Introduction

In recent years, detection and assessment of trace elements has become an arena of growing importance in various clinical disciplines [1]. With special reference to oncological disciplines, early researchers have shown deficiency, or an excess accumulation of certain elements have direct correlation with incidence and progression of certain types of cancer [2, 3]. Among different types of site-specific cancers, oral cancer, which has been shown to be related with micronutrient imbalance and is the eighth most common cancer worldwide, has a remarkable high incidence in India, where the age standardized incidence rate of oral cancer is reported to be 12.6 per 100,000 populations [4]. Annually, about 270,000 cases of oral cancer are reported worldwide, and about 82,000 of them are diagnosed in India [3, 5].

Addictions like smoking or chewing tobacco in any form along with poor sense of oral hygiene are being considered as the predisposing factors of cancer or precancerous disorders [6]. The two most common precancerous disorders of the oral cavity are leukoplakia (premalignant lesion) and oral submucous fibrosis (premalignant condition) [7]. While in case of leukoplakia, a white patch is found in oral mucosa, oral submucous fibrosis is characterized by rigidity and stiffening of the mucosa in association with some specific symptoms like burning sensation of mouth, intolerance to spicy food, and progressive difficulty in mouth opening [8]. The incidence of leukoplakia varied from 1.3 to 2.1 per 1,000 individuals in different parts of India depending on the types of tobacco habits, but the prevalence of this lesion in India was found to be about 17 per 1,000 tobacco users [9]. Oral submucous fibrosis is linked to the chronic placement of betel quid and is found in 0.4% of India's villagers [10]. It has been reported by earlier researchers that among these two potential malignant disorders, the annual transformation rate of OSF into malignancy is much higher (7.6%) than that of leukoplakia (0.3–1%) in India [11, 12].

It is already established that trace elements are of growing importance for clinical oncological sciences. This has prompted the possible establishment of a new scientific discipline as “Medical Elementology” [13]. There are many suggestions in the scientific literature about levels and incidence of different types of cancers such as high levels of arsenic in skin and bladder cancer, cadmium in breast and prostate cancer [14], and copper and iron in respiratory tract, urinary tract, and thorax cancer. However, literature survey revealed scanty information regarding incidence of oral submucous fibrosis and/or oral leukoplakia in relation to elemental profile of the concerned human subjects. Furthermore, it is also established that screening and early diagnosis of precancerous disorders do prevent widespread incidence and prevalence of cancer [15, 16]. Based on this perspective, the present study was designed to ascertain possible correlation of trace elemental profile and the progression or regression of these two precancerous oral diseases towards malignancy of the oral cavity.

Materials and Methods

The study was carried out over a period of four years [2005–2009]. Three hundred patients were randomly selected, and patients with systemic and contagious diseases were excluded. The study design encompassed two sets of male patients in the age group of 20–50 years, one each of leukoplakia and oral submucous fibrosis. Each set of patients were studied in two groups, each consisting of 30 patients. The first two groups were studied in the time period 2005–2007, and the second two were studied in between 2007 and 2009. Preliminary clinical diagnoses of the selected disorders were confirmed by histopathological examination of the representative tissues. The control group consisted of 30 healthy men aged between 20 to 50 years who donated blood voluntarily. The study subjects were briefed about the purpose of the study, and written consent was obtained from each of them.

Blood (5 ml) was drawn from each subject from the anterior cubital vein following clinical procedures and manners and then transported in containers containing heparin to prevent coagulation. The samples were kept at −80°C refrigerators until further processing for trace element estimation was carried out. During processing, the samples were first lyophilized at −80°C and made into powder. Powder (200 mg) was taken and compressed to form a pellet of 13 mm diameter, with a table-top pelletizer using 100 kg/cm2 pressure. The pellets were then subjected to Energy Dispersive X-Ray Fluorescence (EDXRF) technique. The concentration of trace elements in the blood samples was measured by employing EDXRF technique, a highly sensitive, non-destructive, multi-elemental technique, widely used for trace element studies. The main principle of the EDXRF is based on the excitation of characteristic X-rays in the sample that give quantitative information about the elemental concentration.

Student's t test was performed for evaluating statistical significance.

Results

EDXRF analysis showed presence of K, Ca, V, Cr, Mn, Fe, Cu, Zn, Se, Br, Rb, Sr, Co, and Pb in the blood samples collected from the two groups of patients suffering from the two types of oral precancerous disorders, OSF and LKP and the control group (healthy subjects). Out of these elements, Mn, Fe, Zn, Br, and Co showed remarkable alteration in their profile in both leukoplakia and oral submucous fibrosis patients in comparison with that of the control group. While Zn, Br, and Fe showed gross depletion in both the diseased groups, Mn and Co depicted an interesting pattern of alteration. The change in concentration of Mn and Co was radically opposite in the two groups of patients each compared with respect to the control subjects.

Comparison of the average concentration of Zn in the blood samples of both OSF and leukoplakia patients with respect to the disease-free healthy groups showed significant depletion (p < 0.05) in the range of 13.2% and14%, respectively (Fig. 1).

Fig. 1
figure 1

Percentage change in Zn content of OSF and LKP patients w.r.t. controls. Bars marked with a star means significant difference from the controls at 5% confidence level

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It was found that the Cu/Zn ratio of the diseased conditions was raised with respect to the control. In the case of leukoplakia, it was found to be increased by 7%; in case of oral submucous fibrosis, the increase was 21%.

Similar to the data observed in case of Zn, significant depletion in Br concentration was noted in blood from OSF and leukoplakia with respect to their normal counterparts. However, the range of decrease in Br was more pronounced than that observed for Zn. While OSF patients suffered a decrease of 23.5% in Br concentration, leukoplakia patients displayed a Br depletion of 24.1 % with respect to the healthy subjects (Fig. 2; p < 0.05).

Fig. 2
figure 2

Percentage change in Br content of OSF and LKP patients w.r.t. controls. Bars marked with a star means significant difference from the controls at 5% confidence level

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The average whole blood iron level was reasonably lower in both the sets of samples of the diseased group as compared to that of the controls. The fall in mean of Fe level was found to be almost double in OSF (12.9%) than that of leukoplakia (6.4%) as shown in Fig. 3. The trend of such depletion, however, was not statistically significant.

Fig. 3
figure 3

Percentage change in Fe content of OSF and LKP patients w.r.t. controls

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Figures 4 and 5 represent alterations in Mn and Co profile, respectively, in the subjects suffering from the two types of oral lesions. It is interesting to note that Mn and Co displayed a reverse pattern of change in the two types of oral lesions. While OSF patients suffered from an increase in both Mn (21.1%) and Co (28%) concentration with respect to their control counterparts, subjects suffering from leukoplakia presented a decrease in concentration of these two elements when compared to that of the control groups. Depletion of Mn in blood of leukoplakia patients was in the range of 59%, while Co was decreased by only 22.5% with respect to control.

Fig. 4
figure 4

Percentage change in Mn content of OSF and LKP patients w.r.t. controls

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Fig. 5
figure 5

Percentage change in Co content of OSF and LKP patients w.r.t. controls

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Intercorrelation calculations between the elements carried out in order to rule out direct or inverse associations showed noteworthy changes in both OSF and leukoplakia samples (Tables 1 and 2). In both OSF and leukoplakia conditions, positive correlation was noted between Br–Cu and Zn–Fe, while Zn–Cu, Br–Zn, Br–Fe, and Cu–Fe indicated negative correlation. Mn manifested positive correlation with Br, Fe, and Cu, in case of leukoplakia, while in case of oral submucous fibrosis, it showed positive correlation with Zn and Fe. For Co, positive correlation was noted with Zn, Br, Cu, and Fe in leukoplakia in contrast to OSF where Co was positively correlated only to Fe. Negative correlation was noted in case of Zn–Mn in leukoplakia and Mn–Br, Mn–Cu, Co–Zn, Co–Br, and Co–Cu in OSF. In both leukoplakia and oral submucous fibrosis, Co–Mn were found to be positively correlated.

Table 1 Matrix showing intercorrelation between elements for leukoplakia
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Table 2 Matrix showing intercorrelation between elements for OSF
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Discussion

Data of our present study re-establish the role of trace elements in metabolism and their further implications in maintenance of physiological homeostasis. Trace elements such as Zn, Cu, Mn, and Fe are involved in metal-enzyme activity and oxidation-reduction reactions and subsequently regulate the metabolic functions and the antioxidant mechanisms [17, 18].

Zinc is known to be an essential component of DNA-binding proteins with zinc fingers, as well as copper/zinc superoxide dismutase and several proteins involved in DNA repair. Thus, zinc plays an important role in transcription factor function, antioxidant defense, and DNA repair. Deficiencies in zinc can contribute to single and double-stranded DNA breaks and oxidative modifications to DNA that increase risk for cancer development. Significant depletion in Zn concentration in both the two groups of patients suffering from oral premalignant disorders might be considered as a vicious cycle, where decrease in Zn concentration as a consequence of the disorders could be a risk factor to predispose the individuals to further progression of the disease towards cancer [19, 20]. Other researchers also reported that zinc deficiency and zinc-dependent immunologic dysfunctions are present in more than half of the patients with head and neck cancer [21]. As both OSF and leukoplakia involve inflammatory processes, it may be inferred that prolonged inflammation due to such lesions induces oxidative stress. Zn not only serves as a cofactor in numerous transcription factors but also is an active component of many antioxidant enzyme systems viz superoxide dismutase, and carbonic anhydrase or leucine aminopeptidase, which are mainly involved in body homeostasis against neoplastic development [22, 23]. Thus, significant depletion in Zn as observed in the present investigation might be an important signal to reflect the interplay between the underlying oxidant–antioxidant status during progression of the two types of inflammatory precancerous pathological condition.

There is evidence that copper and zinc have pro-oxidant and antioxidant properties, respectively, so that their imbalance may be expected to reflect oxidative stress status [41]. Cu/Zn ratio has been suggested as a good indicator of the extent and prognosis in carcinoma by host of workers as it has been found to be increased in case of malignant lymphomas in children [42] and carcinoma of the gallbladder [43] and breast [44]. In addition to malignancy, inflammatory conditions [45] and even age-related degenerative process [41] have been reported to be associated with an increase of serum Cu/Zn ratio. Data of our present study showing significant increase in Cu/Zn ratio both in OSF and leukoplakia conditions point toward increase in oxidant load in these two precancerous diseases and reestablishes consideration of Cu/Zn ratio as an indicative pathophysiological marker.

Iron, one of the most abundant and essential transition metals in the body, is most commonly associated with the oxygen carrying function of heme iron. Clinically, effect of low iron, which leads to anemia followed by decreased oxygen carrying capacity of blood and related diseases, is of major concern for study related to disease pathology [24]. On the other hand, it is well-known that non-heme iron participates in a range of reactions that are necessary for cell viability and cell proliferation [1]. It is an essential component in DNA synthesis and in respiratory and oxidative metabolism [25, 26]. A trend of depletion in the blood iron level, as observed in both the groups of patients in our study seems similar to the earlier reports of premalignant conditions of oropharynx where iron deficiency has been documented as a risk factor of such condition (Plummer-Vinson syndrome) and was subsequently almost eliminated through improved nutritional status especially with regard to dietary iron [9, 27]. The unusual alterations in epithelium due to iron deficiency has also been depicted as deficiency of keratinized cells, altered nuclear cytoplasmic ratio, abnormal cell maturation, and karyorrhexis [27]. Parallel observation from Khanna et al. [22] reported a decrease in the iron levels in whole blood of the both pre-cancer and cancer group. Hematological abnormalities in oral leukoplakia as reported by Chellacombe [28] might be relevant to the depletion in iron status as observed in our present investigation.

Furthermore, Bhattathiri et al. [29] observed that in patients with submucous fibrosis (all of whom were heavy tobacco and arecanut users too), serum iron decreased, whereas the total tissue collagen content increased significantly in patients with advancement of the disease. Collagen synthesis is effectively mediated through utilization of iron during hydroxylation of proline and lysine, where ferrous ion acts as a cofactor for the enzyme proline hydroxylase. Thus, in our findings too, the possible cause of pronounced decrease in iron concentration in OSF patients than that in leukoplakia patients may be attributed to utilization of iron for collagen synthesis as proposed by the earlier research groups [29, 30], since incidence of OSF has been associated with increased collagen, but further studies in this area are needed to validate the findings.

Significant increase in Mn level observed in case of blood from OSF patients could be attributed to prolonged underlying inflammation in parallel to reports of Morton and Schwartz [31] who observed similar increase in serum Mn of patients during active phase of inflammatory conditions like acute icteric viral hepatitis. Such increased levels, however, returned towards normal in the subsiding phase of the disease clearly reflecting role of Mn in inflammatory processes. In post-necrotic cirrhosis also, it was reported that the serum Mn level has been significantly increased [31]. The liver has a relatively high concentration of Mn, which is primarily excreted in bile. The increase was presumably due to either release of hepatic manganese during parenchymal necrosis or decreased hepatobiliary excretion. Mn concentration in normal human plasma is cyclic with highest concentration found in the early proliferative phase (cyclic days 6 to 8). In line with these observations from the earlier researchers, observed data of our study showing increased Mn concentration in OSF patients may be linked with the inflammatory component or mucositis and the proliferative activity of the fibroblasts as proposed by Rajalalitha and Vali [11]. Interestingly, data of the present investigation study showed remarkable depletion in concentration of Mn in blood of leukoplakia patients as compared to that of the healthy counterparts. This probably reflects the system's need to utilize Mn for its specific role in combating oxidative stress through its association with Mn–SOD activity [32]. The possible biochemical interpretation of such contrasting data may be related to the sharp difference in the etio-pathogenesis of the two diseases where one is primarily a connective tissue disorder (OSF) while the other starts from the epithelium (LKP). However, the precise role of Mn in these two diseases warrants further study.

Co is an important constituent and an integral part of vitamin B12. A deficiency in cobalt results in decreased vitamin B12 supply resulting in nutritional macrocytic anemia [9]. The decreased cobalt concentration observed in blood samples of leukoplakia patients may predispose the patients to anemia and cause persistence of the premalignant condition. This finds support from the work where cobalt deficiency-induced anemia was responsible for in development of malignancy due to decreased reparative capacity of the system [9]. As prolonged inflammation is one of the etiological factors for initiation and progression of the precancerous disorders, effects of inflammatory cytokines such as IL-1 alpha, IL-1 beta, TGF-beta, TNF-alpha, and IL-6 may affect cobalt-induced erythropoietin production. This in turn plays a significant role in the pathogenesis of the anemia of chronic disease and hence causes dysplasia of the epithelium [33]. Thus, in the present investigation in case of leukoplakia, an observed Co-depleted condition, an anemic epithelium cannot counter the insults on it due to the inflammatory load as effectively as healthy epithelium. As such, use of tobacco and other products by those patients acts as an additional inflammatory stress that might have a potential role in persisting the precancerous condition and further promoting it towards malignancy. This is similar with the condition of iron deficiency anemia which acts as a predisposing factor for carcinoma of pharynx [9] and corroborates with our findings of concomitant iron depletion discussed earlier.

Increased concentration of Co noted in OSF patients with respect to the normal healthy subjects might have some relevance with presence of excess Co among other heavy metals in packaged areca nut products which are the chief cause of OSF [34]. Although evidence of increase in Co in relation to oral cancer is lacking, increase in concentration of Co has been noted in other epithelial malignancy [35].

Though essentiality of Br as micronuterient has not yet been established, deficiency of its concentration has been reported in malnourished people [36]. In the present study, it was observed that the general health status of the patient was poor when the patient first reported to the department. Limited findings suggest that bromide may be nutritionally beneficial [37]. Some earlier researchers have observed metabolic perturbations viz. anomalies in carbohydrate, lipid, or mineral metabolism due to Br deficiency in normal subjects [38], while some group reported endocrine disturbances in relation to thyromegaly, adrenal adenomatosis, or microcystic ovary transformation focal proliferative extracapillary glomerulonephritis, etc. in subjects with congenital Br deficiency [39]. It was also reported by earlier researchers that Br deficiency has some bearing with cheilitis, an inflammatory disease of the lips associated with many conditions, including megaloblastic anemia from vitamin B12 deficiency, iron deficiency, etc. [39]. In tune with this, our observation regarding Br depletion in both the two groups of patients (Fig. 2) reflects an indication of possible involvement of Br with the etiology of OSF and Lkp both of which entail inflammatory processes. Furthermore, bromide exposure has been reported to inhibit cell proliferation in breast cancer cells and enhances apoptosis of the neoplastic cells [40]. Reports from Tan et al. [47] portray that among different soil trace elements, Br content in soil has been observed to have inverse association with incidence of cervical cancer, and prediction of such cancer mortality based on trace elements using suitable algorithmic chemometric methods are coming up as recent areas of interest in cancer research. In addition to the above, relevance of the observed data showing decrease in Co concentration in Lkp patients, in addition to depletion in Fe level in both OSF and LKP reconfirms the importance of these elements as markers of the two oral precancerous lesions.

Positive correlation observed between Br and Cu is supported from the fact that an increase in Cu has been well documented to be linked with increase in inflammatory load [41]. Additionally, it has been seen in various case studies that malnutrition added with inflammatory conditions has an association with Br deficiency [39]. As such our present data showing Br depletion in both the precancerous conditions might lead to increase of the inflammatory and oxidant factors. Correlation between zinc and iron shows the importance of these two elements in maintaining the health of the epithelium [19, 27]. Positive correlation of Mn with Br, Cu, and Fe in case of leukoplakia may indicate the overutilization of Mn, a cofactor Mn–SOD enzyme in the condition of depleted Br, Fe, and Cu in this disease [32]. Positive correlation between Co and Fe in both the diseased conditions stresses on the importance of these two elements in maintaining epithelial integrity [9]. Our analysis showing positive correlation between Mn and Fe iron is in good agreement with similar correlation reported earlier in case of hair samples of breast cancer patients [46]. Negative correlation found between some of the elements as detailed in the “Results” section reflects no direct association between these elements in the two selected conditions of precancerous lesions.

This study indicates the utility of monitoring the level of certain trace elements in patients with oral precancerous disorders in order to provide adequate nutrition of relevant elements.