We have known for decades that acetaminophen damages the liver at the higher doses many people take - even at the maximum suggested daily dosage when taken over time as many people do who suffer from arthritis. This stuff is also in most cold and flu medications, as well as in many prescription pain killers.
Now we find that even at dosages lower than those required to cause liver damage, acetaminophen kills brain neurons. Another reason to pull this drug from the market.
Acetaminophen Induces Apoptosis in Rat Cortical Neurons
Inmaculada Posadas, Pablo Santos, Almudena Blanco, Maríangeles Muñoz-Fernández, Valentín CeñaAbstract
Background
Acetaminophen (AAP) is widely prescribed for treatment of mild pain and fever in western countries. It is generally considered a safe drug and the most frequently reported adverse effect associated with acetaminophen is hepatotoxicity, which generally occurs after acute overdose. During AAP overdose, encephalopathy might develop and contribute to morbidity and mortality. Our hypothesis is that AAP causes direct neuronal toxicity contributing to the general AAP toxicity syndrome.
Methodology/Principal Findings
We report that AAP causes direct toxicity on rat cortical neurons both in vitro and in vivo as measured by LDH release. We have found that AAP causes concentration-dependent neuronal death in vitro at concentrations (1 and 2 mM) that are reached in human plasma during AAP overdose, and that are also reached in the cerebrospinal fluid of rats for 3 hours following i.p injection of AAP doses (250 and 500 mg/Kg) that are below those required to induce acute hepatic failure in rats. AAP also increases both neuronal cytochrome P450 isoform CYP2E1 enzymatic activity and protein levels as determined by Western blot, leading to neuronal death through mitochondrial–mediated mechanisms that involve cytochrome c release and caspase 3 activation. In addition, in vivo experiments show that i.p. AAP (250 and 500 mg/Kg) injection induces neuronal death in the rat cortex as measured by TUNEL, validating the in vitro data.
Conclusions/Significance
The data presented here establish, for the first time, a direct neurotoxic action by AAP both in vivo and in vitro in rats at doses below those required to produce hepatotoxicity and suggest that this neurotoxicity might be involved in the general toxic syndrome observed during patient APP overdose and, possibly, also when AAP doses in the upper dosing schedule are used, especially if other risk factors (moderate drinking, fasting, nutritional impairment) are present.
Citation: Posadas I, Santos P, Blanco A, Muñoz-Fernández M, Ceña V (2010) Acetaminophen Induces Apoptosis in Rat Cortical Neurons. PLoS ONE 5(12): e15360. doi:10.1371/journal.pone.0015360Copyright: © 2010 Posadas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Competing interests: The authors have declared that no competing interests exist.
* E-mail: valentin.cena@uclm.es
Introduction
Acetaminophen (paracetamol; AAP) is considered a non-steroidal anti-inflammatory (NSAID) drug, even though in clinical practice and in animal models it shows little anti-inflammatory activity [1]. However, like NSAIDs, AAP is used to treat pain and fever and it has become one of the most popular ‘over-the-counter’ non-narcotic analgesic agents. For instance, this compound has been taken, at least once, by more than 85% of children under the age of 91 months in the UK [2]. In the US, about 79% of the general population regularly takes AAP [3], including more than 35% of pregnant women [2]. The most frequently reported adverse effect associated with AAP is hepatotoxicity, which occurs after acute over dosage (usually doses greater than 10 g are needed) [4] and, less frequently, during long term treatment with doses at the higher levels of the therapeutic range [5] or in the presence of precipitating factors like fasting, nutritional impairment or alcohol intake [4]. Besides hepatic toxicity, no AAP toxic actions have been described in the nervous system, although it is well known that AAP crosses the blood-brain barrier both in rodents and humans [6]. Acetaminophen is mainly metabolised in the liver via conjugation with glucuronic acid and sulphate and then excreted, but, a small fraction is metabolised by cytochrome P-450 [7], [8] forming a chemically reactive metabolite, n-acetyl-p-benzoquinone imine (NAPQI), which reacts with GSH to form a non-toxic conjugate that will be excreted. Once GSH is exhausted, NAPQI binds to cellular proteins, including mitochondrial proteins, leading to hepatocellular death [9]. It has also been described that CYP2E1 is also expressed in the brain [10], suggesting that AAP might be metabolised by neurons producing the toxic metabolite NAPQI, which would lead to neurotoxicity. Although there is a previous report indicating that AAP potentiates staurosporine-mediated toxicity in neuroblastoma [11], information on direct AAP neurotoxicity has not been described.
Mitochondria play a key role in regulating the apoptotic mechanisms and also in some forms of cell death by necrosis [12], [13]. Calcium overload or free radical production induce the mitochondrial inner membrane permeabilization (MIMP) that promotes mitochondrial swelling, outer membrane rupture and release of intermembrane proapoptotic proteins such as cytochrome C (cyt C) and apoptosis inducing factor (AIF) to the cytoplasm [14]. These factors also activate caspases and, subsequently, caspase-activated DNase [15].
In this study, we have studied the effect of AAP on rat cortical neurons in culture and report, for the first time, that this widely used drug has a low but persistent toxicity on neurons through a mitochondrial-dependent mechanism involving cyt C release and caspase 3 activation. In addition, in vivo experiments in rats show that CSF levels achieved following i.p. AAP injection are similar to those drug concentrations that cause neuronal death in vitro and also produce a time-dependent neuronal death in vivo as measured by an increase in the number of TUNEL positive cells in the cortex. These data suggest that neuronal toxicity might be produced by APP, in addition to the well-known hepatic toxicity, and that it might contribute to AAP overdose toxicity.
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