Environmental Lead Pollution and Its Possible Influence on Tooth Loss and Hard Dental Tissue Lesions

Bacground/Aim. Environmental lead (Pb) pollution is a global problem. Hard dental tissue is capable of accumulating lead and other hard metals from the environment. The aim of this study was to investigate any correlation between the concentration of lead in teeth extracted from inhabitants of Panÿevo and Belgrade, Serbia, belonging to different age groups and occurrence of tooth loss, caries and non-carious lesions. Methods. A total of 160 volunteers were chosen consecutively from Panÿevo (the experimental group) and Belgrade (the control group) and divided into 5 age subgroups of 32 subjects each. Clinical examination consisted of caries and hard dental tissue diagnostics. The Decayed Missing Filled Teeth (DMFT) Index and Significant Caries Index were calculated. Extracted teeth were freed of any organic residue by UV digestion and subjected to voltam-metric analysis for the content of lead. Results. The average DMFT scores in Panÿevo (20.41) were higher than in Belgrade (16.52); in the patients aged 31–40 and 41–50 years the difference was significant (p < 0.05) and highly significant in the patients aged 51–60 (23.69 vs 18.5, p < 0.01). Non-carious lesions were diagnosed in 71 (44%) patients from Panÿevo and 39 (24%) patients from Belgrade. The concentrations of Pb in extracted teeth in all the groups from Panÿevo were statistically significantly (p < 0.05) higher than in all the groups from Belgrade. In the patients from Panÿevo correlations between Pb concentration in extracted teeth and the number of extracted teeth, the number of carious lesions and the number of non-carious le-sions showed a statistical significance (p < 0.001, p < 0.01 and p < 0.001, respectively). Conclusion. According to correlations between lead concentration and the number of extracted teeth, number of carious lesions and non-carious lesions found in the patients living in Panÿevo, one possible cause of tooth loss and hard dental tissue damage could be a long-term environmental exposure to lead. zagaĀenoj olovom.


Introduction
The main causes of environmental pollution are production and use of energy, industrial chemicals and increased agricultural activity.As a result, all biological organisms, including humans, live in a chemically polluted environment.The city of Pan evo is a modern, small-sized city and one of the most powerful industrial centers in Serbia, and this inevitably brings with it the frequent occurrence of air pollution and contamination of water and soil.The existence of these risk factors in the environment has a negative impact on public health.An analysis of morbidity in the adult population of Pan evo showed that this population most often suffers from respiratory diseases.The Institute of Public Health in Pan evo has monitored concentrations of SO 2 , NO 2 , NH 3 and soot in the air in a 10-year period (1991-2001).The results showed that concentrations of soot and NH 3 were high above the legal limit during this period.
Environmental lead (Pb) pollution is a global problem.Pb is one of the most important and widely distributed pollutants in the environment [1][2][3] and a great part of this pollution comes from vehicle exhaust fumes through the combustion of leaded petrol.This, and other human activities such as the extensive use of Pb in industry, has resulted in its redistribution in the environment and, hence, the contamination of air, water and food.Consequently, the levels of Pb content in blood, saliva and other human organs 4 are significantly increased.The levels of Pb in blood and saliva reflect recent exposure.Long-term deposition of Pb is much greater in skeletal tissues than in soft tissues 5 .Heavy metals, ie Pb and cadmium (Cd), have no known physiological functions and are toxic even in low concentrations 6 .
Hard dental tissue lesions include caries and non-carious lesions (abrasion, erosion, attrition).The mechanism of caries development has been the subject of many studies during previous years and consequently general and local predisposing factors are now well-known 16 .In recent years, the subject of many studies has been the determination of the effects of heavy metals on the occurrence and incidence of caries.In addition, it has been speculated for some time that environmental pollution, especially by acid fumes, could also be one of the factors involved in the occurrence and incidence of non-carious lesions.A high incidence of tooth-structure damage significantly affects the functional ability of chewing, mental and work capacity of individuals, causes diseases of the digestive tract and other systems and organs and also represents a serious medical, social and economic problem of the global society.
The principal hypothesis of this study was that pollution of the environment by lead cause dlong-lasting changes in teeth through its deposition in dental hard tissues.Therefore, the aim of this study was to find a correlation between the concentration of lead in teeth extracted from inhabitants of Pan evo and Belgrade belonging to different age groups and occurrence of tooth loss, caries and non-carious lesions in the same groups.

Methods
This study was undertaken on 160 patients of both sexes from Pan evo (the experimental group) and 160 patients of both sexes from Belgrade (the control group).The volunteers were selected from patients who visited the Institute of Stomatology at the Faculty of Stomatology, Pan evo, Serbia.The primary criterion for inclusion and subsequent sample collection was that these patients had been living in either Pan evo or Belgrade for a period of at least 15 years prior to the study beginning.They had to be in good general health with no signs of disease or medication use.The volunteers were then divided into five separate age subgroups for each city, with 32 volunteers in each group.
The study proposal was submitted to the Research Ethics Committee (Approval Protocol No. 1323/1-2008, according to Resolution sections 3, 7, and 8 of the National Commission of Ethics in Research).Patients had to sign an informed consent form prior to the inclusion in the study.Additionally, signed permission for collection of samples of extracted teeth (only if extraction was necessary as a therapeutic procedure) had to be obtained from each participant in the study.

Clinical examination
The patients were clinically examined by the standardized procedure for dental examination, using a dental mirror, a straight or proximal dental probe.Dental caries lesions were diagnosed by standard criteria and marked in universal templates for dental status.The Klein-Palmer system Decayed Missing Filled Teeth (DMFT) was applied in assessing the prevalence of dental caries.Also, for each of the age groups of patients a Significant Caries Index (SiC) was calculated, which represents the mean values of the DMFT index for one third of respondents with the highest DMFT values, using tables recommended by the World Health Organisation (WHO) 17 .Information related to clinically diagnosed loss of enamel and dentin of a noncarious etiology, the so-called non-carious lesions, was recorded for each patients.

Collection of samples -extracted teeth
Following a detailed examination, all teeth to be extracted were carefully evaluated for the presence of fillings or caries.The final decision for tooth extraction was reached following careful consideration of periodontal status and restorative possibilities.In many cases the cause for extraction was either subsequent orthodontic therapy or progressive periodontal disease.The weight of each sample was at least 0.5 g which is a cut-off value for valid chemical analysis.ples to a constant mass was done under laboratory conditions for 48 h at 80 o C. Dried samples were then finely ground to grains under 1 mm in diameter.For further voltammetric analyses all organic substances had to be removed from the sample.This was done by UV digestion (MILESTONE SK-10, Milestone, Sorisole, Italy).Batches of 0.5 g of the samples were diluted for 30 minutes using 7 mL of 65% HNO 3 and 1 mL of 30% H 2 O 2 at 200 o C.After cooling to room temperature, the digested samples were transferred directly to the appropriate vessel for further analysis.The concentrations of Pb in the final digested solution of samples were determined by the PS control system for voltammetry 797 VA Computrace (Metrohm, Herisau, Switzerland).This method was chosen because it can distinguish between different oxidation states of metal ions as well as between free and bound metal ions, which provides important information regarding the bioavailability and toxicity of Pb.Validation of the voltammeter was done using the GLP Wizard of the machine.Chemical analysis was done using a Pb-ion standard.The decisive parameters for the validation of the measuring instrument are the accuracy and the scatter of the result.Both values are calculated automatically by the internal software of the 797 VA Computrace.Electronic validation included: current at -or + 200 mV: measured values -or + 2 μA: tolerances from -1.6 μA to -2.4 μA.Peak voltage: measured values -497 mV; tolerances: from -450 mV to +450 mV.Chemical validation included: measuring: 20 mL H 2 O + 0.5 mL KCl + 100 μL Pb standard.Electrolyte was c (KCl) = 3 mol/L, and standard:

Chemical analysis
(Pb) = 1 g/L.Accuracy was 0.95 -1.05 g/L and scatter was ± 3%.Sensitivity of the method was achieved to the level of Pb of 0.0005-2.5 μg/m 3 .
The detection limit for Pb used in this study, as stated by the manufacturer, was 0.02 μg/g.The detection limit for atomic spectroscopy for the same metal was 0.2 μg/g.
The statistical significance was calculated by the Student's t-test and its modification by Bonferroni 18 .The processor CORR from the SAS package, version 6.4 19 , was used to estimate correlations between trait pairs (the number of extracted teeth and concentration of lead; the number of carious lesions and concentration of lead; the number of non carious lesions and concentration of lead) within locality.Correlations were computed as Pearson product-moment correlations.

Results
All the results were statistically processed and shown in tables.Each investigated parameter is represented by mean value and statistical significance and separately marked for both sexes since no sex-related differences were found.
The mean values of the number of extracted teeth in all the five age groups, from Pan evo and from Belgrade are given in Table 1.It was clear that the number of extracted teeth was statistically significantly (p < 0.001) higher in groups III, IV and V from Pan evo than in groups III, IV and V from Belgrade.The number of extracted teeth in the group I from Pan evo was higher (no statistical significance) than in the group I from Belgrade, whereas the number of extracted teeth was higher (no statistical significance) in the group II from Belgrade than in the group II from Pan evo.
Table 2 shows the mean values of the DMFT index for each of the five groups of patients from the experimental and  In the group I the difference was not statistically significant (p > 0.05), while in the groups II and III the difference between the coefficient of DMFT patients from the experimental group and the control group was statistically significant (p < 0.05).The most striking differences were in the group IV, where the recorded value of the DMFT index in the experimental group was 23.69 vs 18.50 in the control group, which was a highly statistically significant difference (p < 0.01).Also in the group V the recorded value of the DMFT index in the experimental group was 24.94 vs 19.00 in the control group, which was an extremely statistically significant difference (p < 0.001).The SiC index values were also higher in all age subgroups from the experimental group than in the control group (Table 2).Non-carious lesions were diagnosed in 71 patients (44%) from the experimental group and 39 patients (24%) from the control group (Table 3).
The mean concentrations of Pb (presented in μg/g) in extracted teeth in all age subgroups from Pan evo and from Belgrade are given in Table 4.The concentrations of Pb in extracted teeth in all the groups from Pan evo were statistically significantly (p < 0.05) higher than in all the groups from Belgrade.When the measured levels of Pb did not reach the threshold values for the method used, they were marked as 'undetectable' in the corresponding tables.Correlation determination was done three times, separately for the patients from Pan evo and from Belgrade, with two variables.The first correlation was done between the number of extracted teeth from 32 patients in each of the 5 subgroups, and the concentration of Pb in 8 teeth from each of the five subgroups.The second correlation was done between the number of carious lesions from 32 patients in each of the 5 subgroups, and the concentration of Pb in 8 teeth from each of the 5 subgroups.The third correlation was done between the number of non-carious lesions from 32 patients in each of the 5 subgroups, and the concentration of Pb in 8 teeth from each of the 5 subgroups.For the patients from Belgrade all correlations were negative and without statistical significance.However, all the correlations for patients from Pan evo showed a statistical significance (the number of extracted teeth and the concentration of Pb in extracted teeth -p < 0.001, the number of carious lesions and the concentration of Pb in extracted teeth -p < 0.01 and the number of non-carious lesions and the concentration of Pb in extracted teeth -p < 0.001).

Discussion
In this study it was noticed that the patients from Pan evo had fewer teeth than those from Belgrade although the patients from both cities had similar oral hygiene habits and visited dentists at approximately the same intervals.
Therefore, one possible cause of tooth loss in the patients from Pan evo aged over 40 years could be the long-term exposure to a polluted environment.
The value of the DMFT index and the SiC index values, both in Pan evo and Belgrade, must be considered as extremely high, given some of the values that WHO has defined as acceptable 20 .Namely, as expressed by DMFT index, 6 is the acceptable value of oral health for members of the group aged 35-44 years, while in the experimental group this value was as much as 20, and in the control group 17.These results are worse than in many well-developed countries (Turkey 12.62, Austria 14.7, Germany 16.1, UK 16.6, Denmark 16.7), however, they are similar to, for example, Norway (20.5) and Canada (20.0) 21,22 .For people over 65 years of age, WHO considers acceptable DMFT to be 12, while in the experimental group this value was as much as 25 and in the control group it was 19.This low level of oral health in patients aged over 60 years resulted from the high DMFT index values in all age groups and also from a large number of extracted teeth.
An alarming fact is that the most frequent component of the DMFT index in the experimental group was extracted teeth, with the proportion of 44% vs 25% in the control group (which is also a high percentage).A large number of people prefer to have teeth extracted rather than undergo some kind of conservative treatment, partly due to fear and partly because of low income.Besides caries, the reasons for the large number of extracted teeth can also be found in a low level of health education.Abrasions of anterior teeth and premolars and wedgeshaped erosions where the loss of tooth substance extends to both the enamel and dentin, were the most frequently diagnosed non-carious lesions in the patients from the experimental group, while abrasions were the most frequently diagnosed non-carious lesions in the patients from the control group.In our sample of younger patients (up to 50 years of age) presented with less non-carious lesions, therefore, the standard error was higher.The explanation for the representation of abrasions, could be related to early loss of molar teeth and, therefore, over-loading of the remaining teeth during mastication 23 .The most frequently cited etiological factor in forming cervical erosions is chemical etching, more precisely, acid dissolution of hard dental tissues 24 .Studies have confirmed that cervical erosions are more frequent in people who are exposed to acids in the workplace or living environment, in competitive swimmers, in people who frequently consume acidic drinks, or use chemicals for oral hygiene which chelate calcium.Due to frequent vomiting, a significant frequency of dental erosive lesions was noticed in patients with gastrointestinal problems, bulimia and anorexia neurosa, in pregnant women and alcoholics [25][26][27][28] .Many different studies in recent years, have confirmed that ''bending'' or ''flexing'' of teeth caused by eccentric occlusal forces is one of the factors which could explain the occurrence of cervical lesions [29][30][31][32][33][34][35] while numerous epidemiological studies consider inappropriate teeth brushing technique as one of the reasons for cervical erosions etiology 31,36 .One of the possible reasons for higher number of non-carious lesions in the older patients from this investigation could be heavy metals accumulation during the years.
This study showed that the concentration of Pb in extracted teeth in all the groups of patients from Pan evo was higher compared to the patients from Belgrade.Additionally, the concentration of Pb increased rapidly for the older patients from both cities, indicating that the concentration of Pb is age-dependent.Other studies also showed that concentrations of Pb are age-dependent.Baranowska et al. 37 and Nowak and Chmielnicka 38 found a positive correlation between age and Pb in human teeth.
Teeth are not a uniform mass of calcified tissues and it has been well-established that Pb is not homogeneously distributed within a fully developed tooth, with Pb levels in dentine being significantly higher than in enamel 39 .Furthermore, Arora et al. 40 presented the spatial distribution of Pb in the roots of human primary teeth while other authors measured the Pb content in whole teeth 41 .
In this study a consistent relationship was demonstrated between a long-term environmental Pb exposure and its incorporation into hard dental tissues.This is similar to the results of de Almeida et al. 42 , although they measured the content of Pb in the surface enamel of deciduous teeth sampled in vivo from children living in uncontaminated and in lead-contaminated areas of Brazil using different methods.It had been shown previously that heavy metals can be incorporated into dental tissues if there is exposure during the process of dentinogenesis 43 .Therefore, unlike bone, in which the mineral phase is subject to turnover, once formed, teeth provide a permanent, cumulative and relatively stable record of environmental exposure 44 .
It should be noted that in the patients living in Pan evo statistically significant correlations between the concentration of Pb and the number of extracted teeth (p < 0.001), the number of carious lesions (p < 0.01) and the number of noncarious lesions (p < 0.001) were found, whereas the same correlations in the patients from Belgrade were of no statistical significance.This evidence suggests that the possible cause of tooth loss and hard dental tissue lesions (carious and non-carious) in the patients from Pan evo could be a longterm environmental exposure to lead.

Conclusion
Significantly higher values of the DMFT index and higher frequency of non-carious lesions were recorded in the patients from Pan evo.The concentration of lead in extracted teeth increased rapidly in the older patients from both Pan evo and Belgrade, indicating that the deposition of lead is age-dependent.According to the correlations between the concentration of lead in extracted teeth and the number of extracted teeth, the number of carious lesions and noncarious lesions found in the patients living in Pan evo, one possible cause of tooth loss and hard dental tissue damage could be a long-term environmental exposure to lead.That means that polluted environment is one of the factors that cannot be ignored, but also requires confirmation by further comprehensive basic research.
All chemical analyses were done in an independent laboratory, Department of Ecotoxicology at the Institute of Public Health, Pan evo.The collected samples of extracted teeth were stored in ultrafiltered deionized water.Before analysis all soft tissue remnants and surface stains were removed.Drying of sam-Strana 753 Ceni -Miloševi D, et al.Vojnosanit Pregl 2013; 70(8): 751-756.

Table 1 Total number and mean values of extracted teeth from Pan evo and Belgrade
*Statistically significant; SE = standard error.

Table 2 Mean values of the Decayed Missing Filled Teeth (DMFT) index and significant caries (SiC) index in the patients from Pan evo and Belgrade
control groups, as well as the SiC index values.The average value of the DMFT index in the experimental group and in the control group was 20.41 and 16.52, respectively.It is notable that the index value increased with the age of patients and that it was higher in each subgroup of patients from the experimental group, compared to the control group.