The Trouble with Tylenol

Cameron Baldes

Paracetamol

             As I was perusing through Facebook, a friend posted an interesting comment that his college professor mentioned that the mechanism of action of Tylenol was not completely understood. At first I did not believe it, Tylenol is one of the most common pain relief medications in the United States. However, the official Tylenol website confirms that the major component of Tylenol is acetaminophen, also known as Paracetamol, and that there is no confirmation of its actual mechanism. However, the website gave clues into what the theories behind the mechanism are. This sounded interesting and so I went looking for the source papers behind those theories. As it turns out, common pain relievers like Tylenol have two types of mechanism: analgesic and antipyretic. The analgesic mechanism is the action that causes pain relief. The antipyretic mechanism is the fever reducing mechanism. A quick search through Pubmed revealed the study that gave insight into the analgesic mechanism of Tylenol, entitled: TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Δ⁹-tetrahydrocannabiorcol.

            The paper describes acetaminophen, (acetyl-p-aminophenol; APAP), as having an unknown mechanism of action but that it somehow involves the spinal cord. The paper in question looks at a particular sensor TRPA1 as TRPA1 is a unique sensor of noxious stimuli and, hence, a potential drug target for analgesics. TRPA1 can cause spinal analgesia and due to APAP’s connection to the spinal cord, the researchers in question thought the two could be connected. Therefore, they examined the connection by looking at wild type and TRPA1 deficient mice and their pain resistance to a hot plate. They measured pain by the latency a mouse had from withdrawing its hand from the hot plate.  They did baseline measures and then all of the mice were injected with APAP to see if the latency would change. The latency dramatically increased in wild type mice while the TRPA1 deficient mice remain unaffected.

NAPQI, N-acetyl-p-benzoquinoneimine,

             

The researchers then moved on to test whether APAP itself was directly interacting with TRPA1 or whether the body was breaking down the compound into a metabolite that was interacting with TRPA1. TRPA1 is a sensory receptor for oxidants and thiol-reactive electrophilic compounds^, and APAP is metabolized in vivo to the highly reactive electrophilic compound NAPQI, N-acetyl-p-benzoquinoneimine, in humans and mice following therapeutic, non-toxic doses of APAP. Thus the researchers sought to test if that was the particular metabolite that was interacting with TRPA1. The researchers looked at cell cultures to see if wild type cells exposed to NAPQI would cause a Calcium increase thus signifying interaction through neuronal pathways. The exposure of the cells to NAPQI caused a statistically significant change signifying that NAPQI is a selective TRPA1 activator at concentrations in the micromolar range (figure A).

p-benzoquinone

            In addition to the oxidative formation of NAPQI, APAP is also converted to p-BQ, p-benzoquinone, in vivo. p-BQ is another highly reactive electrophilic compound that is considerably more stable than NAPQI.  They repeated the experiment and again Calcium levels did increase when the cells were exposed to p-BQ (figure D). They then used p-BQ to expose directly to dorsal root ganglia neurons exposed to a contrast microscope. It is clear that in the cells that have TRPA1 a calcium channel opening does occur by the appearance of green specs against the blue background (figure B). The TRPA negative cells do not show any openings giving a conclusive finding. Thus these findings support a molecular mechanism for APAP to affect pain reception.

            Now that was interesting but that paper also mentions that Acetaminophen causes three times as many cases of liver failure as all other drugs combined, and is the most common cause of acute liver failure in the United States. Why is that?

            Well sources from the previous paper led me to an ancient article published in 1973 entitled, Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. This paper summarizes the physiology behind acetaminophen metabolism. Following a therapeutic dose, it is mostly converted to nontoxic metabolites via Phase II metabolism by conjugation with sulfate and glucuronide, with a small portion being oxidized via the cytochrome P450 enzyme system. Phase II reactions are usually known as conjugation reactions, like methylation, are usually detoxifying in nature, and involve the interactions of the polar functional groups of phase I metabolites. Phase I reactions may occur by oxidation, reduction, hydrolysis, cyclization, and decyclization addition of oxygen or removal of hydrogen, carried out by mixed function oxidases. But I digress; the cytochrome P450 (CYP) converts 5% of acetaminophen to a metabolite, N-acetyl-p-benzoquinoneimine (NAPQI). We saw this metabolite in the previous paper so we understand that it plays a role in the pain relief mechanism of acetaminophen. This particular paper looked at in vivo reactions for how NAPQI is disposed of in the body and in the process determines how the toxicity of acetaminophen occurs. They looked at the liver cells of mice and when NAPQI is directly injected into the cells the levels of glutathione decreased sharply. This suggested an interaction and was later proven correct. Under normal conditions, NAPQI is detoxified by conjugation with glutathione to form cysteine and mercapturic acid conjugates. These conjugates then can harmless leave the body. However, previous studies have shown that acetaminophen overdose leads to increased utilization of p450 conversion. This creates more NAPQI in the body.  The paper investigated cells that were overexposed to NAPQI and saw a net loss in glutathione as the body was not able to replenish it at the rate the molecule was interacting. Thus small amounts of NAPQI stayed in their reactive forms.

Paracetamol Metabolism

            The paper, Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes, published in 1983 finally showed that glutathione was a cofactor for glutathione peroxidase that reduced hydrogen peroxidase in vivo (figure A). However, upon overdose with acetaminophen, glutathione levels drop very low and hydrogen peroxidase is allowed to interaction with membrane proteins. The paper showed a time course study looking at the formation of Malondialdehyde (MDA). The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism. The time course showed that when acetaminophen overdose occurred, glutathione levels decreased sharply and MDA levels spiked (figure B). Lactate dehydrogenase (LDH) was another marker used as cells that leak LDH are undergoing lipid peroxidation. The time course also showed a spike in LDH levels (figure C). This correctly showed that without glutathione as a protector, hydrogen peroxidase is allowed to generate radicals within the cell’s membrane proteins. This was shown to lead to hepatic necrosis. 

Time Course of Lipid Peroxidation Markers

Activated Carbon for Medical Use

            I thought that was the end of the rabbit hole so to speak but again my curiosity was piqued when the paper mentioned charcoal as a short term antidote to acetaminophen overdose. I totally failed to see how a material I use to grill steaks can save my liver and potentially my life. So I went digging. Well the Wikipedia page on acetaminophen refers to charcoal and that leads to a page called activated carbon and by their definition activated carbon is also called activated charcoal, and is a form of carbon that has been processed to make it extremely porous and thus to have a very large surface area available for adsorption or chemical reactions.

            However, the first mention of activated charcoal being used for treatment against acetaminophen overdose in primary literature occurs in a paper published in 1973 entitled: Reduction of Absorption of Paracetamol by Activated Charcoal and Cholestyramine: A Possible Therapeutic Measure. The experiment described is taking hepatic cells and injecting them with acetaminophen. Then the cells are subjected to activated charcoal and a time course is taken throughout the experiment. The levels of acetaminophen in the cells decreased after exposure to activated charcoal. This paper was followed by an in depth paper published in 1976 entitled: Effect of activated charcoal on acetaminophen absorption. This details how activated charcoal is an emergency decontaminant used to trap and absorb poisonous substances in the gastrointestinal tract. The tiny holes that open up in the charcoal after it is treated with oxygen increases its surface area. This allows liquids or gas to pass through the charcoal. The porous carbon attracts other carbon-based impurities as the liquid or gas passes through, and binds to them non-covalently. This allows the carbon to absorb the impurities so that it is not passed on to the body where it would be absorbed instead. Therefore, activated charcoal is a common panacea and is not specific to acetaminophen.

            Charcoal only works before acetaminophen is absorbed by the body. Studies have produced an antidote that does react with excess NAPQI to render it harmless. A study entitled: Reversal of experimental paracetamol toxicosis with N-acetylcysteine, showed a time course study where hepatic cells were treated with overdose levels of acetaminophen and then N-acetylcysteine was administered after a delayed time. The amount of acetaminophen did drop even after the delay in treatment.. Acetylcysteine is the N-acetyl derivative of the amino acid L-cysteine, and is a precursor in the formation of glutathione in the body. Acetylcysteine acts to augment the glutathione reserves in the body and then directly bind to toxic metabolites. Glutathione reacts with the toxic NAPQI metabolite so that it does not damage cells and can be safely excreted.

            Further research is being done to precisely determine the analgesic mechanism of acetaminophen. This post is only a short summary on the toxicology of acetaminophen and its related metabolites, but does give insight into Tylenol’s overall chemical effects within the human body.

 Sources:

            Andersson, D. A. (2011). TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Δ9-tetrahydrocannabiorcol. Nature Communications.

 

Elliot Piperno, DanielA Berssenbruegge, REVERSAL OF EXPERIMENTAL PARACETAMOL TOXICOSIS WITH N-ACETYLCYSTEINE, The Lancet, Volume 308, Issue 7988, 2 October 1976, Pages 738-739, ISSN 0140-6736, 10.1016/S0140-6736(76)90030-1.

(http://www.sciencedirect.com/science/article/pii/S0140673676900301)

Emanuele Albano, Giuseppe Poli, Elena Chiarpotto, Fiorella Biasi, Mario Umberto Dianzani, 

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes, Chemico-Biological Interactions, 

Volume 47, Issue 3, December 1983, Pages 249-263, ISSN 0009-2797, 10.1016/0009-2797(83)90161-8.

(http://www.sciencedirect.com/science/article/pii/0009279783901618)

Dordoni B, Willson RA, Thompson RP, Williams R. Reduction of absorption of paracetamol by activated charcoal and cholestyramine: A possible therapeutic measure. Br Med J. 1973;3:86–7.

            Tylenol Professional. (2012). Retrieved march 12, 2012, from Tylenol: http://www.tylenolprofessional.com/pharmacology.html#MechanismofAction