Metabolite Of Acetaminophen Research Articles

Targeting hepatic thioredoxin reductase by the acetaminophen metabolite N‐acetyl‐p‐benzoquinone (844.15)

Acetaminophen is biotransformed by cytochrome P450's in hepatocytes to an electrophilic metabolite, N‐acetyl‐p‐benzoquinone imine (NAPQI). This reactive species is known to bind to glutathione and a variety of hepatic proteins, an important downstream event mediating acetaminophen hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a critical cellular antioxidant which contains a selenocysteine in its C‐terminal redox center, an accessible target for electrophilic modification. In the present studies, we characterized the reaction of NAPQI with TrxR. Acetaminophen treatment of mice (300 mg/kg, i.p.) caused a marked time‐dependent inhibition of the activities of both cytosolic TrxR1 and mitochondrial TrxR2. Using purified rat liver TrxR1, we found that inactivation of the enzyme was correlated with APAP metabolism by P450s. This suggested that enzyme inhibition was a result of the formation of reactive metabolites of acetaminophen. To examine the mechanism underlying the inhibitory actions of acetaminophen, the effects of NAPQI on purified TrxR were characterized. We found that NAPQI‐mediated enzyme inhibition required reduced TrxR. A recombinant mutant enzyme, in which selenocysteine was replaced with cysteine was only weakly inhibited by NAPQI indicating that selenocysteine residues in the redox motifs of the enzyme are critical for enzyme inactivation. This was supported by our findings that alkylation of TrxR with biotin‐conjugated iodoacetamide, which selectively reacts with selenol or thiol groups on proteins, was inhibited by NAPQI. Moreover, LC‐MS/MS analysis confirmed that NAPQI selectively modified cysteine 59, cysteine 497 and selenocysteine 498 residues in the redox centers of TrxR, resulting in enzyme inhibition. Collectively, these data demonstrate that reactive intermediates of acetaminophen can target TrxR demonstrating a novel mechanism by which this analgesic induces oxidative stress and hepatotoxicity.Grant Funding Source: Supported by AR055073, ES004738, CA132624, and ES005022.

Baccharis trimera Improves the Antioxidant Defense System and Inhibits iNOS and NADPH Oxidase Expression in a Rat Model of Inflammation

Acetaminophen is a common analgesic and antipyretic compound which, when administered in high doses, has been associated with significant morbidity and mortality, secondary to hepatic toxicity. Although this may be due to a direct interaction of reactive acetaminophen metabolites with hepatocyte proteins, recent studies have suggested that reactive species produced by neutrophils also contribute to the pathophysiological process. Researches on the chemical composition of B. trimera show that this plant has bioactive compounds such as flavonoids, related to the organism's protection against free radicals. Therefore, in the present study, using Fischer rats, the effect of B. trimera on the antioxidant defense system, the production of nitric oxide (NO) and on the expression of nitric oxide synthase (iNOS), superoxide dismutase (SOD), catalase (CAT) and of the subunits of the NADPH oxidase in neutrophils was evaluated in a model of phagocytosis induced by zimosan (ZC3b) and in a model of inflammation induced by acetaminophen. The results show that the treatment with B. trimera improves the defense system of antioxidant and restores the balance ROS / NO that is altered in the inflammatory process induced by APAP. In conclusion, B. trimera extracts exert antioxidant properties by scavenging ROS and decrease the expression of genes responsible by reactive species production in neutrophils.

Evaluation of Human Hepatocytes Cultured by Three-dimensional Spheroid Systems for Drug Metabolism

We investigated the utility of three-dimensional (3D) spheroid cultures of human hepatocytes in discovering drug metabolites. Metabolites of acetaminophen, diclofenac, lamotrigine, midazolam, propranolol and salbutamol were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) to measure enzyme activities in this system cultured for 2 and 7 days. Sequential metabolic reactions by Phase I and then Phase II enzymes were found in diclofenac [CYP2C9 and UDP-glucuronyltransferases (UGTs)], midazolam (CYP3A4 and UGTs) and propranolol (CYP1A2/2D6 and UGTs). Moreover, lamotrigine and salbutamol were metabolized to lamotrigine-N-glucuronide and salbutamol 4-O-sulfate, respectively. These metabolites, which are human specific, could be observed in clinical studies, but not in conventional hepatic culture systems as in previous reports. Acetaminophen was metabolized to glucuronide and sulfate conjugates, and N-acetyl-p-benzo-quinoneimine (NAPQI) and its metabolites were not observed. In addition, mRNA of drug-metabolism enzymes [CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, UGT1A1, UGT2B7, sulfotransferase 1A1 (SULT1A1) and glutathione S-transferase pi 1 (GSTP1)], which were measured by qRT-PCR, were expressed in the human hepatocyte spheroids. In conclusion, these results suggest that human hepatocyte spheroids are useful in discovering drug metabolites.

Effect of human glutathione S-transferase hGSTP1-1 polymorphism on the detoxification of reactive metabolites of clozapine, diclofenac and acetaminophen

Recent association studies suggest that genetically determined deficiencies in GSTs might be a risk factor for idiosyncratic adverse drug reactions resulting from the formation of reactive drug metabolites. hGSTP1-1 is polymorphic in the human population with a number of single nucleotide polymorphisms that yield an amino acid change in the encoded protein. Three allelic variants of hGSTP1-1 containing an Ile105Val or Ala114Val substitution, or a combination of both, have been the most widely studied and showed different activity when compared to wild-type hGSTP1-1*A (Ile105/Ala114). In the present study, we studied the ability of these allelic variants to catalyze the GSH conjugation of reactive metabolites of acetaminophen, clozapine, and diclofenac formed by bioactivation in in vitro incubations by human liver microsomes and drug metabolizing P450 BM3 mutants. The results show that effects of the change of amino acid at residue 105 and 114 on conjugation reactions were substrate dependent. A single substitution at residue 105 affects the ability to catalyze GSH conjugation, while when both residue 105 and 114 were substituted the effect was additionally enhanced. Single mutation at position 114 did not show a significant effect. The different hGSTP1-1 mutants showed slightly altered regioselectivities in formation of individual GSH conjugates of clozapine which suggests that the binding orientation of the reactive nitrenium ion of clozapine is affected by the mutations. For diclofenac, a significant decrease in activity in GSH-conjugation of diclofenac 1′,4′-quinone imine was observed for variants hGSTP1-1*B (Val105/Ala114) and hGSTP1-1*C (Val105/Val114). However, since the differences in total GSH conjugation activity catalyzed by these allelic variants were not higher than 30%, differences in inactivation of reactive intermediates by hGSTP1-1 are not likely to be a major factor in determining interindividual difference in susceptibility to adverse drug reactions induced by the drugs studied.

Synthesis and characterization of combined cross-linked laccase and tyrosinase aggregates transforming acetaminophen as a model phenolic compound in wastewaters

Laccase (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1) are ubiquitous enzymes present in nature as they are known to originate from bacteria, fungi, plants, etc. Both laccase and tyrosinase are copper-containing phenoloxidases requiring readily available O2 without auxiliary cofactor for their catalytic transformation of numerous phenolic substrates. In the present study, laccase and tyrosinase have been insolubilized as combined crosslinked enzyme aggregates (combi-CLEA) using chitosan, a renewable and biodegradable polymer, as crosslinker. The combi-CLEA, with specific activity of 12.3U/g for laccase and 167.4U/g for tyrosinase, exhibited high enzymatic activity at pH5–8 and temperature at 5–30°C, significant resistance to denaturation and no diffusional restriction to its active site based upon the Michaelis–Menten kinetic parameters. Subsequently, the combi-CLEA was applied to the transformation of acetaminophen as a model phenolic compound in samples of real wastewaters in order to evaluate the potential efficiency of the biocatalyst. In batch mode the combi-CLEA transformed more than 80% to nearly 100% of acetaminophen from the municipal wastewater and more than 90% from the hospital wastewater. UPLC–MS analysis of acetaminophen metabolites showed the formation of its oligomers as dimers, trimers and tetramers due to the laccase and 3-hydroxyacetaminophen due to the tyrosinase.

Mechanisms of interaction of acetaminophen metabolites in terms of hepatotoxicity

The most serious complication after ingestion of a toxic dose of acetaminophen is liver damage. The aim of study was to review the research on the biochemical and molecular mechanisms of acetaminophen hepatotoxicity. It was shown that effect of the acetaminophen metabolism is the production of N-acetyl-p-benzoquinone, which is formed by bioactivation with the participation of cytochrome P450 enzymes. The combination of N-acetyl-p-benzoquinone and proteins can cause a disruption in protein homeostasis in cell membrane and mitochondrion. It may interfere with cellular signal process. Small and chronic doses of acetaminophen lead to damage of the cell nucleus. These effects can explain the mechanism of hepatotoxicity. N- acetyl- pbenzoquinone can cause an increase in oxidative stress. Lipids peroxidation and proteins oxidation are the main factors, which lead to necrosis of hepatocytes. It was shown that N-acetyl-p-benzoquinone can cause a decrease in GSH level and SH groups. For this reason, N-acetyl-p-benzoquinone was recognized as a key factor of the acetaminophen hepatotoxicity.

Interacción warfarina y acetaminofén: Evaluación para establecer su relevancia clínica

Objetivo: realizar una revisión estructurada de la interacción warfarina y acetaminofén, buscando establecer su relevancia clínica y profundizar en el mecanismo de dicha interacción. 
 Método: revisión estructurada en PubMed/Medline, de artículos en inglés y español, buscando los términos warfarin AND (acetaminophen OR paracetamol) en el título o resumen. La búsqueda se complementó con las referencias de artículos valorados como importantes. Los trabajos se agruparon en: relacionados con el aumento de sangrado por la interacción warfarina-acetaminofén, o relacionados con el mecanismo de la interacción. 
 Resultados: se identificaron 45 artículos, de los cuales se incluyeron 15 en la revisión: 11 relacionados con el aumento del riesgo de sangrado por la interacción y cuatro con el mecanismo de la interacción. La gravedad del efecto (aumento de la probabilidad de sangrado) se consideró moderada; mientras que la probabilidad de aparición fue valorada como definida. Además, se identificó una relación entre la dosis de acetaminofén y el riesgo de sangrado. Por su parte, el N-acetil-para-benzoquinona-imina (metabolito del acetaminofén) inhibe enzimas del ciclo de la vitamina K y tiene un efecto sinérgico con el efecto anticoagulante de la warfarina. 
 Conclusiones: la relevancia clínica de la interacción warfarina - acetaminofén es de riesgo alto, debido a que la gravedad del efecto (aumento del riesgo de sangrado) es moderada y su probabilidad de presentación es definida. Por tanto, estos dos medicamentos pueden ser utilizados conjuntamente, pero se debe realizar una estricta monitorización. El metabolito N-acetil-para-benzoquinona-imina es el responsable del aumento del efecto anticoagulante de la warfarina. (Acta Med Colomb 2013; 38: 22-27)

Effect of acetaminophen on the progression of renal damage in adenine induced renal failure model rats

AimsAcetaminophen is a safe antipyretic and analgesic drug within the clinically recommended dosage range, but overdose can cause fatal liver and or kidney damage. Most of the nonsteroidal anti-inflammatory drugs (NSAIDs) exert their analgesic effect via inhibition of cyclooxygenase, which also results in a reduction of renal blood flow. Therefore, the use of NSAIDs in pain treatment for chronic kidney disease (CKD) patients is of particular concern. Acetaminophen lacks the anti-inflammatory and anti-coagulatory properties of the NSAIDs. In this study, we investigate whether acetaminophen has an impact on the progression of renal failure. Main methodsAcetaminophen (150mg/kg/day or 750mg/kg/day) or indomethacin (5mg/kg/day) was orally administered to adenine-induced chronic renal failure model rats for 4weeks. The plasma concentrations of acetaminophen and its metabolites were measured during the treatment period; renal function and oxidative stress in the rats were also monitored. Key findingsIndomethacin significantly decreased the survival rate of renal failure model rats. In contrast, both low (150mg/kg) and high (750mg/kg) doses of acetaminophen improved the survival rate. The progression of renal failure was attenuated by acetaminophen (750mg/kg) after administration for 2weeks. The metabolites of acetaminophen were found to accumulate in plasma. Plasma glutathione concentration had significantly recovered after acetaminophen administration. SignificanceAcetaminophen has no effect on the progression of renal damage in adenine-induced renal failure model rats. This result is in part due to acetaminophen's antioxidant activity. These results suggest that acetaminophen is a suitable analgesic agent for treating CKD patients.

Severe diabetes and leptin resistance cause differential hepatic and renal transporter expression in mice

BackgroundType-2 Diabetes is a major health concern in the United States and other Westernized countries, with prevalence increasing yearly. There is a need to better model and predict adverse drug reactions, drug-induced liver injury, and drug efficacy in this population. Because transporters significantly contribute to drug clearance and disposition, it is highly significant to determine whether a severe diabetes phenotype alters drug transporter expression, and whether diabetic mouse models have altered disposition of acetaminophen (APAP) metabolites.ResultsTransporter mRNA and protein expression were quantified in livers and kidneys of adult C57BKS and db/db mice, which have a severe diabetes phenotype due to a lack of a functional leptin receptor. The urinary excretion of acetaminophen-glucuronide, a substrate for multidrug resistance-associated proteins transporters was also determined. The mRNA expression of major uptake transporters, such as organic anion transporting polypeptide Slco1a1 in liver and kidney, 1a4 in liver, and Slc22a7 in kidney was decreased in db/db mice. In contrast, Abcc3 and 4 mRNA and protein expression was more than 2 fold higher in db/db male mouse livers as compared to C57BKS controls. Urine levels of APAP-glucuronide, -sulfate, and N-acetyl cysteine metabolites were higher in db/db mice.ConclusionA severe diabetes phenotype/presentation significantly altered drug transporter expression in liver and kidney, which corresponded with urinary APAP metabolite levels.

Acetaminophen differentially enhances social behavior and cortical cannabinoid levels in inbred mice

Supratherapeutic doses of the analgesic acetaminophen (paracetomol) are reported to promote social behavior in Swiss mice. However, we hypothesized that it might not promote sociability in other strains due to cannabinoid CB(1) receptor-mediated inhibition of serotonin (5-HT) transmission in the frontal cortex. We examined the effects of acetaminophen on social and repetitive behaviors in comparison to a cannabinoid agonist, WIN 55,212-2, in two strains of socially-deficient mice, BTBR and 129S1/SvImJ (129S). Acetaminophen (100mg/kg) enhanced social interactions in BTBR, and social novelty preference and marble burying in 129S at serum levels of ≥70 ng/ml. Following acetaminophen injection or sociability testing, anandamide (AEA) increased in BTBR frontal cortex, while behavior testing increased 2-arachidonyl glycerol (2-AG) levels in 129S frontal cortex. In contrast, WIN 55,212-2 (0.1mg/kg) did not enhance sociability. Further, we expected CB(1)-deficient (+/-) mice to be less social than wild-type, but instead found similar sociability. Given strain differences in endocannabinoid response to acetaminophen, we compared cortical CB(1) and 5-HT(1A) receptor density and function relative to sociable C57BL/6 mice. CB(1) receptor saturation binding (Bmax=958±117 fmol/mg protein), and affinity for [(3)H] CP55,940 (K(D)=3±0.8 nM) was similar in frontal cortex among strains. CP55,940-stimulated [(35)S] GTPγS binding in cingulate cortex was 136±12, 156±22, and 75±9% above basal in BTBR, 129S and C57BL/6 mice. The acetaminophen metabolite para-aminophenol (1 μM) failed to stimulate [(35)S] GTPγS binding. Hence, it appears that other indirect actions of acetaminophen, including 5-HT receptor agonism, may underlie its sociability promoting properties outweighing any CB(1) mediated suppression by locally-elevated endocannabinoids in these mice.

Metabolic Acidosis in Acetaminophen Overdose without Concurrent Liver Toxicity

Introduction Metabolic acidosis and coma are common complications of acetaminophen overdose. These affects are usually attributed to massive hepatic necrosis caused by Nacetyl-p-benzoquinoneimine (NAPQI), a toxic metabolite of acetaminophen. We describe an unusual case of lactic acidosis and coma in a patient with acetaminophen toxicity without associated hepatocellular damage as the identifiable cause.

A Comparison of Self-Reported Analgesic Use and Detection of Urinary Ibuprofen and Acetaminophen Metabolites by Means of Metabonomics: The INTERMAP Study

Information on dietary supplements, medications, and other xenobiotics in epidemiologic surveys is usually obtained from questionnaires and is subject to recall and reporting biases. The authors used metabolite data obtained from hydrogen-1 (or proton) nuclear magnetic resonance ((1)H NMR) analysis of human urine specimens from the International Study of Macro-/Micro-Nutrients and Blood Pressure (INTERMAP Study) to validate self-reported analgesic use. Metabolic profiling of two 24-hour urine specimens per individual was carried out for 4,630 participants aged 40-59 years from 17 population samples in Japan, China, the United Kingdom, and the United States (data collection, 1996-1999). (1)H NMR-detected acetaminophen and ibuprofen use was low (∼4%) among East Asian population samples and higher (>16%) in Western population samples. In a comparison of self-reported acetaminophen and ibuprofen use with (1)H NMR-detected acetaminophen and ibuprofen metabolites among 496 participants from Chicago, Illinois, and Belfast, Northern Ireland, the overall rate of concordance was 81%-84%; the rate of underreporting was 15%-17%; and the rate of underdetection was approximately 1%. Comparison of self-reported unspecified analgesic use with (1)H NMR-detected acetaminophen and ibuprofen metabolites among 2,660 Western INTERMAP participants revealed similar levels of concordance and underreporting. Screening for urinary metabolites of acetaminophen and ibuprofen improved the accuracy of exposure information. This approach has the potential to reduce recall bias and other biases in epidemiologic studies for a range of substances, including pharmaceuticals, dietary supplements, and foods.

TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Δ9-tetrahydrocannabiorcol

TRPA1 is a unique sensor of noxious stimuli and, hence, a potential drug target for analgesics. Here we show that the antinociceptive effects of spinal and systemic administration of acetaminophen (paracetamol) are lost in Trpa1(-/-) mice. The electrophilic metabolites N-acetyl-p-benzoquinoneimine and p-benzoquinone, but not acetaminophen itself, activate mouse and human TRPA1. These metabolites also activate native TRPA1 and, as a consequence, reduce voltage-gated calcium and sodium currents in primary sensory neurons. The N-acetyl-p-benzoquinoneimine metabolite L-cysteinyl-S-acetaminophen was detected in the mouse spinal cord after systemic acetaminophen administration. In the hot-plate test, intrathecal administration of N-acetyl-p-benzoquinoneimine, p-benzoquinone and the electrophilic TRPA1 activator cinnamaldehyde produced antinociception that was lost in Trpa1(-/-) mice. Intrathecal injection of a non-electrophilic cannabinoid, Δ(9)-tetrahydrocannabiorcol, also produced TRPA1-dependent antinociception in this test. Our study provides a molecular mechanism for the antinociceptive effect of acetaminophen and discloses spinal TRPA1 activation as a potential pharmacological strategy to alleviate pain.

Effects of the Analgesic Acetaminophen (Paracetamol) and its para‐Aminophenol Metabolite on Viability of Mouse‐Cultured Cortical Neurons

Acetaminophen has been used as an analgesic for more than a hundred years, but its mechanism of action has remained elusive. Recently, it has been shown that acetaminophen produces analgesia by the activation of the brain endocannabinoid receptor CB1 through its para-aminophenol (p-aminophenol) metabolite. The objective of this study was to determine whether p-aminophenol could be toxic for in vitro developing mouse cortical neurons as a first step in establishing a link between acetaminophen use and neuronal apoptosis. We exposed developing mouse cortical neurons to various concentrations of drugs for 24 hr in vitro. Acetaminophen itself was not toxic to developing mouse cortical neurons at therapeutic concentrations of 10-250 μg/ml. However, concentrations of p-aminophenol from 1 to 100 μg/ml produced significant (p < 0.05) loss of mouse cortical neuron viability at 24 hr compared to the controls. The naturally occurring endocannabinoid anandamide also caused similar 24-hr loss of cell viability in developing mouse cortical neurons at concentrations from 1 to 100 μg/ml, which indicates the mechanism of cell death could be through the cannabinoid receptors. The results of our experiments have shown a detrimental effect of the acetaminophen metabolite p-aminophenol on in vitro developing cortical neuron viability which could act through CB1 receptors of the endocannabinoid system. These results could be especially important in recommending an analgesic for children or individuals with traumatic brain injury who have developing cortical neurons.

SIRT3‐dependent deacetylation exacerbates acetaminophen hepatotoxicity

Acetaminophen/paracetamol-induced liver failure--which is induced by the binding of reactive metabolites to mitochondrial proteins and their disruption--is exacerbated by fasting. As fasting promotes SIRT3-mediated mitochondrial-protein deacetylation and acetaminophen metabolites bind to lysine residues, we investigated whether deacetylation predisposes mice to toxic metabolite-mediated disruption of mitochondrial proteins. We show that mitochondrial deacetylase SIRT3(-/-) mice are protected from acetaminophen hepatotoxicity, that mitochondrial aldehyde dehydrogenase 2 is a direct SIRT3 substrate, and that its deacetylation increases acetaminophen toxic-metabolite binding and enzyme inactivation. Thus, protein deacetylation enhances xenobiotic liver injury by modulating the binding of a toxic metabolite to mitochondrial proteins.

In-source formation of N-acetyl- p-benzoquinone imine (NAPQI), the putatively toxic acetaminophen (paracetamol) metabolite, after derivatization with pentafluorobenzyl bromide and GC–ECNICI-MS analysis

Pentafluorobenzyl (PFB) bromide (PFB-Br) is a versatile derivatization reagent for numerous classes of compounds. Under electron-capture negative-ion chemical ionization (ECNICI) conditions PFB derivatives of acidic compounds readily and abundantly ionize to produce intense anions due to [M−PFB] −. In the present article we investigated the PFB-Br derivatization of unlabelled acetaminophen ( N-acetyl- p-aminophenol, NAPAP-d 0; paracetamol; MW 151) and tetradeuterated acetaminophen (NAPAP-d 4; MW 155) in anhydrous acetonitrile and their GC–ECNICI-MS behavior using methane as the buffer gas. In addition to the expected anions [M−PFB] − at m/ z 150 from NAPAP-d 0 and m/ z 154 from NAPAP-d 4, we observed highly reproducibly almost equally intense anions at m/ z 149 and m/ z 153, respectively. Selected ion monitoring of these ions is suitable for specific and sensitive quantification of acetaminophen in human plasma and urine. Detailed investigations suggest in-source formation of N-acetyl- p-benzoquinone imine (NAPQI; MW 149), the putatively toxic acetaminophen metabolite, from the PFB ether derivative of NAPAP. GC–ECNICI-MS of non-derivatized NAPAP did not produce NAPQI. The peak area ratio of m/ z 149 to m/ z 150 and of m/ z 153 to m/ z 154 decreased with increasing ion-source temperature in the range 100–250 °C. Most likely, NAPQI formed in the ion-source captures secondary electrons to become negatively charged (i.e., [NAPQI] −) and thus detectable. Formation of NAPQI was not observed under electron ionization (EI) conditions, i.e., by GC–EI-MS, from derivatized and non-derivatized NAPAP. NAPQI was not detectable in flow injection analysis LC–MS of native NAPAP in positive electrospray ionization (ESI) mode, whereas in negative ESI mode low extent NAPQI formation was observed (<5%). Our results suggest that oxidation of drug derivatives in the ion-sources of mass spectrometers may form intermediates that are produced from activated drugs in enzyme-catalyzed reactions.

ATM Mutant Lymphoid Tumour Cells Exhibit Impaired Activation of the Redox-Sensitive Nrf2-ARE Detoxification Pathway and Are Differentially Sensitive to Nrf2-Activating Compounds

AbstractAbstract 49Inactivation of the tumour suppressor gene, Ataxia Telangiectasia Mutated (ATM), is a frequent event in lymphoid malignancies such as chronic lymphocytic leukaemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and, to a lesser degree, T-cell acute lymphoblastic leukaemia (ALL). ATM mutant tumour cells exhibit defective p53-mediated apoptosis following DNA damaging agents in vitro and consequently exhibit poor clinical responses. Multiple pro-survival responses are also altered by ATM dysfunction and may reveal novel approaches for treatment. Our previous gene expression and transcription factor array analyses in primary CLL cells indicated impaired binding of the NF-E2-related factor 2 (Nrf2) transcription factor to the antioxidant response element (ARE) in ATM null cells following IR-induced DNA double strand breaks compared to ATM wild type cells. Nrf2 is a stress responsive transcription factor, which is activated by phenolic compounds and electrophilic agents, and induces the transcription of a chemo- and redox-protective ARE gene battery. The function of these gene products include regulation of cellular glutathione levels (glutamate-cysteine ligase subunits), glutathione utilising enzymes (glutathione S-transferases) and drug metabolising enzymes such as NAD(P)H:quinone oxidoreductase-1 (NQO1) and thioredoxin reductase (TXNRD1). To confirm ATM-dependent regulation of the Nrf2-ARE pathway, we first used electromagnetic shift analysis (EMSA) and demonstrated differential binding of Nrf2-ARE in an independent panel of primary CLL cell lysates. Binding patterns of lysates to the ARE probe were reduced in both untreated and in IR-treated ATM null compared to wt cells. We subsequently investigated the impact of impaired binding of Nrf2-ARE on the transcription of target genes. Quantitative RT-PCR revealed that in isogenic MEC1 CLL cell lines with (MEC1-ATMshRNA) and without (MEC1-GFPshRNA) stable ATM knockdown, ATM loss led to significant impairment in the induction of expression of the Nrf2-ARE-dependent genes, NQO1 and TXNRD1, 8h after tert Butylhydroquinone (tBHQ)–induced activation compared with wt cells. Furthermore, primary ATM mutant CLL cells also revealed impaired induction of NQO1 following tBHQ treatment. Finally, 3 isogenic CLL cell line pairs derived from PGA, CII and MEC, either with or without stable ATM knockdown, consistently revealed a 20% reduction in basal total glutathione levels in cells with ATM knockdown compared to controls, indicating impaired Nrf2-mediated glutathione synthesis. Given defective Nrf2-ARE pathway in ATM null cells, we reasoned that ATM mutant lymphoid tumour cells would be differentially sensitive to agents which activate the pathway and are detoxified by Nrf2-ARE gene products. N-acetyl-p-benzoquinoneimine (NAPQI), the metabolite of acetaminophen, activates Nrf2 and is detoxified directly by glutathione. We observed differential sensitivity of CII-ATMshRNA cells with maximal (80%) killing at 150μ M NAPQI compared to CII-GFPshRNA cells which showed no such killing after 4 days of treatment. Furthermore, 4 primary ATM mutant CLLs revealed differential sensitivity to 50μ M NAPQI with maximal (80%) killing achieved at 100μ M NAPQI after 72h. In contrast, 4 ATM wildtype CLLs exhibited no sensitivity even at 150μ M NAPQI. Treatment with N-acetylcysteine (NAC) abolished NAPQI sensitivity indicating a glutathione-dependent mechanism. Finally, Annexin V/PI staining revealed an apoptosis-dependent mechanism which was caspase-independent as pan-caspase inhibitor treatment had no impact on NAPQI-induced cell death. Finally, in IR-resistant CII-ATMshRNA cells, 4h pre-treatment with 50μ M NAPQI resulted in almost complete sensitisation to IR-induced killing. In summary, ATM null lymphoid tumour cells exhibit impaired activation of the NRF2-ARE detoxification pathway. Consequently, ATM mutant cells can be differentially targeted for killing by agents that activate the Nrf2-ARE pathway. This targeted approach may provide novel treatment options for otherwise chemo-resistant ATM mutant tumours and additionally reduce morbidity in elderly patients. Disclosures:No relevant conflicts of interest to declare.

Blood Pressure Destabilization on Nonsteroidal Antiinflammatory Agents

It has been conventional wisdom for some time now that nonsteroidal antiinflammatory drugs (NSAIDs), whether selective for inhibition of cyclooxygenase 2 (COX-2) or nonselective, increase blood pressure (BP) or interfere with BP control and that acetaminophen should not have this effect.1,–,4 Controlled clinical trials have shown that NSAIDs have heterogeneous effects on clinic and 24-hour BP in normotensive and hypertensive subjects.3 The inhibition of COX-2 by NSAIDs results in decreased actions of both vasodilatory and natriuretic prostaglandins.3 In most individuals, homeostasis of plasma volume is reestablished by small, nearly undetectable increases in BP,5 whereas in patients with impaired excretory function, more substantial volume retention occurs that may be associated with hypertension, edema, and congestive heart failure.3,5 Article see p1789 Before 2002, little was known about the effects of NSAIDs on 24-hour BP in patients with arthritis who also had hypertension and/or vascular diseases.6,–,8 Because ambulatory BP has improved reproducibility compared with clinic measurements, detection of small but clinically meaningful differences in drug treatment groups9,10 is more likely to occur. Ambulatory monitoring also allows improved evaluation of pharmacodynamic effects of any drug, including the NSAIDs.6,7 For example, in the Celecoxib Rofecoxib Efficacy and Safety in Comorbidities Evaluation Trial (CRESCENT), destabilization of systolic BP control occurred for ≈18 of the 24 hours after dosing of rofecoxib at standard osteoarthritis doses of 25 mg each morning.8 In contrast, no changes from baseline in 24-hour systolic BP were observed with celecoxib or naproxen at …

Drug-dependent clearance of human platelets in the NOD/scid mouse by antibodies from patients with drug-induced immune thrombocytopenia

AbstractDrug-induced immune thrombocytopenia (DITP) is a relatively common and sometimes life-threatening condition caused by antibodies that bind avidly to platelets only when drug is present. How drug-dependent antibodies (DDAbs) are induced and how drugs promote their interaction with platelets are poorly understood, and methods for detecting DDAbs are suboptimal. A small animal model of DITP could provide a new tool for addressing these and other questions concerning pathogenesis and diagnosis. We examined whether the nonobese diabetic/severe combined immunodeficient (NOD/scid) mouse, which lacks xenoantibodies and therefore allows infused human platelets to circulate, can be used to study drug-dependent clearance of platelets by DDAbs in vivo. In this report, we show that the NOD/scid model is suitable for this purpose and describe studies to optimize its sensitivity for drug-dependent human antibody detection. We further show that the mouse can produce metabolites of acetaminophen and naproxen for which certain drug-dependent antibodies are specific in quantities sufficient to enable these antibodies to cause platelet destruction. The findings indicate that the NOD/scid mouse can provide a unique tool for studying DITP pathogenesis and may be particularly valuable for identifying metabolite-specific antibodies capable of causing immune thrombocytopenia or hemolytic anemia.

Differential effect of covalent protein modification and glutathione depletion on the transcriptional response of Nrf2 and NF-κB

Liver injury associated with exposure to therapeutic agents that undergo hepatic metabolism can involve the formation of reactive metabolites. These may cause redox perturbation which can result in oxidative stress as well as protein modification leading to activation or inhibition of cellular transcriptional responses. Nevertheless, the effects of these challenges on more than one transcriptional pathway simultaneously remain unclear. We have investigated two transcription factors known to be sensitive to electrophilic stress and redox perturbation, Nrf2 and NF-κB, in mouse liver cells. Cellular stress was induced by the probes: N-acetyl-p-benzoquinineimine (NAPQI), the reactive metabolite of acetaminophen; dinitrochlorobenzene (DNCB), a model electrophile; and buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase. NAPQI, DNCB and BSO can all cause glutathione (GSH) depletion; however only NAPQI and DNCB can covalently bind proteins. We also employed RNAi to manipulate Keap1 (the inhibitor of Nrf2), Nrf2 itself and NF-κB-p65, to understand their roles in the response to drug stress. All three chemicals induced Nrf2, but NF-κB binding activity was only increased after BSO treatment. In fact, NF-κB binding activity decreased after exposure to NAPQI and DNCB. While RNAi depletion of Keap1 led to reduced toxicity following exposure to DNCB, depletion of Nrf2 and NF-κB augmented toxicity. Interestingly, increased Nrf2 caused by Keap1 depletion was reversed by co-depletion of NF-κB. We demonstrate that Keap1/Nrf2 and NF-κB respond differently to electrophiles that bind proteins covalently and the redox perturbation associated with glutathione depletion, and that crosstalk may enable NF-κB to partly influence Nrf2 expression during cellular stress.