“You’re never fully dressed without a smile”. Who said that anyway? I didn’t say it, because I feel self-conscious about my smile sometimes. Or rather, I feel self conscious about the colour of my teeth. There are times when I’m unhappy with their colour…but what are my options? I could go buy some whitening strips, but sometimes my teeth hurt afterwards. Why is this happening? Should I be concerned? As a Biochemistry student I should be able to figure out what is causing my teeth to be sensitive.
Dental bleaching has been around since the 1860’s, and over the years many different bleaching agents have been used. The two main agents used in dental bleaching products are hydrogen peroxide (H2O2) and carbamide peroxide. These are both highly oxidative compounds that interact with chromogens on tooth surfaces. Coloured compounds, like chromogens, typically consist of conjugated chains altering in single and double bonds, often including heteroatoms, carbonyl groups, and phenyl rings. Figure one gives an example of a chromogen found in many of the foods we eat. Either one of these peroxides is able to break one or more of the double bonds in the conjugated chain via cleavage or oxidation. These peroxide compounds produce a number of highly reactive oxygen species (ROS) depending on the temperature, pH, and light when the reaction is taking place on teeth.
This raises a red flag for me. Having reactive oxygen species floating around in my mouth doesn’t sound like my cup of tea. Those molecules must be what are causing my teeth to be sensitive. I wonder what the biochemical mechanisms are that occur in my dental tissues to induce this pain?
Brand new research published by Wu et al. (2013) from the Department of General Dentistry at the Shanghai Ninth People’s Hospital has provided some insight into a new idea for how teeth are becoming sensitive. They are proposing that H2O2 is able to induce apoptosis in human dental pulp cells by a specific apoptotic pathway. Pulp cells are found in the dental pulp layer of the tooth, and are often referred to as the nerve itself since it is connected to the nerves and blood vessels. Pulp cells are also involved in regenerating dentin. The researchers are aware that H2O2 is able to penetrate into the top two layers of the teeth (enamel and dentin) and reach the pulp chamber. This would be what is leading to sensitivity because the nerves are exposed to ROS.
Wait, I’ve heard of apoptosis before in some of my other classes. That’s cell suicide, also known as programmed cell death. It’s a highly regulated process that occurs during development, or when the cell is under stress. The plasma membrane is maintained during the process and membrane blebbing occurs, forming small vesicles containing the cytosolic contents. Another type of cell death is necrosis, but it is premature cell death. The cytosolic contents of the cell will be released after the plasma membrane loses its integrity, and this will activate the inflammatory response.
Apoptosis can occur via two pathways: the extrinsic path and the intrinsic path. Wu et al. are referring to the intrinsic pathway in their research. A simplified overview of the intrinsic apoptotic pathway is shown in Figure 2. 
This apoptotic pathway involves the outer mitochondrial membrane first becoming permeable after the cell has received a signal to begin apoptosis (like from a ROS). Pro-apoptotic Bcl-2 proteins are causing the membrane permeability. Many proteins from the mitochondria will now be able to go into the cytoplasm. One of the proteins is cytochrome C, which is going to form a complex with an apoptotic protease-activating factor (Apaf-1). This is depicted in Figure 3A. Apaf-1 will undergo a conformational change, and interact with pro-caspase-9, forming an apoptosome, which can be seen in Figure 3B. Caspases are proteases containing a cysteine in the active site and cleave at aspartate residues. Initiator caspases, like caspase-9, will undergo auto-cleavage to activate themselves. Then, they can activate other effector caspases via proteolytic cleavage, so these caspases can continue the apoptotic signal cascade. Caspase-3 is the effector caspase in this process.
Wu et al. thought H2O2 could be triggering this intrinsic apoptotic pathway, and they attempted to build some evidence for their hypothesis. They first cultured human dental pulp cells and treated them with various concentrations of the bleaching agent for four hours. Once the cells were ready, they tested cell viability and saw that H2O2 treated cells were unable to thrive in the environment compared to the untreated group. They concluded this bleaching agent to be cytotoxic to the pulp cells.
What else did they find? After a Flow Cytometry test where they stained cells with Annexin V and propidium iodide (PI) their evidence grew. Annexin V only stain cells undergoing apoptosis. This is because it binds to phosphatidyserine, a lipid that is translocated from the inner side of the plasma membrane to the surface when apoptosis is occurring. PI stain can’t penetrate membranes, so it stains cells that are undergoing necrosis or that are in the late apoptotic phase. The researchers found higher percentages of apoptotic cells in those that were treated with H2O2. The highest concentration they tested was 100µmol/L, and there was a large percentage of these cells undergoing necrosis.
So this means higher concentrations of the ROS will induce our inflammatory response. This list of reasons why I don’t want to use H2O2 on my teeth is starting to grow. But wait, there’s more. Once Wu et al. knew apoptosis was occurring, they wanted to ensure it was by the intrinsic pathway they proposed. They did a Western Blot analysis to look at protein amounts, more specifically the amounts of activated caspase-9 and caspase-3. Both of these proteins were found to have increased expression in cells treated with H2O2 compared to the control group. These proteases are specific to the intrinsic pathway, and therefore they concluded apoptosis is indeed occurring in these human dental pulp cells when they are exposed to H2O2.
My cells are committing suicide when I try to make them whiter. Those poor pulp cells are dying and my dentin isn’t being regenerated. That’s probably why when I drink something cold or hot I feel sensitivity; my soft tissue is becoming more and more exposed. No wonder my teeth hurt after I wear those white strips. At least now I have an understanding of what is happening at a scientific level.
The discovery that the intrinsic apoptotic pathway is being induced is opening the door for a potential new target for improving the effects of bleaching agents on tooth sensitivity. Maybe one day I’ll be able to be “fully dressed” with a smile that doesn’t hurt so much.
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The Tooth Hurts 