APAP-treated Animals Research Articles

The Protective Potential eff ect of Metformin during Acetaminophen Hepatotoxicity through Nrf2 Activation

Acetaminophen (N-acetyl-para-aminophenol [APAP]) is the most commonly used medication for the relief of pain and fever around the world. Although APAP is safe and eff ective at a therapeutic level, acute overdose causes hepatotoxicity and severe liver damage. The hepatotoxicity induced by APAP is a persistent global problem that results in hepatotoxicity cases; acute liver failure and even death worldwide. This toxicity is characterized by extensive oxidant stress which consequently causes hepatocyte cell death. On the other hand, scientifi c studies have proven that MET shows hepatoprotective eff ect against hepatotoxicity induced by APAP through numerous mechanisms, e.g., antioxidant activity that alleviate the hepatotoxicity by activating Nrf2 pathway. The activation of Nrf2 pathway is anticipated to protect cells from oxidative stress that forms during hepatotoxicity. The benefi cial of using MET to protect against hepatotoxicity after APAP has been performed in an in-vitro experiment using Hep2G cell culture. However, up to our knowledge non, an in-vivo study (experimental animal) has been used to approve the benefi t of MET. Justifi cation: Nrf2 pathway activation by MET is one of the most important issues that need to be explained and followed up in laboratory animals since it has been previously studied in-vitro. Our study focuses on studying Nrf2 pathway in-vivo, since the results of in-vivo study are more relevant and similar to that of human beings. Aim of the study: To examine the possible stimulant eff ect of MET on Nrf2 pathway in experimental animals and its role in protecting the liver from oxidative stress that formed during APAP-induced hepatotoxicity. Methodology: Twenty-four wistar rats were divided randomly into four groups (six rats/Grp). Grp1; normal saline orally, Grp2; toxic dose of APAP (1000 mg/kg) orally; Grp3; 200 mg/kg of MET ip after 1-hour of APAP (1000 mg/kg) orally, Grp 4; 200 mg/kg MET ip for 24 hours. Then sera from experimental animals were collected for subsequent assessments of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme activities. Liver tissue was harvested to detect the expression of keap1 which is the negative regulator of Nrf2, and HO-1 and GST A1, which are related to Nrf2 pathway, by western blotting technique. Results: The results were showed a signifi cant elevation in ALT and AST activities in APAP treated group while MET normalized these biomarkers (AST and ALT). Western blotting assay showed that keap1 expression increased in APAP-treated animals while MET showed a signifi cant decrement in keap1 expression. The expression of antioxidant proteins HO-1 and GST A1 are decreased signifi cantly in APAP-treated animals while increased signifi cantly by MET treatment. Conclusion: The present results demonstrated that MET has a hepatoprotective eff ect in experimental animals against hepatotoxicity induced by APAP through activating Nrf2 pathway.

Possible Role of Extracellular Vesicles in Hepatotoxicity of Acetaminophen.

We examined proteomic profiles of rat liver extracellular vesicles (EVs) shed following treatment with a sub-toxic dose (500 mg/kg) of the pain reliever drug, acetaminophen (APAP). EVs representing the entire complement of hepatic cells were isolated after perfusion of the intact liver and analyzed with LC-MS/MS. The investigation was focused on revealing the function and cellular origin of identified EVs proteins shed by different parenchymal and non-parenchymal liver cells and their possible role in an early response of this organ to a toxic environment. Comparison of EV proteomic profiles from control and APAP-treated animals revealed significant differences. Alpha-1-macroglobulin and members of the cytochrome P450 superfamily were highly abundant proteins in EVs shed by the normal liver. In contrast, proteins like aminopeptidase N, metalloreductase STEAP4, different surface antigens like CD14 and CD45, and most members of the annexin family were detected only in EVs that were shed by livers of APAP-treated animals. In EVs from treated livers, there was almost a complete disappearance of members of the cytochrome P450 superfamily and a major decrease in other enzymes involved in the detoxification of xenobiotics. Additionally, there were proteins that predominated in non-parenchymal liver cells and in the extracellular matrix, like fibronectin, receptor-type tyrosine-protein phosphatase C, and endothelial type gp91. These differences indicate that even treatment with a sub-toxic concentration of APAP initiates dramatic perturbation in the function of this vital organ.

Dextromethorphan improves locomotor activity and decreases brain oxidative stress and inflammation in an animal model of acute liver failure.

Hepatic encephalopathy (HE) is a serious clinical problem leading to severe neurological disorders and death. No specific treatment is available for the management of HE-associated neurological damage. This study aimed to evaluate the effect of dextromethorphan (DXM) on oxidative stress and disturbed locomotor activity in an animal model of HE. In the current study, BALB/c mice received acetaminophen (APAP; 1000 mg/kg, intraperitoneally [IP]). Dextromethorphan (0.5, 1, 5, 10 mg/kg, subcutaneously [SC]) was injected in three doses (every 6 h), starting two hours after acetaminophen. Animals' locomotor activity, brain and plasma ammonia levels, as well as biomarkers of oxidative stress and inflammatory cytokines in the brain tissue, were assessed 24 hours after acetaminophen injection. It was found that APAP administration was significantly associated with liver damage and increased plasma biomarkers of liver injury. Ammonia levels in plasma and brain tissue of APAP-treated mice also increased significantly. There was also a significant difference in motor activity between the control and APAP-treated animals. The acute liver injury also increased the brain level of pro-inflammatory cytokines (tumor necrosis factor a [TNF-a], interleukin 6 [IL-6], and interleukin 1b [IL-1b]). It was found that DXM could significantly improve the motor activity of animals in all doses and decrease the biomarkers of inflammation and oxidative stress in the brain tissue of animals with hyperammonemia. The effect of dextromethorphan on oxidative stress and inflammation seems to be a major mechanism for its neuroprotective properties in HE. Based on these data DXM could be applied as an effective pharmacological option against HE-associated brain injury.

Formulating a New Pharmaceutical Drug; Acetaminophen Tablet Containing N-acetyl Cysteine, To Alleviate the Severity of Liver Damage in Rats: Phase I, Animal Study

Background and Aim: Acetaminophen (APAP) is a commonly used analgesic and also the leading cause of medication-induced liver damage. On the other hand, N-acetylcysteine (NAC) is a medication widely used to treat APAP overdose. Despite this interest, a few studies have investigated the co-administration effects of these medications. Therefore, this study aimed to evaluate the effects of NAC and APAP on renal and liver functions in rats when they use concurrently. Methods: Male Wistar rats were orally treated with a single dose of APAP (700 mg/kg) alone or in combination of NAC at the three different doses (200, 500, and 700 mg/kg). After 24 hours, the blood and liver samples were collected for biochemical and histopathological evaluations. Results: Liver damage was well established in the 700 mg/kg APAP-treated rats, as evidenced by elevated the plasma levels of aspartate transaminase (AST) and alanine transaminase (ALT). In addition, the plasma level of blood urea nitrogen (BUN) was significantly increased in the APPA group compared to the control group. Moreover, histological examinations revealed that liver degeneration was evident in APAP-treated animals. NAC only at the highest dose (700 mg/kg) could inhibit ALT elevation, but had no effect on AST and BUN levels. Interestingly, co-administration of NAC (700 mg/kg) with APAP (700 mg/kg) could slightly shift liver histological alterations from the irreversible stage (fibrosis) toward reversible lesions such as necrosis and hemorrhage. Conclusion: The study findings indicate that co-administration of NAC and APAP can reduce the severity of APAP-induced liver damage in rats. *Corresponding Author: Mehran Hosseini; Email: mehranhosseiny@bums.ac.ir; ORCID: https://orcid.org/0000-0002-6793-2035 Please cite this article as: Mehrpour O, Dastjerdi M, Nakhaee S, Amirabadizadeh A, Bijari B, Roomi H, Hosseini M. Formulating a New Pharmaceutical Drug; Acetaminophen Tablet Containing N-acetyl Cysteine, To Alleviate the Severity of Liver Damage in Rats: Phase I, Animal Study. Arch Med Lab Sci. 2021;7:1-8 (e14). https://doi.org/10.22037/amls.v7.35528

Resveratrol promotes liver regeneration in drug-induced liver disease in mice

Studies suggest that the bioactive polyphenolic compound resveratrol (RESV, trans-isomer), found naturally in certain foods such as red grapes and peanuts, may be able to ameliorate liver damage. However, the effects and efficacy of long-term treatment with RESV remain unclear. Here, we used an acetaminophen (APAP; 400 mg/kg/d for 15 days) overdose model to induce liver damage in C56BL/6 mice. Three days after the intoxication was stopped, we observed biochemical, histological and ultrastructural alterations in the livers of these mice. The APAP-treated animals were then given RESV (10 mg/kg/d) for 60 days. Blood and tissue were analyzed at days 7, 30 and 60. Our data show that long-term RESV treatment (60 days) ameliorates the liver injury caused by APAP intoxication, restoring histological features, ultrastructural organization and serum biochemical parameters (albumin, alanine aminotransferase). Ck18- and F4/80-positive cells (indicators of hepatocyte recovery) were reestablished and the number of α-SMA positive cells was normalized after long-term RESV treatment. Additionally, downregulation of the drug transporter BCRP was observed. Electron microscopy revealed that treatment with RESV was effective in restoring the shape and size of hepatic microvilli and normalizing both the number and viability of mitochondria. Taken together, these results indicate that long-term treatment with RESV is effective in alleviating liver injury caused by APAP administration.

<p>The Potential Neuroprotective Role of Citicoline in Hepatic Encephalopathy</p>

PurposeHepatic encephalopathy (HE) is described as impaired brain function induced by liver failure. Ammonia is the most suspected chemical involved in brain injury during HE. Although the precise mechanism of HE is not clear, several studies mentioned the role of oxidative stress in ammonia neurotoxicity. In animal models, the use of some compounds with antioxidant properties was reported to reduce the neurotoxic effects of ammonia, improve energy metabolism, and ameliorate the HE symptoms. Citicoline is a principal intermediate in the biosynthesis pathway of phosphatidylcholine that acts as neurovascular protection and repair effects. Various studies mentioned the neuroprotective and antioxidative effects of citicoline in the central nervous system. This study aims to investigate the potential protective effects of citicoline therapeutic in an animal model of HE.Materials and MethodsMice received acetaminophen (APAP,1g/kg, i. p.) and then treated with citicoline (500 mg/kg, i.p) one and two hours after APAP. Animals were monitored for locomotor activity and blood and brain ammonia levels. Moreover, markers of oxidative stress were assessed in the brain tissue.ResultsThe result of the study revealed that plasma and brain ammonia and the liver injury markers increased, and locomotor activity impaired in the APAP-treated animals. Besides, an increase in markers of oxidative stress was evident in the brain of the APAP-treated mice. It was found that citicoline supplementation enhanced the animal’s locomotor activity and improved brain tissue markers of oxidative stress.ConclusionThese data propose citicoline as a potential protective agent in HE. The effects of citicoline on oxidative stress markers could play a fundamental role in its neuroprotective properties during HE.

N-acetyl cysteine treatment preserves mitochondrial indices of functionality in the brain of hyperammonemic mice.

Aim of the studyAcute or chronic live failure could result in hyperammonemia and hepatic encephalopathy (HE). HE is a clinical complication characterized by severe cognitive dysfunction and coma. The ammonium ion (NH4+) is the most suspected toxic molecule involved in the pathogenesis of HE. NH4+ is a neurotoxic agent. Different mechanisms, including oxidative/nitrosative stress, inflammatory response, excitotoxicity, and mitochondrial impairment, are proposed for NH4+-induced neurotoxicity. N-acetyl cysteine (NAC) is a well-known thiol-reductant and antioxidant agent. Several investigations also mentioned the positive effects of NAC on mitochondrial function. In the current study, the effect of NAC treatment on brain mitochondrial indices and energy status was investigated in an animal model of HE.Material and methodsAcetaminophen (APAP)-induced acute liver failure was induced by a single dose of the drug (800 mg/kg, i.p.) to C57BL/6J mice. Plasma and brain levels of NH4+ were measured. Then, brain mitochondria were isolated, and several indices, including mitochondrial depolarization, ATP level, lipid peroxidation, glutathione content, mitochondrial permeabilization, and dehydrogenase activity, were assessed.ResultsA significant increase in plasma and brain NH4+ was evident in APAP-treated animals. Moreover, mitochondrial indices of functionality were impaired, and mitochondrial oxidative stress biomarkers were significantly increased in APAP-treated mice. It was found that NAC treatment (100, 200, and 400 mg/kg, i.p.) significantly mitigated mitochondrial impairment in the brain of APAP-treated animals.ConclusionsThese data suggest the effects of NAC on brain mitochondrial function and energy status as a pivotal mechanism involved in its neuroprotective properties during HE.

Glutamine protection in an experimental model of acetaminophen nephrotoxicity.

Acetaminophen (APAP) is a widely prescribed analgesic and antipyretic drug. In the present work, we studied the effects of glutamine (Gln) in an in vivo model of APAP-induced nephrotoxicity in male Wistar rats. Renal function, histological characteristics, and Na+,K+-ATPase cortical abundance and distribution were analyzed. The appearance of HSP70 and actin in urine was also evaluated. Myeloperoxidase (MPO) activity in cortical tissue was measured as an index of the inflammatory response. Gln administration 30 min before APAP protected from the renal functional and histological damage promoted by APAP. Rats that received the dual treatment Gln and APAP (Gln/APAP) showed the same level of Na+,K+-ATPase cortical induction as APAP-treated animals, but the enzyme maintained its normal basolateral localization. HSP70 abundance was increased up to the same level in the Gln, APAP, and Gln/APAP groups. Urinary HSP70 and actin were detected only in the APAP-treated animals, reinforcing the protection of renal tubular integrity afforded by the Gln pretreatment. Gln pretreatment also protected from the increment in MPO activity promoted by APAP. Our results support the idea that Gln pretreatment could be a therapeutic option to prevent APAP-induced renal injury.

Identification of microRNA biomarker candidates in urine and plasma from rats with kidney or liver damage.

MicroRNAs (miRNA) are short single‐stranded RNA sequences that have a role in the post‐transcriptional regulation of genes. The identification of tissue specific or enriched miRNAs has great potential as novel safety biomarkers. One longstanding goal is to associate the increase of miRNA in biofluids (e.g., plasma and urine) with tissue‐specific damage. Next‐generation sequencing (miR‐seq) was used to analyze changes in miRNA profiles of tissue, plasma and urine samples of rats treated with either a nephrotoxicant (cisplatin) or one of two hepatotoxicants (acetaminophen [APAP] or carbon tetrachloride [CCL4]). Analyses with traditional serum chemistry and histopathology confirmed that toxicant‐induced organ damage was specific. In animals treated with cisplatin, levels of five miRNAs were significantly altered in the kidney, 14 in plasma and six in urine. In APAP‐treated animals, five miRNAs were altered in the liver, 74 in plasma and six in urine; for CCL4 the changes were five, 20 and 6, respectively. Cisplatin treatment caused an elevation of miR‐378a in the urine, confirming the findings of other similar studies. There were 17 in common miRNAs elevated in the plasma after treatment with either APAP or CCL4. Four of these (miR‐122, −802, −31a and −365) are known to be enriched in the livers of rats. Interestingly, the increase of serum miR‐802 in both hepatotoxicant treatments was comparable to that of the well‐known liver damage marker miR‐122. Taken together, comparative analysis of urine and plasma miRNAs demonstrated their utility as biomarkers of organ injury. Copyright © 2016 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.

Hepatoprotective effect of withanolide-rich fraction in acetaminophen-intoxicated rat: decisive role of TNF-α, IL-1β, COX-II and iNOS

Context: Overdose of acetaminophen (APAP) is common in humans and is often associated with hepatic damage. Withania somnifera (L.) Dunal (Solanaceae) shows multiple pharmacological activities including antioxidant and anti-inflammatory potential.Objective: To evaluate the possible mechanism of hepatoprotective activity of withanolide-rich fraction (WRF) isolated from a methanolic extract of Withania somnifera roots.Materials and methods: Hepatotoxicity was induced by oral administration of APAP (750 mg/kg, p.o.) for 14 d. The control group received the vehicle. APAP-treated animals were given either silymarin (25 mg/kg) or graded doses of WRF (50, 100 and 200mg/kg) 2 h prior to APAP administration. Animals were killed on 15th day and blood and liver tissue samples were collected for the further analysis.Results: In WRF-treated group, there was significant and dose-dependent (p < 0.01 and p < 0.001) decrease in serum bilirubin, ALP, AST and ALT levels with significant and dose-dependent (p < 0.01 and p < 0.001) increase in hepatic SOD, GSH and total antioxidant capacity. The level of MDA and NO decreased significantly (p < 0.01) by WRF treatment. Up-regulated mRNA expression of TNF-α, IL-1β, COX-II and iNOS was significantly down-regulated (p < 0.001) by WRF. Histological alternations induced by APAP in liver were restored to near normality by WRF pretreatment.Conclusion: WRF may exert its hepatoprotective action by alleviating inflammatory and oxido-nitrosative stress via inhibition of TNF-α, IL-1β, COX-II and iNOS.

Acetaminophen Modulates P-Glycoprotein Functional Expression at the Blood-Brain Barrier by a Constitutive Androstane Receptor–Dependent Mechanism

Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4-1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp-mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drug-drug interaction that modulates opioid analgesic efficacy.

Acute Exposure to Ozone Exacerbates Acetaminophen-Induced Liver Injury in Mice

Ozone (O(3)), an oxidant air pollutant in photochemical smog, principally targets epithelial cells lining the respiratory tract. However, changes in gene expression have also been reported in livers of O(3)-exposed mice. The principal aim of the present study was to determine if acute exposure to environmentally relevant concentrations of O(3) could cause exacerbation of drug-induced liver injury in mice. Overdose with acetaminophen (APAP) is the most common cause of drug-induced liver injury in developed countries. In the present study, we examined the hepatic effects of acute O(3) exposure in mice pretreated with a hepatotoxic dose of APAP. C57BL/6 male mice were fasted overnight and then given APAP (300 mg/kg ip) or saline vehicle (0 mg/kg APAP). Two hours later, mice were exposed to 0, 0.25, or 0.5 ppm O(3) for 6 h and then sacrificed 9 or 32 h after APAP administration (1 or 24 h after O(3) exposure, respectively). Animals euthanized at 32 h were given 5-bromo-2-deoxyuridine 2 h before sacrifice to identify hepatocytes undergoing reparative DNA synthesis. Saline-treated mice exposed to either air or O(3) had no liver injury. All APAP-treated mice developed marked centrilobular hepatocellular necrosis that increased in severity with time after APAP exposure. O(3) exposure increased the severity of APAP-induced liver injury as indicated by an increase in necrotic hepatic tissue and plasma alanine aminotransferase activity. O(3) also caused an increase in neutrophil accumulation in livers of APAP-treated animals. APAP induced a 10-fold increase in the number of bromodeoxyuridine-labeled hepatocytes that was markedly attenuated by O(3) exposure. Gene expression analysis 9 h after APAP revealed differential expression of genes involved in inflammation, oxidative stress, and cellular regeneration in mice treated with APAP and O(3) compared to APAP or O(3) alone, providing some indications of the mechanisms behind the APAP and O(3) potentiation. These results suggest that acute exposure to near ambient concentrations of this oxidant air pollutant may exacerbate drug-induced liver injury by delaying hepatic repair.

Hepatoprotective Effects of an Anthocyanin Fraction from Purple-Fleshed Sweet Potato Against Acetaminophen-Induced Liver Damage in Mice

The present study was undertaken to examine the protective effects of an anthocyanin fraction (AF) obtained from purple-fleshed sweet potato on acetaminophen (paraceptamol [APAP])-induced hepatotoxicity in mice and to determine the mechanism involved. Mice pretreated with AF prior to APAP administration showed significantly lower increases in serum alanine aminotransferase and aspartate aminotransferase activities and hepatic malondialdehyde formation than APAP-treated animals without AF. In addition, AF prevented hepatic glutathione (GSH) depletion by APAP, and hepatic GSH levels and GSH S-transferase activities were up-regulated by AF. APAP-induced hepatotoxicity was also prevented by AF, as indicated by liver histopathology findings. In addition, the effects of AF were examined on cytochrome P450 (CYP) 2E1, the major isozyme involved in APAP bioactivation. Treatment of mice with AF significantly and dose-dependently reduced CYP2E1-dependent aniline hydroxylation and CYP2E1 protein levels. Furthermore, AF had an antioxidant effect on FeCl(2)/ascorbate-induced lipid peroxidation in mouse liver homogenates and had superoxide radical scavenging activity. These results suggest that AF protects against APAP-induced hepatotoxicity by blocking CYP2E1-mediated APAP bioactivation, by up-regulating hepatic GSH levels, and by acting as a free radical scavenger.

Two-dimensional database of mouse liver proteins: changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisomer 3-acetamidophenol.

Overdose of acetaminophen (APAP) causes acute hepatotoxicity in rodents and man. The mechanism underlying APAP-induced liver injury remains unclear, but experimental evidence strongly suggests that activation of APAP and subsequent formation of protein adducts are involved in hepatotoxicity. Using proteomics technologies, we constructed a two-dimensional protein database for mouse liver, comprising 256 different gene products and investigated the proteins affected after APAP-induced hepatotoxicity. Adult male mice received a single dose of APAP (100 or 300 mg/kg) or its nontoxic regioisomer 3-acetamidophenol (AMAP, 300 mg/kg). The extent of liver damage was assessed 8 h after administration by increased liver enzyme release and histopathology. Changes in the protein level were studied by comparison of the intensities of the corresponding spots on two-dimensional (2-D) gels. The expression level of about 35 of the identified proteins was modified due to treatment with APAP or AMAP. The observed changes were usually in the order of 10-50% of the control value and were more marked in the high- than in the low-dose of APAP-treated animals. Most of the changes caused by AMAP occurred in a subset of the proteins modified by APAP. Many of the proteins showing changed expression levels are either known targets for covalent modification by N-acetyl-p-benzoquinoneimine (NAPQI) or involved in the regulation of mechanisms that are believed to drive APAP-induced hepatotoxicity.

Effect of ascorbic acid esters on hepatic glutathione levels in mice treated with a hepatotoxic dose of acetaminophen

Acetaminophen (APAP) with or without ascorbyl stearate (AS) or ascorbyl palmitate (AP) was administered by gavage to male Swiss-Webster mice at a dose of 600 mg/kg for each chemical. The biochemical markers of hepatotoxicity, serum transaminases (serum glutamate pyruvate transaminase [SGPT], serum glutamate oxaloacetic transaminase [SGOT]) and serum isocitrate dehydrogenase (SICD) activities were monitored after APAP and APAP + AP or AS dosing. There were significant reductions in serum transaminase and SICD activities in the APAP- + ascorbate ester-treated animals as compared to APAP-positive controls. Oral coadministration of APAP with AP or AS did not prevent the initial hepatic GSH depletion (15 min-4 hr postdosing). However, hepatic GSH content began to rise in the APAP + AS or AP-treated animals at 4 hr and reached control values within 12 hr postdosing. Urinary mercapturate conjugates were also significantly higher in the APAP + AP or AS-treated animals as compared to APAP alone when measured over a 60-min postdosing period. Plasma sulfobromophthalein (BSP) retention was approximately eight times higher in APAP-treated animals as compared to the APAP + ascorbate ester treatments indicating maintenance of hepatic excretory functions in presence of AP or AS. Prior depletion of hepatic GSH by diethyl maleate (DEM) did not alter hepatoprotective effects of AP or AS in the presence of APAP. Hepatic ascorbate levels also peaked at 4 hours after APAP + AP or AS treatments. The possible role of L-ascorbic acid esters in GSH regeneration following co-administration of a hepatotoxic dose and APAP is discussed.

Selective acetaminophen metabolite binding to hepatic and extrahepatic proteins: An in vivo and in vitro analysis

Acetaminophen (APAP) administration (600 mg/kg, po) to fasted male CD-1 mice resulted in cellular damage to liver, lung, and kidney. An affinity purified antibody against covalently bound APAP was used to identify APAP-protein adducts in microsomal and cytosolic extracts from these target organs. The proteins were resolved on SDS-PAGE, transblotted to nitrocellulose membranes, and analyzed immunochemically. Covalent binding of APAP to intracellular proteins was only observed in those organs which exhibited cellular damage; no APAP adducts were detected in tissues which did not undergo necrosis. In all target tissues the arylation of proteins was not random but highly selective with two adducts of 44 and 58 kDa accounting for the majority of the total APAP-bound proteins which were detected immunochemically. In addition, a third major APAP-protein adduct of 33 kDa was also observed in kidney cytosol. The severity of tissue damage and the amount of adducts present in these tissues could be significantly reduced when mice were pretreated with the mixed function oxidase inhibitor, piperonyl butoxide, prior to APAP dosing. Immunochemical analysis of plasma from APAP-treated animals indicated the presence of several protein adducts by 4 hr following drug administration. These adducts did not appear to be of plasma origin. Incubation of cytosolic proteins from liver, lung, kidney, spleen, brain, and heart with an APAP metabolite generating liver microsomal system demonstrated that the cytosolic 58-kDa protein target was native to all tissues tested. By contrast, the 58-kDa protein target did not appear to be endogenous to plasma since it was not detected when plasma was incubated in vitro with the liver microsomal system. These studies indicate that, although the 58-kDa proteins appear to be endogenous to both target and nontarget tissues, the 58-kDa APAP-protein adducts are detectable only in tissues which become damaged by APAP.