Effects of vitamin K1 treatment on plasma concentrations of long-acting anticoagulant rodenticide enantiomers following inhalation of
contaminated synthetic cannabinoids
Douglas L Feinstein, Daniel G Nosal, Swetha Ramanathan, Jifang Zhou, Luying Chen, Ronald C Hershow, Richard B van Breemen, Erik Wright, John W Hafner & Israel Rubinstein
Background: An outbreak of synthetic cannabinoid (SC)-associated coagulopathy and bleeding in Illinois, USA was determined to be due to inhalation of SC contaminated with brodifacoum (BDF), difenacoum (DiF), and bromadiolone (BDL), highly potent long-acting anticoagulant rodenticides (LAARs). Treatment with high-dose vitamin K1 (VK1) prevented mortality; however, plasma LAAR levels were not measured risking recurrence of coagulopathy and bleeding due to premature discontinuation. The
goal of this study was to determine if plasma LAAR levels were reduced following standard of care treatment to normalize coagulopathy.
Methods: Blood samples were collected from a cohort of 32 patients, and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis used to quantify plasma LAAR levels including enantiomers.
Results: BDF was detected in 31 samples; 30 also contained DiF and 18 contained BDL. Initial plasma levels were 581 ± 87, 11.0 ± 1.9, and 14.9 ± 5.9 ng/mL for BDF, DiF, and BDL, respectively (mean ± SE). At discharge plasma, BDF levels remained elevated at 453 ± 68 ng/mL. Plasma half-lives for BDF, DiF, and BDL were 7.5 ± 1.3, 7.2 ± 1.9, and 1.8 ± 0.3 days, respectively. The half-life for trans-BDF enantiomers (5.7 ± 0.8 days) was shorter than for cis-enantiomers (7.6 ± 1.9 days). BDF half-lives were shorter, and coagulopathy normalized faster in patients receiving intravenous VK1 as compared to oral VK1.
Patients prescribed VK1 at discharge had fewer re-admittances.
Conclusions: These results demonstrate that plasma LAAR levels at discharge were elevated in poisoned patients despite normal coagulation, and that the route of VK1 administration affected LAAR pharmacokinetics and INR normalization. We propose plasma LAAR levels and coagulation be monitored concomitantly during follow-up of patients with LAAR poisoning.
KEY POINTS
1. In patients treated with high-dose vitamin K1 for LAAR poisoning, plasma levels remained 40-fold above safe levels upon discharge from hospital.
2. LAAR half-lives, normalization of coagulopathy, and readmittances were reduced by treatment with intravenous vitamin K1.
The long lasting rodenticide brodifacoum induces neuropathology in adult male rats.
The Chicago CounterAct Center:
Sergey Kalinin, Natalia Marangoni, Katarzyna Kowal, Arunangsu Dey, Kinga Lis, Sergey Brodsky, Richard van Breemen, Zane Hauck, Richard Ripper, Israel Rubinstein, Guy Weinberg, Douglas L Feinstein
Superwarfarins are very long lasting rodenticides effective in warfarin-resistant rodents at extremely low doses. The consequences of chronic superwarfarin levels in tissues, due to biological half-lives on the order of 20 days, have not been examined. We now characterized the neurological effects of brodifacoum (BDF), one of the most widely used superwarfarins, in adult male Sprague Dawley rats. Dosing curves established the acute oral lethal dose for BDF as 221 ± 14 mg/kg. Measurement of tissue BDF levels showed accumulation throughout the body, including the CNS (central nervous system), with levels diminishing over several days. Immunocytochemical staining showed that both astrocyte and microglial activation was increased 4 days after BDF administration, as were levels of carbonylated proteins, and neuronal damage assessed by fluorojade B staining. Direct toxic effects of BDF on neurons and glia were observed using enriched cultures of cerebellar neurons and cortical astrocytes. Proteomic analysis of cerebellar lysates revealed that BDF altered expression of 667 proteins in adult rats. Gene ontology and pathway analysis identified changes in several functional pathways including cell metabolism, mitochondria function, and RNA handling with ribosomal proteins comprising the largest group. In vitro studies using primary astrocytes showed that BDF suppressed de novo protein synthesis. These findings demonstrate that superwarfarin accumulation increases indices of neuroinflammation and neuropathology in adult rodents, suggesting that methods which minimize BDF toxicity may not address delayed neurological sequelae.
MEDICAL COUNTERMEASURES FOR THE TREATMENT OF BRODIFACOUM (BDF) POISONING
The Chicago CounterAct Center:
Matt Lindeblad, Kathy Kowalski, Sergey Kalinin, Alex Lyubimov, Rudi Rubinstein, Guy Weinberg, Richard van Breemen3 Douglas L. Feinstein
Superwarfarins (SWs) are modified forms of the commonly used anti coagulant Warfarin, and cause bleeding by reducing vitamin K levels. SWs are used throughout the world as rodenticides. The SW most commonly currently used is brodifacoum
(BDF). BDF is about 100 times more potent than warfarin, because it is more fatty and stays in the body longer. In addition, it appears that the biological half life of BDF is increased due to enterohepatic recirculation (EH). SWs like BDF not only block
clotting but also accumulate in the CNS where they can induce long lasting neuropathology. We are therefore developing a model of BDF poisoning in rabbits in order to identify novel treatments to reduce acute toxicity and long term consequences.
BRODIFACOUM POISONING: A CLEAR AND PRESENT DANGER TO PUBLIC HEALTH IN THE USA.
The Chicago CounterAct Center:
Douglas L Feinstein, Sergey Brodsky, Guy Weinberg, Richard van Breeman, and Israel Rubinstein
According to the US Centers for Disease Control and Prevention, “chemical threat agents can be poisonous vapors, aerosols, liquids or solids that have toxic effects on people. These chemicals can be naturally occurring in the environment or synthetically produced. Chemical releases can be unintentional, as in the case of an industrial accident, or intentional, as in the case of a terrorist attack.” To this end, superwarfarins, such as brodifacoum (BDF), are highly toxic anticoagulant rodenticides that are considered chemical threats to the US population by the Department of Human and Health Services (HHS) because of their potential misuse by terrorists. In this commentary we highlight recent examples of accidental and intentional exposure to this commonly used rodenticide.
THE EMERGING THREAT OF SUPERWARFARINS: HISTORY, DETECTION, MECHANISMS, AND COUNTERMEASURES
The Chicago CounterAct Center: Feinstein DL, Akpa BS, Ayee MA, Boullerne AI, Braun D, Brodsky SV, Gidalevitz D, Hauck Z, Kalinin S, Kowal K, Kuzmenko I, Lis K, Marangoni N, Martynowycz MW, Rubinstein I, van Breemen R, Ware K, Weinberg G.
Superwarfarins were developed following the emergence of warfarin resistance in rodents. Compared to warfarin, superwarfarins have much longer half-lives and stronger affinity to vitamin K epoxide reductase and therefore can cause death in warfarin-resistant rodents. By the mid-1970s, the superwarfarins brodifacoum and difenacoum were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raise the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1-2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K-dependent and -independent effects, which are not mitigated by vitamin K therapy. In this review, we summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches
LC-MS/MS ANALYSIS OF BRODIFACOUM ISOMERS IN RAT TISSUE
Hauck ZZ, Feinstein DL, van Breemen RB
Brodifacoum (BDF) is a second-generation anticoagulant rodenticide structurally related to warfarin but containing two chiral centers. Highly stable, BDF can contaminate food and water supplies causing accidental poisoning of humans and nontarget animals. To determine the distribution of BDF isomers in serum and tissues, a quantitative method was developed and validated according to FDA guidelines based on high-performance liquid chromatography-tandem mass spectrometry. A single liquid-liquid extraction step provided recoveries exceeding 93%. Reversed-phase chromatographic separations required <6 min, and quantitative analysis utilized a triple-quadrupole mass spectrometer equipped with negative ion electrospray and selected reaction monitoring. The standard curve had a linear regression coefficient of 0.999 and intra- and inter-assay variations of <10%. The chromatographic method enabled the resolution and measurement of pairs of BDF diastereomers in commercial materials as well as in rat tissues. This method is suitable for measuring BDF exposure as well as basic science studies of the distribution and elimination of BDF diastereomers to various tissues.
MEMBRANE CHOLESTEROL MODULATES SUPERWARFARIN TOXICITY
Marangoni MN, Martynowycz MW, Kuzmenko I, Braun D, Polak PE, Weinberg G, Rubinstein I, Gidalevitz D, Feinstein DL.
Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.
BRODIFACOUM INDUCES EARLY HEMOGLOBINURIA AND LATE HEMATURIA IN RATS: NOVEL RAPID BIOMARKERS OF POISONING
Ware KM, Feinstein DL, Rubinstein I, Weinberg G, Rovin BH, Hebert L, Muni N, Cianciolo RE,
Satoskar AA, Nadasdy T, Brodsky SV.
Brodifacoum (BDF) is a superwarfarin that is used primarily as a rodenticide. There have been increasing numbers of reports of human cases of accidental or intentional BDF ingestion with high mortality rate. Its broad availability and high lethality suggest that BDF should be considered a potential chemical threat. Currently, there is no biomarker for early detection of BDF ingestion in humans; patients typically present with severe coagulopathy. Since we demonstrated earlier that warfarin can induce acute kidney injury with hematuria, we tested whether BDF would also lead to change in urinary biomarkers. BDF was administered to Sprague Dawley rats via oral gavage. N-acetylcysteine (NAC) was given per os in drinking water 24 h prior to BDF. Urinalysis was performed at different times after BDF administration. Anticoagulation and serum creatinine levels were analyzed in the blood. We observed that within a few hours the animals developed BDF-dose-dependent transient hemoglobinuria, which ceased within 24 h. This was accompanied by a transient decrease in hematocrit, gross hemolysis and an increase in free hemoglobin in the serum. At later times, animals developed true hematuria with red blood cells in the urine, which was associated with BDF anticoagulation. NAC prevented early hemoglobinuria, but not late hematuria associated with BDF. We propose that transient early hemoglobinuria (associated with oxidative stress) with consecutive late hematuria (associated with anticoagulation) are novel biomarkers of BDF poisoning, and they can be used in clinical setting or in mass casualty with BDF to identify poisoned patients.