Cadmium is one of the main chemical pollutants found in the daily environment of developed countries. Cigarettes are a significant source of that metal, which makes it important in terms of oral cavity health. The aim of this study was to determine if increased supply of zinc in chronic exposure to cadmium might protect the sublingual gland structure against oxidative damage. The experiment took 12 months and was conducted on 72 adult male rats. They were randomized into 9 groups. Eight groups received cadmium in drinking water (as CdCl2) at 5 or 50 mg Cd/dm3 and/or zinc (as ZnCl2) at 30 or 60 mg Zn/dm3. The control group received regular water. In the sublingual gland of all animal groups, levels of oxidative parameters were measured. The oxidative stress index was calculated as a TOS/TAS ratio. Cadmium exposure at 5 mg and 50 mg Cd/dm3 induced oxidative stress in the sublingual glands of the rats. Cadmium reduced the TAS and GSH levels and increased LPO, H2O2, TOS, and OSI. In cadmium exposure conditions, increasing the supply of zinc by 79% or 151%, as compared to the standard dietary intake of this microelement, completely prevented the reduction of TAS and GSH levels and accumulation of LPO, H2O2, and TOS in the examined gland at both exposure levels to that metal. The outcome data confirm the protective effect of increased zinc intake on the sublingual gland tissue in chronic cadmium exposure.
Cadmium is one of the main chemical pollutants found in the daily environment of developed countries [
Cadmium is accumulated in various tissues and organs and may have serious consequences for the general population health. Chronic exposure to cadmium may damage the kidneys, bones, liver, lungs, and other organs, including sublingual glands [
The available literature of the subject indicates that many toxic effects of cadmium can be prevented or at least reduced by increasing the supply of zinc [
Thus far, the impact of zinc on the sublingual gland of an organism exposed to cadmium has not been studied. In consideration thereof, this study involved experimental analysis to determine if the increased supply of zinc in chronic exposure to cadmium might protect the sublingual gland structure against oxidative damage. For this purpose, in the sublingual gland of rats which received cadmium and/or zinc and of control animals, the oxidative stress markers were assayed.
The experiment took 12 months and was conducted on 72 adult male Wistar rats with an initial body weight of 220 g. Throughout the experiment period, the animals were kept in standard conditions (air temperature 18–21°C, relative humidity 50 ± 10%, and 12-hour circadian rhythm) and were provided with unlimited access to balanced granulated LSM fodder (Motycz near Lublin) and drinking water.
The research protocol was approved by the Local Ethics Committee for Animal Experiments in Białystok (Poland) and performed in accordance with the ethical principles and institutional guidelines and International Guide for the Use of Animals in Biomedical Research.
The rats were randomized into 9 groups, and in each group was 8 animals. In research, 2 groups received Zn alone, 2 groups were treated with Cd alone, and 4 groups received Zn supplementation during exposure to Cd. Zn and Cd were administered in drinking water at the concentrations of 30 or 60 mg Zn/L (as ZnCl2; Merck) and 5 or 50 mg Cd/L (as CdCl2·2 1/2H2O; POCH; Gliwice, Poland) alone (30 mg Zn/L, 60 mg Zn/L, 5 mg Cd/L, and 50 mg Cd/L groups) and in combination (5 mg Cd/L + 30 mg Zn/L, 5 mg Cd/L + 60 mg Zn/L, 50 mg Cd/L + 30 mg Zn/L, and 50 mg Cd/L + 60 mg Zn/L groups) for up to 12 months. The control group received drinking water without cadmium or zinc. During the experiment, the daily intake of fluids and body weight gain were controlled. Both the fluid intake and body weight gains were similar across all rat groups. Rat exposure to cadmium at 5 mg/dm3 is an equivalent of environmental exposure of humans to that metal, particularly smokers; at 50 mg of Cd/dm3, it is equivalent to occupational exposure and exposure arising from high pollution and heavy smoking. Administering zinc at 30 mg or 60 mg/dm3 to the animals increased the daily intake of this bioelement by 79% and 151%, respectively, in comparison with standard dietary intake. This dose was chosen based on findings of other authors and observations of the Department of Toxicology of the Medical University of Białystok [
After ending the exposure, the animals were put under barbiturate anesthesia (Vetbutal 30 mg/kg of body weight i.p.) and various types of biological material were collected for analysis, including sublingual glands which were flushed in PBS, drained on blotting paper, and secured for further analysis by deep freezing at −80°C. After thawing, the dissected glands were weighted and 20% homogenates were prepared using a glass tissue homogenizer (Schuett Homogen, Göttingen, Germany) in a cold 50 mM potassium phosphate buffer with pH = 7.4. In order to prevent automatic oxidation of the analyzed material, 0.5 M BHT acetonitrile was added to the samples (10
The total antioxidative capacity (TAS) and the total oxidative status (TOS) of the homogenates were determined using ImAnOx (TAS) ELISA kit and PerOx (TOS) ELISA kit by Immundiagnostik AG (Germany). The TAS values assayed in the control samples provided with the kit were 191.88 ± 10.7 and 264.33 ± 15.6
The glutathione (GSH) levels were assayed using the Glutathione Assay Kit, Cayman Chemical (USA). The precision of the method expressed as CV was <1.5%.
The lipid peroxidation (LPO) levels (Bioxytech® LPO-586™) and hydrogen peroxide (H2O2) (Bioxytech® H2O2-560™) were assayed using kits supplied by OxisResearch (USA). The precision of the method expressed as CV was <4.5%.
All assays performed using commercial kits were performed as per the manufacturers’ instructions, and the measured parameters were adjusted for protein concentration.
The obtained results were analyzed statistically using Statistica 10 software (StatSoft; Tulsa, USA). In order to assess the statistical significance of differences between the study groups, a one-way analysis of variance (ANOVA) was performed using Duncan’s post hoc test. The independent and interactive impact of cadmium and zinc on the stress index levels was assessed using a two-way analysis of variance (ANOVA/MANOVA). Also, Spearman’s rank correlation test was performed for the assessed parameters in the tissue of the studied gland. The differences between groups and correlations between variables were considered statistically significant at
The GSH levels in the sublingual gland of the rats are provided in Table
The impact of zinc on GSH, LPO, and H2O2 levels in the sublingual gland of rats exposed to cadmium.
Nonenzymatic antioxidant |
Oxidative stress index | ||
---|---|---|---|
LPO |
H2O2 | ||
Control | 1.881 ± 0.800 | 0.122 ± 0.011 | 21.320 ± 2.343 |
30 mg Zn/dm3 | 1.926 ± 0.181 | 0.095 ± 0.008 | 19.110 ± 0.617 |
60 mg Zn/dm3 | 1.838 ± 0.570 | 0.093 ± 0.015 | 15.740 ± 1.336a |
5 mg Cd/dm3 | 1.433 ± 0.086a‡b‡c |
0.263 ± 0.034a†b‡c† | 26.930 ± 1.000a |
5 mg Cd/dm3 + 30 mg Zn/dm3 | 1.933 ± 0.136d† | 0.113 ± 0.020d† | 17.480 ± 0.720d‡ |
5 mg Cd/dm3 + 60 mg Zn/dm3 | 2.334 ± 0.144a†b |
0.104 ± 0.012d† | 18.020 ± 1.248d‡ |
50 mg Cd/dm3 | 1.340 ± 0.046a†b‡c†e‡f‡ | 0.376 ± 0.066a‡b‡c‡d |
41.800 ± 2.877a‡b‡d‡f‡ |
50 mg Cd/dm3 + 30 mg Zn/dm3 | 1.715 ± 0.067f‡g |
0.233 ± 0.045a |
21.860 ± 1.480d |
50 mg Cd/dm3 + 60 mg Zn/dm3 | 1.739 ± 0.052f‡g |
0.167 ± 0.023d |
17.540 ± 1.963d‡g‡ |
The values are arithmetic means ± SEM.
Exposure of rats to 5 mg or 50 mg Cd/dm3 resulted in the increase of LPO levels 2.2-folds (
Supplementation of zinc at 30 mg/dm3 had no impact on the H2O2 levels in the analyzed gland, but a higher concentration of that bioelement would reduce it by 26%. Exposure of rats to 5 mg or 50 mg Cd/dm3 resulted in the increase of H2O2 levels by 26% (
The TOS levels in the sublingual gland of rats are provided in Table
The impact of zinc on TOS and TAS levels, and the TOS/TAS ratio in the sublingual gland of rats exposed to cadmium.
TOS |
TAS |
TOS/TAS | |
---|---|---|---|
Control | 21.846 ± 3.751 | 3.466 ± 0.468 | 7.709 ± 1.971 |
30 mg Zn/dm3 | 16.393 ± 1.157 | 7.843 ± 0.590a‡ | 2.181 ± 0.236a† |
60 mg Zn/dm3 | 17.951 ± 0.985 | 6.138 ± 0.839a‡b |
3.272 ± 0.435a |
5 mg Cd/dm3 | 36.284 ± 3.438a‡b‡c‡ | 1.770 ± 0.080a |
20.565 ± 1.796a‡b‡c‡ |
5 mg Cd/dm3 + 30 mg Zn/dm3 | 19.700 ± 1.557d‡ | 7.879 ± 0.379a‡c |
2.515 ± 0.519a†d‡ |
5 mg Cd/dm3 + 60 mg Zn/dm3 | 20.974 ± 1.626d‡ | 12.009 ± 0.559a‡b‡c‡d‡e‡ | 1.777 ± 0.157a†d‡ |
50 mg Cd/dm3 | 44.153 ± 3.943a‡b‡c‡d |
1.930 ± 0.213a |
24.208 ± 2.543a‡b‡c‡d |
50 mg Cd/dm3 + 30 mg Zn/dm3 | 21.205 ± 2.054d‡g‡ | 5.382 ± 0.627a |
4.093 ± 0.411d‡g‡ |
50 mg Cd/dm3 + 60 mg Zn/dm3 | 26.684 ± 2.313b |
5.104 ± 0.454a |
5.433 ± 0.533d‡g‡ |
The values are arithmetic means ± SEM.
The TAS levels in the sublingual gland of rats which, throughout the experiment period, received only zinc at 30 mg or 60 mg/dm3 were 2.3- and 1.8-folds higher (
In the animals which, throughout the experiment, received only zinc at 30 mg and 60 mg/dm3, the TOS/TAS ratio was 3.5-folds (
Independent and interactive impact of cadmium and zinc on the levels of selected oxidative stress indices in the rat sublingual gland.
ANOVA/MANOVA | GSH | LPO | H2O2 | TOS | TAS | TOS/TAS |
---|---|---|---|---|---|---|
Independent impact of Cd | 6.389 |
24.84‡ | 20.38‡ | 35.05‡ | 0.635 | 62.04‡ |
Independent impact of Zn | 9.526† | 14.09‡ | 37.82‡ | 33.31‡ | 53.18‡ | 149.5‡ |
Interaction effect of Cd and Zn | 9.461† | 7.091† | 13.67‡ | 11.56† | 3.049 | 50.87‡ |
The values reflect the
The independent and interactive impact of cadmium and zinc on the levels of selected stress indices was assessed using a two-way analysis of variance (ANOVA/MANOVA).
Analysis of Spearman’s rank correlation between the assessed parameters in the sublingual gland tissue.
TAS | TOS | TOS/TAS | GSH | H2O2 | |
---|---|---|---|---|---|
TAS | — | ||||
TOS | −0.485‡ | — | |||
TOS/TAS | −0.915‡ | 0.756‡ | — | ||
GSH | 0.580‡ | −0.431‡ | −0.588‡ | — | |
H2O2 | −0.523‡ | 0.388‡ | 0.510‡ | −0379† | — |
LPO | −0.583‡ | 0.416‡ | 0.570‡ | −0.503‡ | 0.501‡ |
The values reflect the rank correlation coefficient
Cadmium is a toxic metal commonly found in the daily environment. Due to the increasing number of reports of the harmful impact of low exposure to those toxic elements published worldwide, the researchers are focused on finding methods to reduce dietary cadmium intake and mitigate its impact on the organism [
Similar to other heavy metals, cadmium has the ability to accumulate in living organisms. The largest accumulation of cadmium occurs in organs rich in metallothionein (MT), that is, the liver and kidneys [
The oral cavity, serving as the initial section of the gastrointestinal tract, is an integral element of the organism. Its liquid environment is the saliva which hosts a number of biochemical reactions necessary to maintain the oral cavity healthy. Exposure to cadmium also affects its condition. Consequences of chronic exposure to that toxic element were also observed both in hard tissue, such as teeth [
Fischer et al. [
Kakei et al. [
Smoking is a major source of exposure to cadmium, especially for people living in areas with low environmental levels of that metal, who have no occupational contact with cadmium [
Thus far, little attention has been paid to the destructive effect of cadmium on salivary glands. The available study outcomes indicate that exposure to that metal may also cause structural and functional changes in those glands, impairing their function and development [
Cadmium toxicity derives from its prooxidative properties. Although cadmium is unable to directly generate RFT through Fenton or Haber-Weiss reaction, it does induce stress indirectly through depleting glutathione and other antioxidant in cells, including vitamins, antioxidant enzymes, and bioelement, among those zinc [
In this study, the long-term exposure to cadmium at both levels induced oxidative stress in the sublingual gland. In the analyzed gland, there was a reduction of the level of nonenzymatic antioxidant (GSH) and of the total antioxidant status (TAS) and an increase of total oxidant status (TOS) and stress indices (LPO, H2O2, and TOS/TAS). The TAS level was positively correlated to the GSH level (
The impact of cadmium on the induction of oxidative stress in the saliva originating from the submandibular gland of rats was studied by Abdollahi et al. [
LPO is a lipid peroxidation marker [
An important indicator of oxidative cellular damage is also the hydrogen peroxide, which is a natural product of cellular metabolism [
Aside from LPO and H2O2, another indicator of increased oxidative stress in the cells is TOS and the mathematically calculated TOS/TAS ratio [
The available study outcomes suggest that antioxidants such as vitamins and polyphenols may protect the organism exposed to cadmium against oxidative stress. Zinc has also been found to have antioxidant properties and has been confirmed to provide efficient protection against many toxic effects of cadmium on the organism, including the damage of the kidneys, liver, and bones [
Thus far, the protective role of this bioelement against the consequences of oxidative damage in the sublingual gland of a rat exposed to cadmium has not been studied. This study is a pioneer venture aimed at answering the question whether the increase of zinc supply by 79% and 151%, as compared to standard dietary intake, has protective effect against accumulation of H2O2 and LPO and low-molecular-weight thiol that is GSH in the analyzed salivary gland.
In the previous own research, we have shown that administering 30 mg Zn/dm3 to animals increases the GPx activity, while increasing the supply of that bioelement by 151% (60 mg Zn/dm3) increases the activity of both CAT and GPx and reduces the H2O2 levels, thus confirming zinc’s antioxidant effect [
This study has shown that administering zinc to animals in both doses during exposure to 5 mg or 50 mg Cd/dm3 completely inhibited the cadmium-induced increase of LPO and H2O2 levels and the reduction of GSH levels in the sublingual gland tissue, allowing us to conclude that zinc supplementation mitigates the oxidative stress in that gland. Furthermore, two-way analysis of variance (ANOVA/MANOVA) has shown that the beneficial effect of zinc supplementation on GSH, LPO, and H2O2 levels arises from zinc’s independent activity and its interaction with cadmium, whereby the independent impact of that bioelement is stronger than the interactive activity between zinc and cadmium.
The role of zinc for oral cavity health has not been extensively studied yet [
Zinc is also used as an antibacterial agent in toothpastes and mouthwashes to control the formation of dental plaque, reduce tartar, and eliminate halitosis [
Uckardes et al. [
Hegde et al. [
Saliva is a biological fluid playing an important role for the health of the oral cavity and can be used not only in diagnostics but also in monitoring the progress of various diseases, including cancer and periodontitis [
Wei et al. [
In other studies, Kurku et al. [
The key outcome of our own research is the confirmation that administering zinc to animals at both concentration levels during exposure to cadmium significantly increases the TAS and reduces the TOS, as well as the TOS/TAS stress index in the sublingual glands of rats, which suggests a reduction of oxidative stress in that gland. Two-way analysis of variance (ANOVA/MANOVA) has shown that the beneficial effect of zinc supplementation on TAS and TOS levels and TOS/TAS ratio arises from zinc’s independent activity and its interaction with cadmium, whereby the independent impact of that bioelement is stronger than the interactive activity between zinc and cadmium.
In light of the available literature data on the protective effect of zinc, it should be noted that this study is the first attempt to analyze the impact of that bioelement on the sublingual gland tissue during exposure to cadmium. Summing up the outcomes of our own research, it should be concluded that a confirmed increase, in the applied experimental model for both the moderate environmental exposure of humans to cadmium and the occupational exposure, of the zinc supply by 79% and 151% reduces the oxidative stress in sublingual glands of rats and clearly shows a protective effect of that bioelement on the tissue of the analyzed gland.
The outcome of this study contributes new, important data confirming the protective effect of increased zinc intake on the sublingual gland tissue in chronic exposure to cadmium.
The data of the materials and methods and conclusions to support the findings of this study are included within the article. If any other data may be needed, please contact the corresponding author upon request.
There is no conflict of interest.