A fatal poisoning due to consumption of crushed Abrus precatorius seeds: an autopsy case report.

Abrus precatorius is an ornamental plant that belongs to the Leguminoceae family. It contains toxalbumin, named abrin, in all of its parts. However, the seeds are more toxic when consumed in crushed form. Deaths due to abrus seed poisoning are rare. We are reporting a case of suicidal ingestion of crushed abrus precatorius seeds by a 37-year-old female. She presented to the hospital with complaints of multiple episodes of vomiting and diarrhea. She was resuscitated with fluid boluses, followed by a stomach wash and activated charcoal, and referred to our tertiary hospital, where she presented with giddiness, abdominal pain, bloody diarrhea, and epigastric tenderness. On subsequent days, she developed altered sensorium, renal failure, and electrolyte imbalance. She was managed conservatively. Blood investigation revealed elevated leucocyte count, increased urea and creatinine levels, and elevated liver enzymes. She died five days after ingesting the seeds. On autopsy examination, the brain and lungs were congested and edematous. The peritoneal cavity contained around 500mL of straw-colored fluid. Petechial hemorrhages were present over the lungs, heart, and liver surfaces. The mucosa of the gastrointestinal tract was hemorrhagic, the liver showed steatosis, and the kidneys showed congestion and obscuration of the corticomedullary junction. Histopathologically, the lungs showed mild congestion with alveolar edema, the liver showed necrosis with steatosis, and the kidney showed acute tubular necrosis. The toxicology screening was positive for abrin. This case highlights not only the rare fatality following abrus precatorius poisoning but also the toxic nature of this plant.

Expanding role of ribosome-inactivating proteins: From toxins to therapeutics.

Ribosome-inactivating proteins (RIPs) are toxic proteins with N-glycosidase activity. RIPs exert their action by removing a specific purine from 28S rRNA, thereby, irreversibly inhibiting the process of protein synthesis. RIPs can target both prokaryotic and eukaryotic cells. In bacteria, the production of RIPs aid in the process of pathogenesis whereas, in plants, the production of these toxins has been attributed to bolster defense against insects, viral, bacterial and fungal pathogens. In recent years, RIPs have been engineered to target a particular cell type, this has fueled various experiments testing the potential role of RIPs in many biomedical applications like anti-viral and anti-tumor therapies in animals as well as anti-pest agents in engineered plants. In this review, we present a comprehensive study of various RIPs, their mode of action, their significance in various fields involving plants and animals. Their potential as treatment options for plant infections and animal diseases is also discussed.

A highly sensitive electrochemiluminescence method for abrin detection by a portable biosensor based on a screen-printed electrode with a phage display affibody as specific labeled probe.

Abrin is a highly toxic ribosome-inactivating protein, which could be used as a biological warfare agent and terrorist weapon, and thus needs to be detected efficiently and accurately. Affibodies are a new class of engineered affinity proteins with small size, high affinity, high stability, favorable folding and good robustness, but they have rarely played a role in biological detection. In this work, we establish a novel electrochemiluminescence (ECL) method for abrin detection with a phage display affibody as the specific probe for the first time, to our knowledge, and a portable biosensor based on a screen-printed electrode (SPE) as the testing platform. On the basis of the double antibody sandwich structure in our previous work, we used a phage display affibody instead of monoclonal antibody as a new specific labeled probe. Due to numerous signal molecules labeled on M13 phages, significant signal amplification was achieved in this experiment. Under optimized conditions, a linear dependence was observed from 0.005 to 100 ng/mL with a limit of detection (LOD) of 5 pg/mL. This assay also showed good reproducibility and specificity, and performed well in the detection of simulated samples. Considering its high sensitivity, interference resistance and convenience, this new biosensing system based on phage display affibodies and a portable ECL biosensor holds promise for in situ detection of toxins and pollutants in different environments.

A Self-Driven Microfluidic Chip for Ricin and Abrin Detection.

Ricin and abrin are phytotoxins that can be easily used as biowarfare and bioterrorism agents. Therefore, developing a rapid detection method for both toxins is of great significance in the field of biosecurity. In this study, a novel nanoforest silicon microstructure was prepared by the micro-electro-mechanical systems (MEMS) technique; particularly, a novel microfluidic sensor chip with a capillary self-driven function and large surface area was designed. Through binding with the double antibodies sandwich immunoassay, the proposed sensor chip is confirmed to be a candidate for sensing the aforementioned toxins. Compared with conventional immunochromatographic test strips, the proposed sensor demonstrates significantly enhanced sensitivity (≤10 pg/mL for both toxins) and high specificity against the interference derived from juice or milk, while maintaining good linearity in the range of 10-6250 pg/mL. Owing to the silicon nanoforest microstructure and improved homogeneity of the color signal, short detection time (within 15 min) is evidenced for the sensor chip, which would be helpful for the rapid tracking of ricin and abrin for the field of biosecurity.

Correlation of abrin-mediated inhibition of protein synthesis and apoptosis.

The plant toxin, abrin, a type-II ribosome inactivating protein, is extremely lethal, the human fatal dose being ~1 µg/kg body weight. Abrin has been classified as an agent for bioterrorism, which is of concern. Conversely, the high toxic property of abrin has been employed in generating immunotoxins, whereas its toxin moiety is conjugated to cell surface marker-specific antibodies for cell-targeted killing. Different cell types exhibit variable levels of sensitivity to abrin toxicity; therefore, adequate knowledge of the molecular mechanism that governs the activity of the protein would be a safeguard. To gain insights into this, two cell lines requiring strikingly different concentrations of abrin for inactivating ribosomes were studied. Employing conjugates of the wild-type and active site mutant of abrin A chain with the ricin B chain, it was found that abrin-induced apoptosis was dependent on inhibition of protein synthesis (PSI) leading to ER-stress in Ovcar-3 cells, but not in KB cells. Abrin was also observed to cause direct DNA damage in KB cells, while in Ovcar-3 cells abrin-induced DNA damage was found to be dependent on caspases. Overall, the study demonstrates that the correlation of abrin-mediated PSI and apoptosis is cell-specific and abrin can induce more than one pathway to cause cell death. © 2018 IUBMB Life, 71(3):357-363, 2019.

Neutrophil mediated inflammatory lung damage following single Sub lethal inhalation exposure to plant protein toxin abrin in mice.

Abrin, a highly toxic plant protein found in the seeds of Abrus precatorius plant. To date, there is no antidote against abrin intoxication. Abrin is toxic by all routes of exposure, but inhalation exposure is the most toxic of all routes. Present study was conducted to evaluate the acute inhalation toxicity of aerosolized abrin in BALB/c mice. Animals were exposed to 0.2 and 0.8LC50 doses of aerosolized abrin and evaluated at 1 and 3 day post toxin exposure. Bronchoalveolar fluid from lungs was used for evaluation of markers for lung injury. Abrin inhalation exposure caused rise in LDH activity, protein content, increase in ß-glucuronidase and myeloperoxidase activity. Increase in CRP activity, MMP-9 expression and recruitment of CD11b + inflammatory cells in lungs was also observed which was associated with severe inflammation and lung damage. Histopathological findings support the lung damage after abrin exposure. Our results indicate lung injury after single aerosol inhalation exposure, associated with excessive inflammation, oxidative stress, pulmonary edema followed by lung damage. These results could supplement treatment strategies and planning for therapeutic approaches against aerosolized abrin inhalation exposure.

Acute disseminated encephalomyelitis due to abrus precatorius poisoning - A case report.

Abrus precatorius, commonly known as 'Rosary pea' or 'Jequirity pea' and known as 'Shisham, Batrah-Hindi or Ain Alfreeth' in the Middle East, grows wild in the tropical and subtropical areas of the world. The seeds of the plant contain one of the most potent toxins known to man. Poisoning with abrus seeds is a rare occurrence as the harder outer coat of the seeds generally resists digestion and such reports are scarce in the literature. We present here a case of a 22 year old lady who developed severe vomiting, diarrhoea and malena at the initial stages and later seizures and acute disseminated encephalomyelitis due to deliberate chewing and swallowing of abrus seeds. She was rescued with several sessions of membrane plasmapheresis and supportive care. The neuropathological process of acute disseminated encephalomyelitis due to abrus poisoning was reversed by plasmapheresis.

Detection of Abrin by Electrochemiluminescence Biosensor Based on Screen Printed Electrode.

For the convenience of fast measurement in the outdoor environment, a portable electrochemiluminescence biosensor with the screen-printed electrode as the reaction center was developed, which possesses the characteristics of high sensitivity, small scale, simplified operation and so on, and has been used for in situ detection of abrin. First, combining with magnetic separation technique, the "biotin-avidin" method was used to immobilize the polyclonal antibody (pcAb) on the magnetic microspheres surface as the capture probe. Secondly, the Ru(bpy)32+-labeled monoclonal antibody (mcAb) was used as the specific electrochemiluminescence signal probe. Then, the "mcAb-toxin-pcAb" sandwich model was built to actualize the quantitative detection of abrin on the surface of the screen-printed electrode. The linear detection range was 0.5-1000 ng/mL; the regression equation was Y = 89.251lgX + 104.978 (R = 0.9989, n = 7, p < 0.0001); and the limit of detection (LOD) was 0.1 ng/mL. The sensing system showed high sensitivity, excellent specificity and good anti-interference ability, and could be used for the analysis of trace abrin in various environmental samples with good recovery and reproducibility. Compared with the traditional electrochemiluminescence sensing device, its miniaturization and portability gives it potential to satisfy the requirement of in situ detection.

Abrin P2 suppresses proliferation and induces apoptosis of colon cancer cells via mitochondrial membrane depolarization and caspase activation.

To explore the cytotoxic mechanism of abrin P2 on human colon cancer HCT-8 cells, abrin P2 was isolated from the seed of Abrus precatorius L. It was found that abrin P2 exhibited cytotoxicity toward 12 different human cancer cell lines. Our results demonstrated that abrin P2 suppressed the proliferation of human colon cancer cells (HCT-8 cells) and induced cell cycle arrest at the S and G2/M phases. The mechanism by which abrin P2 inhibited cell proliferation was via the down-regulation of cyclin B1, proliferating cell nuclear antigen and Ki67, as well as the up-regulation of P21. In addition, abrin P2 induced a dose- and time-dependent increase in the rate of HCT-8 cell apoptosis. Treatment with both Z-VAD-FMK, a broad-spectrum caspase inhibitor, and abrin P2 demonstrated that abrin P2 induced HCT-8 cell apoptosis via the activation of caspases. Together, our results revealed that abrin P2-induced apoptosis in HCT-8 cells was associated with the activation of caspases-3/-8/-9, the reduction in the Bcl-2/Bax ratio, the loss of mitochondrial membrane potential, and the increase in cytochrome c release. We further showed that abrin P2 administration effectively suppressed the growth of colon cancer xenografts in nude mice. This is the first report that abrin P2 effectively inhibits colon cancer cell growth in vivo and in vitro by suppressing proliferation and inducing apoptosis.

Unraveling the Roots of Selectivity of Peptide Affinity Reagents for Structurally Similar Ribosomal Inactivating Protein Derivatives.

Peptide capture agents have become increasingly useful tools for a variety of sensing applications due to their ease of discovery, stability, and robustness. Despite the ability to rapidly discover candidates through biopanning bacterial display libraries and easily mature them to Protein Catalyzed Capture (PCC) agents with even higher affinity and selectivity, an ongoing challenge and critical selection criteria is that the peptide candidates and final reagent be selective enough to replace antibodies, the gold-standard across immunoassay platforms. Here, we have discovered peptide affinity reagents against abrax, a derivative of abrin with reduced toxicity. Using on-cell Fluorescence Activated Cell Sorting (FACS) assays, we show that the peptides are highly selective for abrax over RiVax, a similar derivative of ricin originally designed as a vaccine, with significant structural homology to abrax. We rank the newly discovered peptides for strongest affinity and analyze three observed consensus sequences with varying affinity and specificity. The strongest (Tier 1) consensus was FWDTWF, which is highly aromatic and hydrophobic. To better understand the observed selectivity, we use the XPairIt peptide-protein docking protocol to analyze binding location predictions of the individual Tier 1 peptides and consensus on abrax and RiVax. The binding location profiles on the two proteins are quite distinct, which we determine is due to differences in pocket size, pocket environment (including hydrophobicity and electronegativity), and steric hindrance. This study provides a model system to show that peptide capture candidates can be quite selective for a structurally similar protein system, even without further maturation, and offers an in silico method of analysis for understanding binding and down-selecting candidates.

Gaps in forensic toxicological analysis: The veiled abrin.

Abrus precatorius is an herbaceous, flowering plant that is widely distributed in tropical and subtropical regions. Its toxic component, known as abrin, is classified as one of the potentially significant biological warfare agents and bioterrorism tools due to its high toxicity. Abrin poisoning can be utilized to cause accidents, suicides, and homicides, which necessitates attention from clinicians and forensic scientists. Although a few studies have recently identified the toxicological and pharmacological mechanisms of abrin, the exact mechanism remains unclear. Furthermore, the clinical symptoms and pathological changes induced by abrin poisoning have not been fully characterized, and there is a lack of standardized methods for identifying biological samples of the toxin. Therefore, there is an urgent need for further toxicopathologic studies and the development of detection methods for abrin in the field of forensic medicine. This review provides an overview of the clinical symptoms, pathological changes, metabolic changes, toxicologic mechanisms, and detection methods of abrin poisoning from the perspective of forensic toxicology. Additionally, the evidence on abrin in the field of forensic toxicology and forensic pathology is discussed. Overall, this review serves as a reference for understanding the toxicological mechanism of abrin, highlighting the clinical applications of the toxin, and aiding in the diagnosis and forensic identification of toxin poisoning.

Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis.

BACKGROUND: Ricin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. METHODS: Ricin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin-biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. RESULTS: The LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90-62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. CONCLUSION: The developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.

Sensitive Detection and Differentiation of Biologically Active Ricin and Abrin in Complex Matrices via Specific Neutralizing Antibody-Based Cytotoxicity Assay.

Ricin and abrin are highly potent plant-derived toxins, categorized as type II ribosome-inactivating proteins. High toxicity, accessibility, and the lack of effective countermeasures make them potential agents in bioterrorism and biowarfare, posing significant threats to public safety. Despite the existence of many effective analytical strategies for detecting these two lethal toxins, current methods are often hindered by limitations such as insufficient sensitivity, complex sample preparation, and most importantly, the inability to distinguish between biologically active and inactive toxin. In this study, a cytotoxicity assay was developed to detect active ricin and abrin based on their potent cell-killing capability. Among nine human cell lines derived from various organs, HeLa cells exhibited exceptional sensitivity, with limits of detection reaching 0.3 ng/mL and 0.03 ng/mL for ricin and abrin, respectively. Subsequently, toxin-specific neutralizing monoclonal antibodies MIL50 and 10D8 were used to facilitate the precise identification and differentiation of ricin and abrin. The method provides straightforward and sensitive detection in complex matrices including milk, plasma, coffee, orange juice, and tea via a simple serial-dilution procedure without any complex purification and enrichment steps. Furthermore, this assay was successfully applied in the unambiguous identification of active ricin and abrin in samples from OPCW biotoxin exercises.

A Glycoprotein-Based Surface-Enhanced Raman Spectroscopy-Lateral Flow Assay Method for Abrin and Ricin Detection.

Abrin and ricin, both type II ribosome-inactivating proteins, are toxins of significant concern and are under international restriction by the Chemical Weapons Convention and the Biological and Toxin Weapons Convention. The development of a rapid and sensitive detection method for these toxins is of the utmost importance for the first emergency response. Emerging rapid detection techniques, such as surface-enhanced Raman spectroscopy (SERS) and lateral flow assay (LFA), have garnered attention due to their high sensitivity, good selectivity, ease of operation, low cost, and disposability. In this work, we generated stable and high-affinity nanotags, via an efficient freezing method, to serve as the capture module for SERS-LFA. We then constructed a sandwich-style lateral flow test strip using a pair of glycoproteins, asialofetuin and concanavalin A, as the core affinity recognition molecules, capable of trace measurement for both abrin and ricin. The limit of detection for abrin and ricin was 0.1 and 0.3 ng/mL, respectively. This method was applied to analyze eight spiked white powder samples, one juice sample, and three actual botanic samples, aligning well with cytotoxicity assay outcomes. It demonstrated good inter-batch and intra-batch reproducibility among the test strips, and the detection could be completed within 15 min, indicating the suitability of this SERS-LFA method for the on-site rapid detection of abrin and ricin toxins.

Trends in the analysis of abrin poisoning for forensic purposes.

Abrus precatorius is a poisonous plant known since ancient times. Accidental poisoning is more common due to the intake of plant seeds containing deadly abrin which is a highly toxic and a thermolabile plant toxalbumin. Abrin has also been reported to be a potential chemical agent that can be used as bioweapon in military or terrorism. Abrin is a ribosome inactivating protein that contains multiple isotoxic forms of protein subunits called chain A and B. The identification of this toxalbumin in the plant is important to determine cause of death in poisoning cases. Therefore, the present review focuses on the structure, mode of administration, tokicokinetics, extraction procedures and forensic analysis of abrin and other constituents. It is observed that most of the researchers have utilized immunological methods for the detection of plant components. This technique has proved to be more sensitive, reliable and accurate for the detection of extremely low concentrations of toxin.

Silibinin ameliorates abrin induced hepatotoxicity by attenuating oxidative stress, inflammation and inhibiting Fas pathway.

Abrin is a toxin from the seeds of Abrus precatorius. Abrin is considerably more toxic than ricin and a potent bio-warfare agent. The mechanism of abrin induced hepatotoxicity remains unclear. Silibinin has antioxidant, anti-inflammatory and hepatoprotective activities. But, its therapeutic potential in abrin toxicity is unknown. In view of these facts, the purpose of this study was to delineate the mechanisms and ameliorative role of silibinin against abrin induced hepatotoxicity. Parameters related to liver functions, oxidative stress, inflammation, Fas pathway and histopathology were evaluated in the liver of BALB/c mice after abrin exposure. Abrin intoxication resulted in hepatotoxicity, oxidative stress, inflammation, altered histopathology and increased Fas pathway signaling. Silibinin improves survival of abrin-exposed mice by decreasing serum liver enzymes and reinstating the antioxidant capacity. Silibinin also inhibits abrin-induced inflammation and Fas pathway. Present study for the first time demonstrates the hepatoprotective potential of silibinin against abrin toxicity.

Neutralizing Monoclonal Antibody, mAb 10D8, Is an Effective Detoxicant against Abrin-a Both In Vitro and In Vivo.

Abrin is a types II ribosome-inactivating protein (RIP) isolated from Abrus precatorious seeds, which comprises a catalytically active A chain and a lectin-like B chain linked by a disulfide bond. Four isotoxins of abrin have been reported with similar amino-acid composition but different cytotoxicity, of which abrin-a is the most potent toxin. High lethality and easy availability make abrin a potential bioterrorism agent. However, there are no antidotes available for managing abrin poisoning, and treatment is only symptomatic. Currently, neutralizing antibodies remain the most effective therapy against biotoxin poisoning. In this study, we prepared, identified, and acquired a high-affinity neutralizing monoclonal antibody (mAb) 10D8 with a potent pre- and post-exposure protective effect against cytotoxicity and animal toxicity induced by abrin-a or abrin crude extract. The mAb 10D8 could rescue the mouse injected intraperitoneally with a 25 × LD50 dose of abrin-a from lethality and prevent tissue damages. Results indicated that 10D8 does not prevent the binding and internalization of abrin-a to cells but inhibits the enzymatic activity of abrin-a and reduces protein synthesis inhibition of cells. The high affinity, good specificity, and potent antitoxic efficiency of 10D8 make it a promising candidate for therapeutic antibodies against abrin.

A New Method for Abrin Detection Based on the Interaction between Target Molecules and Fluorescently Labeled Aptamers on Magnetic Microspheres.

A quantitative structure-activity relationship (QSAR) model for the structure and affinity of abrin aptamers was established. A higher affinity abrin aptamer based on the established QSAR model was screened by site-directed mutagenesis. The fluorescence quenching effect between magnetic microspheres and fluorescent molecules was studied for the first time. A new method for abrin detection based on the interaction between target molecules and fluorescently labeled aptamers on magnetic microspheres was developed, with the detection limit of 5 ng mL-1. This method can overcome the influence of complex environmental interferents in abrin detection and can meet the analysis requirements for simulated samples such as water, soil, and food.

A Novel Humanized Anti-Abrin A Chain Antibody Inhibits Abrin Toxicity In Vitro and In Vivo.

Abrin, a type-II ribosome inactivating protein from the seed of Abrus precatorius, is classified as a Category B bioterrorism warfare agent. Due to its high toxicity, ingestion by animals or humans will lead to death from multiple organ failure. Currently, no effective agents have been reported to treat abrin poisoning. In this study, a novel anti-abrin neutralizing antibody (S008) was humanized using computer-aided design, which possessed lower immunogenicity. Similar to the parent antibody, a mouse anti-abrin monoclonal antibody, S008 possessed high affinity and showed a protective effect against abrin both in vitro and in vivo, and protected mice that S008 was administered 6 hours after abrin. S008 was found that it did not inhibit entry of abrin into cells, suggesting an intracellular blockade capacity against the toxin. In conclusion, this work demonstrates that S008 is a high affinity anti-abrin antibody with both a neutralizing and protective effect and may be an excellent candidate for clinical treatment of abrin poisoning.

Label-free differentiation and quantification of ricin, abrin from their agglutinin biotoxins by surface plasmon resonance.

Here we describe an affinity molecule-directed surface plasmon resonance (SPR) immunosensor for a label-free, differentiation and quantification of ricin and abrin from their structural highly like agglutinin biotoxins. By an introduction of protein G as the affinity capturing molecule, we fulfilled a complete strategy contains (i) screening monoclonal antibodies to be paired in a sandwiched format, (ii) differentiate quantification from the agglutinin, (iii) ascertain of active from inactive biotoxin, and (iv) structural identification of captured biotoxins on a single chip. By the aid of an enrichment step from immunomagnetic beads, we could accurately measure ricin or abrin with a concentration lowered to 0.6 ng/mL (10 pM) in different complex matrices such as stevia, protein powder, and human plasma, with linear ranges of two or three orders of magnitude, and satisfied recovery. We then differentially quantified the mixed crude extracts from castor beans and jequirity peas, and real samples from the fourth OPCW biotoxin exercise to prove the practical availability. We further provided a SPR-mass spectrometric evidence directly obtained from Protein G affinity chip via a noncovalent molecule surface for the first time for definitely structural identification for crude extracts.