Dissertations / Theses on the topic 'Acetaminophen toxicity'

Background To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice. Methods Using plasma from APAP poisoned mice, either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed, we screened commercially available murine microRNA libraries (SABiosciences, Qiagen Sciences, MD) to evaluate for unique miRNA profiles between these two dosing parameters. Results We distinguished numerous, unique plasma miRNAs both up- and down-regulated in lethally compared to sublethally dosed mice. Of note, many of the greatest up- and down-regulated miRNAs, included, but were not limited to, 574-5p, 466g, 466f-3p, 375, 29c, and 148a. There was a statistically significant increase in alanine aminotransferase levels in the lethal compared to sublethal APAP dosing groups at the 12 h time point ( P < 0.001). There was 90% mortality in the lethally compared to sublethally dosed mice at the 48 h time point ( P = 0.011). Conclusion We identified unique plasma miRNAs both up- and down-regulated in lethally dosed APAP poisoned mice.

Acetaminophen and N,N-dimethylformamide (DMF) are compounds which are extremely toxic to the liver. Acetaminophen is a drug which is well known for its analgesic and antipyretic properties. However, the abuse potential of this agent as a non-narcotic analgesic in alcoholics is well known. It is also the leading cause of overdose in England. DMF toxicity results mainly from occupational exposure. At present there are no known reports of an antidote for DMF poisoning, while N-acetylcysteine, the antidote for acetaminophen poisoning, is known to produce adverse effects. The present study evaluates the potential of melatonin as an antidote for acetaminophen and DMF poisoning. This study also investigates the mechanism underlying acetaminophen addiction and abuse. Initial studies involved in vitro techniques in an attempt to remove the complexities of organ interactions. The photodegradation studies, using ultraviolet (UV) light, revealed that melatonin accelerates the rate of acetaminophen degradation in the presence of air, and reduces the rate of degradation in the presence of nitrogen. This study also revealed that melatonin is rapidly degraded in the presence of air, following UV irradiation. The effect of DMF on hydroxyl radical generation was also determined. DMF was shown to act as a free radical scavenger, rather that a generator of free radicals. The in vitro studies were followed by lipid peroxidation determination. DMF (0.4ml/kg and 0.8ml/kg) did not produce any significant increases in lipid peroxidation in the liver. Three different doses of acetaminophen (30mg/kg, 100mg/kg, and 500mg/kg) were administered to rats for seven days. Acetaminophen (500mg/kg) was shown to significantly increase (p<0.05) lipid peroxidation in the liver. Melatonin (2.5mg/kg) was not able to significantly reduce the damage. The lower doses of acetaminophen (30mg/kg and 100mg/kg) did not increase lipid peroxidation. Electron microscopy studies showed that DMF adversely affects the liver, and in particular, the endoplasmic reticulum. Co administration of melatonin (2.5mg/kg) was able to reduce the damage. Further experiments need to be performed before an accurate assessment can be made on the ability of melatonin as an antidote for DMF and acetaminophen poisoning. Several experiments were done in an attempt to uncover the biochemical mechanism underlying acetaminophen addiction and abuse. The first experiment targeted the liver enzyme tryptophan-2,3-dioxygenase (TDO). This enzyme is the major determinant of tryptophan levels in vivo. Acetaminophen administration (100mg/kg for three hours) was shown to significantly inhibit (p<0.05) the activity of TDO, indicating increased peripheral levels of tryptophan. This experiment was followed up with determination of brain serotonin and pineal melatonin. Brain serotonin was determined using the ELISA technique. Melatonin was estimated using this technique as well as with pineal organ culture. Acetaminophen administration (100mg/kg for three hours) significantly increased (p<0.05) brain serotonin levels. Using organ culture where exogenous (3H) tryptophan is metabolised to (3H) melatonin, acetaminophen (100mg/kg for three hours) was shown to significantly increase (p<0.05) pineal melatonin concentrations. However, the ELISA technique did not reveal any changes in endogenous pineal melatonin levels. The final experiment was the determination of urinary 5-hydroxyindole acetic acid (5- HIAA), the major metabolite of serotonin, following acetaminophen administration (100mg/kg for three hours). Acetaminophen was shown to significantly reduce 5-HIAA levels (p<0.05) suggesting reduced catabolism of serotonin. The findings of this study indicate that acetaminophen mimics the actions of an antidepressant. This compelling finding has important clinical implications, and needs to be examined further.

Thesis (M.S.)--University of Toledo, 2010.
Typescript. "Submitted to the Graduate Faculty as a partial fulfillment of the requirement for the Master of Science in Pharmacology and Toxicology." "A thesis entitled"--at head of title. Title from title page of PDF document. Bibliography: p. 68-81.

Acetaminophen, known as paracetamol in the UK and Tylenol in the United States, is a widespread and commonly used painkiller all over the world. Taken in large enough doses, however, it can cause fatal liver damage. In the U.S., 56000 people are admitted to hospital each year due to acetaminophen overdose and its related effects, at great cost to healthcare services. In this thesis we present a number of different models of acetaminophen metabolism and toxicity. Previously, models of acetaminophen toxicity have been complex and due to this complexity, do not lend themselves well to more advanced mathematical analysis such as the perturbation analysis presented later in this thesis. We begin with a simple model of acetaminophen metabolism, studying a single liver cell and performing numerical and sensitivity analysis to further understand the most important mechanisms and pathways of the model. Through this we identify key parameters that affect the total toxicity in our model. We then proceed to perform singular perturbation analysis, studying the behaviour of the model over different timescales, finding a number of key timescales for the depletion and subsequent recovery of various cofactors as well as critical dose above which we see toxicity occurring. Later in the thesis, this model is used to model metabolism in a spheroid cell culture, examining the difference spatial effects have on metabolism across a 3D cell culture. We then present a more complex model, examining the difference the addition of an adaptive response to acetaminophen overdose from the Nrf2 signalling pathway, has on our results. We aim to reproduce an unexplained result in the experimental data of our colleagues, and so analyse the steady states of our model when subjected to an infused dose, rather than a bolus one. We identify another critical dose which leads to GSH depletion in the infused dose case and find that Nrf2 adaptation decreases toxicity and model sensitivity. This model is then used as part of a whole-body PBPK model, exploring the effects that the distribution of the drug across the bloodstream and different organs has. We explore the affects of that a delay in up-regulation from the Nrf2 pathway has on the model, and find that with rescaled parameters we can qualitatively reproduce the results of our collaborators. Finally, we present the results of in vitro work that we have undertaken, the aim of which was to find new parameters for the model in human hepatocytes, rather than from rodent models, and find a new value for a parameter in our model from human cell lines.

Increasingly, drug-induced liver injury is one of the main reason for drugs to be withdrawn from the market even after passing toxicity studies in pre-clinical and clinical trials because of risks of toxicity and ineffective treatments. Human immortalised hepatocyte cell lines used in drug testing are widely available, inexpensive and easy to culture. However, these cell lines are commonly known to have poor predictive capabilities and improved in vitro hepatic models are required for predicting hepatotoxicity of large numbers of compounds in drug discovery. In this study, the primary goal was to develop an improved in vitro human hepatic model using a combination of the C3A human hepatic cell line and human umbilical vein endothelial cells (HUVECs), for prediction of acetaminophen (APAP) hepatotoxicity. Initial experiments showed that co-culture of HUVEC:C3A in EGM-2, an endothelial medium, was essential to support both cell types, and that co-cultures maintained the initial cell seeding ratio of 1:1 (HUVEC:C3A) after 3 days. Phenotyping of co-cultured cells using platelet endothelial cell adhesion molecule (PECAM-1/CD31) for HUVECs, and hepatic epithelial (EpCAM) markers for C3As demonstrated that at ratio 1:1 (HUVEC:C3A), there is cross-talk between HUVECs and C3As and cells in co-culture showed properties of self-organisation. This interaction resulted in improved hepatic metabolic activity in vitro in respect of albumin synthesis and cytochrome P450 activity. Treatment with low (5 mM), intermediate (10 mM) and high doses (20 mM) of APAP, showed that prediction of hepatotoxicity using specific kits for cell viability and mitochondria function, was significantly improved in C3As in the presence of HUVECs, thus demonstrating an in vitro human hepatic co-culture could be an invaluable model for drug toxicity studies. We observed that the intermediate APAP dose had no effect on cell viability and mitochondrial function in co-cultures, whilst by comparison both lactate levels and oxidative stress were perturbed in mono-cultures. Co-cultures also up-regulated expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in HUVECs following APAP exposure, which may be important in modulating the toxic effect of APAP on C3As. To further improve the in vitro liver-like model, Matrigel™ was incorporated to promote vascular formation by HUVECs and support hepatic organization, migration and function of C3As. In HUVEC mono-cultures, Matrigel™-promoted vascularization, haptotaxis and self-organization and in HUVEC:C3A co-cultures formation of structures reminiscent of liver sinusoids and maintenance of hepatic albumin synthesis and CYP3A4 activity. Time-lapse imaging showed haptotactic migration of hepatocytes towards endothelial cells, with Matrigel™ likely having a chemotactic effect on HUVECs and C3As, resulting in interconnected vascular network. APAP inhibited angiogenesis in HUVEC mono-cultures whereas APAP had no effect in HUVEC:C3A co-cultures. In conclusion, the development of an in vitro human organotypic co-culture model of HUVECs and C3As significantly enhanced hepatic function, demonstrated by significant improvement in hepatic metabolism, evidence of greater resistance to APAP toxicity, and improved cell-cell communication. Co-cultures markedly modulated APAP hepatotoxicity compared with C3A mono-cultures. Furthermore, co-culture of HUVECs and C3As using a complex basement membrane biomatrix (Matrigel™) produced a self-assembling interconnected vascular network, improved hepatocyte function as well as reproducibility of responses to APAP toxicity. The application of the described co-culture models may improve the accuracy, efficacy and predictive power of drug toxicity testing strategies in drug development.

Drug toxicity may cause liver injury, resulting in damage to cells and tissues. This damage can lead to cytotoxic events that may result in an activation of poly (ADP-ribose) polymerase (PARP). A study was conducted to determine if cocaine and acetaminophen toxicity lead to DNA damage and to the activation of the repair protein, PARP in the liver using the hepatotoxicants: cocaine and acetaminophen (APAP). A dose-response analysis for cocaine concluded that a dose as low as 20 mg/kg resulted in elevated ALT levels. A higher dose of 60 mg/kg was tested for analyses but resulted in severe hemorrhaging. The dose-response analyses for APAP resulted in no elevated liver enzyme levels for a 75 mg/kg and 150 mg/kg dose. A dose of 50 mg/kg for cocaine, and a dose of 300 mg/kg for APAP were used to analyze temporal trends for both toxicants. Both cocaine and APAP produced incremental increases in ALT at the 2 hour, 6 hour, 18 hour, and 24 hour time points, respectively. PARP activity analysis for cocaine measured the highest activity at the 2hr and 6hr time points. PARP analysis for acetaminophen measured gradual increases until the 18 hour time point where the highest level of PARP activity was measured. A PARP inhibition analysis was conducted with cocaine and (APAP) to understand the impact of a PARP inhibitor, 1,5-dihydroxyisoquinoline (DIQ), on PARP activity in the liver. A 50 mg/kg dose of cocaine or a 300 mg/kg dose of APAP was administered, followed by a 10 mg/kg dose of DIQ at 1) the time of initial toxicant dose (0 hour), or 2) 1 hour after initial toxicant dose (1hr). The PARP inhibition analysis for cocaine and APAP was conducted at 6 and 18 hours post initial dose, respectively, when the highest levels of PARP were observed. Inhibition analyses determined that ALT declined significantly when DIQ was administered immediately following the initial toxicant dose for both toxicants. DIQ administered 1 hr after initial toxicant dose resulted in slightly higher ALT than the 0 hr time point. Decreases in PARP activity were observed at the 0 hr time point, with slightly higher PARP levels observed at the 1 hr time point. Decreased PARP activity was observed following DIQ treatment with both, a concurrent drug treatment and treatment following drug administration. Cocaine and APAP treatment did not cause DNA fragmentation. A liver glutathione (GSH) analysis conducted for cocaine and APAP did not correlate with DIQ alteration of PARP activity. The mechanism of DIQ effects on drug-induced hepatotoxicity appears to be GSH independent. DIQ was effective in reducing drug-induced hepatotoxicity and preserving organ function.

Thesis (M.S.)--Central Connecticut State University, 1999.
Thesis advisor: Carol A. Jones. " ... in partial fulfillment of the requirements for the degree of Master of Science in Chemistry." Includes bibliographical references (leaves 42-51).

The liver is responsible for lipid and glucose metabolism, protein and bile synthesis and the biotransformation of xenobiotics. These functions, performed by hepatocytes, are dependent on heterotypic interactions with other liver cell types and the stratified microarchitecture of the organ. In vitro liver models provide insights into the role of each cell type and perturbations upon external stimuli. Despite the dissimilarities to in vivo and rapid dedifferentiation, most liver studies utilize hepatocyte monocultures. These models lack heterotypic interactions causing inaccurate assessments of toxicity and disease. Only a limited number of 3D hepatic models incorporate the major liver cell types, and these cultures primarily focus on the hepatocyte response. We have developed 3D liver models that include all major hepatic cell types and recapitulate the layered architecture of the organ. These models maintain hepatic functions for up to four weeks and can be used to isolate the role and response of each cell type. We used these models to study two critical aspects of the organ -- acute hepatotoxicity and liver fibrosis. There are tens of thousands of chemicals with undetermined effects on the human body. High concentrations of xenobiotics can cause acute liver damage and failure. Liver impairment can result in multiple organ failure, hepatic encephalopathy and death. Therefore, it becomes critically important to investigate hepatotoxicity in a time, cost and resource effective manner. Our 3D liver models were validated for hepatotoxicity testing with acetaminophen, a prototypic drug. We then adapted and optimized the models for high-throughput hepatotoxicity testing with automated procedures and primary human hepatic cells. Liver fibrosis and cirrhosis are well-established consequences of chronic chemical exposure, infection and alcoholism. The initiating factors, end stages and resolution of fibrosis have been extensively studied. However, there is minimal information on the role of the local microenvironment in the progression of the disease from diseased to healthy tissue. We designed 3D liver cultures with a mechanical gradient to gradually model this transition through spatial and temporal perspectives. These findings demonstrate the versatility and accuracy of these 3D hepatic models in the investigation of liver toxicity and fibrosis.
Ph. D.

Class of 2014 Abstract
Specific Aims: To determine the cost of treatment of oral and intravenous n-acetylcysteine (IV NAC) in acute acetaminophen (APAP) toxicity using the length of treatment and length of hospital stay. Methods: A retrospective chart review of Arizona Poison and Drug Information Center electronic records from 2009-2012 and January-June 2013 were evaluated. The following information was collected: age, sex, use oral or intravenous NAC, length of treatment, length of hospital stay (intensive care unit (ICU) and non-ICU) and use of antiemetic. The mean length of stay (MLOS) was calculated for each group as well as the cost of IV and oral NAC. These means were then compared using t-test for independent groups to test for significance. The average total cost of IV and oral NAC treatment was calculated by using monetary values from primary literature. A sensitivity analysis was performed to test the possible effects of an increase or decrease of the final costs by 5 to 10%. Main Results: Patients (≥18 yrs) being treated with IV or oral NAC for acute APAP toxicity (≤8 hours prior to ingestion) were included in this study. A total of 47 patients met the inclusion criteria. Length of hospital stay was shorter in patients receiving IV NAC (42.5% 24-24hr; 37.5% 48-72hr) compared to patients receiving oral NAC (28.6% 48-72hr, 71.4% >72hrs; p<0.001). Total cost of ICU/non-ICU stay in patients receiving IV NAC ($8,720/$3010) was less than patients receiving oral NAC ($12,321/$4703); however, cost of IV NAC-extended (37hrs) in ICU/non-ICU ($13,153/$5535) was greater than oral NAC. The sensitivity analysis performed demonstrated that an increase or a decrease by 5 to 10% in change of cost does not affect our final conclusion. Conclusion: The cost of treatment of IV NAC is lower due to shorter LOS of patients treated with IV NAC (p<0.001). However, when an extended course of treatment is medically necessary for patients on IV NAC then the cost of treatment with IV NAC exceeds the cost of treatment with oral NAC.

L'obésité et les maladies du foie associées (NAFLD) augmentent le risque et la sévérité de l’hépatotoxicité induite par certains xénobiotiques, mais les mécanismes impliqués sont encore mal compris. Pour l'éthanol et le paracétamol (APAP), le rôle du cytochrome P450 2E1 (CYP2E1) hépatique est suspecté car l'activité de cette enzyme est augmentée au cours de ces pathologies dysmétaboliques. Le 1er objectif de notre travail expérimental a été de mettre au point un modèle cellulaire de NAFLD caractérisé non seulement par l'accumulation de triglycérides mais aussi par l’augmentation de l'activité du CYP2E1. Pour cela, des cellules humaines HepaRG différenciées ont été incubées pendant une semaine avec de l'acide stéarique ou de l'acide oléique, en présence de 3 concentrations différentes d'insuline. Les triglycérides cellulaires et l'expression de gènes induits au cours de la stéatose étaient similaires avec les deux acides gras. Cependant, l'activité du CYP2E1 était significativement augmentée uniquement par le stéarate et ceci était associé à une diminution de l'activité du CYP3A4, une autre caractéristique des NAFLD. L’activité du CYP2E1 dans les cellules HepaRG était réduite par l'insuline d'une manière concentration-dépendante et cet effet était reproduit sur des hépatocytes humains en culture primaire. Ainsi, l'activité du CYP2E1 était la plus élevée dans les cellules HepaRG cultivées avec du stéarate et sans insuline. Le 2ème but de notre étude était ensuite d'évaluer la cytotoxicité de l’APAP sur des cellules HepaRG présentant ou non une stéatose et une induction du CYP2E1. Des expériences avec une large gamme de concentrations d’APAP (de 1 à 20 mM) indiquaient que la perte cellulaire d'ATP et du glutathion (GSH) était presque toujours plus forte en présence de stéarate. Dans les cellules prétraitées avec le chlorméthiazole (CMZ, un inhibiteur du CYP2E1), la moindre diminution d’ATP était plus importante en présence de stéarate, avec de faibles (2,5 mM) ou de fortes (20 mM) concentrations d’APAP. Cependant, en l'absence d'insuline, la moindre chute d’ATP induite par le CMZ était significativement plus forte uniquement pour 20 mM d’APAP. Étonnamment, suite au prétraitement par le CMZ, il n'y avait pas de protection vis-à-vis de la diminution du GSH et de la formation des adduits APAP-protéines. Enfin, les concentrations du métabolite APAP-glucuronide étaient significativement augmentées en présence d'insuline. Ainsi, lorsqu’elle est étudiée dans des conditions spécifiques de culture, la lignée cellulaire HepaRG semble être un modèle intéressant de NAFLD, notamment en ce qui concerne les activités du CYP2E1 et du CYP3A4. Nos données suggèrent aussi que l’induction du CYP2E1 observée au cours des NAFLD pourrait être secondaire à l'accumulation de certains acides gras et à la présence d’une faible signalisation insulinique dans le foie. Ainsi, ce modèle cellulaire peut être utilisé pour mettre en évidence les principaux facteurs métaboliques et hormonaux favorisant hépatotoxicité de l’APAP chez les personnes obèses. Cette thèse inclut également une revue de la littérature sur l’hépatotoxicité de l’APAP dans le contexte de l’obésité et des NAFLD (Michaut et al., Liver Int 2014)
Obesity and nonalcoholic fatty liver disease (NAFLD) are able to increase the risk and the severity of hepatotoxicity induced by some xenobiotics including drugs, but the involved mechanisms are still poorly understood. For toxic compounds such as ethanol and acetaminophen (APAP), a role of hepatic cytochrome P450 2E1 (CYP2E1) is suspected since the activity of this enzyme is consistently enhanced during obesity and NAFLD. The first aim of our experimental study was to set up a cellular model of NAFLD characterized not only by triglyceride accumulation but also by higher CYP2E1 activity. To this end, differentiated human HepaRG cells were incubated during one week with stearic acid, or oleic acid, in the presence of 3 different concentrations of insulin. Cellular triglycerides and the expression of lipid-responsive genes were similar with both fatty acids. However, CYP2E1 activity was significantly increased only by stearate and this was associated with lower CYP3A4 activity, another metabolic feature reported in NAFLD. CYP2E1 activity in HepaRG cells was reduced by insulin in a concentration-dependent manner and this effect was reproduced in cultured primary human hepatocytes. Hence, the highest CYP2E1 activity was observed in HepaRG cells with stearate and without insulin. Next, the second aim of our study was to assess APAP cytotoxicity in HepaRG cells presenting or not lipid accretion and CYP2E1 induction. Experiments with a large range of APAP concentrations (1 to 20 mM) showed that the cellular loss of ATP and glutathione (GSH) was almost always stronger in the presence of stearic acid. In cells pretreated with the CYP2E1 inhibitor chlormethiazole (CMZ), recovery of cellular ATP was significantly higher in the presence of stearic acid with both low (2.5 mM) and high (20 mM) concentrations of APAP. However, in the absence of insulin, CMZ-induced ATP recovery was significantly greater only for 20 mM of APAP. Surprisingly, there was no recovery of cellular GSH and no reduction of APAP-protein adducts following CMZ pretreatment. Finally, levels of APAP-glucuronide were significantly enhanced in the presence of insulin. Hence, when studied in specific conditions of culture, the HepaRG cell line can be a valuable model of human NAFLD, especially regarding CYP2E1 and CYP3A4 activity. Our data also suggest that higher CYP2E1 activity in NAFLD could be secondary to the hepatic accumulation of some fatty acids and to the presence of low insulin signaling. This cellular model can be thus used to unveil the main metabolic and hormonal factors favoring APAP hepatotoxicity in obese individuals. This thesis also includes a review on APAP hepatotoxicity in the context of obesity and NAFLD (Michaut et al., Liver Int 2014)

Bibliography: leaves 116-138
[14], 138 leaves, 5 leaves of plates : ill ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical & Experimental Pharmacology, 1988

碩士
中山醫學大學
營養學研究所
101
Obesity is associated with increased tissue and systemic inflammation and oxidative stress. Obesity induced by high-fat diets enhances oxidative stress and glutathione disulfide content, and reduces the level of glutathione and activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase. Several experimental and epidemiological studies have demonstrated that acetaminophen has been associated with elevated levels of oxidative stress, which also causes impaired liver functions. Acetaminophen is the most widely used pharmaceutical analgesic and antipyretic agent in the world. However, the literature regarding the effect of acetaminophen-induced changes of cytokines and oxidative stress in obese rats remains unclear. Therefore, the aim of this study is to evaluation of the effect of acetaminophen-induced changes of cytokines and oxidative stress in normal and high fat diet (HFD)-induced obese rats. The data indicated that body weight, perirenal adipose tissue, and epididymal adipose tissue in high fat diet-low dose-acetaminophen (HLA) and high fat diet-high dose-acetaminophen (HHA) groups were significantly decreased as compared to the HFD group. Serum parameter levels of total cholesterol, LDL-cholesterol, aspartate aminotransferase, alanine aminotransferase, interleukin-6, and interleukin-1β in HHA group was significantly increased as compared to the HFD group. The serum parameter levels of alanine aminotransferase and aspartate aminotransferase in HHA group was significantly increased as compared to the normal diet-high dose-acetaminophen (NHA) group. In the trolox equivalent antioxidant capacity (TEAC) and malondialdehyde (MDA), hepatic TEAC in HLA and HHA groups were significantly decreased as compared to the HFD group. Moreover, hepatic and serum levels of MDA in HHA group was significantly increased as compared to the HFD group. In the hepatic antioxidant enzymatic activities, the levels of glutathione, glutathione reductase, and catalase in HHA group was significantly decreased as compared to the HFD group. The pathological results show that the scores of portal inflammation and intralobular degeneration and inflammation in HLA and HHA groups were significantly increased as compared to normal diet-low dose-acetaminophen (NLA) and NHA groups. In the molecular mechanism, hepatic gene expressions of CYP2E1, MCP-1, IL-6, Stat1, Stat3, Jak2, and SOCS3 in HHA group were significantly increased as compared to the HFD group. Hepatic antioxidant related genes of HO-1, catalase, γ-GCS, GPx1, GPx4, SOD1, SOD2, and Nrf2 in HHA group was significantly decreased as compared to the NHA group. Hepatic pro-oxidant related genes of p47phox and Nox1 in HHA group was significantly increased as compared to the NHA group. These results demonstrated that the intake of acetaminophen can enhance oxidative stress and cause impaired liver functions in rats fed a high-fat diet.

The increased production of highly reactive oxygen and nitrogen species plays a significant role in development of a number of liver disorders associated with hepatocellular death and impaired cell regeneration. Liver injury induced by drug toxicity, ischemia-reperfusion, excessive alcohol consumption and different types of viral hepatitis is in large part mediated by oxidative stress. Liver damage due to oxidative stress induced by drugs, including acetaminophen, accounts for 5% of all hospital admissions and for almost half of all acute liver failures. One of the features of hepatocellular death mediated by oxidative stress is Ca²⁺ overload due its release from intracellular organelles and activation of ion channels on the plasma membrane. Ca²⁺ is fundamental for normal cellular functioning. Ca²⁺ signalling, mediated by the rise in free cytoplasmic Ca²⁺ concentration ([Ca²⁺]c)[c subscript], regulates many cellular events. However, a sustained rise in [Ca²⁺]c [c subscript] can be detrimental, leading to mitochondrial dysfunction and cell death through apoptosis and necrosis. Although it is well recognised that Ca²⁺ plays a significant role in oxidative stress-induced liver damage, the molecular identities of the ion channels that provide a pathway for Ca²⁺ entry in hepatocytes remain unidentified. One of the potential candidates that could be responsible for such Ca²⁺ entry pathway in hepatocytes is Transient Receptor Potential Melastatin 2 (TRPM2) channel. TRPM2 is a non-selective cation channel permeable to Na⁺ and Ca²⁺. The main physiological activator of TRPM2 channel is ADP-ribose, which binding to NUDT9-H motif in the TRPM2 C-terminus leads to the opening of the channel pore. It is known that oxidative stress promotes generation and release of ADPR from mitochondria and nuclei into the cytoplasmic space, thus promoting activation of TRPM2-mediated Ca²⁺ entry. In this thesis, we hypothesised that oxidative stress-induced Ca²⁺ entry in hepatocytes is mediated by TRPM2 channels, and used acetaminophen overdose as a model of oxidative stress-induced liver damage. We show that hepatocytes express long isoform of TRPM2, which mediates ADPR- and H₂O₂-induced Ca²⁺ entry and the cation current in these cells. Furthermore, we show that TRPM2 channels are activated in hepatocytes treated with high concentrations of acetaminophen and are responsible for Ca²⁺ overload in acetaminophen-induced liver toxicity. Experiments using TRPM2 KO mice provide first evidence of a pivotal role of TRPM2 channels in acetaminophen-induced liver injury, showing that lack of TRPM2 expression largely protects liver from acetaminophen overdose. An important finding that TRPM2 channels translocate from intracellular compartments to the plasma membrane provides explanation for a slow development of Ca²⁺ entry in response to H₂O₂ and acetaminophen. Finally, we show that substances previously known to protect liver from acetaminophen-induced damage are, in fact, inhibitors of TRPM2 current. Chlorpromazine, an antipsychotic drug, reversibly blocks TRPM2 channel pore, and curcumin, a chemical found in common spice, potently blocks activation of TRPM2 current by ADPR. The results presented in this thesis provide a fundamental knowledge about the role of TRPM2 channels in oxidative stress-induced liver injury, but also open a new chapter in search for the new drugs and drug targets for the treatment of a number of oxidative stress-related liver pathologies.
Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2015

Erratum tipped in before chapter 1.
Bibliography: leaves 226-248.
xv, 248 leaves : ill. (chiefly col.) ; 30 cm.
Shows that pretreatment with peroxisome proliferators protects mice against the acute hepatotoxicity of paracetamol, in addition to a number of other toxicants.
Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1999