Laura P. James

Mechanisms of Acetaminophen-Induced Liver Necrosis

Although considered safe at therapeutic doses, at higher doses, acetaminophen produces a centrilobular hepatic necrosis that can be fatal. Acetaminophen poisoning accounts for approximately one-half of all cases of acute liver failure in the United States and Great Britain today. The mechanism occurs by a complex sequence of events. These events include: (1) CYP metabolism to a reactive metabolite which depletes glutathione and covalently binds to proteins; (2) loss of glutathione with an increased formation of reactive oxygen and nitrogen species in hepatocytes undergoing necrotic changes; (3) increased oxidative stress, associated with alterations in calcium homeostasis and initiation of signal transduction responses, causing mitochondrial permeability transition; (4) mitochondrial permeability transition occurring with additional oxidative stress, loss of mitochondrial membrane potential, and loss of the ability of the mitochondria to synthesize ATP; and (5) loss of ATP which leads to necrosis. Associated with these essential events there appear to be a number of inflammatory mediators such as certain cytokines and chemokines that can modify the toxicity. Some have been shown to alter oxidative stress, but the relationship of these modulators to other critical mechanistic events has not been well delineated. In addition, existing data support the involvement of cytokines, chemokines, and growth factors in the initiation of regenerative processes leading to the reestablishment of hepatic structure and function.

Acetaminophen‐Induced Hepatotoxicity: Role of Metabolic Activation, Reactive Oxygen/Nitrogen Species, and Mitochondrial Permeability Transition

Large doses of the analgesic acetaminophen cause centrilobular hepatic necrosis in man and in experimental animals. It has been previously shown that acetaminophen is metabolically activated by CYP enzymes to N‐acetyl‐p‐benzoquinone imine. This species is normally detoxified by GSH, but following a toxic dose GSH is depleted and the metabolite covalently binds to a number of different proteins. Covalent binding occurs only to the cells developing necrosis. Recently we showed that these cells also contain nitrated tyrosine residues. Nitrotyrosine is mediated by peroxynitrite, a reactive nitrogen species formed by rapid reaction between nitric oxide and superoxide and is normally detoxified by GSH. Thus, acetaminophen toxicity occurs with increased oxygen/nitrogen stress. This manuscript will review current data on acetaminophen covalent binding, increased oxygen/nitrogen stress, and mitochondrial permeability transition, a toxic mechanism that is both mediated by and leads to increased oxygen/nitrogen stress.

Severe Toxicity Following Synthetic Cannabinoid Ingestion

Objective. To report a case of seizures and supraventricular tachycardia (SVT) following confirmed synthetic cannabinoid ingestion. Background. Despite widespread use of legal synthetic cannabinoids, reports of serious toxicity following confirmed use of synthetic cannabinoids are rare. We report severe toxicity including seizures following intentional ingestion of the synthetic cannabinoid JWH-018 and detail confirmation by laboratory analysis. Case Report. A healthy 48 year old man had a generalized seizure within thirty minutes of ingesting an ethanol mixture containing a white powder he purchased from the Internet in an attempt to get high. Seizures recurred and abated with lorazepam. Initial vital signs were: pulse, 106/min; BP, 140/88 mmHg; respirations, 22/min; temperature, 37.7 °C. A noncontrast computed tomography of the brain and EEG were negative, and serum chemistry values were normal. The blood ethanol concentration was 3.8 mg/dL and the CPK 2,649 U/L. Urine drug screening by EMIT was negative for common drugs of abuse, including tetrahydrocannabinol. On hospital day 1, he developed medically refractory SVT. The patient had no further complications and was discharged in his normal state of health 10 days after admission. The original powder was confirmed by gas chromatography mass spectrometry to be JWH-018, and a primary JWH-018 metabolite was detected in the patients urine (200 nM) using liquid chromatography tandem mass spectrometry. Discussion. Synthetic cannabinoids are legal in many parts of the world and easily obtained over the Internet. Data on human toxicity are limited and real-time confirmatory testing is unavailable to clinicians. The potential for toxicity exists for users mistakenly associating the dose and side effect profiles of synthetic cannabinoids to those of marijuana. Conclusion. Ingestion of JWH-018 can produce seizures and tachyarrhythmias. Clinicians, lawmakers, and the general public need to be aware of the potential for toxicity associated with synthetic cannabinoid use.

Ontogeny of dextromethorphan O- and N-demethylation in the first year of life.

The exponential increase in the number of drugs used to treat infant and childhood illnesses necessitates an understanding of the ontogeny of drug biotransformation for the development of safe and effective therapies. Healthy infants received an oral dose (0.3 mg/kg) of dextromethorphan (DM) at 0.5, 1, 2, 4, 6, and 12 months of age. DM and its major metabolites were measured in urine. CYP2D6 genotype was determined by polymerase chain reaction‐restriction fragment length polymorphism. Genotyping data indicated a strong correlation between CYP2D6 genotype and DM O‐demethylation (β=−0.638; 95% CI: −0.745, −0.532; P<0.001). CYP2D6 activity was detectable and concordant with genotype by 2 weeks of age, showed no relationship with gestational age, and did not change with post natal age up to 1 year. In contrast, DM N‐demethylation developed significantly more slowly over the first year of life. Genotype and the temporal acquisition of drug biotransformation are critical determinants of a drug response in infants.

Acetaminophen Toxicity in Mice Lacking NADPH Oxidase Activity: Role of Peroxynitrite Formation and Mitochondrial Oxidant Stress

Previous data have indicated that activated macrophages may play a role in the mediation of acetaminophen toxicity. In the present study, we examined the significance of superoxide produced by macrophages by comparing the toxicity of acetaminophen in wild-type mice to mice deficient in gp91phox, a critical subunit of NADPH oxidase that is the primary source of phagocytic superoxide. Both groups of mice were dosed with 300 mg/kg of acetaminophen or saline and sacrificed at 1, 2, 4 or 24 h. Glutathione in total liver and in mitochondria was depleted by approximately 90% at 1 h in wild-type and knock out mice. No significant differences in toxicity (serum transaminase levels or histopathology) were observed between wild-type and mice deficient in gp91phox. Mitochondrial glutathione disulfide, as a percent of total glutathione, was determined as a measure of oxidant stress produced by increased superoxide, leading to hydrogen peroxide and/or peroxynitrite. The percent mitochondrial glutathione disulfide increased to approximately 60% at 1 h and 70% at 2 h in both groups of mice. Immunohistochemical staining for nitrotyrosine was present in vascular endothelial cells at 1 h in both groups of mice. Acetaminophen protein adducts were present in hepatocytes at 1 h in both wild-type and knock out animals. These data indicate that superoxide from activated macrophages is not critical to the development of acetaminophen toxicity and provide further support for the role of mitochondrial oxidant stress in acetaminophen toxicity.

Cytochrome P450-mediated Oxidative Metabolism of Abused Synthetic Cannabinoids Found in "K2/Spice": Identification of Novel Cannabinoid Receptor Ligands

Abuse of synthetic cannabinoids (SCs), such as [1-naphthalenyl-(1-pentyl-1H-indol-3-yl]-methanone (JWH-018) and [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM2201), is increasing at an alarming rate. Although very little is known about the metabolism and toxicology of these popular designer drugs, mass spectrometric analysis of human urine specimens after JWH-018 and AM2201 exposure identified monohydroxylated and carboxylated derivatives as major metabolites. The present study extends these initial findings by testing the hypothesis that JWH-018 and its fluorinated counterpart AM2201 are subject to cytochrome P450 (P450)-mediated oxidation, forming potent hydroxylated metabolites that retain significant affinity and activity at the cannabinoid 1 (CB1) receptor. Kinetic analysis using human liver microsomes and recombinant human protein identified CYP2C9 and CYP1A2 as major P450s involved in the oxidation of the JWH-018 and AM2201. In vitro metabolite formation mirrored human urinary metabolic profiles, and each of the primary enzymes exhibited high affinity (Km = 0.81–7.3 μM) and low to high reaction velocities (Vmax = 0.0053–2.7 nmol of product · min−1 · nmol protein−1). The contribution of CYP2C19, 2D6, 2E1, and 3A4 in the hepatic metabolic clearance of these synthetic cannabinoids was minimal (fm = <0.2). In vitro studies demonstrated that the primary metabolites produced in humans display high affinity and intrinsic activity at the CB1 receptor, which was attenuated by the CB1 receptor antagonist (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (O-2050). Results from the present study provide critical, missing data related to potential toxicological properties of “K2” parent compounds and their human metabolites, including mechanism(s) of action at cannabinoid receptors.

Pharmacokinetics of Acetaminophen-Protein Adducts in Adults with Acetaminophen Overdose and Acute Liver Failure

Acetaminophen (APAP)-induced liver toxicity occurs with formation of APAP-protein adducts. These adducts are formed by hepatic metabolism of APAP to N-acetyl-p-benzoquinone imine, which covalently binds to hepatic proteins as 3-(cystein-S-yl)-APAP adducts. Adducts are released into blood during hepatocyte lysis. We previously showed that adducts could be quantified by high-performance liquid chromatography with electrochemical detection following proteolytic hydrolysis, and that the concentration of adducts in serum of overdose patients correlated with toxicity. The following study examined the pharmacokinetic profile and clinical associations of adducts in 53 adults with acute APAP overdose resulting in acute liver failure. A population pharmacokinetic analysis using nonlinear mixed effects (statistical regression type) models was conducted; individual empiric Bayesian estimates were determined for the elimination rate constant and elimination half-life. Correlations between clinical and laboratory data were examined relative to adduct concentrations using nonparametric statistical approaches. Peak concentrations of APAP-protein adducts correlated with peak aminotransferase concentrations (r = 0.779) in adults with APAP-related acute liver failure. Adducts did not correlate with bilirubin, creatinine, and APAP concentration at admission, international normalized ratio for prothrombin time, or reported APAP dose. After N-acetylcysteine therapy, adducts exhibited first-order disappearance. The mean elimination rate constant and elimination half-life were 0.42 ± 0.09 days–1 and 1.72 ± 0.34 days, respectively, and estimates from the population model were in strong agreement with these data. Adducts were detected in some patient samples 12 days postingestion. The persistence and specificity of APAP-protein adducts as correlates of toxicity support their use as specific biomarkers of APAP toxicity in patients with acute liver injury.

Unrecognized acetaminophen toxicity as a cause of indeterminate acute liver failure

Despite extensive investigations, the cause of liver injury in 14% of patients with acute liver failure remains unknown (indeterminate). In a pilot study using a novel assay, highly specific acetaminophen‐cysteine adducts were detected in 7 of 36 indeterminate patients (19%). To extend these observations, sera from 110 subjects enrolled in the Acute Liver Failure Study Group registry with indeterminate acute liver failure were analyzed with a similar but more efficient and sensitive adduct assay. As positive controls, another 199 patients with known or presumed acetaminophen‐induced liver failure were assessed for the presence and quantity of adducts. Clinical, laboratory, and outcome data were compared for the two groups. On the basis of previous data for known therapeutic exposures and acetaminophen overdoses, an adduct concentration ≥1.0 nmol/mL of serum indicated a definite acetaminophen overdose. Among the 110 indeterminate cases, 18% had assay values ≥1.0 with a median level of 9.2 nmol/mL; 94.5% of the positive controls (known acetaminophen cases) had values ≥1.0 nmol/mL. Regardless of the initial diagnosis, subjects with elevated adduct levels demonstrated the clinical profile and hyperacute biochemical injury pattern associated with acetaminophen overdose: a predominance of female gender, very high aminotransferase levels, and low bilirubin levels. Conclusion: These data confirm and extend previous observations regarding the high (18%) prevalence of unrecognized or uncertain acetaminophen toxicity among subjects with indeterminate acute liver failure. N‐Acetylcysteine use was limited in this group, presumably because of the lack of a specific diagnosis of acetaminophen toxicity. (HEPATOLOGY 2011;53:567‐576.)

Acetaminophen-Induced Hepatotoxicity in Mice Occurs with Inhibition of Activity and Nitration of Mitochondrial Manganese Superoxide Dismutase

In overdose the analgesic/antipyretic acetaminophen (APAP) is hepatotoxic. Toxicity is mediated by initial hepatic metabolism to N-acetyl-p-benzoquinone imine (NAPQI). After low doses NAPQI is efficiently detoxified by GSH. However, in overdose GSH is depleted, NAPQI covalently binds to proteins as APAP adducts, and oxygen/nitrogen stress occurs. Toxicity is believed to occur by mitochondrial dysfunction. Manganese superoxide dismutase (MnSOD) inactivation by protein nitration has been reported to occur during other oxidant stress-mediated diseases. MnSOD is a critical mitochondrial antioxidant enzyme that prevents peroxynitrite formation within the mitochondria. To examine the role of MnSOD in APAP toxicity, mice were treated with 300 mg/kg APAP. GSH was significantly reduced by 65% at 0.5 h and remained reduced from 1 to 4 h. Serum alanine aminotransferase did not significantly increase until 4 h and was 2290 IU/liter at 6 h. MnSOD activity was significantly reduced by 50% at 1 and 2 h. At 1 h, GSH was significantly depleted by 62 and 80% at nontoxic doses of 50 and 100 mg/kg, respectively. No further GSH depletion occurred with hepatotoxic doses of 200 and 300 mg/kg APAP. A dose response decrease in MnSOD activity was observed for APAP at 100, 200, and 300 mg/kg. Immunoprecipitation of MnSOD from livers of APAP-treated mice followed by Western blot analysis revealed nitrated MnSOD. APAP-MnSOD adducts were not detected. Treatment of recombinant MnSOD with NAPQI did not produce APAP protein adducts. The data indicate that MnSOD inactivation by nitration is an early event in APAP-induced hepatic toxicity.

Single dose pharmacokinetics of linezolid in infants and children.

Background. Linezolid is an oxazolidinone antibiotic with excellent in vitro activity against a number of Gram-positive organisms including antibiotic-resistant isolates. The safety and pharmacokinetics of intravenously administered linezolid were evaluated in children and adolescents to examine the potential for developmental dependence on its disposition characteristics. Methods. Fifty-eight children (3 months to 16 years old) participated in this study; 44 received a single 1.5-mg/kg dose and 14 received a single 10-mg/kg dose of linezolid administered by intravenous infusion. Repeated blood samples (n = 10 in children ≥12 months;n = 8 in children 3 to 12 months) were obtained during 24 h after drug administration, and linezolid was quantitated from plasma by high performance liquid chromatography with mass spectrometry detection. Plasma concentration vs. time data were evaluated with a model independent approach. Results. Linezolid was well-tolerated by all subjects. The disposition of linezolid appears to be age-dependent. A significant although weak correlation between age and total body clearance was observed. The mean (±sd) values for elimination half-life, total clearance and apparent volume of distribution were 3.0 ± 1.1 h, 0.34 ± 0.15 liter/h/kg and 0.73 ± 0.18 liter/kg, respectively. Estimates of total body clearance and volume of distribution were significantly greater in children than historical values of adult data. As such maximum achievable linezolid plasma concentrations were slightly lower in children, and concentrations 12 h after a single 10-mg/kg dose were below the MIC90 for selected pathogens with in vitro susceptibility to the drug. Conclusion. Based on these data a linezolid dose of 10 mg/kg given two to three times daily would appear appropriate for use in pediatric therapeutic clinical trials of this agent.