Hepatoprotective Effect of Ï-Glutathione in a Murine Model of

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Hepatoprotective Effect of #-GSH in a Murine Model of Acetaminophen-Induced Liver Toxicity. Swati S. More, Jaime Nugent, Ashish Pramod Vartak, Steffan M. Nye, and Robert Vince Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00291 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 11, 2017

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Chemical Research in Toxicology

Hepatoprotective Effect of ψ-GSH in a Murine Model of Acetaminophen-Induced Liver Toxicity. Running title: Potential antidote against Tylenol Hepatotoxicity

Swati S. More*, Jaime Nugent, Ashish P. Vartak, Steffan M. Nye and Robert Vince Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA

*Corresponding author: Swati S. More, Center for Drug Design, 516 Delaware Street SE, 7-146 PWB, MMC 204, Minneapolis, MN 55455, USA Phone: (612) 626-1660; Fax: (612) 625-8154; E-mail: [email protected]

ACS Paragon Plus Environment

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Table of Contents (TOC) graphic


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Abstract Ψ-glutathione (ψ-GSH) is an orally bioavailable and metabolism-resistant glutathione analogue that has been shown previously to substitute glutathione in most of its biochemical roles. Described here in its entirety is the preclinical evaluation of ψ-GSH as a rescue agent for acetaminophen (APAP) overdose: an event where time is of essence. Employing a murine model, four scenarios commonly encountered in emergency medicine are reconstructed. ψ-GSH is juxtaposed against N-acetylcysteine (NAC), the sole clinically available drug, in each of the scenarios. While both agents appear to be equally efficacious when timely administered, ψ-GSH partly retains its efficacy even in the face of substantial delay in administration. Thus implied is the ability of ψ-GSH to intercept secondary toxicology following APAP insult. Oral availability and complete lack of toxicity as evaluated by liver function tests and survival analysis underscored ψ-GSH as a safer and more efficacious alternative to NAC. Finally, the pharmacodynamic mimicry of GSH by ψ-GSH is illustrated through the isolation and chemical characterization of an entity that can arise only through direct encounter of ψ-GSH with NAPQI, the primary toxic metabolite of APAP.

Key Words: hepatotoxicity, acetaminophen, oxidative stress, glutathione, antidote, N-acetylcysteine


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INTRODUCTION Acetaminophen (APAP) is “generally regarded as safe” when dosed appropriately, but its high concentration in OTC formulations presents the likelihood of accidental overdose. An FDAmandated labeling requirement has made APAP hepatotoxicity well known to the general population.1 The APAP amount “per dose” of a “regular strength” product is now limited to 325 mg. Yet, APAP overdose still leads all other causes of acute liver failure in the US and causes about 56,000 emergency room visits per year.2 The majority of APAP dose is subject to glucuronidation (52–57%) and sulfate conjugation (30–44%), producing pharmacologically inert products that undergo biliary excretion. Less than 5% is excreted unchanged. The remainder 5–10% is oxidized at the phenolic function by a variety of CYP450 enzymes3,

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into the electrophilic N-acetyl-p-benzoquinoneimine (NAPQI).

Under normal homeostasis, enough glutathione is available to scavenge NAPQI. The resulting conjugate is converted into cysteine and mercapturic acid conjugates before being excreted. Supratherapeutic doses, however, rapidly saturate phase-II processes and CYP450 oxidation now becomes the dominant metabolic pathway. Intracellular GSH reserves soon get depleted. Now unopposed, NAPQI causes intra- and extracellular havoc by disrupting redox homeostasis and leading to prevalently necrotic cell-death.5 Clinical address of APAP-overdose consists of the restoration of redox homeostasis through replenishment of metabolically available thiol.6 In that regard, N-acetylcysteine (NAC) is an effective, life-saving tool. It acts by (1) providing the cysteine precursor for GSH biosynthesis, (2) directly neutralizing reactive oxygen and peroxynitrites and (3) enhancing the sulfateconjugation of APAP. 7 An influential prognostic parameter is the time from ingestion of APAP to the administration of NAC ("time to NAC"), which in turns depends on the delay in the patient presenting to the ER. While NAC is practically 100% effective in preventing mortality and reducing hepatic injury when administered within 8 hours,8 its efficacy markedly drops beyond this time point. This narrow window is particularly problematic, as many accidental overdoses occur during binge consumption and abuse of narcotic or non-narcotic formulations that also “happen to contain APAP” (Ironically, APAP is added to deter abuse under the pretext of it enhancing the narcotic’s analgesic properties). A clear need exists for an antidote that can still be effective when


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administered after a substantial delay. The lower efficacy of NAC in cases of delayed ER presentation or treatment correlates with existing liver damage. After all, one of the important mechanisms of NAC mediated liver resuscitation is repletion of GSH levels by supplying cysteine for the rate-limiting glutamyl cysteine ligation that is mediated by glutamyl cysteine ligase (GCL). Early phases of APAP insult involve activation of c-Jun N-terminal kinases (JNKs) and GSK-3β, both reducing cyclin D1 activity and thereby globally impeding protein synthesis. Levels of GCL are consequently reduced after APAP overdose.9 It is this phenomenon that renders the provision of L-cysteine directly or through its prodrugs10, 11 rather ineffective at the stage when APAP toxicity has already ravaged the liver. Glutathione itself is not orally bioavailable12 due to degradation by γ-glutamyl transpeptidase (GGT), which also limits the utility of GSH pro-drugs such as diesters. NAC administration (particularly i.v.) is commonly associated with anaphylactoid reactions.13 Such reactions need to be addressed with diphenhydramine w/out corticosteroids and additional supportive care. A small percentage of cases are severe enough to warrant NAC discontinuation or pause. We have previously synthesized ψ-GSH, a peptidomimetic of GSH, where the Glu-Cys amide bond is replaced with a ureide isostere, a single change that addresses many of the aforementioned problems.14 ψ-GSH’s ureide linkage resists GGT attack while being of little consequence to protein-ligand interactions or the thiol oxidation potential when compared to GSH. The plasma residence time of ψ-GSH is manifold greater than GSH. The utility of ψ-GSH in opposing Alzheimer’s disease progression in a transgenic mouse model has been established making it an advanced drug candidate.15 As ψ-GSH acts through pharmacodynamic mimicry of GSH, the possible ability of ψGSH to counteract APAP hepatotoxicity warrants thorough evaluation. This account describes a series of experiments that juxtapose ψ-GSH and NAC as antidotes for APAP overdose in mouse. Specific aspects studied here are liver biochemistry and histology after APAP overdose followed by rescue by either of the two agents. Importantly, we have examined the effect of a delay in ad-


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ministration of either agent on rescue efficacy. Here, for the first time, we have isolated and characterized APAP-ψ-GSH adduct and determined its in vivo significance.


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Materials and Methods Chemicals and Reagents All the chemicals used in this study were purchased from Sigma-Aldrich, unless otherwise stated. ψ-GSH was synthesized in our laboratory by previously described procedures 14,15. The identity and purity of every batch of ψ-GSH were verified by mass spectrometry and reverse phase HPLC, and was always > 97%. Histological analysis of mouse liver samples was conducted by the Comparative Pathology Shared Resource (CPSR) at the University of Minnesota. General Synthetic Procedures All commercially available chemicals were used as supplied unless otherwise indicated. All reactions were performed under inert atmosphere of argon with oven-dried glassware. 1H and 13C NMR spectra were recorded on a Varian 600 MHz NMR spectrometer. High resolution mass spectra were acquired on an Agilent Bio TOF-II spectrometer with positive and negative ion electrospray ionization sources. PPG or PEG were used as internal standards. Preparation of APAP-ψ-GSH Conjugate The APAP-ψ-GSH conjugate was synthesized according to the method of Thatcher and Murray16; (modified by Roušar et al.)17. A suspension of APAP in anhydrous chloroform was treated with freshly prepared silver oxide. The suspension was stirred and filtered under argon gas. The filtrate was then treated with ψ-GSH (dissolved in 0.1 M sodium phosphate buffer, pH = 7.4). After a short while, the organic phase was removed and the aqueous phase was evaporated under reduced pressure at 40 °C to a yellow-brown sticky residue. The APAP-ψ-GSH conjugate was purified by preparative liquid chromatography (pHPLC). A portion (approximately 0.5 g) of the brown-yellow gel was diluted with distilled water and assayed using pHPLC. The conditions of separation were as follows: (a) the mobile phase was a mixture of water, methanol and acetic acid in a volumetric ratio of 87:12: 1; (b) the stationary phase was a preparative column Phenomenex PSI (250 × 25 mm; C18; 7 µm). The experiments were performed using an isocratic pHPLC system (25 °C) consisting of a preparative pump LCP3102, injection valve equipped with a 10-


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mL external sample loop and a UV detector (Saphire). The detection wavelength was set at 254 nm. Fractions of mobile phase that contained APAP-ψ-GSH were collected and identified by +-ve ion ESI followed by scanning with an LCQ ion-trap analyzer (Agilent Technologies, Waltham, MA, USA) in the range m⁄Z 50–800. Briefly: evaporated fraction samples were dissolved in methanol and analyzed by direct infusion at the flow rate 10 µl/min. ESI source was tuned to provide maximum intensity for [M+H]+ion at m⁄Z 458.13 (source voltage set to 4 kV, capillary voltage to 24 V, tube lens voltage to 0 V, capillary temperature to 175 °C and sheath gas flow rate m

to 40 units). The tandem mass spectra for precursor [M+H]+ ion were measured in ⁄Z range of 125–800 using collision dissociation with 35% collision energy. Purity of the substance was measured by HPLC/UV at similar detection and separation conditions as in case of pHPLC, except the mobile phase flow was 0.7 ml/min instead of 15 ml/min and stationary phase was 250×4.5 mm; C18; 5 µm. The separation was performed at 37 °C on HPLC/UV equipped with LC-20ADXR pump, SPD-20A UV detector, Rheodyne injection valve with 10 µl external injection loop (Rheodyne, Oak Harbor, WA, USA). 1H NMR (D2O, 600 MHz) δ ppm 7.36–7.05 (m, 2H), 6.70–6.67 (dd, J1 = 6.6 Hz, J2 = 1.8 Hz, 2H), 4.08 (dd, J1 = 13.2 Hz, J2 = 6 Hz, 2H), 3.56– 3.20 (m, 5H), 2.39–1.73 (m + singlet at 2.19, 5H), 1.66–1.48 (m, 1H).Note. An α-H (typically the cysteinyl) was not visible because of water peak in the 4.9–4.7 region. The compound was not soluble in other solvent mixture incl. DMSO + TFA. 13C NMR (D2O, 125 MHz) δ ppm 174.4, 172.8, 169.9, 169.7, 156.6, 137.2, 123.00, 123.3, 118.5, 115.5, 54.4, 54.0, 42.5, 34.1, 30.2, 28.1, 23.3. Identification of the APAP-ψ-GSH adduct in mouse liver samples Frozen liver tissue was homogenized 1:5 (w/v) in 20 mM ammonium acetate (pH 7) buffer and then centrifuged to collect supernatant for analysis of APAP–ψ-GSH adducts. The supernatant was mixed with equal volume of acetonitrile and centrifuged in case of insoluble debris prior to evaluation by HPLC. Samples were resolved on an Agilent 1200 Infinity series HPLC system over a reverse-phase Phenomenex Gemini C18 column (5 µ, 4.6 × 250 mm) using isocratic elution solvent system 10% B in A; solvent A = water with 0.1% acetic acid, solvent B = MeCN;


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flow rate = 1.0 mL/min.; and absorbance at 254 nm. The chemically synthesized adduct was used as a reference. Determination of ψ-GSH was carried out on a LC-MS/MS system consisting of an AB Sciex QTrap 5500 mass spectrometer and an Agilent 1260 Infinity HPLC. The chromatographic separation of analytes was achieved using a Thermo Aquasil C18 column (150 × 2.1 mm, 3 µm). The two eluents were: (A) H2O with 0.1% formic acid; and (B) acetonitrile with 0.1% formic acid. The mobile phase was delivered at a flow rate of 0.3 mL/min using an gradient elution: 0–1.5 min, 0-35% B (v/v); 1.5-1.7 min, 35-95% B (v/v); 1.7-2.7 min, 95-95% B (v/v); 2.7-2.9 min, 955% B (v/v); 2.9-6.0 min, 5-5% B, (v/v). Only eluate from 1.2-2.2 min was diverted into the mass spectrometer for analysis. MS/MS detection of the ψ-GSH was conducted using ESI ion source with MRM detection in positive mode. The curtain gas was set at 25 psi. The ionspray voltage was set at 5000 V, and the temperature at 700 °C. The nebulizer gas (GS1) was set at 60 psi and turbo gas (GS2) were set at 50 psi. Table 1 summarized the individual MS/MS parameters for p-GSH, including declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP). A MRM transition of m/z 309.1 to m/z 105.0 was selected to monitor p-GSH. The selection of this MRM transition avoids cross-channel interference with the endogenous GSH. Animals Male Swiss Webster Mice of ages between 6 and 10 weeks (acquired from Harlan) were used in the present study. All experimental procedures and animal handling were executed in accordance with the national ethics guidelines, approved and complied with all protocol requirements at the University of Minnesota, Minneapolis, MN (IACUC). For all experiments, animals were housed three per cage in our facility, in a controlled environment (22 °C, RH 50–60%, light from 07:00– 19:00 hours). Food and water were made available ad libitum. Injections Prior to drug administration, mice were fasted overnight, weighed and separated into different


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treatment groups (N = 8-12 per treatment group). Food was kept out of the cages until after the final drug injection. The APAP solution (20 mg/mL) was prepared in warm saline and was injected intraperitoneally at a dose of 370 mg/kg (2.45 mmol/kg). At the specified time after APAP injection, ψ-GSH or NAC was injected in mice by i.p., i.v. or oral routes. Groups of saline, ψGSH only and NAC only were included as controls. Food was returned to the cages at this point and animals were monitored every 30 min for the first two hours. Blood and liver tissues were collected at 3 or 24 hours after the APAP overdose and submitted for liver enzyme analysis to Veterinary Diagnostic Laboratory, University of Minnesota. Quantitation of reduced glutathione from mouse livers Manufacturer’s instructions from the Cayman GSH assay kit (catalog #703002, Ann Arbor, MI) were followed with the following modifications: liver tissue was homogenized in 5 ml of 50 mM ice cold 2-(N-morpholino)ethanesulphonic acid (MES) buffer per gram of tissue and centrifuged at 10,000 × g for 15 min at 4 °C. Supernatants were analyzed for the protein content by BCA assay and deproteinated by addition of an equal volume of metaphosphoric acid. To a 50ul of each deproteinated homogenate, 2.5ul of 4M triethanolamine reagent was added and diluted 10 times with MES buffer for determination of total GSH. For GSSG quantitation, diluted deproteinated samples were mixed with 1M 2-vinylpyridine solution (10 µl/1 ml of homogenate) and incubated for one hour at room temperature. Protein Carbonyl Determination Excised liver tissue was homogenized in an ice-cold phosphate buffer (50 mM phosphate, pH = 7.00 with 1 mM EDTA and supplemented with protease inhibitors), and centrifuged at 10 000 × g at 4 °C for 15 min. The clear supernatant (100 µl) thus obtained was diluted with 100 µl of 2,4dinitrophenylhydrazine (DNPH, 10 mM) in 2.5 N HCl. Blanks consisted of 2.5 N HCl alone. Samples were incubated in the dark at ambient temperature for 1 h with intermittent agitation. Then, 200 µl of 20% TCA solution was added to each incubate to precipitate all proteins. Samples were cooled on ice for 5 min before centrifugation for 10 min at 10 000 × g at 4 °C. The protein pellet was washed 3 times with EtOH–EtOAc (1:1, 1 mL) mixture with centrifugation step


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after each washing (10 000 × g at 4 °C for 10 min) to remove unreacted DNPH. The resultant pellet was then dissolved in 6M aqueous guanidine HCl. The mixtures were held at 37 °C for 30 min to promote protein dissolution and centrifuged to remove any insoluble material. The clear supernatant was then analyzed for protein oxidation products by measuring absorbance at 390 nm. Total protein was determined through the BCA assay utilizing a kit (Pierce, Rockford, IL). Carbonyl content was normalized to the protein content and represented as percentage increase over the samples obtained from saline-treated mice. Lipid Peroxidation Assay The extent of lipid peroxidation was calculated based on the levels of thiobarbituric (TBA) acidreactive entities (TBARS assay). Homogenized liver samples from protein carbonyl assay (50 µl) were mixed with ice-cold phosphate buffer (50 (µl) used for homogenization into a 96-well plate. Each well was exposed to 50 µl of 50% w/v trichloroacetic acid and 75 µl of 1.3% w/v 2thiobarbituric acid solution and incubated for 15 min at 60 °C for 40 min on an incubated shaker. Samples were then cooled on ice prior to addition of 20 µl of SDS solution (0.2 g/ml) and the light absorption was measured at 530 nm and 630 nm using a plate reader against a blank reference. Difference in the absorbance at 530 and 630 nm was proportional to the levels of lipid oxidized products. Total protein was determined through the BCA protein assay. TBARS where then proportioned to total protein content and represented as percentage of the values in saline-treated control mice. Data Analysis Data is expressed as the mean values with the standard error of the mean. Data were analyzed using student t tests or by one way analysis of variance (ANOVA), wherever appropriate. Statistical significance was held at 0.05. RESULTS Protective effect of ψ-GSH on APAP induced hepatotoxicity. Overnight-fasted male Swiss

Webster mice (HSD) were administered 370 mg/kg APAP (2.45 mmol/kg) intraperitoneally to induce


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hepatic damage. Elevation of the alanine aminotransferase (ALT) level was used as a convenient gauge of hepatic toxicity, supplemented with histology of the damaged tissue. As expected, APAP caused extensive liver damage noted as a more than 40-fold increase in ALT level when compared with that in mice treated with the vehicle alone (Figure 1). Histopathology upon hematoxylin and eosin (H&E) staining indicated moderate to severe centrilobular necrosis (Figure 2 and Table 1). Administration of the clinically used rescue agent, NAC, at 1,200 mg/kg (7.35 mmol/kg) i.p. 30-min post APAP overdose protected the mice almost completely against APAP-induced liver injury. Liver morphology of such mice was normal with necrosis confined to narrow zones of the subcapsular region. Lower doses of NAC failed to rescue elevated ALT levels after APAP overdose (Supporting Information, Figure S1). Mortality rate of 10-15% was observed in APAP + NAC (7.35 mmol/kg) combination group. ψ-GSH when similarly administered at ¾, ½ and Molar equivalents of the NAC dose above also prohibited liver ALT elevation in mice, with marginally better protection at 0.75 molar equivalent dose (5.5 mmol/kg) compared to the equivalent dose. This dose was selected for further experimentation. Notably, the combination of APAP and ψ-GSH was well tolerated by all the mice with no significant histological findings. Oral administration of ψ-GSH at 7.35 mmol/kg also prevented drastic elevation of liver ALT level in APAP-overdosed mice (Figure 1). As the usual route of administration of the antidote in APAP-overdose patients is intravenous, we conducted the aforementioned APAP overdose experiment with i.v. administration of ψ-GSH. The MTD of NAC when given i.v. was found to be 1.3 mmol/kg. Rescue doses of ψ-GSH at 2×, 1× and 0.5× the molar equivalent of the MTD of NAC were employed (Figure 3). A dose-dependent reduction in ALT elevation was noted. NAC and ψ-GSH at the 1.3 mmol/kg dose caused quantitatively (ALT level) and histologically similar effects, with the very important difference of NAC administration at that dose post-APAP overdose being lethal to 20–25% of the mice. Delayed administration of ψ-GSH and NAC post APAP overdose. To better mirror clinical situations, we studied the effect of a delay in administration of the antidotes. An 80-fold liver ALT elevation was observed 24 hours post APAP overdose (Figure 4). ψ-GSH treatment 1, 2 and 4 hours after APAP overdose lowered these elevations by 95%, 89% and 36%, respectively, with


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a 100% survival rate. In contrast, NAC treatment following the same schedule lowered ALT elevations by 88% and 79% at hours 1 and 2, but was inconsequential at the 4 hour time-point. The survival rate in the NAC group was only 20%. Effects of NAC vs. ψ-GSH treatment on liver glutathione levels and oxidative stress markers. One of the effects of APAP induced liver-toxicity, apart from elevation of serum ALT, is macromolecular oxidation whose surrogate indicators are the total glutathione, lipid and protein oxidation product content (Figure 5). Three hours post APAP overdose, liver ‘glutathione’ (total thiol) content was found to be lowered to 38% of the corresponding saline-only control group. Partial recovery (to 70%) was observed in the surviving members of this group after 24 hours. In contrast, the mice that received ψ-GSH (5.5 mmol/kg) immediately after APAP overdose lost no liver thiol at the 3-hour mark, and had higher total thiol at 24 hours (115%) in comparison with the control group. GSH recovery by ψ-GSH at merely 30 min post antidote was higher than that of NAC (7.35 mmol/kg) treated animals and the GSH levels were comparable to saline controls at 2 h time point. Rise in total concentration of intracellular GSH was paralleled by reduction in free ψGSH concentration (Figure 5C). NAC treatment, however, did not have any effect on liver glutathione at both of the time points tested. We examined the possibility of GSH recycling machinery to recycle ψ-GSH. It appears that glutathione reductase and glutathione peroxidase enzymes process ψ-GSH in a fashion analogous to GSH, although at a lower rate (Supporting Information, Figure S2). Importantly though, ψ-GSH was recognized by glutathione-S-transferase, a key enzyme involved in formation of APAP-GSH adduct, as an alternative substrate. Another measure of APAP-induced liver damage is the protein and lipid oxidation product content. Figure S3 in the Supporting Information shows the protein carbonyl levels and the extent of lipid peroxidation in APAP-treated mice livers. Protein carbonyls (detected as 2,4-dinitrophenylhydrazones) were elevated significantly in APAP treated mice (1.46-fold; p < 0.0001) relative to saline treated mice. Intraperitoneal NAC treatment caused a modest reduction of protein carbonyls (1.16-fold) relative to saline controls, in contrast to ψGSH treated mice livers (0.78-fold). Thiobarbituric acid reactive substance (TBARS) content is a surrogate indicator of the extent of lipid peroxidation, particularly that of the production of the


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end product malondialdehyde (MDA, Figure S3B). Similar to the effect on protein carbonyls, NAC treatment partially reduced MDA elevation (1.37-fold relative to saline control) when compared to the APAP only group (2.18-fold over saline controls). ψ-GSH completely prevented MDA elevation (0.90-fold relative to saline controls), (p < 0.0001). Chemical characterization of the putative acetaminophen-ψ-GSH conjugate and its identification in mouse liver homogenate. The method of Thatcher17 (modified by Rousar18) for the synthesis of the analogous APAP-GSH adduct was directly adopted. NAPQI (produced by silver-oxide treatment of APAP) with ψ-GSH. The crude reaction mass was directly subjected to C18-silica gel chromatography. The structure of the product was elucidated by NMR and mass spectroscopy. Similar to that found for GSH, the cysteinyl thiol of ψ-GSH was bound covalently to the ortho-position of NAPQI. RP-HPLC indicated that the product was >97% pure. With this chemical standard in hand, attempts were made to detect the APAP-ψ-GSH adduct in liver samples of mice rescued by ψ-GSH after APAP overdose. Under the conditions described under Material and Methods, a peak at 8.04 minutes appeared in the mouse sample. This was also the retention time of the chemical standard (Figure 6). Confirmation of the peak’s identity was then obtained by spiking the liver homogenate with the authentic sample of the APAP-ψGSH adduct.

DISCUSSION APAP hepatotoxicity occurs only at unreasonably high doses or when combined with alcohol and other hepatotoxins. It is the presence of APAP in almost all popular OTC cold/flu and fever medications that creates circumstances where a toxic dose of APAP is more likely to be ingested when compared with aspirin or NSAIDS. The toxic level may be reached cumulatively or by concurrent use of multiple formulations containing APAP. The most frequently prescribed anal-


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gesics: hydrocodone (e.g., Norco®, Vicodin®) and oxycodone (Percocet®) contain 300–325 mg of APAP per dose. Ingestion above the recommended ‘safe’ dose of 4000 mg/24 h often occurs in patients with moderate–severe pain. Illicit consumption of cough syrups (typically containing APAP in addition to dextromethorphan) in large quantities in pursuit of a psychotropic effect is another cause for unintended APAP overdose. Hepatotoxicity notwithstanding, APAP when taken as recommended is preferred over most NSAIDs due to its lack of gastrointestinal adverse effects.18 It then becomes important that the acute and delayed effects of APAP induced hepatotoxicity be fully addressed. The nature of a typical APAP-overdose case in an ER setting creates an urgent and yet unmet need for an antidote that can satisfactorily remedy the APAP-distressed liver even when administered long after the toxic APAP dose. NAC, the sole available antidote is effective when administered within 8–10 hours. There are problems: histamine-dependent life-threatening adverse reactions such as persistent bronchospasm occur at a frequency high enough that diphenhydramine prophylaxis is part of the rescue protocol. NAC is currently understood to exert its hepatoprotective effect through (i) provision of cysteine for GSH synthesis (in limited capacity), (ii) increasing the levels of the neurotransmitter hypotaurine (Htau), which also is a radical scavenger (e.g., HOO•, O2•, HO•) antioxidant aside from its main role as a neurotransmitter (iii) direct chemical scavenging of non-radical electrophiles such as NAPQI and ONOO‒ through its ionizabe –SH function that at physiological pH readily dissociates into the nucleophilic thiolate (–S‒) and (iv) stimulation of pyruvate dehydrogenase (PDH), thereby improving/restoring the mitochondrial TCA cycle. The latter three effects were often overlooked, but are now increasingly regarded as critical to successful hepatoprotection after considerable delay post APAP overdose. It would then seem that effects (ii)–(iv) are exerted to an insufficient extent by NAC. Mole for mole, GSH is demonstrably superior to NAC 7 in inducing effects (ii)–(iv). The problem is γ-GTT. A notably efficient and widely distributed enzyme that has stymied many attempts at the direct use of GSH or its ester prodrugs in an intact mammal. We have previously demonstrated that ψ-GSH overcomes this barrier, substitutes GSH well as a substrate for all


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GSH-dependent enzymes and is ferried into the cell by GSH transport mechanisms.14 Consequently, ψ-GSH has much superior pharmacokinetics. Its toxicology is unnoteworthy in mice at doses as high as 2000 mg⁄kg. We have established its utility in an Alzheimer’s mouse model and it is thus now a lead candidate for drug development. Herein, we sought to examine the ability of ψ-GSH to exert hepatoprotection in mouse post APAP overdose. Four sets of experiments were designed to model variables in APAP rescue: state of the patient at presentation, time elapsed between presentation at the ER and the APAP overdose, and route of NAC administration. This experiment examines the relative efficacy of ψ-GSH and NAC when administered 30 minutes after APAP overdose i.e., when APAP metabolism is still underway and NAPQI is being produced (Figure 1). As expected, both antidotes when administered i.p. were effective in reducing the extent of APAP induced liver ALT level elevation at all doses employed. ψ-GSH conferred marginally better than NAC at the 5.5 mmol⁄Kg dose, but we opine that this difference is of no practical significance since the parameter studied is merely liver ALT elevation. Histopathology of mice rescued with NAC or ψ-GSH did not indicate centrilobular necrosis or general hepatocyte degeneration. Mortality rate of 10-15% was observed for combination of APAP and NAC (7.35 mmol/kg). The second experiment models the most common route of NAC administration employed in emergency care: intravenous (Figure 3). The maximally tolerated dose (MTD) of NAC was employed, while ψ-GSH was dosed below, at and above a dose equimolar to the MTD of NAC. ψ-GSH protected the mice to extents similar to that when administered i.p. In contrast, the combination of NAC and APAP appeared to be lethal to 20-25% of the animals receiving it, clearly suggesting additive toxicity. Similar results are reported previously by Corcoran and Wong (1986)19 and subsequently by McConnachie et al.,20 however the underlying mechanism of NAC-induced enhancement of APAP toxicity is yet to be understood. In a number of cases, the patient is conscious at arrival to the ER and NAC may be given orally. The third experiment where the antidotes were administered through oral gavage models is such a situation. This situation was modeled in the second set of experiments where the dosing was by oral gavage (Figure 1). Mole for mole, ψ-GSH appears to be clearly superior to NAC at


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reducing ALT level increase post APAP overdose. It must be stressed that there is a time lag between NAC administration and GSH synthesis. It now is known that NAC does not directly react with NAPQI, at least in the intact animal. Any direct neutralization of NAPQI must thus arise from augmented GSH synthesis. In contrast, ψ-GSH is a structural and pharmacodynamic GSH mimetic. Although this makes it very likely that ψ-GSH could directly quench NAPQI, we had no material evidence of this property. We therefore chemically synthesized an adduct between NAPQI and ψ-GSH. 1H and 13C NMR spectra indicate that the site of covalent linkage is the ortho-position of the phenolic hydroxy (quinonic C=O in NAPQI) function, similar to the study conducted by Leeming et al.21 This adduct could be detected in liver samples of mice rescued from APAP toxicity with ψ-GSH (Figure 6). The fourth experiment models the worrisome (and unfortunately the most common) case of the patient presenting to the ER after a substantial delay post APAP overdose. By such a time, most of the NAPQI produced from APAP has already reacted with cellular proteins, intracellular GSH reserves are depleted, and as reported,22 severe oxidative stress is underway. When administered 1 and 2 hours post APAP overdose (Figure 4), ψ-GSH conferred excellent (but not complete) hepatoprotection against ALT elevation (implying reversal of redox balance) while protection due to NAC was modest. When administered 4 hours post APAP overdose, ψ-GSH was able to rescue 100% of the animals receiving it. In contrast, 80% of those in the NAC treated dose at this time point died. Covalent binding of NAPQI to cellular proteins causes oxidative stress through various mechanisms; it would appear that ψ-GSH counteracts those mechanisms. Histology indicated that ψ-GSH had checked the occurrence of centrilobular necrosis, thus facilitating tissue repair and explaining the full survival rate. Finally, we conducted a biochemical analysis of the APAP-overdosed mouse livers in all groups (Figure 5). Notably, only ψ-GSH had prevented APAP-induced hepatic GSH depletion at the 3 hour time-point. Ψ-GSH may cause GSH level restoration through two mechanisms: (a) it may sacrificially shield the growing cellular glutathione from oxidative damage, evident from the reduction in intracellular ψ-GSH levels, and (b) it may actively replenish GSH levels through mechanisms other than provision of cysteine. One plausible pathway is ψGSH lending reduction


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potential to the cell through cytoplasmic or mitochondrial oxidoreductases, which are famously non-specific with their substrates. The net effect would then be provision of NADPH for cellular mechanism to restore redox levels, GSH concentration being an important component. Levels of protein and lipid oxidation products were dramatically lower in the ψ-GSH treated group than those in the NAC treated group. Substrate activity of ψ-GSH toward GSH-dependent enzymes such as GR, GPx and GST is expected to help recycling of bioactive reduced form of ψ-GSH. The metabolic advantage that ψ-GSH has over GSH14 would undermine the reduced affinity observed with these enzymes. However, there are several reports23, 24 stating that the levels of GSH peroxidase and GSH reductase are reduced after APAP overdose. Thus reserving the pool of endogenous GSH and provide an alternative GSH mimetic is a valid strategy for developing antidotes. Advantages over NAC are apparent in this study since no lethal toxicity of ψ-GSH and APAP combination, complete protection of liver from APAP toxicity and more efficacious than NAC as a delayed antidote. Accidental APAP over-consumption is reported more frequently in the elderly population, the culprits being cumulative doses of APAP from various OTC products and lately, the indiscrete prescription of opiate painkillers containing APAP. Aging itself includes increased oxidative stress and reduction in glutathione stores of all tissues. With age, the glutathione synthesis pathway is perturbed owing to altered homeostasis that causes changes in the catalytic efficiency and levels of enzymes in the GSH biosynthetic pathway (such as GCL).25 Supply of NAC to such patients may prove to be of limited utility since the cellular machinery for vigorous GSH synthesis is deteriorated. APAP-overdose patients are somewhat analogous since severe oxidative stress also hampers cell organelles involved in maintaining conditions for efficient GSH biosynthesis. An effective, metabolically stable GSH mimetic with a wide therapeutic window is the logical answer to this scenario. The experiments described here suggest that ψ-GSH could fulfill this criteria and could be a possible alternative to NAC as an antidote to APAP overdose.

Supporting Information


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Figure demonstrating dose dependent rescue of acetaminophen overdose by intraperitoneal Nacetylcysteine administration; reaction kinetics of ψ-GSH with glutathione-dependent enzymes (glutathione reductase, glutathione peroxidase and glutathione S-transferase); and figure describing the effects of ψ-GSH treatment on increased protein and lipid oxidation products after acetaminophen overdose. This material is available free of charge via the Internet at http://pubs.acs.org. Funding This work was supported by the Center for Drug Design (CDD) research Endowment Funds at the University of Minnesota, Minneapolis. Acknowledgement We profusely thank Dr. Herbert Nagasawa, a Member of the Center for Drug Design, University of Minnesota, for his constant support and valuable discussions regarding this project. We thank Dr. Gerry O’Sullivan, Director of Comparative Pathology Shared Resource, Masonic Cancer Center, University of Minnesota, for histopathology reports of the liver samples. Authors would also like to thank Ms. Jessica Williams for conducting enzymatic assays. List of Abbreviations APAP, acetaminophen; NAC, N-acetylcysteine; GSH, glutathione; NAPQI, N-acetyl-pbenzoquinonimine; ALT, alanine transaminase.

References (1)http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/uc m424898.pdf. (2) Ghanem, C. I., Perez, M. J., Manautou, J. E., and Mottino, A. D. (2016) Acetaminophen from liver to brain: New insights into drug pharmacological action and toxicity. Pharmacol. Res. (3) Dong, H., Haining, R. L., Thummel, K. E., Rettie, A. E., and Nelson, S. D. (2000) Involvement of human cytochrome P450 2D6 in the bioactivation of acetaminophen. Drug Metab. Dispos. 28, 1397-1400. (4) Prescott, L. F. (1983) Paracetamol overdosage. Pharmacological considerations and clinical management. Drugs 25, 290-314.


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(10)

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(21) (22)

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Gujral, J. S., Knight, T. R., Farhood, A., Bajt, M. L., and Jaeschke, H. (2002) Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis? Toxicol. Sci. 67, 322-328. Davis, M. (1986) Protective agents for acetaminophen overdose. Semin. Liver Dis. 6, 138-147. Saito, C., Zwingmann, C., and Jaeschke, H. (2010) Novel mechanisms of protection against acetaminophen hepatotoxicity in mice by glutathione and N-acetylcysteine. Hepatology 51, 246-254. Wolf, S. J., Heard, K., Sloan, E. P., and Jagoda, A. S. (2007) Clinical policy: critical issues in the management of patients presenting to the emergency department with acetaminophen overdose. Ann. Emerg. Med. 50, 292-313. Shinohara, M., Ybanez, M. D., Win, S., Than, T. A., Jain, S., Gaarde, W. A., Han, D., and Kaplowitz, N. (2010) Silencing glycogen synthase kinase-3beta inhibits acetaminophen hepatotoxicity and attenuates JNK activation and loss of glutamate cysteine ligase and myeloid cell leukemia sequence 1. J. Biol. Chem. 285, 8244-8255. Roberts, J. C., Nagasawa, H. T., Zera, R. T., Fricke, R. F., and Goon, D. J. (1987) Prodrugs of L-cysteine as protective agents against acetaminophen-induced hepatotoxicity. 2-(Polyhydroxyalkyl)- and 2-(polyacetoxyalkyl)thiazolidine-4(R)carboxylic acids. J. Med. Chem. 30, 1891-1896. Wlodek, L., and Rommelspacher, H. (1997) 2-Methyl-thiazolidine-2,4-dicarboxylic acid as prodrug of L-cysteine. Protection against paracetamol hepatotoxicity in mice. Fundam. Clin. Pharmacol. 11, 454-459. Wendel, A., and Jaeschke, H. (1982) Drug-induced lipid peroxidation in mice--III. Glutathione content of liver, kidney and spleen after intravenous administration of free and liposomally entrapped glutathione. Biochem. Pharmacol. 31, 3607-3611. Schmidt, L. E. (2013) Identification of patients at risk of anaphylactoid reactions to Nacetylcysteine in the treatment of paracetamol overdose. Clin. Toxicol. (Phila.) 51, 467472. More, S. S., and Vince, R. (2012) Potential of a gamma-glutamyl-transpeptidase-stable glutathione analogue against amyloid-beta toxicity. ACS Chem. Neurosci. 3, 204-210. More, S. S., Vartak, A. P., and Vince, R. (2013) Restoration of glyoxalase enzyme activity precludes cognitive dysfunction in a mouse model of Alzheimer's disease. ACS Chem. Neurosci. 4, 330-338. Thatcher, N. J., and Murray, S. (2001) Analysis of the glutathione conjugate of paracetamol in human liver microsomal fraction by liquid chromatography mass spectrometry. Biomed. Chromatogr. 15, 374-378. Rousar, T., Parik, P., Kucera, O., Bartos, M., and Cervinkova, Z. (2010) Glutathione reductase is inhibited by acetaminophen-glutathione conjugate in vitro. Physiol. Res. 59, 225-232. Matsui, H., Shimokawa, O., Kaneko, T., Nagano, Y., Rai, K., and Hyodo, I. (2011) The pathophysiology of non-steroidal anti-inflammatory drug (NSAID)-induced mucosal injuries in stomach and small intestine. J. Clin. Biochem. Nutr. 48, 107-111. Corcoran, G. B., and Wong, B. K. (1986) Role of glutathione in prevention of acetaminophen-induced hepatotoxicity by N-acetyl-L-cysteine in vivo: studies with Nacetyl-D-cysteine in mice. J. Pharmacol. Exp. Ther. 238, 54-61. McConnachie, L. A., Mohar, I., Hudson, F. N., Ware, C. B., Ladiges, W. C., Fernandez, C., Chatterton-Kirchmeier, S., White, C. C., Pierce, R. H., and Kavanagh, T. J. (2007) Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice. Toxicol. Sci. 99, 628-636. Leeming, M. G., Gamon, L. F., Wille, U., Donald, W. A., and O'Hair, R. A. (2015) What Are the Potential Sites of Protein Arylation by N-Acetyl-p-benzoquinone Imine (NAPQI)? Chem. Res. Toxicol. 28, 2224-2233. Masubuchi, Y., Nakayama, J., and Sadakata, Y. (2011) Protective effects of exogenous glutathione and related thiol compounds against drug-induced liver injury. Biol. Pharm.


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Bull. 34, 366-370. Acharya, M., and Lau-Cam, C. A. (2010) Comparison of the protective actions of Nacetylcysteine, hypotaurine and taurine against acetaminophen-induced hepatotoxicity in the rat. J. Biomed. Sci. 17, S35. Adamson, G. M., and Harman, A. W. (1989) A role for the glutathione peroxidase/reductase enzyme system in the protection from paracetamol toxicity in isolated mouse hepatocytes. Biochem. Pharmacol. 38, 3323-3330. Toroser, D., and Sohal, R. S. (2007) Age-associated perturbations in glutathione synthesis in mouse liver. Biochem. J. 405, 583-589.

Table 1. Histochemical analysis of mouse liver samples Treatment

Histological findings in excised liver tissue

Saline APAP only

NSF* Moderate–severe centrilobular necrosis (CN), mild–moderate vacuolation, minimal–moderate reactive leukocytes mmol Narrow linear zones of subcapsular necrosis, but CN absent. APAP + NAC (7.35 /kg) Mild–severe centrilobular and midzonal vacuolar change. mmol NSF, minimal CN, minimal vacuolation, necrosis confined to kg / ) APAP + ψ-GSH (7.35 rare, singular areas. APAP + ψ-GSH (5.50 mmol/kg) NSF, rare to mild CN, minimal vacuolation. APAP + ψ-GSH (3.67 mmol/kg) NSF, mild centrilobular vacuolation. *no significant findings


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Figure legends Figure 1. Effect of intraperitoneal ψ-GSH and NAC administration 30 min post acetaminophen (APAP) overdose on liver ALT levels (N = 8). ψ-GSH APAP overdose caused 40 fold increase in the ALT levels. ψ-GSH was administered at ¾ , ½ and molar equivalent doses to NAC and all the treatments reduced the APAP-induced elevated ALT levels to that of saline control group. Mortality rate of 10-15% was observed in APAP + NAC combination group and number of animals for that group was raised to N =10 (** p < 0.01, *** p < 0.001; when compared to APAP only group). Figure 2. H and E stained liver samples, imaged under brightfield at 200x magnification, 100um scale bar. (A) Acetaminophen (APAP) treated mouse displaying hepatocyte degeneration and necrosis around the Central Vein (CV), centrilobular vacuolation and degeneration (Black arrow) and leukocyte infiltration (White arrow). (B) APAP treated mouse recovered with NAC displaying no necrosis around the CV but having moderate vacuolar change (Black arrow). Mouse treated with APAP and recovered with ψ-GSH (C) and mouse treated with saline only (D) display similar liver morphology with some mild vacuolation and degeneration seen in the ψ-GSH mouse (Black arrow). Figure 3. Effect of intravenous ψ-GSH and NAC on elevated liver ALT levels after acetaminophen (APAP) overdose. Administration of NAC at dose equivalent to its MTD (1.3 mmol/kg) and ψ-GSH at 2×, 1× and 0.5× the molar equivalent of the NAC MTD exhibited a dose-dependent reduction in ALT levels. However, APAP + NAC combination was lethal to 20-25% of the animals and N for that group was raised to N = 12 (* p < 0.05, ** p , 0.01, *** p < 0.001; when compared to APAP only group). Figure 4. Delayed administration of antidotes, ψ-GSH and NAC, post acetaminophen (APAP) overdose (N = 8). ψ-GSH exhibited complete protection of hepatic toxicity as gauged by ALT levels up to 2 h and near complete protection at 4 hours post APAP administration. Combination of APAP + ψ-GSH was safe at all time points tested. NAC, although, exhibited complete to near complete protection at 1, 2 and 4 h time points post APAP overdose, survival rate at 4 h was only


22

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20%. Number of animals was increased to 12 for this treatment group (* p < 0.05, ** p < 0.01; when compared to APAP only group). Figure 5. ψ-GSH treatment is more efficacious in reducing the increased liver oxidative stress after acetaminophen (APAP) overdose than NAC. Mice received intraperitoneal ψ-GSH (5.5 mmol/kg) or NAC (7.35 mmol/kg), 30 min post APAP (2.45 mmol/kg) administration. Reduced GSH (A and B) levels were restored to saline control levels by ψ-GSH treatment, while NAC was less efficient at all the time points tested. (C) Levels of ψ-GSH were measured by LCMS/MS and showed steady drop over time (** p < 0.01, *** p < 0.001; when compared to APAP only group). Figure 6. HPLC chromatogram of the liver homogenate from mice overdosed with acetaminophen (APAP) in the presence and absence of ψ-GSH. APAP-ψ-GSH adduct was detected at a retention time of 8.04 min and was compared to its chemically synthesized authentic sample.

Figures Figure 1.


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Figure 2.

Figure 3


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Figure 4.


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Figure 5.


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Figure 6.


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Table of Contents (TOC) graphic 84x52mm (300 x 300 DPI)


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Figure 1. Effect of intraperitoneal ψ-GSH and NAC administration 30 min post acetaminophen (APAP) overdose on liver ALT levels (N = 8). ψ-GSH APAP overdose caused 40 fold increase in the ALT levels. ψGSH was administered at at ¾, ½ and molar equivalent doses to NAC and all the treatments reduced the APAP-induced elevated ALT levels to that of saline control group. Mortality rate of 10-15% was observed in APAP + NAC combination group and number of animals for that group was raised to N =10 (** p < 0.01, *** p < 0.001; when compared to APAP only group). 82x54mm (300 x 300 DPI)



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Figure 2. H and E stained liver samples, imaged under brightfield at 200x magnification, 100um scale bar. (A) Acetaminophen (APAP) treated mouse displaying hepatocyte degeneration and necrosis around the Central Vein (CV), centrilobular vacuolation and degeneration (Black arrow) and leukocyte infiltration (White arrow). (B) APAP treated mouse recovered with NAC displaying no necrosis around the CV but having moderate vacuolar change (Black arrow). Mouse treated with APAP and recovered with ψ-GSH (C) and mouse treated with saline only (D) display similar liver morphology with some mild vacuolation and degeneration seen in the ψ-GSH mouse (Black arrow). 177x133mm (300 x 300 DPI)


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Figure 3. Effect of intravenous ψ-GSH and NAC on elevated liver ALT levels after acetamino-phen (APAP) overdose. Administration of NAC at dose equivalent to its MTD (1.3 mmol/kg) and ψ-GSH at 2×, 1× and 0.5× the molar equivalent of the NAC MTD exhibited a dose-dependent reduction in ALT levels. However, APAP + NAC combination was lethal to 20-25% of the animals and N for that group was raised to N = 12 (* p < 0.05, ** p , 0.01, *** p < 0.001; when compared to APAP only group). 82x56mm (300 x 300 DPI)



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Figure 4. Delayed administration of antidotes, ψ-GSH and NAC, post acetaminophen (APAP) overdose (N = 8). ψ-GSH exhibited complete protection of hepatic toxicity as gauged by ALT levels up to 2 h and near complete protection at 4 hours post APAP administration. Combination of APAP + ψ-GSH was safe at all time points tested. NAC, although, exhibited complete to near complete protection at 1, 2 and 4 h time points post APAP overdose, survival rate at 4 h was only 20%. Number of animals was increased to 12 for this treatment group (* p < 0.05, ** p < 0.01; when compared to APAP only group). 145x62mm (300 x 300 DPI)


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Figure 5. ψ-GSH treatment is more efficacious in reducing the increased liver oxidative stress after acetaminophen (APAP) overdose than NAC. Mice received intraperitoneal ψ-GSH (5.5 mmol/kg) or NAC (7.35 mmol/kg), 30 min post APAP (2.45 mmol/kg) administration. Reduced GSH (A and B) levels were restored to saline control levels by ψ-GSH treatment, while NAC was less efficient at all the time points tested. (C) Levels of ψ-GSH were measured by LC-MS/MS and showed steady drop over time (** p < 0.01, *** p < 0.001; when compared to APAP only group). 177x159mm (300 x 300 DPI)



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Figure 6. HPLC chromatogram of the liver homogenate from mice overdosed with acetaminophen (APAP) in the presence and absence of ψ-GSH. APAP-ψ-GSH adduct was detected at a retention time of 8.04 min and was compared to its chemically synthesized authentic sample. 270x171mm (96 x 96 DPI)


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