Anticoagulant Rodenticide Toxicity in Terrestrial Raptors: Tools to Estimate the Impact on Populations in North America and Globally.

Anticoagulant rodenticides (ARs) have caused widespread contamination and poisoning of predators and scavengers. The diagnosis of toxicity proceeds from evidence of hemorrhage, and subsequent detection of residues in liver. Many factors confound the assessment of AR poisoning, particularly exposure dose, timing and frequency of exposure, and individual and taxon-specific variables. There is a need, therefore, for better AR toxicity criteria. To respond, we compiled a database of second-generation anticoagulant rodenticide (SGAR) residues in liver and postmortem evaluations of 951 terrestrial raptor carcasses from Canada and the United States, 1989 to 2021. We developed mixed-effects logistic regression models to produce specific probability curves of the toxicity of ∑SGARs at the taxonomic level of the family, and separately for three SGARs registered in North America, brodifacoum, bromadiolone, and difethialone. The ∑SGAR threshold concentrations for diagnosis of coagulopathy at 0.20 probability of risk were highest for strigid owls (15 ng g-1) lower and relatively similar for accipitrid hawks and eagles (8.2 ng g-1) and falcons (7.9 ng g-1), and much lower for tytonid barn owls (0.32 ng g-1). These values are lower than those we found previously, due to compilation and use of a larger database with a mix of species and source locations, and also to refinements in the statistical methods. Our presentation of results on the family taxonomic level should aid in the global applicability of the numbers. We also collated a subset of 440 single-compound exposure events and determined the probability of SGAR-poisoning symptoms as a function of SGAR concentration, which we then used to estimate relative SGAR toxicity and toxic equivalence factors: difethialone, 1, brodifacoum, 0.8, and bromadiolone, 0.5. Environ Toxicol Chem 2024;43:988-998. © 2024 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Sanitary measures considerably improve the management of resistant Norway rats on livestock farms.

BACKGROUND: Norway rats (Rattus norvegicus) need to be controlled to prevent transmission of pathogens and damages to stored products and material, leading to considerable economic risks and losses. Given increasing resistance in Norway rats, the most persistent, bio-accumulative and toxic anticoagulant rodenticides are widely used for management, which presents hazards to the environment especially for non-target species. We investigated how sanitary measures improved management of Norway rats on 12 paired livestock farms in a region of Germany with a high population of resistant rats for reducing application of rodenticides. We recorded food intake, and tracked activity and resistance frequency during the pre-treatment, treatment and post-treatment periods. RESULTS: In the post-treatment period, farms using sanitary measures had a higher control success with > 13% more bait boxes without feeding than farms not using sanitary measures. In addition, the reoccurrence of rats was delayed by 85 days. With increasing accessibility to buildings and more precise positioning of the boxes, control success improved, especially when rats could not spread from water-bearing ditches through the sewer system, and when rat-hunting animals were present. Resistant animals were more common indoors than outdoors, and there were more resistant rats recorded before and during treatment than in the post-treatment period. CONCLUSION: The control success was substantially higher and reoccurrence was delayed using sanitary measures on farms. Sanitary measures can reduce resistance indirectly due to delayed re-colonization and establishment of resistant populations inside buildings. Hence, sanitary measures help to reduce economic losses, rodenticides required for rat management and environmental risk especially in the resistance area. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of anticoagulant rodenticide poisoning on spatial behavior of farm dwelling Norway rats.

Commensal rodent species cause damage to crops and stored products, they transmit pathogens to people, livestock and pets and threaten native flora and fauna. To minimize such adverse effects, commensal rodents are predominantly managed with anticoagulant rodenticides (AR) that can be transferred along the food chain. We tested the effect of the uptake of the AR brodifacoum (BR) by Norway rats (Rattus norvegicus) on spatial behavior because this helps to assess the availability of dead rats and residual BR to predators and scavengers. BR was delivered by oral gavage or free-fed bait presented in bait stations. Rats were radio-collared to monitor spatial behavior. BR residues in rat liver tissue were analyzed using liquid chromatography coupled with tandem mass spectrometry. Norway rats that had consumed BR decreased distances moved and had reduced home range size. Treatment effects on spatial behavior seemed to set in rapidly. However, there was no effect on habitat preference. Ninety-two percent of rats that succumbed to BR died in well-hidden locations, where removal by scavenging birds and large mammalian scavengers is unlikely. Rats that ingested bait from bait stations had 65% higher residue concentrations than rats that died from dosing with two-fold LD50. This suggests an overdosing in rats that are managed with 0.0025% BR. None of the 70 BR-loaded rats was caught/removed by wild predators/scavengers before collection of carcasses within 5-29 h. Therefore, and because almost all dead rats died in well-hidden locations, they do not seem to pose a significant risk of AR exposure to large predators/scavengers at livestock farms. Exposure of large predators may originate from AR-poisoned non-target small mammals. The few rats that died in the open are accessible and should be removed in routine searches during and after the application of AR bait to minimize transfer of AR into the wider environment.

Differences in teratogenicity of some vitamin K antagonist substances used as human therapeutic or rodenticide are due to major differences in their fate after an oral administration.

All vitamin K antagonist active substances used as rodenticides were reclassified in 2016 by the European authorities as active substances "toxic for reproduction", using a "read-across" alternative method based on warfarin, a human vitamin K antagonist drug. Recent study suggested that all vitamin K antagonist active substances are not all teratogenic. Using a neonatal exposure protocol, warfarin evokes skeletal deformities in rats, while bromadiolone, a widely used second-generation anticoagulant rodenticide, failed to cause such effects. Herein, using a rat model we investigated the mechanisms that may explain teratogenicity differences between warfarin and bromadiolone, despite their similar vitamin K antagonist mechanism of action. This study also included coumatetralyl, a first-generation active substance rodenticide. Pharmacokinetic studies were conducted in rats to evaluate a potential difference in the transfer of vitamin K antagonists from mother to fetus. The data clearly demonstrate that warfarin is highly transferred from the mother to the fetus during gestation or lactation. In contrast, bromadiolone transfer from dam to the fetus is modest (5% compared to warfarin). This difference appears to be associated to almost complete uptake of bromadiolone by mother's liver, resulting in very low exposure in plasma and eventually in other peripheric tissues. This study suggests that the pharmacokinetic properties of vitamin K antagonists are not identical and could challenge the classification of such active substances as "toxic for reproduction".

Vitamin K antagonist rodenticides display different teratogenic activity.

Vitamin K antagonists (VKA) are not recommended during pregnancy because warfarin (a first-generation VKA) is associated with a malformation syndrome "the fetal warfarin syndrome" (FWS). VKA are also used for rodent management worldwide. Recently, the Committee for Risk Assessment responsible for the European chemical legislation for advances on the safe use of chemicals had classed 8 anticoagulant used as rodenticides in the reprotoxic category 1A or 1B. This classification emerges from a read-across prediction of toxicity considering the warfarin malformation syndrome. Herein, our study explores the teratogenicity of warfarin at the human therapeutic dose and that of bromadiolone, a second-generation anticoagulant rodenticide. Using a rat model, our study demonstrates that warfarin used at the therapeutic dose is able to induce teratogenicity, while in the same conditions bromadiolone does not induce any teratogenic effect, challenging the classification of all VKA as reprotoxic molecules.

Brodifacoum Toxicity in American Kestrels (Falco sparverius) with Evidence of Increased Hazard on Subsequent Anticoagulant Rodenticide Exposure.

A seminal question in ecotoxicology is the extent to which contaminant exposure evokes prolonged effects on physiological function and fitness. A series of studies were undertaken with American kestrels ingesting environmentally realistic concentrations of the second-generation anticoagulant rodenticide (SGAR) brodifacoum. Kestrels fed brodifacoum at 0.3, 1.0, or 3.0 µg/g diet wet weight for 7 d exhibited dose-dependent hemorrhage, histopathological lesions, and coagulopathy (prolonged prothrombin and Russell's viper venom times). Following termination of a 7-d exposure to 0.5 µg brodifacoum/g diet, prolonged blood clotting time returned to baseline values within 1 wk, but brodifacoum residues in liver and kidney persisted during the 28-d recovery period (terminal half-life estimates >50 d). To examine the hazard of sequential anticoagulant rodenticide (AR) exposure, kestrels were exposed to either the first-generation AR chlorophacinone (1.5 µg/g diet) or the SGAR brodifacoum (0.5 µg/g diet) for 7 d and, following a recovery period, challenged with a low dose of chlorophacinone (0.75 µg/g diet) for 7 d. In brodifacoum-exposed kestrels, the challenge exposure clearly prolonged prothrombin time compared to naive controls and kestrels previously exposed to chlorophacinone. These data provide evidence that the SGAR brodifacoum may have prolonged effects that increase the toxicity of subsequent AR exposure. Because free-ranging predatory and scavenging wildlife are often repeatedly exposed to ARs, such protracted toxicological effects need to be considered in hazard and risk assessments. Environ Toxicol Chem 2020;39:468-481. © 2020 SETAC.

Efficacy of rodenticide baits with decreased concentrations of brodifacoum: Validation of the impact of the new EU anticoagulant regulation.

Anticoagulants are the most frequently used rodenticides at the global scale. Because of their persistency, bioaccumulation and potential for secondary intoxication, they have faced increasing legislative regulations. Recently, the European Union Regulation (EU) 2016/1179 resulted in the production and application of rodenticides with nearly half dose (<30 ppm) of anticoagulants. However, published data on the biological efficacy of rodenticides with decreased doses are scarce in the EU. Therefore, this work compared the efficacy of the original high-dose (50 ppm) and new low-dose (25 ppm) brodifacoum-based baits in the offspring of wild-caught house mice (Mus musculus L.). In the no-choice laboratory feeding tests, 100% animals died in all treated groups and 0% died in the control groups. The achieved time to death did not differ between the original and low-dose baits across both types of feeding trials/regimes. The low-dose baits (25 ppm) were consequently tested under field conditions in two populations showing 95.7% and 99.8% efficacy. The obtained results highlighted the good efficacy of the new baits based on low-dose brodifacoum in non-resistant mouse populations. However, further validation is required regarding the remaining anticoagulant compounds and resistant rodent populations.

The relative toxicity of brodifacoum enantiomers.

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.

Exposure of non-target small mammals to anticoagulant rodenticide during chemical rodent control operations.

The extensive use of anticoagulant rodenticides (ARs) results in widespread unintentional exposure of non-target rodents and secondary poisoning of predators despite regulatory measures to manage and reduce exposure risk. To elucidate on the potential vectoring of ARs into surrounding habitats by non-target small mammals, we determined bromadiolone prevalence and concentrations in rodents and shrews near bait boxes during an experimental application of the poison for 2 weeks. Overall, bromadiolone was detected in 12.6% of all small rodents and insectivores. Less than 20 m from bait boxes, 48.6% of small mammals had detectable levels of bromadiolone. The prevalence of poisoned small mammals decreased with distance to bait boxes, but bromadiolone concentration in the rodenticide positive individuals did not. Poisoned small mammals were trapped up to 89 m from bait boxes. Bromadiolone concentrations in yellow-necked mice (Apodemus flavicollis) were higher than concentrations in bank vole (Myodes glareolus), field vole (Microtus agrestis), harvest mouse (Micromys minutus), and common shrew (Sorex araneus). Our field trials documents that chemical rodent control results in widespread exposure of non-target small mammals and that AR poisoned small mammals disperse away from bating sites to become available to predators and scavengers in large areas of the landscape. The results suggest that the unintentional secondary exposure of predators and scavengers is an unavoidable consequence of chemical rodent control outside buildings and infrastructures.

Do bromadiolone treatments to control grassland water voles (Arvicola scherman) affect small mustelid abundance?

BACKGROUND: The use of pesticides can affect non-target species by causing population declines through indirect intoxication. Small mustelids (SMs; weasels, Mustela nivalis L.; stoats, Mustela erminea L.) consume water voles (WVs, Arvicola scherman S.) and can be exposed to bromadiolone, an anticoagulant rodenticide used in some countries to reduce WV damage to grasslands. Here, we investigated whether bromadiolone affected SM abundance. RESULTS: We monitored SM abundance using footprint tracking tunnels in spring and autumn at ten sites. Among these sites, four were treated with bromadiolone, while six were not treated. We found reduced SM abundance at these four sites from spring to autumn (treated sites, mean ± SE SM abundance change = -1.68 ± 0.42; untreated sites, 0.29 ± 0.25). Using a linear model, we observed that SM abundance decreased as a function of the quantity of bromadiolone applied during the 3 months before the autumn estimate. We found that WV abundance increased at treated sites (linear model, treated sites, mean ± SE WV abundance change = 1.4 ± 0.4; untreated sites, 0.33 ± 0.25). Thus, at treated sites, SM abundance declined despite increased food availability. By analyzing residues in vole livers and SM scats we showed that SMs may be exposed to bromadiolone at the sites where this compound was used. CONCLUSION: This study is the first to document the relationship between SM abundance and bromadiolone usage for small mammal control. Declines in SM abundance were observed at treated sites, where bromadiolone residue was found in SM scats. This correlative approach suggests that bromadiolone treatment may lead to seasonal SM declines and associated WV increases. © 2018 Society of Chemical Industry.

[Evaluation of Brodifacoum-induced Toxicity by Metabonomics Approach Based on HPLC-TOF-MS].

OBJECTIVES: To analyse the metabolic changes in urine of rats with brodifacoum intoxication, and to reveal the molecular mechanism of brodifacoum-induced toxicity on rats. METHODS: By establishing a brodifacoum poisoning rats model, the urine metabolic profiling data of rats were acquired using high performance liquid chromatography-time of flight mass spectrometry （HPLC-TOF-MS）. The orthogonal partial least squares analysis-discrimination analysis （OPLS-DA） was applied for the multivariate statistics and the discovery of differential metabolites closely related to toxicity of brodifacoum. RESULTS: OPLS-DA score plot showed that the urinary metabolic at different time points before and after drug administration had good similarity within time period and presented clustering phenomenon. Comparing the urine samples of rats before drug administration with which after drug administration, twenty-two metabolites related to brodifacoum-induced toxicity were selected. CONCLUSIONS: The toxic effect of brodifacoum worked by disturbing the metabolic pathways in rats such as tricarboxylic cycle, glycolysis, sphingolipid metabolism and tryptophan metabolism, and the toxicity of brodifacoum is characterized of accumulation effect. The metabonomic method based on urine HPLC-TOF-MS can provide a novel insight into the study on molecular mechanism of brodifacoum-induced toxicity.

The Long-Lasting Rodenticide Brodifacoum Induces Neuropathology in Adult Male Rats.

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 µg/kg. Measurement of tissue BDF levels showed accumulation throughout the body, including the 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.

Anticoagulant rodenticide exposure and toxicosis in four species of birds of prey in Massachusetts, USA, 2012-2016, in relation to use of rodenticides by pest management professionals.

Restrictions on second-generation anticoagulant rodenticides (SGARs) in the United States, which were partially implemented in 2011, prohibit the sale of SGAR products through general consumer outlets to minimize use by non-professional or non-agricultural applicators. This study analyzed liver tissue from four species of birds of prey admitted to a wildlife clinic in Massachusetts, USA, from 2012-2016 for residues of anticoagulant rodenticides (ARs). Ninety-four birds were analyzed; 16 were symptomatic for AR toxicosis, and 78 asymptomatic. Ninety-six percent of all birds tested were positive for SGARs: 100% of those diagnosed with AR toxicosis ante-mortem and/or post-mortem and 95% of subclinically exposed birds. Brodifacoum was found in 95% of all birds. Sixty-six percent of all birds contained residues of two or more SGARs. A significant increase in exposures to multiple SGARs occurred in later years in the study. Pesticide use reports (PURs) filed with the Massachusetts Department of Agricultural Resources were reviewed to determine the frequency of use of different ARs by pest management professionals (PMPs) across five years. This study finds that the three SGARs favored by PMPs-bromadiolone, difethialone, brodifacoum-were present in combination in the majority of birds, with increases in multiple exposures driven by increased detections of bromadiolone and difethialone. Continued monitoring of AR residues in nontarget species following full implementation of sales and packaging restrictions in the US is needed in order to elucidate the role of PMP use of SGARs in wildlife exposures and to evaluate the effectiveness of current mitigation measures.

Mortality of Western Burrowing Owls ( Athene cunicularia hypugaea) Associated with Brodifacoum Exposure.

Western Burrowing Owls ( Athene cunicularia hypugaea) frequently occupy periurban areas, where they may be exposed to pest control agents. This short communication describes necropsy findings and detected brodifacoum rodenticide levels for four Western Burrowing Owls in Lake Havasu City, Arizona, US, 2013-15. Levels detected ranged from 0.077 mg/kg to 0.497 mg/kg. Brodifacoum, one of several second-generation anticoagulant rodenticides recently removed from the general consumer market, is still available for use by licensed pesticide applicators. Despite recent regulatory actions, second-generation anticoagulant pesticides continue to threaten predatory species in periurban areas.

Synthesis and biological activity of the novel indanedione anticoagulant rodenticides containing fluorine.

Here, 3 fluorinated intermediates of drug were synthesized: (M1), (M2), (M3). Three new anticoagulant rodenticides were designed which were based on 4-hydroxycoumarin or 1,3-indandione, added acute toxicity groups containing fluorine. The structures of synthesized compounds were analyzed and proved by FT-IR spectroscopy and 1H nuclear magnetic resonance (1H-NMR). The compounds were also evaluated for their anticoagulant and acute biologic activity. In addition, both the acute orally toxicity and the feeding indexes of R1 and R2 were tested. The result of the experiment proved that the new synthesis of 1, 3 - indan diketone for maternal new anticoagulant rodenticide can replace the current 4 - hydroxyl coumarin as the mother of the second generation anticoagulant rodenticide and 1, 3 - indan diketone for maternal new anticoagulant rodenticides will have a good development prospect.

Evaluation of Brodifacoum-induced Toxicity by Metabonomics Approach Based on HPLC-TOF-MS / 法医学杂志

OBJECTIVES@#To analyse the metabolic changes in urine of rats with brodifacoum intoxication, and to reveal the molecular mechanism of brodifacoum-induced toxicity on rats.@*METHODS@#By establishing a brodifacoum poisoning rats model, the urine metabolic profiling data of rats were acquired using high performance liquid chromatography-time of flight mass spectrometry （HPLC-TOF-MS）. The orthogonal partial least squares analysis-discrimination analysis （OPLS-DA） was applied for the multivariate statistics and the discovery of differential metabolites closely related to toxicity of brodifacoum.@*RESULTS@#OPLS-DA score plot showed that the urinary metabolic at different time points before and after drug administration had good similarity within time period and presented clustering phenomenon. Comparing the urine samples of rats before drug administration with which after drug administration, twenty-two metabolites related to brodifacoum-induced toxicity were selected.@*CONCLUSIONS@#The toxic effect of brodifacoum worked by disturbing the metabolic pathways in rats such as tricarboxylic cycle, glycolysis, sphingolipid metabolism and tryptophan metabolism, and the toxicity of brodifacoum is characterized of accumulation effect. The metabonomic method based on urine HPLC-TOF-MS can provide a novel insight into the study on molecular mechanism of brodifacoum-induced toxicity.

Comparison of the effects of difenacoum and brodifacoum on the ultrastructure of rat liver cells.

We used transmission electron microscopy to examine the cytotoxic effects of the second-generation anticoagulant rodenticides difenacoum and brodifacoum on rat liver. A single dose of difenacoum or brodifacoum was administered to rats by gastric gavage and liver samples were taken after 24 h, four days or seven days. In the livers of rats treated with difenacoum for 24 h, hepatocytes typically showed increased numbers of lysosomes, as well as enlargement of both the perinuclear space and the cisternae of the rough endoplasmic reticulum (RER), while sinusoids were irregularly shaped and contained Kupffer cells. Similar irregularities occurred in brodifacoum-treated rats at the same time point, but additionally increased numbers of vacuoles, damaged mitochondrial cristae, and clumping of chromatin were observed in hepatocytes, and hemolysed erythrocytes were noted in the sinusoids. Comparable findings were made in each group of rats after four days. After seven days of difenacoum treatment, hepatocytes suffered loss of cytoplasmic material and mitochondrial shrinkage, while RER cisternae became discontinuous. In contrast, exposure to brodifacoum for seven days caused the formation of numerous vacuoles and lipid droplets, disordered mitochondrial morphology, chromatin clumping and invagination of the nuclear envelope in hepatocytes. Sinusoids in the livers of rodenticide-treated rats contained an accumulation of dense material, lipid droplets, cells with pycnotic nuclei and hemolysed erythrocytes. Overall, our results show that brodifacoum causes more severe effects in liver cells than difenacoum. Thus our microscopic data along with additional biochemical assays point to a severe effect of rodenticide on vertebrates.

Plasma metabolic profiling analysis of toxicity induced by brodifacoum using metabonomics coupled with multivariate data analysis.

Brodifacoum is one of the most widely used rodenticides for rodent control and eradication; however, human and animal poisoning due to primary and secondary exposure has been reported since its development. Although numerous studies have described brodifacoum induced toxicity, the precise mechanism still needs to be explored. Gas chromatography mass spectrometry (GC-MS) coupled with an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was applied to characterize the metabolic profile of brodifacoum induced toxicity and discover potential biomarkers in rat plasma. The toxicity of brodifacoum was dose-dependent, and the high-dose group obviously manifested toxicity with subcutaneous hemorrhage. The blood brodifacoum concentration showed a positive relation to the ingestion dose in toxicological analysis. Significant changes of twenty-four metabolites were identified and considered as potential toxicity biomarkers, primarily involving glucose metabolism, lipid metabolism and amino acid metabolism associated with anticoagulant activity, nephrotoxicity and hepatic damage. MS-based metabonomics analysis in plasma samples is helpful to search for potential poisoning biomarkers and to understand the underlying mechanisms of brodifacoum induced toxicity.

Membrane Cholesterol Modulates Superwarfarin Toxicity.

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.