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Dog tooth decay stages


Dog tooth decay stages, including early decay (ED), early moderate decay (EMD), late moderate decay (LMD), and advanced decay (AD), were classified according to the American Dental Association (ADA) criteria.^[@R5]^ The stage of tooth decay and the occurrence of secondary caries were recorded in the first molar and premolar regions, respectively. Tooth surfaces affected by dental caries were further subdivided into smooth or irregular surfaces. Caries in the smooth surfaces was defined as 'cavity/tooth defect', whereas that in the irregular surface was defined as 'dental defect'.^[@R12],[@R13]^ Dental plaque samples were obtained from tooth surfaces using a sterile disposable periodontal probe. The plaque samples were collected from smooth surfaces of the first molar (F~1~) and premolar (F~2~) regions. Each sample was taken using a 10-mm-long periodontal probe at the following three points: mid-buccal (D~1~), mid-lingual (D~2~), and distal of mesial caries (D~3~). The same examiner collected all the plaque samples.

The participants were invited to collect the saliva sample from the anterior floor of the mouth immediately after brushing.^[@R2]^ Salivary samples were taken using a saliva-collecting device (Salivette^®^, Sarstedt AG &, Co., Nümbrecht, Germany) provided with a disposable plastic syringe. All saliva samples were transferred into clean microcentrifuge tubes and stored at −20 °C until analysis.

Measurements of chemical elements in plaque {#s2-3}

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Before saliva collection, plaque samples were taken from the tooth surfaces using a sterile disposable periodontal probe. The samples were transferred into small screw-cap vials and stored at −20 °C until the analysis. The samples were dried at 60 °C for 24 hours and pulverized using an Retsch Mill^®^ MM400 (Retsch, Inc., Haan, Germany).

The total amount of each element in the plaque was determined by measuring the concentrations of the respective element in the dry samples (mg kg^−1^). For this, samples of the prepared pulverized dental plaque were analyzed using ICP-OES (ICP Spectrometer-80, Varian, Palo Alto, CA) coupled with an autosampler (CETAC, Omaha, NE) by the Laboratory of Advanced Microchemistry of the University of Rzeszow, Poland. Measurements were carried out in the range from 0.02 to 5.00 mg l^−1^. The following analytical parameters were used: instrument temperature---1,520 °C, gas flow---1.0 l min^−1^, air flow---1.5 l min^−1^, nebulizer---45.0 nmol min^−1^, lens aperture---1.0, plasma power---1.6 kW. During measurement, the intensity of the argon plasma was recorded at a wavelength of 247.04 nm, and the peak areas of the elements were recorded. The concentrations were calculated by means of calibration curves of pure standards prepared using a solution containing different amounts of a particular element. A certified standard for all measurements was also used.

Statistical analysis {#s2-4}

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Statistical analysis was performed using the STATISTICA 10.0 package. One-way analysis of variance (ANOVA) was applied, followed by Tukey's HSD post hoc test. Differences in values of chemical elements and biochemical parameters were considered to be statistically significant at *P*<,0.05.

Results {#s3}

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In total, nine elements were analyzed, namely: As, Cd, Cr, Cu, Fe, Mg, Mn, Pb and Zn. The content of selected elements was also measured in three parts of the liver, which consisted of a central, and left and right lobes. Concentration of each of the elements in each part of the liver was expressed as mg/kg of wet mass of the analysed tissue. Moreover, the level of selected biochemical markers, namely: glucose, lactate and cholesterol in the serum, and activity of alanine aminotransferase and aspartate aminotransferase (ALT and AST) in the liver tissue homogenate, were also determined.

Metals {#s3-1}

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Our study revealed significantly different concentrations of the selected elements in analysed tissues. The highest concentrations of zinc were observed in the central and left lobes, whereas the lowest were found in the right lobes ([Table 1](#T1){ref-type="table"}). Cd was the only element, where significant differences were found between different parts of the analysed tissue (*P*<,0.05). Moreover, the highest concentration of cadmium was determined in the right lobes of the liver. No significant differences were observed between the analysed parts of the liver. The highest concentration of lead was determined in the central lobe, while the lowest was observed in the left lobe ([Table 1](#T1){ref-type="table"}). The highest concentration of arsenic was detected in the right lobe of the liver, while the lowest was found in the central part. No significant differences were observed between different parts of the liver. However, the highest and the lowest concentration of copper was found in the central and left lobes of the liver, respectively ([Table 1](#T1){ref-type="table"}). The highest concentration of lead and the lowest of cadmium were found in the central lobe of the spleen ([Table 1](#T1){ref-type="table"}). Only cadmium was significantly different between different parts of the spleen. No significant differences were found between the analysed parts of the spleen. No significant differences were found between different parts of the liver in the content of all analysed metals, however, the lowest was found in the central and the highest in the left and right lobes of the liver, respectively.

###### Comparison of the mean metal concentrations in the central, left, right lobes and spleen of male C57BL/6 mice of control group (C) and experimental group (E), in pg/g tissue.

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**Elements** **Central lobe** **Left lobe** **Right lobe** **Spleen**

***Mean ± SE*** ***p-value*** ***Mean ± SE*** ***p-value*** ***Mean ± SE*** ***p-value***

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