Chronic exposure to 35% carbamide peroxide tooth bleaching agent induces histological and hematological alterations, oxidative stress, and inflammation in mice
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Previous studies have demonstrated the side effects of tooth whiteners on the gastric mucosa. However, the impact of dental bleaching products on the liver, kidney, and heart remains obscure. The present study investigated the toxic potential of 35% carbamide peroxide (CPO) containing tooth whitening product (TWP) on the liver, kidney, heart, and stomach of mice, pointing to the role of oxidative stress and inflammation. Mice received 250 or 500 mg/kg body weight CPO-TWP orally for 3 weeks and samples were collected for analyses. Both doses of CPO-TWP induced a significant increase in circulating liver, kidney, and heart function markers. CPO-TWP-administered mice showed several histological alterations and a significant increase in liver, kidney, heart, and stomach lipid peroxidation levels along with diminished glutathione, superoxide dismutase, and catalase. In addition, administration of CPO-TWP provoked anemia, leukocytosis, and a significant increase in circulating levels of pro-inflammatory cytokines. In conclusion, exposure to 35% CPO-TWP induced functional, histological, and hematological alterations, oxidative stress, and inflammation in mice. Therefore, the frequent use of tooth bleaching agents should be monitored very carefully to avoid the application of excess amounts as well as the intake.
KeywordsCarbamide peroxide Oxidative stress Inflammation Toxicity Tooth bleaching
Dental bleaching is widely used for tooth whitening or removal of tooth discoloration. In-office or at-home tooth whitening represents effective and conservative bleaching approaches performed by dentists (Majeed et al. 2015). Several tooth whitening products (TWP) with different concentrations of hydrogen peroxide (H2O2) or carbamide peroxide (CPO) are available in the market (Matis et al. 2013). CPO is used in a tray-based technique for teeth bleaching and dissociates into H2O2 and urea (da Costa et al. 2012; da Silva Marques et al. 2012). In order to reduce the time of tooth exposure, high concentrations of the bleaching agent are used during the bleaching process (Rezende et al. 2016). Despite the high success rate of bleaching agents in removing tooth discoloration, the wrong use or application of excessive amounts might cause undesirable side effects (Cherry et al. 1993; Dahl and Becher 1995; Paula et al. 2015). The degree of side effects depends on the used bleaching technique, frequency, concentration of H2O2 or CPO and the regularity of controls made by the dental professionals (Matis et al. 2006). Tooth sensitivity is the common adverse effect of bleaching agents containing a high concentration of H2O2 (Matis et al. 2009). When compared with CPO, H2O2 has a greater risk and can cause gingival necrosis or chemical burns (Al Shethri et al. 2003; Pugh Jr. et al. 2005).
The in vitro and in vivo toxicities of tooth bleaching agents have been reported in different studies. In vitro exposure of the odontoblast-like MDPC-23 cells to successive treatments of 10% CPO gel promoted severe toxic effects (Lima et al. 2013). Ten percent CPO applied to dentin and enamel-dentin discs induced morphologic alterations and significantly reduced the rate of cell metabolism of odontoblast-like cells (Lima et al. 2010; Soares et al. 2011). The exact mechanism underlying the toxicity of tooth bleaching products is not understood; however, several studies have pointed to the role of reactive oxygen species (ROS) and oxidative stress. H2O2 released from the bleaching agents can diffuse through the dental tissue to the pulp space and exert toxic effects (Gokay et al. 2004; Gokay et al. 2000). In addition, the daily application of 10% CPO induced mild inflammation in the dental pulp of premolars (Fugaro et al. 2004). In vivo, ulceration of the gastric mucosa of rats has been demonstrated as a hazardous effect of acute exposure to CPO and tooth whiteners containing CPO (Dahl and Becher 1995). The administration of a tooth whitener containing 6% H2O2 induced histological alterations in the gastric mucosa of rats (Paula et al. 2015). Cherry et al. have demonstrated loss of righting reflex, labored breathing, bloody urine, and other manifestations in female rats exposed to acute ingestion of tooth whiteners (Cherry et al. 1993). In a clinical trial involved a small group of 15 users, the continuous use of TWP for 6 months to remove tetracycline stains did not induce any adverse effects on the oral cavity (Leonard Jr. et al. 2003).
To date, the in vivo impact of TWP on different organs other than the gastric mucosa has not been investigated. Since TWP may be swallowed during the bleaching procedure, studies investigating whether ingestion of these tooth whiteners resulted in toxic effects are needed. Therefore, this study was designed to evaluate the effect of chronic administration of 35% CPO containing TWP on the liver, kidney, heart and stomach of mice, pointing to the role of oxidative stress and inflammation.
Materials and methods
Experimental animals and treatments
Eight-week male Swiss albino mice weighing 25–27 g, obtained from the animal house of the College of Pharmacy, King Saud University (Riyadh, Saudi Arabia), were used in this study. The mice were housed in standard cages at normal temperature (23 ± 2 °C) and 12 h light/dark cycle and supplemented with a standard diet and water ad libitum. All experiments and protocols were approved by the Ethics Committee for Animal Experimentation of King Saud University.
Group I (control): mice received distilled water orally for 3 weeks.
Group II (250 mg/kg CPO-TWP): mice received 250 mg/kg body weight 35% carbamide peroxide containing tooth whitening product (CPO-TWP; Opalescence®) dissolved in distilled water orally for 3 weeks.
Group III (500 mg/kg CPO-TWP): mice received 500 mg/kg body weight 35% CPO-TWP dissolved in distilled water orally for 3 weeks.
The dose of CPO-TWP was adjusted according to any change in the body weight. The two doses were selected based on previous studies where a dose of 500 mg/kg body weight CPO-TWP has been used (Dahl and Becher 1995). We applied the 500 mg/kg as a high dose and the 250 mg/kg CPO-TWP as a lower dose. The solution of CPO-TWP was freshly prepared before administration and was protected from light to prevent the decomposition of peroxides.
At the end of the experiment, mice were sacrificed under anesthesia and samples were collected immediately. Blood samples were collected on EDTA for hematological assays and other samples were left to coagulate for serum separation. The mice were dissected; and the liver, kidney, heart, and stomach were excised and washed in cold phosphate buffered saline (PBS). Samples from the collected organs were homogenized in cold phosphate buffered saline (PBS; 10% w/v) centrifuged and the clear homogenate was collected for biochemical assays. Other samples were fixed in 10% neutral buffered formalin for histological processing.
Determination of liver, kidney, and heart function markers
Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine, urea, and creatine kinase-MB were determined in serum samples using reagent kits purchased from Spinreact (Spain), according to the manufacturer’s instructions.
Determination of tumor necrosis factor alpha and interleukin-1beta
TNF-α and IL-1β were determined in serum of control and experimental mice using specific ELISA kits (R&D Systems, USA), following the manufacturer’s instructions.
Determination of lipid peroxidation and antioxidants
The collected liver, kidney, heart, and stomach samples were washed in cold PBS, weighed, and homogenized in cold PBS (10% w/v). The homogenate was centrifuged and the clear supernatant was used to assay malondialdehyde (MDA; (Ohkawa et al. 1979)), reduced glutathione (GSH; (Beutler et al. 1963)), superoxide dismutase (SOD; (Marklund and Marklund 1974)) and catalase (CAT; (Aebi 1984)).
Determination of hematological parameters
Erythrocytes (RBCs) count, hemoglobin (HB) content, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and leukocytes (WBCs) count were determined in blood samples collected in EDTA tubes using the Genius Kt-6400 hematology analyzer (Diamond Diagnostics Inc., USA).
The liver, kidney, heart, and stomach samples were fixed in 10% neutral buffered formalin for 24 h, dehydrated, cleared, and embedded in paraffin wax. Four micrometer sections were cut using RM2245 semi-motorized rotary microtome (Leica Biosystems, Germany). The sections were processed for hematoxylin and eosin (H&E) staining and then examined using a Olympus BX51 microscope.
The data were analyzed using Graphpad Prism version 5 (San Diego, CA, USA) and expressed as mean ± standard error of the mean (SEM). The means were compared using the one-way ANOVA followed by Tukey’s test post hoc analysis. A P value < 0.05 was considered significant.
CPO-TWP induces liver, kidney, and heart dysfunction in mice
Effect of 35% CPO-TWP on liver, heart, and kidney function in mice
25.61 ± 2.98
69.15 ± 3.41**
76.77 ± 6.36***
40.79 ± 3.83
79.58 ± 4.32**
93.82 ± 5.09***
58.83 ± 4.97
95.34 ± 6.62**
103.75 ± 7.57**
0.49 ± 0.03
0.96 ± 0.06**
1.14 ± 0.07***
17.48 ± 2.07
40.43 ± 4.14**
44.48 ± 3.37**
65.83 ± 3.65
116.94 ± 6.49**
132.60 ± 7.36***
Serum creatinine and urea levels were also increased significantly in mice received 250 mg/kg (P < 0.01; P < 0.01) and 500 mg/kg (P < 0.001; P < 0.01) 35% CPO-TWP as represented in Table 1. The heart function marker CK-MB was significantly increased in the serum of mice exposed to either 250 mg/kg (P < 0.01) or 500 mg/kg (P < 0.001) 35% CPO-TWP when compared with the control group (Table 1).
CPO-TWP causes histological alterations in the liver, kidney, heart, and stomach of mice
CPO-TWP provokes oxidative stress in the liver, heart, kidney, and stomach of mice
CPO-TWP induces inflammation in mice
CPO-TWP induces hematological alterations in mice
Teeth-bleaching techniques might have different side effects, depending on the frequency and the technique used (Matis et al. 2006). Accordingly, Paula et al. have demonstrated the effect of a tooth-whitening product containing 6% H2O2 on the histology of gastric mucosa in rats (Paula et al. 2015). The use of 6% H2O2-containing product was associated with several histological lesions, including foci of necrosis and loss of the surface layer of the mucosal epithelium (Paula et al. 2015). In addition, Dahl and Becher reported that acute exposure to CPO induced dose-dependent ulcerations of the gastric mucosa (Dahl and Becher 1995). However, studies on the side effects of CPO-containing TWP in the liver, kidney, and heart are scarce. Herein, we investigated the impact of Opalescence 35% CPO on the liver, kidney, heart, and stomach of mice. We assumed that the toxic effects of CPO on different organs might be induced via induction of oxidative stress and inflammation.
Oral administration of 35% CPO-TWP induced hepatotoxicity evidenced by the significant increase in circulating levels of ALT, AST, and ALP. These enzymes are located inside hepatocytes and released into the circulation following hepatocyte damage. Thus, the circulating levels of these enzymes are used as sensitive markers of hepatocyte injury. Given the central role of liver in the clearing of drugs and toxic substances, hepatotoxic events are the most common adverse drug reactions (Reuben et al. 2010). The hepatotoxic effect of CPO-TWP in mice has been further confirmed by the histological findings. Microscopic examination revealed histological alterations in the liver of mice, including congested blood vessels, vacuolations and inflammatory cell infiltration. In addition to hepatocyte damage, mice received 35% CPO-TWP exhibited increased serum levels of creatinine, urea, and CK-MB, demonstrating kidney as well as heart injury. In support of these results, the kidney of 35% CPO-induced mice showed glomerular degeneration and inflammatory cell infiltration, and several histological derangements of the cardiomyocytes, congestions, and inflammatory cell infiltration have been observed in the examined heart sections. The acute administration of 500 mg/kg CPO-TWP has been previously shown to exert no effect on the liver histology of rats, while the kidney appeared more hyperemic (Dahl and Becher 1995). However, the chronic administration of 250 and 500 mg/kg 35% CPO-TWP in the present study induced histological alterations in both the kidney and liver of mice. Therefore, both the biochemical and histological findings pointed to the role of CPO in inducing hepato-, nephro-, and cardiotoxicity. Furthermore, the stomach of mice received 35% CPO-TWP showed congested blood vessels and thick mucus secretions. The impact of CPO-TWP on the gastric mucosa has been previously reported by Dahl and Becher (Dahl and Becher 1995) who demonstrated dose-dependent ulcerations following acute exposure.
Next, we determined the levels of lipid peroxidation in the liver, kidney, heart, and stomach of CPO-induced mice. Excess production of ROS has been implicated in different disease conditions, including liver (Mahmoud et al. 2017b, 2017c), kidney (Abd El-Twab et al. 2016; Al-Rasheed et al. 2018), and heart injury (Al-Rasheed et al. 2017; Hozayen et al. 2019). ROS can induce damage to the cellular macromolecules, including DNA, proteins, and lipids, resulting in cell death. Peroxidation of membrane lipids is one of the deleterious effects of ROS. Given the presence of carbon-carbon double bonds in the polyunsaturated fatty acids of membrane lipids, they are vulnerable to damage by ROS (Ghosh et al. 2015). Lipid peroxidation can lead to disruption of the cell membranes and inactivation of membrane-bound proteins (Birben et al. 2012). In our study, oral administration of 35% CPO-TWP induced a significant increase in MDA, a lipid peroxidation product, in liver, kidney, and heart of mice. Therefore, it is worthy assumed that oxidative stress plays a key role, at least in part, in CPO-TWP toxicity in mice. Oxidative stress is a condition resulting from the imbalance between ROS levels and cellular antioxidant defense system (Liu et al. 2014). The antioxidant defenses, both enzymatic and non-enzymatic, confer protection against the deleterious effect of ROS and other oxidants. ROS can promote carbonylation of proteins and loss of their functions (Suzuki et al. 2010). This oxidative protein damage can decrease the cellular antioxidant capacity via altering their function and structure (Pavlović et al. 2016). Therefore, reduced antioxidant defenses in the cells are a marker of oxidative stress. Here, CPO-induced mice showed significantly diminished GSH content, and activity of the antioxidant enzymes SOD and CAT. In addition, we demonstrated increased lipid peroxidation and decreased antioxidants in the stomach of CPO-induced mice. Although CPO-TWP has been reported to induce gastric ulcerations in rats (Dahl and Becher 1995), the mechanism underlying its effect on the gastric mucosa remains obscure. By showing the potential of CPO-TWP to induce oxidative stress, our findings provided an explanation of its deleterious effects on the gastric mucosa. The pro-oxidant potential of CPO-TWP could be explained in terms of the H2O2 produced during its dissociation. The resultant H2O2 represents around one-third of the concentration of CPO, and urea breaks down into ammonia and water (da Costa et al. 2012; da Silva Marques et al. 2012).
CPO-induced oxidative stress in the present investigation was associated with increased circulating levels of the pro-inflammatory cytokines, TNF-α and IL-1β. Increased levels of these inflammatory mediators point to the role of inflammation in CPO-TWP toxicity in mice. In a recent study, a 38% H2O2 containing teeth bleaching product provoked moderate pulp inflammation after 24 h of its application in rats, while severe inflammation and necrosis were observed after 10 days (Silva-Costa et al. 2018). Inflammation has been evidenced by the increased immunohistochemical staining of TNF-β and IL-1β (Silva-Costa et al. 2018). The exact mechanism underlying the pro-inflammatory potential of TWP is unknown. However, the pro-oxidant effect of tooth whiteners might be involved in the induced inflammation. Excessive ROS can activate the redox-sensitive nuclear factor-kappaB (NF-κB), a transcription factor that controls the expression and production of multiple inflammatory mediators, including TNF-α and IL-1β (Mahmoud and Al Dera 2015). Increased pro-inflammatory cytokines in conjunction with oxidative stress has been reported in different animals models of hepatic (Alqahtani and Mahmoud 2016; Mahmoud 2014; Mahmoud et al. 2017a), renal (Abd El-Twab et al. 2016), and cardiac (Al-Rasheed et al. 2016; Al-Rasheed et al. 2017; Hozayen et al. 2019) damage as we previously reported.
In addition to oxidative stress and inflammation, CPO-TWP administration was associated with hematological alterations. Assessment of hematological parameters has been used as an earlier index of drug toxicity (Alya et al. 2015; Germoush et al. 2018). Mice received CPO-TWP for 3 weeks showed a significant decrease in the number of erythrocytes, HB content and Hct. On the other hand, MCV and MCH were non-significantly affected, demonstrating that the decreased RBCs count is a result of lysis. Oxidative stress might be linked to the decreased RBCs count in CPO-induced mice. Under oxidative stress conditions, ROS can induce lipid peroxidation and decrease the deformability of RBCs membrane, resulting in cell lysis (Kolanjiappan et al. 2002). In contrast to RBCs, CPO-induced mice exhibited a significant and dose-dependent leukocytosis which was positively correlated with the increased levels of serum pro-inflammatory cytokines.
In conclusion, this study confers new information on the toxicity of 35% CPO-containing tooth whiteners in experimental mice. The deleterious effect of tooth whiteners on the gastric mucosa has been previously reported; however, our results showed that CPO-TWP can induce liver, kidney and heart injury. CPO-TWP provoked functional and histological alterations, lipid peroxidation, and decreased antioxidant defenses in different organs of mice. Furthermore, CPO-TWP induced inflammation and hematological alterations. Thus, the use of CPO containing tooth whiteners during bleaching therapy should be monitored very carefully by the dentist to avoid the application of excess amounts as well as the intake. However, it should be noted that the doses used in this study are higher than the daily exposure to TWP in humans. Therefore, further studies are needed to evaluate the impact of lower doses.
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group No (RG-1439-60).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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