Synthesis of Ferulenol by Engineered Escherichia coli: Structural Elucidation by Using the In Silico Tools.

4-Hydroxycoumarin (4HC) has been used as a lead compound for the chemical synthesis of various bioactive substances and drugs. Its prenylated derivatives exhibit potent antibacterial, antitubercular, anticoagulant, and anti-cancer activities. In doing this, E. coli BL21(DE3)pLysS strain was engineered as the in vivo prenylation system to produce the farnesyl derivatives of 4HC by coexpressing the genes encoding Aspergillus terreus aromatic prenyltransferase (AtaPT) and truncated 1-deoxy-D-xylose 5-phosphate synthase of Croton stellatopilosus (CstDXS), where 4HC was the fed precursor. Based on the high-resolution LC-ESI(±)-QTOF-MS/MS with the use of in silico tools (e.g., MetFrag, SIRIUS (version 4.8.2), CSI:FingerID, and CANOPUS), the first major prenylated product (named compound-1) was detected and ultimately elucidated as ferulenol, in which information concerning the correct molecular formula, chemical structure, substructures, and classifications were obtained. The prenylated product (named compound-2) was also detected as the minor product, where this structure proposed to be the isomeric structure of ferulenol formed via the tautomerization. Note that both products were secreted into the culture medium of the recombinant E. coli and could be produced without the external supply of prenyl precursors. The results suggested the potential use of this engineered pathway for synthesizing the farnesylated-4HC derivatives, especially ferulenol.

Synthesis and Biological Screening of New 4-Hydroxycoumarin Derivatives and Their Palladium(II) Complexes.

Two newly synthesized 4-hydroxycoumarin bidentate ligands (L1 and L2) and their palladium(II) complexes (C1 and C2) were screened for their biological activities, in vitro and in vivo. Structures of new compounds were established based on elemental analysis, 1H NMR, 13C NMR, and IR spectroscopic techniques. The obtained compounds were tested for their antioxidative and cytotoxic activities and results pointed to selective antiradical activity of palladium(II) complexes towards •OH and -•OOH radicals and anti-ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical) activity comparable to that of ascorbate. Results indicated the effect of C1 and C2 on the enzymatic activity of the antioxidative defense system. In vitro cytotoxicity assay performed on different carcinoma cell lines (HCT166, A375, and MIA PaCa-2), and one healthy fibroblast cell line (MRC-5) showed a cytotoxic effect of both C1 and C2, expressed as a decrease in carcinoma cells' viability, mostly by induction of apoptosis. In vivo toxicity tests performed on zebrafish embryos indicated different effects of C1 and C2, ranging from adverse developmental effect to no toxicity, depending on tested concentration. According to docking studies, both complexes (C1 and C2) showed better inhibitory activity in comparison to other palladium(II) complexes.

Demonstrating Ligandability of the LC3A and LC3B Adapter Interface.

Autophagy is the common name for a number of lysosome-based degradation pathways of cytosolic cargos. The key components of autophagy are members of Atg8 family proteins involved in almost all steps of the process, from autophagosome formation to their selective fusion with lysosomes. In this study, we show that the homologous members of the human Atg8 family proteins, LC3A and LC3B, are druggable by a small molecule inhibitor novobiocin. Structure-activity relationship (SAR) studies of the 4-hydroxy coumarin core scaffold were performed, supported by a crystal structure of the LC3A dihydronovobiocin complex. The study reports the first nonpeptide inhibitors for these protein interaction targets and will lay the foundation for the development of more potent chemical probes for the Atg8 protein family which may also find applications for the development of autophagy-mediated degraders (AUTACs).

Monitoring the hepatic residues of cis- and trans-diastereoisomers of second generation anticoagulant rodenticides reveals a different bioaccumulation of diastereoisomers in the food chain of the Réunion harrier (Circus maillardi).

The Réunion harrier is an endangered raptor and endemic species to the Réunion Island. Second generation anticoagulant rodenticides (SGARs) are widely used pesticides on the island in order to control rodent populations. The latter are responsible for the transmission of leptospirosis to humans, the damage of sugarcane crops, and the decline of endemic endangered birds. SGARs are very persistent chiral pesticides and consequent secondary exposure or poisoning of the Réunion harrier has been observed (73% of prevalence in a group of 58 harriers). Commercial formulations of SGARs are a mixture of trans- and cis-diastereoisomers. Both diastereoisomers of all SGARs have been shown to inhibit coagulation function with the same potency. On the other hand, they have been shown to have a significant difference in terms of tissue-persistence. This difference has led to residue levels in rats with a significantly lower proportion of one of the isomers compared to the bait composition. In this study, residue levels of the diastereoisomers of all SGARs were evaluated in the livers of 58 harrier carcasses. The respective concentrations and proportions of cis- and trans- diastereoisomers of all SGARs are presented. cis-Brodifacoum and trans-bromadiolone had the highest concentrations (up to 438 and 573 ng/g ww respectively), while trans-brodifacoum was less than 46 ng/g and cis-bromadiolone was barely detected. cis-Difenacoum showed the highest prevalence and the highest concentration was 82 ng/g ww, while trans-difenacoum was never detected. This study demonstrated that only cis-brodifacoum and trans-bromadiolone (and cis-difethialone, but with a low prevalence) had hepatic concentrations above a toxic threshold. The cis- and trans-diastereoisomers of SGARs had differential bioaccumulation in the food chain of the Réunion harrier compared to commercial baits. This suggests that a change of the proportions of SGARs diastereoisomers in baits could reduce the risk of secondary poisoning of predators, but maintain primary toxicity.

Determination of anticoagulant rodenticides in faeces of exposed dogs and in a healthy dog population.

BACKGROUND: Exposure to anticoagulant rodenticides (ARs) in dogs is among the most common causes of poisoning in small animal practice, but information about toxicokinetic of these rodenticides in dogs is lacking. We analysed blood and faeces from five accidentally exposed dogs and 110 healthy dogs by reversed phase ultra-high performance liquid chromatography-tandem mass spectrometry. The aim of the study was to estimate elimination of brodifacoum, bromadiolone and difenacoum after acute exposure, calculate the half-lives of these rodenticides in dogs, estimate faecal elimination in a litter of puppies born, and further to identify the extent of AR exposure in a healthy dog population. RESULTS: Three dogs were included after single ingestions of brodifacoum; two dogs ingested bromadiolone and one dog ingested difenacoum. Maximum concentrations in faeces were found after day 2-3 for all ARs. The distribution half-lives were 1-10 days for brodifacoum, 1-2 days for bromadiolone and 10 days for difenacoum. Brodifacoum and difenacoum had estimated terminal half-lives of 200-330 days and 190 days, respectively. In contrast, bromadiolone had an estimated terminal half-life of 30 days. No clinical signs of poisoning or coagulopathy were observed in terminal elimination period. In blood, the terminal half-life of brodifacoum was estimated to 8 days. Faeces from a litter of puppies born from one of the poisoned dogs were examined, and measurable concentrations of brodifacoum were detected in all samples for at least 28 days after parturition. A cross-sectional study of 110 healthy domestic dogs was performed to estimate ARs exposure in a dog population. Difenacoum was detected in faeces of one dog. Blood and faecal samples from the remaining dogs were negative for all ARs. CONCLUSIONS: Based on the limited pharmacokinetic data from these dogs, our results suggest that ARs have a biphasic elimination in faeces using a two-compartment elimination kinetics model. We have shown that faecal analysis is suitable and reliable for the assessment of ARs exposure in dogs and a tool for estimating the AR half-lives. Half-lives of ARs could be a valuable indicator in the exposed dogs and provides important information for veterinarians monitoring AR exposure and assessment of treatment length in dogs.

Mass spectrometry characterization of anticoagulant rodenticides and hydroxyl metabolites.

RATIONALE: Anticoagulant rodenticides (ARs) are used worldwide for rodent population control to protect human health and biodiversity, and to prevent agricultural and economic losses. Rodents may develop a metabolic resistance to ARs. In order to help understand such metabolic resistance, mass spectrometry was used to position the hydroxylated group of hydroxyl metabolites of second-generation ARs (SGARs). METHODS: Most AR pesticides are derived from the 4-hydroxycoumarin/thiocoumarin family. We used low-resolution and high-resolution mass spectrometry to understand the fragmentation pathways of the ARs and their respective metabolites, and to better define the structure of their tandem mass spectrometry product ions. RESULTS: Seven specific product ions were evidenced for five ARs, with their respective chemical structures. Those ions were obtained as well from the mass spectra of the hydroxyl metabolites of four SGARs, difenacoum (DFM), brodifacoum (BFM), difethialone (DFTL) and flocoumafen (FLO), with different positions of the hydroxyl group. CONCLUSIONS: The differences in chemical structure between DFM on the one hand and BFM, FLO and DFTL on the other could explain the differences in bioavailability between these two groups of molecules. The defined product ions will be used to investigate the part played by the metabolic issue in the field resistance of SGARs.

Synthesis of Three Bioactive Aromatic Compounds by Introducing Polyketide Synthase Genes into Engineered Escherichia coli.

Intermediates in aromatic amino acid biosynthesis can serve as substrates for the synthesis of bioactive compounds. In this study we used two intermediates in the shikimate pathway of Escherichia coli, chorismate and anthranilate, to synthesize three bioactive compounds: 4-hydroxycoumarin (4-HC), 2,4-dihydroxyquinoline (DHQ), and 4-hydroxy-1-methyl-2(1H)-quinolone (NMQ). We introduced genes for the synthesis of salicylic acid from chorismate to supply the substrate for 4-HC and the gene encoding N-methyltransferase for the synthesis of N-methylanthranilate from anthranilate. Polyketide synthases and coenzyme (Co)A ligases were tested to determine the optimal combination of genes for the synthesis of each compound. We also tested several constructs and identified the best one for increasing levels of endogenous substrates for chorismate, anthranilate, and malonyl-CoA. With the use of these strategies, 255.4 mg/L 4-HC, 753.7 mg/L DHQ, and 17.5 mg/L NMQ were synthesized. This work provides a basis for the synthesis of diverse coumarin and quinoline derivatives with potential medical applications.

Frequent detection of anticoagulant rodenticides in raptors sampled in Taiwan reflects government rodent control policy.

Anticoagulant rodenticides (ARs) are known to cause extensive secondary exposure in top predators in Europe and North America, but there remains a paucity of data in Asia. In this study, we collected 221 liver samples from 21 raptor species in Taiwan between 2010 and 2018. Most birds were recovered from rescue organizations, but some free-ranging individuals were obtained from bird-strike prevention measures at airports. ARs were detected in 10 species and more than half of the total samples. Common rodent-eating Black-winged Kites (Elanus caeruleus) had the highest prevalence (89.2%) and highest average sum concentration (0.211 ±â€¯0.219 mg/kg), which was similar between free-ranging birds at airports and injured birds from rescue organizations. Scavenging Black Kites (Milvus migrans) and snake-eating Crested Serpent-eagles (Spilornis cheela) had the second highest prevalence or sum concentration, respectively. Seven different AR compounds were detected, of which brodifacoum was the most common and had the highest average concentration, followed by flocoumafen and bromadiolone. The frequency of occurrence in the three most numerous species (Black-winged Kite, Crested Goshawk [Accipiter trivirgatus], and Collared Scops-owl [Otus lettia]) was significantly higher in autumn than summer, which is consistent with the timing of the Taiwanese government's supply of free ARs to farmers. Regional differences in the detection of individual compounds also tended to reflect differences in human population density and use patterns (in agriculture or urban-dominated environments). Clinical poisoning was confirmed in Black Kites with sum concentrations as low as 0.026 mg/kg; however, further study of interspecific differences in AR sensitivity and potential population effects are needed. In addition, continued monitoring remains important given the Taiwanese government has modified their farmland rodent control policy to gradually reduce free AR supplies since 2015.

Development of a series of 4-hydroxycoumarin platinum(IV) hybrids as antitumor agents: Synthesis, biological evaluation and action mechanism investigation.

A series of new 4-hydroxycoumarin platinum(IV) complexes were designed, synthesized and evaluated as antitumor agents. All the title compounds display moderate to effective antitumor activities toward the tested cell lines and two prominent compounds were screened out with activities comparable to cisplatin and oxaliplatin. The mechanism investigation demonstrates that the platinum(IV) compounds could be reduced to bivalence and exert significant genotoxicity to tumor cells. Meanwhile the coumarin moiety endows the title compounds with cyclooxygenase inhibitory competence which might favour the reduction of tumor-related inflammation and further influence tumor proliferation. The coumarin platinum(IV) complex could effectively induce apoptosis of SKOV-3 cells through up-regulating the expression of caspase3 and caspase9. Furthermore, the conversion of platinum(II) drugs to platinum(IV) form via the conjunction with 4-hydroxycoumarin enhances the drug uptake in whole cells and DNA simultaneously. Moreover, the 4-hydroxycoumarin platinum(IV) complex could combine with human serum albumin via van der Waals force and hydrogen bond, which would influence their transport and bioactivities in vivo.

Synthesis, characterization and unravelling the molecular interaction of new bioactive 4-hydroxycoumarin derivative with biopolymer: Insights from spectroscopic and theoretical aspect.

In the progress of small molecule as drug candidates, 4-hydroxycoumarin based compounds bearing a crucial place as potent antibiotic agents with appreciable safety in drug invention. Being synthetically and easily obtainable, 4-hydroxycoumarin related compounds with planar structure have been promoted predominantly as DNA targeting agent. Nevertheless, here we elucidate the synthesis, characterization and theoretical study of bio-active small molecule 4-hydroxy-3,4'-bichromenyl-2,2'-dione (4HBD). Then we have illuminated the binding interactions of 4HBD with calf thymus DNA (ctDNA), which is particularly designed for biological application. Extensive investigations of the binding of 4HBD with ctDNA are provided by utilizing multi-spectroscopic and molecular docking approaches, including UV-vis absorbance, steady-state, time-resolved fluorescence spectroscopy and circular dichroism study. The calculated binding and quenching constant value from quantitative data analysis of absorption and emission spectroscopy shows that 4HBD binds to the ctDNA groove. Further confirmation of the same is found by comparative displacement and iodide quenching studies. Negative enthalpy, negative free energy and positive entropy change imply a hydrophobic force monitors the association of 4HBD with the biomacromolecule. Interestingly the small molecule (4HBD) shows potential anti-bacterial activity against the model pathogenic gram-negative (Escherichia coli and Pseudomonas aeruginosa) and gram-positive (Bacillus subtilis and Staphylococcus aureus) bacteria. The noncytotoxic nature of the 4HBD is demonstrated in vitro with the help of MTT assay by normal kidney epithelial (NKE), breast cancer cells (MCF-7) and human prostate cancer cell (PC3) lines. Hemolytic assay exhibits insignificant hemolysis of human erythrocyte cells at the minimum inhibitory concentration (MIC) of these tested bacteria. In this regard the present invention of 4-hydroxycoumarin based antimicrobial and noncytotoxic 4HBD molecule holds future promise in the development of new antibiotics.

Elevated difenacoum metabolism is involved in the difenacoum-resistant phenotype observed in Berkshire rats homozygous for the L120Q mutation in the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene.

BACKGROUND: Soon after difenacoum began to be used, resistance to this rodenticide was detected in rats in northeast Hampshire and northwest Berkshire in England. Resistance to difenacoum has been reported to be stronger in rats from Berkshire than in rats from Hampshire. Surprisingly, after the discovery of the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene, rats from Berkshire and Hampshire were all shown to be homozygous for the L120Q mutation in Vkorc1. RESULTS: This study aimed to evaluate the resistance of Berkshire rats to confirm their extreme resistance and determine mechanisms supporting this resistance. For this purpose, we created a quasicongenic rat F7 strain by using a Berkshire rat as a donor to introduce the L120Q mutation in Vkorc1 into the genetic background of an anticoagulant-susceptible recipient strain. The use of F7 rats enabled demonstration of (i) the level of resistance to difenacoum conferred by the L120Q mutation, (ii) co-dominance of the L120 and Q120 alleles, (iii) the extreme resistance of Berkshire rats compared with Q120/Q120 rats as a consequence of additional resistance mechanisms, and (iv) the involvement of cytochrome P 450 (CYP450) enzymes in this extreme resistance. CONCLUSION: This study demonstrated that elevated CYP450 oxidative metabolism leading to accelerated difenacoum detoxification is involved in the Berkshire phenotype. © 2017 Society of Chemical Industry.

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.

Evidence of a target resistance to antivitamin K rodenticides in the roof rat Rattus rattus: identification and characterisation of a novel Y25F mutation in the Vkorc1 gene.

BACKGROUND: In spite of intensive use of bromadiolone, rodent control was inefficient on a farm infested by rats in Zaragoza, Spain. While metabolic resistance was previously described in this rodent species, the observation of a target resistance to antivitamin K rodenticides had been poorly documented in Rattus rattus. RESULTS: From rats trapped on the farm, cytochrome b and Vkorc1 genes were amplified by PCR and sequenced in order to identify species and detect potential Vkorc1 mutations. VKORC1-deduced amino acid sequences were thus expressed in Pichia pastoris, and inhibition constants towards various rodenticides were determined. The ten rats trapped on the farm were all identified as R. rattus. They were found to be homozygous for the g.74A>T nucleotide replacement in exon 1 of the Vkorc1 gene, leading to p.Y25F mutation. This mutation led to increased apparent inhibition constants towards various rodenticides, probably caused by a partial loss of helical structure of TM4. CONCLUSION: The p.Y25F mutation detected in the Vkorc1 gene in R. rattus trapped on the Spanish farm is associated with the resistance phenotype to bromadiolone that has been observed. It is the first evidence of target resistance to antivitamin K anticoagulants in R. rattus.

Novel macromolecules derived from coumarin: synthesis and antioxidant activity.

The rational design of 4-hydroxycoumarins with tailor-made antioxidant activities is required nowadays due to the wide variety of pharmacologically significant, structurally interesting of coumarins and researcher orientation toward green chemistry and natural products. A simple and unique coumarins have been achieved by reaction of 4-hydroxycoumarin with aromatic aldehyde accompanied with the creation of a macromolecules have 2-aminothiazolidin-4-one. The molecular structures of the compounds were characterized by the Fourier transformation infrared and Nuclear magnetic resonance spectroscopies, in addition to CHN analysis. The scavenging abilities of new compounds against stable DPPH radical (DPPH•) and hydrogen peroxide were done and the results show that the compounds exhibited high antioxidant activates.

Spatial and temporal exposure patterns in non-target small mammals during brodifacoum rat control.

Worldwide pest rodents on livestock farms are often regulated using anticoagulant rodenticides (ARs). Second generation ARs in particular can cause poisoning in non-target species due to their high toxicity and persistence. However, research on exposure of small mammals is rare. We systematically investigated spatial and temporal exposure patterns of non-target small mammals in a large-scale replicated study. Small mammals were trapped at different distances to bait stations on ten farms before, during and after brodifacoum (BR) bait application, and liver samples of 1178 non-target small mammals were analyzed for residues of eight ARs using liquid chromatography coupled with tandem mass spectrometry. BR residues were present in 23% out of 742 samples collected during and after baiting. We found clear spatial and temporal exposure patterns. High BR residue concentrations mainly occurred within 15m from bait stations. Occurrence and concentrations of residues significantly decreased with increasing distance. This pattern was found in almost all investigated taxa. After baiting, significantly more individuals contained residues than during baiting but concentrations were considerably lower. Residue occurrence and concentrations differed significantly among taxa, with the highest maximal residue concentrations in Apodemus species, which are protected in Germany. Although Sorex species are known to be insectivorous we regularly found residues in this genus. Residues of active agents other than brodifacoum were rare in all samples. The confirmation of substantial primary exposure in non-target small mammals close to the baiting area indicates considerable risk of secondary poisoning of predators, a pathway that was possibly underestimated until now. Our results will help to develop risk mitigation strategies to reduce risk for non-target small mammals, as well as their predators, in relation to biocidal AR usage.

Bioaccumulation of chlorophacinone in strains of rats resistant to anticoagulants.

BACKGROUND: Anticoagulants are the only available compounds in the EU to control rat populations. Resistance to anticoagulant rodenticides (antivitamin K or AVK) is described and widespread across Europe. The present objective was to determine whether resistance was associated with an increased potential for bioaccumulation of AVK in the liver. Rats were selected from three major resistant genetically identified strains across Europe: Y139C (Germany), Y139F (France) and L120Q (United Kingdom). The rats were housed in individual cages and fed chlorophacinone wheat bait (50 mg kg(-1) ). Animals were assigned to groups for euthanasia either on day 1, 4, 9 or 14 (resistant rats) or on days 1 and 4 (susceptible rats). RESULTS: Chlorophacinone accumulated from day 1 to day 4 in all strains (maximum 160 µg liver(-1)) and remained stable thereafter. There was no significant difference between strains. Extensive metabolism of chlorophacinone was also found, and was similar (in nature and proportion of metabolites) across strains (3 OH-metabolites identified). Only the survival time differed significantly (L120Q > Y139C = Y139F > susceptible). CONCLUSIONS: Accumulation of chlorophacinone occurs from day 1 to day 4, and an equilibrium is reached, suggesting rapid elimination. Resistant and susceptible rats accumulate chlorophacinone to the same extent and only differ in terms of survival times. Resistant rats may then be a threat for non-target species for prolonged periods of time.

High exposure rates of anticoagulant rodenticides in predatory bird species in intensively managed landscapes in Denmark.

The extensive use of anticoagulant rodenticides (ARs) for rodent control has led to widespread secondary exposure in nontarget predatory wildlife species. We investigated exposure rates and concentrations of five ARs in liver samples from five raptors and six owls from Denmark. A total of 430 birds were analysed. ARs were detected in 84-100 % of individual birds within each species. Multiple AR exposure was detected in 73 % of all birds. Average number of substances detected in individual birds was 2.2 with no differences between owls and raptors. Difenacoum, bromadiolone, and brodifacoum were the most prevalent substances and occurred in the highest concentrations. Second-generation ARs made up 96 % of the summed AR burden. Among the six core species (sample size >30), summed AR concentrations were lower in rough-legged buzzard (Buteo lagopus) and long-eared owl (Asio otus) than in barn owl (Tyto alba), buzzard (B. buteo), kestrel (Falco tinnunculus), and tawny owl (Strix aluco). There was a strong tendency for seasonal variations in the summed AR concentration with levels being lowest during autumn, which is probably related to an influx of less-exposed migrating birds from northern Scandinavia during autumn. High hepatic AR residue concentrations (>100 ng/g wet weight), which have been associated with symptoms of rodenticide poisoning and increased mortality, were recorded high frequencies (12.9-37.4 %) in five of the six core species. The results suggest that the present use of ARs in Denmark, at least locally, may have adverse effects on reproduction and, ultimately, population status in some raptors and owls.

Study on the interaction between NCP-(4-hydroxycoumarins) and bovine serum albumin by spectroscopic techniques.

The interaction between N-confused porphyrins-(4-hydroxycoumarins) diad (NCP-(4-hydroxycoumarins)) and bovine serum albumin (BSA) was studied using fluorescence and ultraviolet spectroscopy at different temperatures under imitated physiological conditions. The experimental results showed that the fluorescence of BSA was quenched by NCP-(4-hydroxycoumarins) through a combined quenching procedure. The binding constants, binding sites and corresponding thermodynamic parameters between NCP-(4-hydroxycoumarins) and BSA at different temperatures were obtained. According to Förster non-radiation energy transfer theory, the binding distance between BSA and NCP-(4-hydroxycoumarins) was calculated to be about 2.1 nm. The effect of NCP-(4-hydroxycoumarins) on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy. In addition, the effect of some metal ions Cu(2+), Ca(2+), Mg(2+), and Ni(2+) on the binding constant between NCP-(4-hydroxycoumarins) and BSA was examined.

Accidental discharge of brodifacoum baits into a freshwater lake: a case study.

Approximately 700 kg of cereal bait containing 20 ppm of the anticoagulant rodenticide brodifacoum was spilled into a southern New Zealand lake in 2010 from a helicopter being used to transport containers of brodifacoum bait for an aerial baiting operation. In the month after the spill no residual brodifacoum was detected in samples of lake water, sediment, benthic invertebrates, eels, and two birds.