. Estanes, M. Uribe, Diagnostic value of the copper/zinc ratio in digestive cancer: a case control study, Arch. Med. Res. 28 (1997) 259. [11] T. Magalova, V. Bella, A. Brtkova, I. Beno, M. Kudlackova, K. Volovova, Copper, zinc and superoxide dismutase in precancerous, benign diseases and gastric, colorectal and breast cancer, Neoplasma 46 (1999) 100. [12] D. Ferrigno, G. Buccheri, T. Camilla, Serum copper and zinc content in nonsmall cell lung cancer: abnormalities and clinical correlates, Monaldi Arch. Chest Dis. 54 (1999) 204. [13] A.S. Prasad, Zinc: an overview, Nutrition 11 (1) (1995) 93. [14] V. Vijayan, R.K. Choudhury, B. Mallick, S. Sahu, S.K. Choudhury, H.P. Lenka, T.R. Rautray, P.K. Nayak, External particle induced X-ray emission, Current Sci. 85 (2003) 772. [15] S. Sahu, S.K. Choudhury, B. Mallick, T.R. Rautray, V. Vijayan, R.K. Choudhury, Design of a rotating vane chopper for external PIXE analysis, in: Proceedings of Indian Particle Accelerator Conference, Indore, India, 2003, p. 695. [16] R.K. Choudhury, V. Vijayan, N.C. Mohanty, Scientific study of metallic compositions of Orissa state museum specimens, Orissa Rev. 59 (2003) 48.
2883
[17] T.R. Rautray, V. Vijayan, S. Panigrahi, Analysis of Indian pigment gallstones, Nucl. Instr. and Meth. B 255 (2007) 409. [18] J.L. Campbell, T.L. Hopmann, J.A. Maxwell, Z. Nezedly, The Guelph PIXE software package III: alternative proton database, Nucl. Instr. and Meth. B 170 (2000) 193. [19] ANGES software package, IAEA, Vienna. Available from