Evaluation of salivary protein patterns among a rural population exposed and non-exposed to arsenic-contaminated drinking water in areas of Tucumán (Argentina): a pilot study

Abstract Arsenic contamination in the environment and groundwater is a major global public health problem. Several researchers suggest that the toxicity of arsenic could be related to oral cancer development, usually resulting from potentially malignant lesions. During pathological processes, salivary proteins suffer modifications, which could lead to the discovery of new biomarkers. Objective To analyze the protein profile in human saliva samples from a rural population exposed to high levels of arsenic in drinking water and its association with potentially malignant lesions. Methodology This observational, analytic and cross-sectional design included 121 patients from the state of Graneros (Tucumán, Argentina). Arsenic concentration in drinking water was determined and, according to the values obtained, individuals were divided into 2 groups: exposed group and non-exposed group. Saliva samples were obtained, and total protein concentration was measured by Bradford method. Finally, Laemmli SDS-polyacrylamide gel electrophoresis was conducted to obtain the protein profile. Results Total protein concentration in saliva was lower in the exposed group than in the non-exposed group. Average areas of 20 and 42 KDa bands were significantly lower in exposed group than non-exposed group. Conclusion Chronic intake of high arsenic concentrations in drinking water produces changes in the salivary protein profile, which is associated with the presence of potentially malignant lesions.


Introduction
Arsenic is a metalloid found ubiquitously in the environment and its contamination of groundwater is considered a major global public health problem. It affects an estimated 140 million people in more than 50 countries, including the United States, Taiwan, Mexico, Mongolia, Argentina, India, Chile and others. Arsenic is present throughout the environment in its inorganic forms, and the level of exposure varies greatly by geographical location. The current World Health Organization (WHO) and Environmental Protection Agency (EPA) recommended limit for arsenic levels in drinking water is 10 μg/L. 1,2 Arsenic is considered as a class 1 human carcinogen by the International Agency for Research on Cancer (IARC) and the chronic exposure to arsenic is associated with chronic endemic regional hydroarsenicism (HACRE, in Spanish), which is characterized by skin lesions (leukoderma and/or keratosis), cardiovascular disease, neurotoxicity, type II diabetes, oral cancer (OC), and others. 3,4 OC is a malignant neoplasia that arises on the lip or oral cavity. It is traditionally defined as a squamous cell carcinoma (OSCC), because 90% of cancers are histologically originated in the squamous cells. 5 The main risk factors to the development of OC are tobacco, alcohol and betel nut intake, and human papillomavirus (HPV) infection. However, they do not explain the high OC incidence. Several researchers suggest that the toxicity of heavy metals present in the floor, such as lead, nickel, arsenic, and others, could be related to that. Despite the well-documented effect of arsenic exposure in the development of skin, lung and bladder cancer, evidence of a relationship with OC is scarce. [6][7][8] OC presents precursor lesions that are not considered neoplasms, although they are related to a higher probability of developing into OSCC, known as potentially malignant lesions (PML). Those are: oral submucous fibrosis, erythroplakia, leukoplakia, chronic candidiasis, congenital dyskeratosis, lichen planus and actinic cheilitis. 9,10 Saliva is a fluid produced and poured into the oral cavity by the salivary glands. After secretion into the oral cavity, it mixes with a serum transudate that emanates from the gingival sulcus. The singular contribution of gingival fluid allows markers to be present in the saliva which derive from the circulation. 11 Proteins are another component of saliva that suffers modifications during pathological processes. These changes affect the protein profiles produced, which could lead to the discovery of new biomarkers and new approaches for diagnosis and detection of PML.
For certain malignant diseases, there are markers that can be detected in saliva such as anti-p53 antibodies in patients with OSCC. 12 This pilot study aimed to analyze the protein profile present in human saliva samples from a rural population exposed to high levels of arsenic in drinking water and its association with PML. According to the values obtained, individuals were divided into 2 groups: exposed group (EG) with arsenic concentration higher than 0.05 mg/L in drinking water;

Methodology
and non-exposed group (NEG) with levels lower than 0.05 mg/L in drinking water (cutoffs established by the Argentine Food Code). 14 Biochemical parameters To standardize the collection according to the circadian rhythm, unstimulated saliva samples were obtained after nighttime fasting, between 8 and 10 am, to prevent food ingestion, which would cause salivary secretion by increasing neurotransmitter activity due to taste and mastication stimuli. Before collection, patients washed their mouths with distilled water to remove any possible debris or contaminating material.  Table 1  concentrates the main economic activity and a large part of the population does not work. All subjects are considered vulnerable, since they have no access to the health system, so care and monitoring of oral-dental pathologies are infrequent.

Results
Participants of this research were divided into two groups: EG (n=55) and NEG (n=66). Risk factors analysis for the development of PML (smoking, hazardous drinking and harmful drinking alcohol consumption) showed no significant differences between both groups (data not shown). Furthermore, patients in EG showed a lower total protein concentration in saliva than NEG (6.6±1.9 and 8.7±4.1, respectively). Table 2  Oral pathologies in soft and hard tissue present in EG were chronic periodontitis (common cause of tooth loss), actinic cheilitis and stained teeth. 16 Whereas, in NEG the pathologies found were biofilm-gingival, frictional keratosis and chronic periodontitis ( Figure   2). Table 3 shows the main pathologies found in soft and hard tissues of the oral cavity.   FK + Dental biofilm-induced gingivitis 0 4 (6.06%) EG: exposed group (n=55); NEG: non exposed group (n=66). CP: chronic periodontitis; AC: actinic cheilitis; FK: frictional keratosis.

Table 3-Oral pathologies in soft and hard tissue
Evaluation of salivary protein patterns among a rural population exposed and non-exposed to arsenic-contaminated drinking water in areas of Tucumán (  A: Representative SDS-polyacrylamide gel electrophoresis results obtained from saliva samples of EG (1, 2, 3, 4, 7, and 8) and NEG (5,6) groups. B: Densitometric analysis of the detected bands. The values were expressed as mean±SD. * p<0.05 was considered significant. EG: exposed group, NEG: non exposed group, MW: molecular weight.   EG (n=55). * p<0.05 was considered significant. Table 4-Correlation between 20 kDa and 42 kDa bands between Actinic cheilitis and Chronic periodontitis Evaluation of salivary protein patterns among a rural population exposed and non-exposed to arsenic-contaminated drinking water in areas of Tucumán (  33 In the population studied, 50% of patients exposed to high levels of arsenic in drinking water were diagnosed with actinic cheilitis. They were reported to both work and live in rural areas. The synergism between exposure to As and agrochemicals (pesticides) could have multiplied the effect along the oral carcinogenesis process. 34 This study suggests that the chronic intake of high arsenic concentrations in drinking water decreases protein saliva levels and changes in their profile. This is associated with a high percentage of potentially malignant lesions. Therefore, saliva analysis could be used as a diagnostic sample in these patients, due to their advantages compared to other samples, since its obtention is easy, safe, non-invasive, economic and better tolerated.
This preliminary study has some limitations; its cross-sectional design only allows association, but no causality. Since it is a pilot study, the results cannot be generalized. The sample size is relatively small, and some results cannot be applied indiscriminately.
Therefore, further studies are needed to confirm the results, such as those aimed at establishing the relationship between protein concentration in saliva and arsenic levels in water; investigating markers of chronic exposure in hair and nails; and identifying altered proteins (20 and 42 kDa bands) by techniques used for the analysis of saliva as mass spectrometry or liquid chromatography.

Conclusion
Chronic intake of high arsenic concentrations in drinking water produces changes in the salivary protein profile and this is associated with the presence of potentially malignant lesions.