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. Author manuscript; available in PMC: 2020 May 15.
Published in final edited form as: Toxicol Lett. 2019 Feb 16;306:61–65. doi: 10.1016/j.toxlet.2019.02.011

The relative toxicity of brodifacoum enantiomers

Douglas L Feinstein 1,2, Kamil Gierzal 1, Asif Iqbal 3, Sergey Kalinin 1, Richard Ripper 1,2, Matthew Lindeblad 3, Alexander Zahkarov 3, Alexander Lyubimov 3, Richard van Breemen 4, Guy Weinberg 1,2, Israel Rubinstein 2,5
PMCID: PMC6408973  NIHMSID: NIHMS1522462  PMID: 30779948

Abstract

Brodifacoum (BDF) is a potent, long-acting anticoagulant rodenticide that can cause fatal poisoning in humans. The chemical structure of BDF includes 2 chiral carbons, resulting in 2 pairs of diastereomers, BDF-cis (R/S and S/R) and BDF-trans (R/R and S/S). However, the relative potency of these molecules is not known. The purpose of this study was to compare the in vitro and in vivo toxic effects of the 2 BDF diastereomer pairs. In adult Sprague-Dawley rats BDF-cis was significantly more toxic than BDF-trans (LD50 values of 219 versus 316 μg/kg, respectively) while racemic BDF had intermediate potency (266 μg/kg). In adult New Zealand white rabbits, BDF-cis had a longer half-life than BDF-trans which could contribute to its observed increased toxicity. Lastly, BDF-cis (10 μM), but not BDF-trans, damaged cultured SH-SY5Y human neuroblastoma cells by attenuating mitochondrial reductive capacity. Taken together, these data suggest that different toxic manifestations of BDF poisoning in mammals could be attributed, in part, to differences in relative enantiomer concentrations present in racemic formulations of this commercially-available toxicant.

Keywords: superwarfarins, brodifacoum, enantiomer, diastereomer, anticoagulation

Graphical abstract

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Introduction

A recent nationwide epidemic of severe, life-threatening synthetic cannabinoid-associated coagulopathy and massive internal bleeding caused by contamination of these products with the long acting anti-coagulant rodenticide (LAAR) brodifacoum (BDF) has raised serious public health concerns (Connors, 2018). BDF was developed in the 1970’s to eliminate warfarin-resistant rats in the environment (Hadler and Shadbolt, 1975; Redfern and Gill, 1980; Redfern et al., 1976). The primary mechanism of action of LAARs is similar to that of warfarin, namely binding to and inhibition of VKORC1 (vitamin K epoxide reductase complex subunit 1) (Oldenburg et al., 2006), leading to a decrease in concentrations of vitamin K which is a necessary cofactor for gamma-glutamyl carboxylase (GGC) mediated carboxylation and activation of several clotting factors and other proteins. BDF, one of the most-commonly used LAARs, has significantly stronger binding affinity for VKORC1 than warfarin (Gebauer, 2007). In contrast to warfarin, BDF has an extremely long biological half-life (~20 days) due to minimal metabolism and limited clearance. In addition to its anticoagulant actions, BDF exerts direct neurotoxic effects on neurons which may be related to intercalation into and perturbation of cell and sub-cellular membranes (Marangoni et al., 2016). These deleterious effects were not observed with warfarin suggesting that they are related to unique hydrophobic characteristics of the BDF molecule.

As the case for the parent compound warfarin, BDF exists as multiple stereoisomers. In warfarin, the presence of a single stereocenter results in a mixture of R- and S- enantiomers (Gulseth et al., 2009; Takahashi and Echizen, 2001) (Figure 1). The 2 chiral centers in BDF (see Figure 1) lead to 4 isomers, a trans (R/R and S/S) and a cis (R/S and S/R) diastereomeric pair. In the case of warfarin, studies using individual isomers demonstrate that the S-enantiomer has 3 to 5-fold greater potency as an anticoagulant than the R-enantiomer (Breckenridge et al., 1974). Other studies have demonstrated selective metabolism of warfarin enantiomers by different cytochromes (Barnette et al., 2017), which is of high clinical relevance when formulating patient dosage to take into account cytochrome polymorphisms which influence metabolism. Although BDF does not undergo significant metabolism, it has been reported that the trans (R/R, S/S) diastereomers have a shorter half-life (68.7 hr) than the cis (R/S, S/R) diastereomers (120 hrs) (Damin-Pernik et al., 2017). Whether BDF diastereomers differ in anti-coagulant potency or other properties has not been reported. In this study, using enriched diastereomeric pairs, we show that cis-isomers have greater toxicity than trans-isomers both in vitro and in vivo.

Figure 1. Chemical structure of BDF and parent compound warfarin.

Figure 1.

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Asterisks indicate the 2 chiral carbons present in BDF

Materials and Methods

Chemicals

For in vivo studies, a stock solution of racemic brodifacoum (BDF-rac, CPC Scientific, Sunnyvale, CA) was dissolved into 100% DMSO to provide a final concentration of 3.5 mM as determined by measurement of absorbance at 260. For cell studies, BDF-rac was dissolved into 100% ethyl acetate to a final concentration of 5.0 mm, then diluted into cell culture media to the final desired concentrations. Difenacoum (DiF, Sigma, St Louis, MO) was used as internal standard for HPLC-MS/MS determination.

HPLC-MS/MS analysis of BDF and enantiomer enrichment

Separation of BDF diastereomeric pairs was carried out as described (Hauck et al., 2016) using a Shimadzu (Kyoto, Japan) LC-20AD chromatography system equipped with a YMC (Allentown, PA) HPLC C18 semi-preparative column (10 mm × 250 mm, 5 μm) with UV detection at 280 nm. A linear gradient was used at room temperature with a flow rate of 4.5 mL/min with the following program: 60% to 90% acetonitrile/water over 20 min, 3 min hold at 90% acetonitrile/water, and re-equilibration for 3 min at 60% acetonitrile/water. The injection volume was 200 μL of 3.5 mg/mL BDF in ethanol/ethyl acetate (6:1, v/v). Data acquisition and integration were carried out using Shimadzu Lab Solutions software. The retention times of the diastereomeric pairs were ~17.2 and ~18.0 min, representing the R/S and S/R (“cis”) and R/R and S/S (“trans”) isomers, respectively. After semi-preparative reversed phase purification, the mobile phase was evaporated to dryness at room temperature under a stream of dry nitrogen, and each pair of diastereomers was stored in solid form at −20 °C until use.

To confirm BDF enantiomer enrichment, an aliquot of collected BDF fractions (in 100% DMSO) was diluted to 10 ng/mL final concentration in methanol: water at 1:1 ratio. The diluted BDF fractions were injected (10 μL) into Waters Xbridge C18 HPLC column (3.5 μm 2.1 × 50 mm). The column was equilibrated with acetonitrile and 0.1% formic acid in water at ratio 60:40 (v/v). The acetonitrile content was increased to 65% over 1.5 min, and then held for 3.5 min. The column was washed with 95% acetonitrile for 1 min and re-equilibrated at initial composition for ~1 min before the next analysis. The flow rate was 0.3 ml/min. Autosampler and column temperatures were set at 4 and 45 °C, respectively. BDF was detected using negative ion electrospray tandem mass spectrometry on a Shimadzu (Kyoto, Japan) LCMS-8050 triple quadrupole mass spectrometer with selection reaction monitoring of the transitions m/z 523 to m/z 135 for BDF and m/z 443 to m/z 293 for the internal standard DiF. The retention times for diastereomeric BDF peak 1 and peak 2 were approximately 3.9 and 4.3 min, respectively.

Animals

Adult (250–300 gm) male Sprague Dawley rats were purchased from Harlan (Envigo, Huntingdon, UK). After acclimation, rats were administered BDF at the indicated doses. The BDF stock solution was diluted into sterile saline to a final volume of 500 μL to provide the required dose. Animals were restrained manually in the supine position and BDF administered by i.p. injection to the right lower quadrant using a 25G needle. After BDF administration, the rats were housed in a chemical hazard room, to allow collection of waste and bedding contaminated with BDF. All procedures were approved by local IACUC. Pharmacokinetic studies were carried out in adult male New Zealand White rabbits (Envigo, Huntingdon, UK). Following BDF administration (200 μg/kg, once by gavage), blood samples were collected via medial artery of the ear at indicated times, BDF extracted as described (Lindeblad et al., 2018), and then an aliquot measured by using HPLC-MS/MS for BDF enantiomeric pair quantification.

Cell culture and treatments

Human neuroblastoma SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) /F-12 (Gibco Thermo Fisher, Waltham, MA) supplemented with 2 mM L-glutamine, and antibiotic-antimycotic solution (10,000 I.U. penicillin/mL, 10 mg/mL streptomycin, 25 μg/mL amphotericin diluted 100-fold) and containing 15% (v/v) heat-inactivated fetal bovine serum (FBS, Sigma, St Louis, MO). Cells were maintained at 37°C in a humidified chamber containing 95% air and 5% CO2. Cells were allowed to reach >80% confluence and then used for studies. For experimental treatments, the indicated concentrations of BDF enantiomers, or equivalent amount of vehicle (DMSO) were added to cells in 96-well plates. The final amount of DMSO present corresponded to 0.60%, equivalent to the amount present when the BDF concentration was 20 mM. Cell viability was measured as MTS reductive activity using CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI).

Data analysis

All statistical analyses were carried out using GraphPad Prism v7.0 (GraphPad Software, San Diego, CA). Effects of BDF on cell death were analyzed by 2-way ANOVA and Tukey post hoc comparisons. BDF enantiomer serum half-lives were determined by fitting the data to a one-phase decay with plateau (final serum level) set to 0. Significance was taken at p values < 0.05. LD50 ± s.e.m. values were determined by Dixon analysis (Dixon and Mood, 1946), using the formula LD50= [Xf + K*D] ± S*D (mean ± SD) where Xf is the final test dose, K and S are constants provided by the Dixon sequence, and D is the dosing interval. For BDF-trans and BDF-rac, k= 0.315; for BDF-cis k = 0.372. For BDF-trans and BDF-rac S is 0.67, for BDF-cis S is 0.61. The dosing interval D was 50 μg/ml. Calculated LD50 values were compared by 1-way ANOVA and Tukey’s multiple comparison.

Results

Enrichment of BDF enantiomeric forms

Racemic BDF (BDF-rac) was applied to an HPLC C18 semi-preparative column and eluted with a linear 60–90% acetonitrile:water gradient, which separated BDF into 2 peaks corresponding to the cis (R/S and S/R, peak1) and trans (R/R and S/S, peak 2) diastereomers. Fractions containing BDF-cis or BDF-trans were collected, evaporated to dryness, then resuspended into 100% DMSO. Aliquots were re-run under the same conditions to confirm enrichment. Quantitation of peak areas showed that BDF-cis (Fig 2B) contained about 2.2% BDF-trans; and BDF-trans (Fig 2C) contained 3.6% BDF-cis.

Figure 2. Enrichment for BDF enantiomeric pairs.

Figure 2.

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Racemic BDF (BDF-rac) was separated as described in materials and methods, fractions corresponding to peaks 1 and 2 were collected, and rerun to confirm enrichment. (A) Chromatograph of BDF-rac; (B) Rerun of peak 1 corresponding to BDF-cis; (C) Rerun of peak 2 corresponding to BDF-trans.

BDF enantiomers show differential toxicity

BDF toxicity was examined in adult male Sprague Dawley rats (Fig 3). The LD50 calculated for racemic BDF was 266 ± 34 mg/kg, slightly higher than our previously reported value of 221 ± 14 mg/ml (Kalinin et al., 2017). Administration of BDF-trans was less toxic, with an LD50 of 316 ± 34 mg/kg; and BDF-cis was most toxic with an LD50 of 219 ± 18 μg/kg.

Figure 3. Dixon plot analysis of acute BDF toxicity.

Figure 3.

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Adult male Sprague Dawley rats (about 300 gm) were administered an i.p. injection (500 μL) to provide the final indicated doses of (A) BDF-rac (n=6 rats), (B) BDF-trans (n=7 rats), or (C) BDF-cis (n=6 rats). The x-axis ‘Test’ indicates each individual test that was done at the indicated BDF concentration. Results of Dixon analysis are shown in the figures, and are mean ± SD. Symbols: o, survived; x, died. *, P < 0.05 versus BDF-cis or BDF-trans; ***, P < 0.0001 versus BDF-trans (1-way ANOVA Tukey’s multiple comparison).

BDF enantiomer serum half-lives

One factor that could contribute to increased toxicity of BDF-cis is a longer biological half-life. To allow multiple sampling from the same animal, we carried out a pharmacokinetic analysis using NZW rabbits (Fig 4). When analyzed as a single-phase decay, the half-life for BDF-rac was determined to be 2.4 days (Fig 4A). However, the calculated half-lives differed between diastereomers, with BDF-trans showing a half-life of 2.1 days, and BDF-cis a longer half-life of 2.8 days (Fig 4B).

Figure 4. Serum half-lives for BDF diastereomers.

Figure 4.

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NZW rabbits (n=8) were given a single administration of racemic BDF (200 μg/kg, by gavage). Blood samples were taken at the indicated times, and serum BDF-cis and –trans levels determined by UHPLC/MS-MS. Total BDF levels were calculated as sum of BDF-cis and BDF-trans (A) Time course of total BDF levels; (B) Time course of BDF-cis (red circles) and BDF-trans (blue circles) levels. Data represent mean ± SE of n=5–8 samples per time point due to deaths occurring during the study (one at day 8; 2 at day 10). The data were fit for one phase decay with plateau set to 0, and the best fit curves are indicated.

BDF enantiomers induce neuronal damage

Direct neurotoxic actions of BDF diastereomers were tested using human SH-SY5Y neuroblastoma cells. In the presence of 10 mM BDF (Fig 5A), reduction of the tetrazolium compound MTS (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was significantly reduced by BDF-rac and BDFcis, with decreases between 20–30% observed after 5 to 6 hr incubation. In contrast incubation with 10 mM BDF-trans did not alter MTS reduction during this time period. At the higher concentration of 20 mM (Fig 5B), MTS reduction was similarly reduced by all BDF species.

Figure 5. BDF induces neurotoxicity.

Figure 5.

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The indicated concentrations of BDF or equivalent amount of vehicle were added to human SH-Y-5Y neuroblastoma cells. Immediately after, a time course of mitochondrial reductive capacity was measured by assessment of MTS reduction at the indicated time points. Data are mean ± SE of n=4–6 independent samples. *, P<0.05; **< P<0.005; ***, P<0.0005, 2-way ANOVA and Tukey’s range test.

Discussion

The novel findings of this study are that in adult Sprague-Dawley rats the LD50 of BDF-cis diastereomers was ~50% lower than that for BDF-trans diastereomers (219 versus 316 μg/kg, respectively) while racemic BDF had intermediate potency (266 μg/kg). In adult New Zealand white rabbits, the serum half life of BDF-cis was approximately 40% longer than BDF-trans (2.8 versus 2.1 days, respectively) which could contribute to its observed increased toxicity. It should be noted that due to the limited number of samples collected at early time points, half-life calculations will be more weighted by the later time points and may not accurately reflect early kinetics. However, at least within the first few days following admininstration of racemic BDF the BDF-trans diastereomers show more rapid decrease than the cis forms from serum. Lastly, BDF-cis (10 μM), but not BDF-trans, damaged cultured SH-SY5Y human neuroblastoma cells by attenuating reductive capacity. Taken together, these data suggest that different toxic manifestations of BDF poisoning in mammals could be attributed, at least in part, to differences in the relative enantiomer concentrations in various racemic formulations of this commercially-available toxicant. Further studies including using the 4 individual BDF isomers will be required to confirm or refute this hypothesis.

It is well established that depletion of clotting factors that leads to uncontrolled bleeding and death is the predominant mechanism underlying BDF-induced toxicity in mammals (King and Tran, 2015). However, several studies suggest that BDF also elicits anticoagulant-independent tissue injury. For instance, BDF is highly potent in Sprague-Dawley rats (LD50 ~200 μg/kg) yet, unlike rabbits, minimal bleeding is observed in various organs at necropsy (Kalinin et al., 2017). Likewise, BDF-poisoned bobcats (Lynx rufus) residing in southern California die predominantly from severe systemic inflammation elicited by parasite mites whereas BDF-poisoned mountain lions (Puma concolor) in the same area die from massive internal bleeding (Fraser et al., 2018; Serieys et al., 2018). In addition, we found that exposure of primary neuronal cells to BDF induced mitochondrial dysfunction and cell death (Marangoni et al., 2016) and that BDF (and other superwarfarins but not warfarin) can intercalate into and disrupt cell membranes (Ayee et al., 2016), which could dysregulate cell function and induce cell death. Although the mechanism(s) underlying these disparate toxic effects are uncertain, the presence of distinct BDF isomers in racemic BDF may play a role.

In conclusion, we found that in rats LD50 of BDF-cis is ~50% lower than that for BDF-trans while racemic BDF has intermediate potency. In rabbits, BDF-cis had a longer half-life than BDF-trans which could account, in part, for its increased toxicity. Lastly, BDF-cis, but not BDF-trans, damaged cultured SH-SY5Y human neuroblastoma cells by attenuating overall NAD(P)H dependent reductive capacity, suggesting a reduction of cellular metabolic processes. Taken together, these data suggest that different toxic manifestations of BDF poisoning could be attributed, in part, to differences in the relative enantiomer levels present in the commercially available formulations.

Highlights.

  • The long acting anti-coagulant brodifacoum has 4 isomers, however their relative toxicity has not been determined

  • HPLC was used to obtain 2 pairs of BDF diastereomers

  • Toxicity studies in adult male rats show the BDF-cis diastereomers are more toxic than the BDF-trans forms

  • Serum levels of BDF-cis showed a longer half-life than that of BDF-trans, which could contribute to the greater toxicity of BDf-cis

Acknowledgments

Funding Information

This work was supported by NIH Grant U01NS083457, VA merit grants (to I.R. and G.W.), and a VA research career scientist award (to D.L.F.).

Footnotes

Declarations

DLF and IR are co-founders of EnSol Therapeutics, LLC. GW is co-founder of ResQ Pharmaceuticals, Inc. All other authors declare that they have no competing financial or non-financial interests.

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