Hepatic Mrp4 induction following acetaminophen exposure is dependent on Kupffer cell function

During acetaminophen (APAP) hepatotoxicity, increased expression of multidrug resistance-associated proteins 2, 3, and 4 (Mrp2-4) occurs. Mrp4 is the most significantly upregulated transporter in mouse liver following APAP treatment. Although the expression profiles of liver transporters following APAP hepatotoxicity are well characterized, the regulatory mechanisms contributing to these changes remain unknown. We hypothesized that Kupffer cell-derived mediators participate in the regulation of hepatic transporters during APAP toxicity. To investigate this, C57BL/6J mice were pretreated with clodronate liposomes (0.1 ml iv) to deplete Kupffer cells and then challenged with APAP (500 mg/kg ip). Liver injury was assessed by plasma alanine aminotransferase and hepatic transporter protein expression was determined by Western blot and immunohistochemistry. Depletion of Kupffer cells by liposomal clodronate increased susceptibility to APAP hepatotoxicity. Although increased expression of several efflux transporters was observed after APAP exposure, only Mrp4 was found to be differentially regulated following Kupffer cell depletion. At 48 and 72 h after APAP dosing, Mrp4 levels were increased by 10- and 33-fold, respectively, in mice receiving empty liposomes. Immunohistochemistry revealed Mrp4 staining confined to centrilobular hepatocytes. Remarkably, Kupffer cell depletion completely prevented Mrp4 induction by APAP. Elevated plasma levels of TNF-alpha and IL-1beta were also prevented by Kupffer cell depletion. These findings show that Kupffer cells protect the liver from APAP toxicity and that Kupffer cell mediators released in response to APAP are likely responsible for the induction of Mrp4.     

Indocyanine green clearance varies as a function of N-acetylcysteine treatment in a murine model of acetaminophen toxicity

Standard assays to assess acetaminophen (APAP) toxicity in animal models include determination of ALT (alanine aminotransferase) levels and examination of histopathology of liver sections. However, these assays do not reflect the functional capacity of the injured liver. To examine a functional marker of liver injury, the pharmacokinetics of indocyanine green (ICG) were examined in mice treated with APAP, saline, or APAP followed by N-acetylcysteine (NAC) treatment.Male B6C3F1 mice were administered APAP (200 mg/kg IP) or saline. Two additional groups of mice received APAP followed by NAC at 1 or 4 h after APAP. At 24 h, mice were injected with ICG (10 mg/kg IV) and serial blood samples (0, 2, 10, 30, 50 and 75 min) were obtained for determination of serum ICG concentrations and ALT. Mouse livers were removed for measurement of APAP protein adducts and examination of histopathology. Toxicity (ALT values and histology) was significantly increased above saline treated mice in the APAP and APAP/NAC 4 h mice. Mice treated with APAP/NAC 1 h had complete protection from toxicity. APAP protein adducts were increased in all APAP treated groups and were highest in the APAP/NAC 1 h group. Pharmacokinetic analysis of ICG demonstrated that the total body clearance (Cl_T) of ICG was significantly decreased and the mean residence time (MRT) was significantly increased in the APAP mice compared to the saline mice. Mice treated with NAC at 1 h had Cl_T and MRT values similar to those of saline treated mice. Conversely, mice that received NAC at 4 h had a similar ICG pharmacokinetic profile to that of the APAP only mice. Prompt treatment with NAC prevented loss of functional activity while late treatment with NAC offered no improvement in ICG clearance at 24 h. ICG clearance in mice with APAP toxicity can be utilized in future studies testing the effects of novel treatments for APAP toxicity.     

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.     

Effects of acetaminophen on hepatic gene expression in mice

Acetaminophen (APAP) is one of the most commonly used drugs for the safe and effective treatment of fever and pain. However, it is a well-established hepatotoxin. The objective of this study was to identify alternation in various genes in liver of mice after administration of low and high doses of APAP. Male C57BL/6J mice received APAP (30 or 300 mg/kg, i.p.). They were sacrificed after 6 hr and 24 hr for assessment of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total RNA isolation, cDNA microarray analysis and histopathological analysis of liver injury. Low dose of APAP did not cause hepatotoxicity in mice. However, it was toxic at a high dose. Using microarray technology, we selected changed genes more than 1.5 fold. Gene expression changes were recorded even at a low dose treatment with APAP. Six (6) hr after APAP treatment at low dose, 6 genes were up-regulated and 25 genes were down-regulated. However, 24 hr after treatment at low dose 8 genes were up-regulated and 34 genes were down-regulated. 6 hr after of high dose treatment 29 genes were down-regulated and none was up-regulated. A 24 hr treatment with high dose up-regulated 6 genes and down-regulated 18 genes. These expression patterns provide information on high versus low dose mechanisms of APAP toxicity. Gene expression signatures recorded after a nontoxic dose of APAP strongly support the validity of gene expression changes as meaningful markers of hepatotoxicity.     

Pharmacokinetic profile of (+/-)-gossypol in male Sprague-Dawley rats following single intravenous and oral and subchronic oral administration

The pharmacokinetic profile of (+/-)-gossypol was determined in male Sprague-Dawley rats following a single intravenous or oral 10 mg/kg dose and after receiving a daily oral 10 mg/kg dose for 14 days. The intravenous plasma (+/-)-gossypol level data were fitted with a three-compartment, open-model system. The apparent half-life of elimination of (+/-)-gossypol following intravenous administration was 11.44 hr, corresponding to an elimination rate constant of 0.05 hr-1. The total plasma clearance (Cl), volume of distribution (Vd), and AUCplasma following a single intravenous administration were 0.16 liter/hr/kg, 0.05 liter/kg, and 63.09 mg.hr/liter, respectively. The bioavailability of a single oral dose of (+/-)-gossypol in rats was 60%. The change in plasma (+/-)-gossypol concentration after a single or after multiple doses showed a biphasic pattern. A single oral dose of (+/-)-gossypol, however, was eliminated five times faster than the daily administered chemical. Thus, a single oral dose of (+/-)-gossypol was eliminated at a rate constant of 0.01 hr-1, corresponding to half-life of 64.76 hr. Subchronic oral administration of (+/-)-gossypol showed an apparent half-life of 101.91 hr-1, corresponding to a rate constant of 0.007 hr-1. The results indicate that multiple oral dosing of (+/-)-gossypol resulted in its longer retention in body tissue than a single oral dose. This study suggests that pharmacokinetics of (+/-)-gossypol may play, at least in part, a role in the reproductive toxicity of subchronic but not single oral dosing.     

Aminotriazole Alleviates Acetaminophen Poisoning via Downregulating P450 2E1 and Suppressing Inflammation

Aminotriazole (ATZ) is commonly used as a catalase (CAT) inhibitor. We previously found ATZ attenuated oxidative liver injury, but the underlying mechanisms remain unknown. Acetaminophen (APAP) overdose frequently induces life-threatening oxidative hepatitis. In the present study, the potential hepatoprotective effects of ATZ on oxidative liver injury and the underlying mechanisms were further investigated in a mouse model with APAP poisoning. The experimental data indicated that pretreatment with ATZ dose- and time-dependently suppressed the elevation of plasma aminotransferases in APAP exposed mice, these effects were accompanied with alleviated histological abnormality and improved survival rate of APAP-challenged mice. In mice exposed to APAP, ATZ pretreatment decreased the CAT activities, hydrogen peroxide (H₂O₂) levels, malondialdehyde (MDA) contents, myeloperoxidase (MPO) levels in liver and reduced TNF-α levels in plasma. Pretreatment with ATZ also downregulated APAP-induced cytochrome P450 2E1 (CYP2E1) expression and JNK phosphorylation. In addition, posttreatment with ATZ after APAP challenge decreased the levels of plasma aminotransferases and increased the survival rate of experimental animals. Posttreatment with ATZ had no effects on CYP2E1 expression or JNK phosphorylation, but it significantly decreased the levels of plasma TNF-α. Our data indicated that the LD₅₀ of ATZ in mice was 5367.4 mg/kg body weight, which is much higher than the therapeutic dose of ATZ in the present study. These data suggested that ATZ might be effective and safe in protect mice against APAP-induced hepatotoxicity, the beneficial effects might resulted from downregulation of CYP2E1 and inhibiton of inflammation.     

Liquiritigenin, an aglycone of liquiritin in Glycyrrhizae radix, prevents acute liver injuries in rats induced by acetaminophen with or without buthionine sulfoximine

Glycyrrhizae radix has been used as one of the oldest and most frequently employed botanicals in both western and oriental countries. Previously, we showed that liquiritigenin (LQ), an aglycone of liquiritin in G. radix, exerts cytoprotective effects against heavy metal-induced toxicity in vitro. This study investigated in vivo protective effects of LQ against acute liver injuries induced by acetaminophen (APAP) or APAP plus buthionine sulfoximine (BSO). Liver injuries were assessed by blood biochemistry and histopathology in rats administered with LQ purified from the acid hydrolyates of liquiritin singly (p.o. or i.v., 2-4 days) or in combination with dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate (DDB), a synthetic derivative of Schisandrin C in Fructus shizandrae, and exposed to APAP or APAP + BSO. LQ treatments (oral) effectively decreased liver injuries induced by a single dose of APAP, as evidenced by decreases in hepatic necrosis and inflammation as well as plasma alanine aminotransferase and lactate dehydrogenase activities. LQ, when intravenously applied, enhanced hepatoprotective effect with a greater potency. APAP + BSO led to severe liver injuries, resulting in lethality. LQ pretreatments significantly reduced the potentiated liver necrosis, decreasing mortality. In spite of the improvement in blood biochemistry, DDB failed to protect the liver from injuries induced by APAP or APAP + BSO. Combined treatments of rats with LQ and DDB showed some additive protective effect. The present study demonstrates that LQ efficaciously protects the liver from acute injuries induced by APAP or from APAP-induced severe injuries during GSH deficiency, indicating that LQ is one of the principal cytoprotective components comprised in G. radix.     

Nobiletin attenuates acetaminophen‐induced hepatorenal toxicity in rats

The study aimed to examine the effects of nobiletin on the toxicity model induced with acetaminophen (APAP). For this purpose, 24 adult male rats were equally divided into four groups. The groups were the control group (group 1); dimethyl sulfoxide only, the APAP group (group 2) received a single dose of APAP 1000 mg/kg on the 10th day of experiment; the Nobiletin group (group 3), nobiletin (10 mg/kg) for 10 days; and the APAP + Nobiletin group (group 4), nobiletin (10 mg/kg) for 10 days with a single dose of APAP (1000 mg/kg) administered on the 10th day and the experiment ended after 48 hours. At the end of the study, a significant increase in malondialdehyde, interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), and tumor necrosis factor‐α (TNF‐α) levels and a significant decrease in glutathione levels, glutathione peroxidase activities and nuclear factor erythroid‐derived 2‐like 2 (Nrf‐2) and heme oxygenase‐1 (HO‐1) expressions were observed with APAP application in liver and kidney tissues. Serum aspartate transaminase (AST), alanine transaminase (ALT), urea, and creatinine levels were also significantly increased in the APAP group. However, nobiletin treatment in group 4 reversed oxidative stress and inflammatory and histopathological signs caused by APAP. It is concluded that nobiletin may be a beneficial substance that confers hepatorenal protection to APAP‐induced toxicity via antioxidant and anti‐inflammatory mechanisms.     

Comparison of the Effects of Antidepressants and Their Metabolites on Reuptake of Biogenic Amines and on Receptor Binding

1. The present survey compares the effects of antidepressants and their principal metabolites on reuptake of biogenic amines and on receptor binding. The following antidepressants were included in the study: the tricyclic antidepressants amitriptyline, dothiepin, and lofepramine and the atypical antidepressant bupropion, which all have considerable market shares in the UK and/or US markets; the selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline; and the recently approved antidepressants venlafaxine and nefazodone.2. Amitriptyline has similar in vitro reuptake inhibitory potencies for 5-HT and NA, whereas the metabolite nortriptyline is preferentially a NA reuptake inhibitor. Both amitriptyline and nortriptyline are also 5-HT₂ receptor antagonists.3. Dothiepin has equipotent 5-HT and NA reuptake inhibitory activity, whereas northiaden shows a slight selectivity for NA reuptake inhibition. Dothiepin and northiaden are also 5-HT₂ receptor antagonists. The slow elimination rate of northiaden (36–46 hr) compared to dothiepin (14–24 hr) suggests that northiaden contributes significantly to the therapeutic effect of dothiepin.4. Lofepramine is extensively metabolized to desipramine. Desipramine plays an important role in the antidepressant activity of lofepramine, as the plasma elimination half-life of lofepramine (4–6 hr) is much shorter than that of desipramine (24 hr). Both compounds are potent and selective inhibitors of NA reuptake.5. The five approved SSRIs, citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline, are potent 5-HT reuptake inhibitors, and the demethyl metabolites, norfluoxetine, demethylsertraline, and demethylcitalopram, also show selectivity. Paroxetine and sertraline are the most potent inhibitors of 5-HT reuptake, whereas citalopram is the most selective. Fluoxetine is the least selective and the metabolite of fluoxetine, norfluoxetine, is a more selective and more potent 5-HT reuptake inhibitor than the parent compound and has an extremely long half-life (7–15 compared to 1–3 days). Thus the metabolite plays an important role for the therapeutic effect of fluoxetine. Fluoxetine is also a 5-HT_2C receptor antagonist. Demethylsertraline is a weaker and less selective 5-HT reuptake inhibitor in vitro than sertraline, but demethylsertraline has a very long half-life (62–104 hr) compared to the parent compound (24 hr) and it might play a role in the therapeutic effects of sertraline. Demethylcitalopram has about a 10 times lower 5-HT reuptake inhibitory potency in vitro than citalopram, and the elimination half-lives are approximately 1.5 and 2 days, respectively.6. Bupropion and hydroxybupropion are weak inhibitors of biogenic amine reuptake. The mechanisms of action responsible for the clinical effects of bupropion are not fully understood, but it has been suggested that both dopaminergic and noradrenergic components play a role and that the hydroxybupropion metabolite contributes significantly to the antidepressant activity.7. Venlafaxine and O-demethylvenlafaxine are weak inhibitors of 5-HT and NA reuptake, and the selectivity ratios are close to one. O-Demethylvenlafaxine is eliminated more slowly than venlafaxine (plasma half-lives of 5 and 11 hr, respectively), and it is likely that it contributes to the overall therapeutic effect of venlafaxin.8. Nefazodone and -hydroxynefazodone are equipotent 5-HT and NA reuptake inhibitors. Both compounds are also 5-HT₂ receptor antagonists. Both parent compound and metabolite have short elimination half-lives.     

Identification of novel toxicity-associated metabolites by metabolomics and mass isotopomer analysis of acetaminophen metabolism in wild-type and Cyp2e1-null mice

CYP2E1 is recognized as the most important enzyme for initiation of acetaminophen (APAP)-induced toxicity. In this study, the resistance of Cyp2e1-null mice to APAP treatment was confirmed by comparing serum aminotransferase activities and blood urea nitrogen levels in wild-type and Cyp2e1-null mice. However, unexpectedly, profiling of major known APAP metabolites in urine and serum revealed that the contribution of CYP2E1 to APAP metabolism decreased with increasing APAP doses administered. Measurement of hepatic glutathione and hydrogen peroxide levels exposed the importance of oxidative stress in determining the consequence of APAP overdose. Subsequent metabolomic analysis was capable of constructing a principal components analysis (PCA) model that delineated a relationship between urinary metabolomes and the responses to APAP treatment. Urinary ions high in wild-type mice treated with 400 mg/kg APAP were elucidated as 3-methoxy-APAP glucuronide (VII) and three novel APAP metabolites, including S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid (VI, formed by a Cys-APAP transamination reaction in kidney), 3,3'-biacetaminophen (VIII, an APAP dimer), and a benzothiazine compound (IX, originated from deacetylated APAP), through mass isotopomer analysis, accurate mass measurement, tandem mass spectrometry fragmentation, in vitro reactions, and chemical treatments. Dose-, time-, and genotype-dependent appearance of these minor APAP metabolites implied their association with the APAP-induced toxicity and potential biomarker application. Overall, the oxidative stress elicited by CYP2E1-mediated APAP metabolism might significantly contribute to APAP-induced toxicity. The combination of genetically modified animal models, mass isotopomer analysis, and metabolomics provides a powerful and efficient technical platform to characterize APAP-induced toxicity through identifying novel biomarkers and unraveling novel mechanisms.     

Hydroxysafflor yellows alleviate thrombosis and acetaminophen-induced toxicity in vivo by enhancing blood circulation and poison excretion

BackgroundHydroxysafflor yellow A (HSYA) from the flower of Carthamus tinctorius (Safflower) has been reported to have various pharmacological effects. However, little is known about the bioactivities of other chemical constituents in Safflower and the relationship between enhancement of blood circulation and hepatoprotection by HSYA.PurposeThe present research was to evaluate the antithrombotic and hepatoprotective activities of HSYA and C, examine their mechanisms of actions, including influence on the excretion velocity of acetaminophen, and the relationship between the antithrombotic, hepatoprotective, and other bioactivities.MethodsThe hepatoprotective activities were examined by acetaminophen (APAP)-induced zebrafish toxicity and carbon tetrachloride (CCl₄)-induced mouse liver injury. The concentrations of APAP in zebrafish and APAP that was excreted to the culture media were quantified by UHPLC-MS. The anti-thrombosis effect of HSYA and C were examined by the phenylhydrazine (PHZ)-induced zebrafish thrombosis.ResultsHSYA and HSYC showed robust protection on APAP-induced toxicity and PHZ-induced thrombosis. The hepatoprotective effects of HSYA and C were more potent than that of the positive control, acetylcysteine (61.7% and 58.0%, respectively, vs. 56.9% at 100 µM) and their antithrombosis effects were more robust than aspirin (95.1% and 86.2% vs. 52.7% at 100 µM). HSYA and C enhanced blood circulation, rescued APAP-treated zebrafish from morphological abnormalities, and mitigated APAP-induced toxicity in liver development in liver-specific RFP-expressing transgenic zebrafish. HSYC attenuated CCl₄-induced mouse liver injury and regulated the levels of HIF-1α, iNOS, TNF-α, α-SMA, and NFκB in liver tissues. HSYA was also protective in a dual thrombotic and liver toxicity zebrafish model. By UHPLC-MS, HSYA accelerated the excretion of APAP.ConclusionHSYA and C are the bioactive constituents of Safflower that are responsible for the herbal drug's traditional use in promoting blood circulation to remove blood stasis. Safflower and its chalcone constituents may protect from damage due to exogenous or disease-induced endogenous toxins by enhancing the excretion velocity of toxins.     

New Therapeutic Approach: Diphenyl Diselenide Reduces Mitochondrial Dysfunction in Acetaminophen-Induced Acute Liver Failure

The acute liver failure (ALF) induced by acetaminophen (APAP) is closely related to oxidative damage and depletion of hepatic glutathione, consequently changes in cell energy metabolism and mitochondrial dysfunction have been observed after APAP overdose. Diphenyl diselenide [(PhSe)₂], a simple organoselenium compound with antioxidant properties, previously demonstrated to confer hepatoprotection. However, little is known about the protective mechanism on mitochondria. The main objective of this study was to investigate the effects (PhSe)₂ to reduce mitochondrial dysfunction and, secondly, compare in the liver homogenate the hepatoprotective effects of the (PhSe)₂ to the N-acetylcysteine (NAC) during APAP-induced ALF to validate our model. Mice were injected intraperitoneal with APAP (600 mg/kg), (PhSe)₂ (15.6 mg/kg), NAC (1200 mg/kg), APAP+(PhSe)₂ or APAP+NAC, where the (PhSe)₂ or NAC treatment were given 1 h following APAP. The liver was collected 4 h after overdose. The plasma alanine and aspartate aminotransferase activities increased after APAP administration. APAP caused a remarkable increase of oxidative stress markers (lipid peroxidation, reactive species and protein carbonylation) and decrease of the antioxidant defense in the liver homogenate and mitochondria. APAP caused a marked loss in the mitochondrial membrane potential, the mitochondrial ATPase activity, and the rate of mitochondrial oxygen consumption and increased the mitochondrial swelling. All these effects were significantly prevented by (PhSe)₂. The effectiveness of (PhSe)₂ was similar at a lower dose than NAC. In summary, (PhSe)₂ provided a significant improvement to the mitochondrial redox homeostasis and the mitochondrial bioenergetics dysfunction caused by membrane permeability transition in the hepatotoxicity APAP-induced.     

Non-human primate (baboon) anti-gross cystic disease fluid protein-15 antibody infusion in four women with metastatic breast carcinoma

Summary Four women with metastatic breast carcinoma and elevated plasma levels of human breast gross cystic disease fluid protein of 15,000 dalton monomer size (GCDFP-15) were treated IV with non-human primate (baboon) anti-GCDFP-15 antibody. Three patients were given a single IV infusion of antibody, while the fourth patient received four sequential IV infusions. Antibody dosage patients, after antibody infusion the plasma level of GCDFP-15 decreased to 0 ng/ml and remained there as long as free circulating anti-GCDFP-15 antibody was present. The plasma half-life of the antibody ranged between 1 and 40 h and the duration of detectable free antibody ranged from 6 to 240 h. No toxicity was observed for the dosage range of antibody tested. No anti-baboon antibody response was detected. In the patient who received four sequential infusions of antibody partial regression of subcutaneous metastatic nodules occurred. The other three patients showed no clinically detectable changes from the antibody infusion.Funded in part by a grant from the William J. Matheson Foundation     

p-Aminophenol-induced liver toxicity: tentative evidence of a role for acetaminophen

p-Aminophenol (PAP) is a widely used industrial chemical and a metabolite of analgesics, such as acetaminophen (APAP). It was found recently that PAP, a known nephrotoxicant, could cause acute hepatotoxicity in mice but not in rats. The mechanism of hepatotoxicity is not known. The aim of this study was to investigate the role of N-acetylation of PAP to APAP in PAP-induced toxicity. Male C57BL/6 mice injected intraperitoneally (i.p.) with various doses of PAP were sacrificed at 12 hours for measurement of serum glutamic-pyruvic transaminase (GPT) and sorbitol dehydrogenase (SDH) levels and determination of the extent of hepatic nonprotein sulfhydryl (NPSH) and glutathione (GSH) depletion. Plasma levels of APAP and its metabolites were measured by HPLC after PAP administration. p-Aminophenol depleted NPSH in a dose- and time-dependent manner. Depletion of NPSH in mouse liver occurred at PAP doses above 400 mg/kg. Buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, potentiated the PAP-induced hepatotoxicity. Ascorbate, a reducing agent, did not affect PAP-induced hepatotoxicity and NPSH depletion. After PAP treatment, APAP and its sulfate and glucuronide conjugates as well as GSH conjugates (APAP-cysteine and APAP-mercapturate) were detected in the plasma. The results suggest the roles of GSH and N-acetylation of PAP to APAP in PAP-induced hepatotoxicity.     

Variation in the biological half-life of methylmercury in humans: Methods,measurements and meaning

BackgroundUnderstanding methylmercury (MeHg) toxicity requires a complete understanding of its fundamental toxicokinetic and toxicodynamic characteristics in the human body. The biological half-life (t_1/2) of MeHg is a kinetic property that directly influences the body burden of Hg that results from repeated exposures such as can occur with fish and seafood consumption. The t_1/2 of MeHg in humans is approximately 50 days, equivalent to an elimination rate (k_el) of 0.014 day⁻¹. However, numerous studies report a wide range of half-life values (t_1/2 < 30 to >120 days), demonstrating that significant variation in the biological process of MeHg elimination exists. This variation is a source of considerable uncertainty in deriving a meaningful reference dose for MeHg applicable to all individuals in a population.Scope of reviewFirst, we summarize fundamentals of MeHg toxicokinetics, emphasizing the central role that biological half-life plays in MeHg dosimetry. We next present important considerations for how kinetic analyses are performed. We provide an example of how MeHg half-life variation directly influences the body burden and, in certain contexts, can result in MeHg levels exceeding the US EPA Reference Dose. We then survey existing studies that report MeHg half-life determinations in people.Major conclusionsRecent advances in methods of determining MeHg kinetics in people have made individualized assessment of MeHg elimination rates more accurate and readily obtainable.General significanceCharacterization of MeHg half-life, particularly in vulnerable individuals, such as pregnant women and children, will diminish the remaining toxicokinetic uncertainty surrounding MeHg exposures and will better inform the risk assessment process.     

Involvement of Toll-like receptor 4 in acetaminophen hepatotoxicity

The objective of this study was to determine whether Toll-like receptor 4 (TLR4) has a role in alcohol-mediated acetaminophen (APAP) hepatotoxicity. TLR4 is involved in the inflammatory response to endotoxin. Others have found that ethanol-mediated liver disease is decreased in C3H/HeJ mice, which have a mutated TLR4 resulting in a decreased response to endotoxin compared with endotoxin-responsive mice. In the present study, short-term (1 wk) pretreatment with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, caused no histologically observed liver damage in either C3H/HeJ mice or endotoxin-responsive C3H/HeN mice, despite an increase in nitrotyrosine levels in the livers of C3H/HeN mice. In C3H/HeN mice pretreated with the alcohols, subsequent exposure to APAP caused a transient decrease in liver nitrotyrosine formation, possibly due to competitive interaction of peroxynitrite with APAP producing 3-nitroacetaminophen. Treatment with APAP alone resulted in steatosis in addition to congestion and necrosis in both C3H/HeN and C3H/HeJ mice, but the effects were more severe in endotoxin-responsive C3H/HeN mice. In alcohol-pretreated endotoxin-responsive C3H/HeN mice, subsequent exposure to APAP resulted in further increases in liver damage, including severe steatosis, associated with elevated plasma levels of TNF-alpha. In contrast, alcohol pretreatment of C3H/HeJ mice caused little to no increase in APAP hepatotoxicity and no increase in plasma TNF-alpha. Portal blood endotoxin levels were very low and were not detectably elevated by any of the treatments. In conclusion, this study implicates a role of TLR4 in APAP-mediated hepatotoxicity.     

Tissue distribution,metabolism, and elimination of perfluorooctanoic acid in male and female rats

The elimination, tissue distribution, and metabolism of [1-¹⁴C]perfluorooctanoic acid (PFOA) was examined in male and female rats for 28 days after a single ip dose (9.4 μmol/kg, 4 mg/kg). A sex difference in urinary elimination of PFOA-derived ¹⁴C was observed. Female rats eliminated PFOA-derived radioactivity rapidly in the urine with 91% of the dose being excreted in the first 24 hr. In the same period, male rats eliminated only 6% of the administered ¹⁴C in the urine. The sex-related difference in urinary elimination resulted in the observed difference in the whole-body elimination half-life (t_1/2) of PFOA in males (t_1/2 = 15 days) and females (t_1/2 < 1 day). Analysis of PFOA-derived ¹⁴C in tissues showed that the liver and plasma of male rats and the liver, plasma, and kidney of female rats were the primary tissues of distribution. The relatively high concentration of PFOA in the male liver was further examined using an in situ nonrecirculating liver perfusion technique. It was shown that 11% of the PFOA infused was extracted by the liver in a single pass. The ability of the liver to eliminate PFOA into bile was examined in rats whose renal pedicles were ligated to alleviate sex differences in the urinary excretion of PFOA. In a 6-hr period following IP administration of PFOA, there was no apparent difference in biliary excretion, where both males and females eliminated less than 1% of the PFOA dose via this route. We hypothesized that the sex difference in the persistence of PFOA was due to a more rapid formation of a PFOA-containing lipid (i.e., a PFOA-containing mono-, di-, or triacylglycerol, cholesteryl ester, methyl ester, or phospholipid) in the male rat. Also, the increased urinary elimination of PFOA in females may have been due to increased metabolism to a PFOA-glucuronide or sulfate ester. However, no evidence that PFOA is conjugated to form a persistent hybrid lipid was obtained, nor were polar metabolites of PFOA in urine or bile detected. In addition, daily urinary excretion of fluoride in male and female rats before or after PFOA treatment were similar, suggesting that the parent compound is not defluorinated. Thus, the more rapid elimination of PFOA from female rats is not due to formation of a PFOA metabolite.     

beta-hydroxy-beta-methylbutyrate (HMB) kinetics and the influence of glucose ingestion in humans

The dietary supplement, beta-hydroxy-beta-methylbutyrate (HMB), has been shown to decrease muscle proteolysis during the stress of exercise and disease. The aim of this investigation was to determine the time course kinetics of HMB and to determine whether oral glucose ingestion alters the kinetics. In Study 1, eight males (32 +/- 10 yrs) participated in two randomize trials: 1) oral ingestion of 1g of HMB with water in capsule form (HMB), and 2) placebo. Blood samples were obtained prior to ingestion of treatment and at 30, 60, 90, 120, 150, and 180 min for the measurement of plasma HMB. Additional blood samples were obtained at 6, 9, and 12 hr. Urine was collected prior to ingestion and at 3, 6, 9, and 12 h for the measurement of urinary HMB. In Study 2, eight males (25 +/- 6 yrs) followed the same study design and testing procedure as for Study 1. Treatments were 1) modified glucose tolerance test (75 g glucose) (GLU), 2) oral ingestion of 3 g of HMB with water (HMB), and 3) ingestion of 3 g of HMB with 75 g of glucose (HMB+GLU). Blood samples were analyzed for insulin, glucose, and HMB. Additional blood samples were obtained at 24h and 36h for the measurement of HMB. Additional urine samples were collected at 24h and 36h. In Study 1, plasma HMB peaked at 120 nmol/ml at 2.0 +/- 0.4 hr in HMB trial. Half-life was 2.37 +/- 0.1 hr. Following the consumption of 1g of HMB, approximately 14% of the HMB consumed accumulated in the urine. In Study 2, plasma glucose and insulin levels were significantly greater in GLU and HMB+GLU treated subjects compared to HMB treated subject at minutes 30, 60 and 90. Plasma HMB peaked at 487.9 +/- 19.0 nmol/ml at 1.0 +/- 0.1 hr in the HMB treated subjects and at 352.1 +/- 15.3 nmol/ml at 1.94 +/- 0.2 hr when subjects consumed HMB+GLU. The time to reach peak was different (P <0.001) between HMB and HMB+GLU. The plasma HMB half-life was less (P = 0.08) 2.38 +/- 0.1 hr in HMB trial compared to 2.69 +/- 0.2 hr in HMB+GLU trial. Area under the plasma HMB curve during the first 3 hr was less (P = 0.002) in the HMB+GLU trial compared to the HMB trial. From 3 h through 36 h the area under the HMB curve tended to be less (P = 0.106) for the HMB+GLU compared to the HMB alone. HMB accumulation in the urine as well as the area under the curve were similar with both HMB (94875.8 +/- 15159.5 nmol/36 hrs) and HMB+GLU (80678.2 +/- 3863.1 nmol/36 hrs). The percentage of the HMB dose that accumulates in the urine was 27% for HMB+GLU and 29% for HMB alone. In conclusion, HMB plasma levels peak within 60 to 120 min depending on the amount of HMB consumed and whether glucose is consumed with HMB. The plasma half-life is approximately 2.5 hr. Plasma HMB reaches baseline levels at approximately 9 hr following ingestion. However, 70 to 85% of the ingested oral HMB is retained in the body for further metabolism.     

Identification of Novel Translational Urinary Biomarkers for Acetaminophen-Induced Acute Liver Injury Using Proteomic Profiling in Mice

Drug-induced liver injury (DILI) is the leading cause of acute liver failure. Currently, no adequate predictive biomarkers for DILI are available. This study describes a translational approach using proteomic profiling for the identification of urinary proteins related to acute liver injury induced by acetaminophen (APAP). Mice were given a single intraperitoneal dose of APAP (0–350 mg/kg bw) followed by 24 h urine collection. Doses of ≥275 mg/kg bw APAP resulted in hepatic centrilobular necrosis and significantly elevated plasma alanine aminotransferase (ALT) values (p<0.0001). Proteomic profiling resulted in the identification of 12 differentially excreted proteins in urine of mice with acute liver injury (p<0.001), including superoxide dismutase 1 (SOD1), carbonic anhydrase 3 (CA3) and calmodulin (CaM), as novel biomarkers for APAP-induced liver injury. Urinary levels of SOD1 and CA3 increased with rising plasma ALT levels, but urinary CaM was already present in mice treated with high dose of APAP without elevated plasma ALT levels. Importantly, we showed in human urine after APAP intoxication the presence of SOD1 and CA3, whereas both proteins were absent in control urine samples. Urinary concentrations of CaM were significantly increased and correlated well with plasma APAP concentrations (r = 0.97; p<0.0001) in human APAP intoxicants, who did not present with elevated plasma ALT levels. In conclusion, using this urinary proteomics approach we demonstrate CA3, SOD1 and, most importantly, CaM as potential human biomarkers for APAP-induced liver injury.     

Unequal Twice-Daily,Sustained-Release Theophylline Dosing in Chronic Obstructive Pulmonary Disease

Three regimens of sustained-release theophylline (SRT), Theostat® were administered to 12 male patients with chronic obstructive pulmonary disease in a randomized cross-over trial. Each 7-day treatment consisted of

treatment A—8 mg/kg at 0700 hr and 4 mg/kg at 1900 hr

treatment B—6 mg/kg at 0700 hr and 6 mg/kg at 1900 hr

treatment C—4 mg/kg at 0700 hr and 8 mg/kg at 1900 hr.

Peak expiratory flow (PEF) was recorded each day at 0700, 1100, 1500, 1900 and 2300 hr and theophylline plasma levels were determined on the 7th day of each treatment sequence. Cosinor analysis of the data revealed significant circadian rhythms in PEF for each treatment: the mesor (24-hr average) was significantly higher with C and acrophases (Φ, peak time of PEF rhythm) were located at 1426 hr for A and 1425 hr for C; a shift of the acrophase to an earlier timing was detected for B (Φ = 0958 hr. These findings suggest that an unequal, twice-daily SRT dosing with the greater amount of drug at night may be beneficial in the treatment of COPD.