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.

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.

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.

Characterization of Lung Injury following Abrin Pulmonary Intoxication in Mice: Comparison to Ricin Poisoning.

Abrin is a highly toxic protein obtained from the seeds of the rosary pea plant Abrus precatorius, and it is closely related to ricin in terms of its structure and chemical properties. Both toxins inhibit ribosomal function, halt protein synthesis and lead to cellular death. The major clinical manifestations following pulmonary exposure to these toxins consist of severe lung inflammation and consequent respiratory insufficiency. Despite the high similarity between abrin and ricin in terms of disease progression, the ability to protect mice against these toxins by postexposure antibody-mediated treatment differs significantly, with a markedly higher level of protection achieved against abrin intoxication. In this study, we conducted an in-depth comparison between the kinetics of in vivo abrin and ricin intoxication in a murine model. The data demonstrated differential binding of abrin and ricin to the parenchymal cells of the lungs. Accordingly, toxin-mediated injury to the nonhematopoietic compartment was shown to be markedly lower in the case of abrin intoxication. Thus, profiling of alveolar epithelial cells demonstrated that although toxin-induced damage was restricted to alveolar epithelial type II cells following abrin intoxication, as previously reported for ricin, it was less pronounced. Furthermore, unlike following ricin intoxication, no direct damage was detected in the lung endothelial cell population following abrin exposure. Reduced impairment of intercellular junction molecules following abrin intoxication was detected as well. In contrast, similar damage to the endothelial surface glycocalyx layer was observed for the two toxins. We assume that the reduced damage to the lung stroma, which maintains a higher level of tissue integrity following pulmonary exposure to abrin compared to ricin, contributes to the high efficiency of the anti-abrin antibody treatment at late time points after exposure.

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.

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.

Differential toxicity of abrin in human cell lines of different organ origin.

Abrus precatorius is a highly toxic seed containing the poison abrin. Similar in properties to ricin, this toxin binds to ribosomes causing cessation of protein synthesis and cell death. With an estimated human lethal dose of 0.1-1 µg/kg, it has been the cause of fatalities due to accidental and intentional ingestion. In present study, we profiled seven human cell lines of different organ origin, for their sensitivity against abrin toxicity. These cell lines are, A549, COLO 205, HEK 293, HeLa, Hep G2, Jurkat, SH-SY5Y and derived from lung, intestine, kidney, cervix, liver, immune and nervous system respectively. MTT, NR, CVDE and LDH assays have been used to determine their response against abrin toxin. Among these cell lines A549 was the most sensitive cell line while Hep G2 was found least sensitive cell lines. Hep G2 cells are shown to have mitochondrial resistance and delayed generation of oxidative stress compared to A549 cells. Remarkable variation in sensitivity against abrin toxicity prompted the evaluation of Bcl2, Bax and downstream caspases in both cells. Difference in Bcl2 level has been shown to play important role in variable sensitivity. Findings of present study are helpful for selection of suitable cellular model for toxicity assessment and antidote screening.

E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide targets abrin and ricin toxicity: Hitting two toxins with one stone.

The efficacy of small molecule inhibitors (SMIs) against the enzymatic activity of Shiga toxin prompted the evaluation of their efficacy on related toxins viz. ricin and abrin. Ricin, like Shiga toxin, is listed as a category B bioweapon and belongs to the type II family of ribosome inactivating proteins (RIPs). Abrin though structurally and functionally similar to ricin, is considerably more toxic. In the present study, 35 compounds were evaluated in A549 cells in in vitro assays, of which 5 offered protection against abrin and 2 against ricin, with IC50 values ranging between 30.5-1379 µM and 300-341 µM, respectively. These findings are substantiated by fluorescence based thermal shift assay. Moreover, the binding of the promising compounds to the toxin components has been validated by Surface Plasmon Resonance assay and in vitro protein synthesis assay. In vivo studies reveal complete protection of mice with compound 4 E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide against orally administered lethal doses of, both, abrin and ricin. The present study thus proposes the emergence of E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide as a lead compound against RIPs.

CCD Based Detector for Detection of Abrin Toxin Activity.

Abrin is a highly potent and naturally occurring toxin produced in the seeds of Abrus precatorius (Rosary Pea) and is of concern as a potential bioterrorism weapon. There are many rapid and specific assay methods to detect this toxic plant protein, but few are based on detection of toxin activity, critical to discern biologically active toxin that disables ribosomes and thereby inhibits protein synthesis, producing cytotoxic effects in multiple organ systems, from degraded or inactivated toxin which is not a threat. A simple and low-cost CCD detector system was evaluated with colorimetric and fluorometric cell-based assays for abrin activity; in the first instance measuring the abrin suppression of mitochondrial dehydrogenase in Vero cells by the MTT-formazan method and in the second instance measuring the abrin suppression of green fluorescent protein (GFP) expression in transduced Vero and HeLa cells. The limit of detection using the colorimetric assay was 10 pg/mL which was comparable to the fluorometric assay using HeLa cells. However, with GFP transduced Vero cells a hundred-fold improvement in sensitivity was achieved. Results were comparable to those using a more expensive commercial plate reader. Thermal inactivation of abrin was studied in PBS and in milk using the GFP-Vero cell assay. Inactivation at 100 °C for 5 min in both media was complete only at the lowest concentration studied (0.1 ng/mL) while treatment at 63 °C for 30 min was effective in PBS but not milk.

Dose dependent acute toxicity of abrin in Balb/c mice after intraperitoneal administration.

Abrin toxin is one of the most potent and deadly plant toxin obtained from the seeds of Abrus precatorious. It is more toxic than ricin which is classified as Schedule 1 agent by OPCW and Category B bioterrorism agent by Centre for Disease Control (CDC). Dose dependent acute toxicity of abrin is still a matter of investigation. The present study was carried out to assess the toxicity of abrin from sub lethal to supralethal doses (0.5X, 1X, 2X and 5XLD50) after intraperitoneal administration. After 8 and 24h of abrin exposure, hematological, biochemical, inflammatory and oxidative stress associated parameters were analyzed. Liver histology was also done to analyze the effect of abrin. Abrin exerts its toxicity in a dose and time dependent manner. Increases in neutrophil counts, lipid peroxidation with decreased lymphocyte counts, are the initiating factor irrespective of time and dose. At higher doses of abrin there was a decrease in hemoglobin level and RBC count which is reflected by increased levels of serum ammonia and bilirubin. Neutrophil infiltration in the liver and lipid peroxidation cause liver toxicity (increased production of ALT and ALP); oxidative stress (depletion of GSH and total antioxidant status); inflammation (increased production of TNF-α and IFN-γ). Further, at higher doses of abrin, intensity of oxidative stress, inflammation and liver toxicity are more pronounced which may have been maintained by the self-sustaining loop of toxicity leading to death of the animals.

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.

Integration of transcriptomics, proteomics and metabolomics data to reveal the biological mechanisms of abrin injury in human lung epithelial cells.

BACKGROUND: Abrin toxin (AT) is a potent plant toxin that belongs to the type Ⅱ ribosome inactivating protein family and is recognized as an important toxin agent for potential bioweapons. Exposure to AT by way of aerosol is the most lethal route, but the mechanism of injury requires further investigation. MATERIALS AND METHODS: In the present study, we performed a comprehensive analysis of transcriptomics, proteomics and metabolomics on the potential mechanism of abrin injury in human lung epithelial cells. RESULTS: In total, 6838 genes, 314 proteins and 178 metabolites showed significant changes in human lung epithelial cells after AT treatment. Using molecular function, pathway, and network analysis, the genes and proteins regulated in AT-treated cells were mainly attributed to amino acid metabolism, lipid metabolism, and genetic information processing. Furthermore, a comprehensive analysis of the transcripts, proteins, and metabolites was performed. The results revealed that the correlated genes, proteins, and metabolism pathways regulated in AT-treated human lung epithelial cells were involved in tryptophan metabolism, biosynthesis of amino acids, and protein digestion and absorption. CONCLUSION: This study provides large-scale omics data to develop new strategies for the prevention, rapid diagnosis, and treatment of AT poisoning, especially AT from aerosol.

Biological Toxins as the Potential Tools for Bioterrorism.

Biological toxins are a heterogeneous group produced by living organisms. One dictionary defines them as "Chemicals produced by living organisms that have toxic properties for another organism". Toxins are very attractive to terrorists for use in acts of bioterrorism. The first reason is that many biological toxins can be obtained very easily. Simple bacterial culturing systems and extraction equipment dedicated to plant toxins are cheap and easily available, and can even be constructed at home. Many toxins affect the nervous systems of mammals by interfering with the transmission of nerve impulses, which gives them their high potential in bioterrorist attacks. Others are responsible for blockage of main cellular metabolism, causing cellular death. Moreover, most toxins act very quickly and are lethal in low doses (LD50 < 25 mg/kg), which are very often lower than chemical warfare agents. For these reasons we decided to prepare this review paper which main aim is to present the high potential of biological toxins as factors of bioterrorism describing the general characteristics, mechanisms of action and treatment of most potent biological toxins. In this paper we focused on six most danger toxins: botulinum toxin, staphylococcal enterotoxins, Clostridium perfringens toxins, ricin, abrin and T-2 toxin. We hope that this paper will help in understanding the problem of availability and potential of biological toxins.

Whole-Cell Multiparameter Assay for Ricin and Abrin Activity-Based Digital Holographic Microscopy.

Ricin and abrin are ribosome-inactivating proteins leading to inhibition of protein synthesis and cell death. These toxins are considered some of the most potent and lethal toxins against which there is no available antidote. Digital holographic microscopy (DHM) is a time-lapse, label-free, and noninvasive imaging technique that can provide phase information on morphological features of cells. In this study, we employed DHM to evaluate the morphological changes of cell lines during ricin and abrin intoxication. We showed that the effect of these toxins is characterized by a decrease in cell confluence and changes in morphological parameters such as cell area, perimeter, irregularity, and roughness. In addition, changes in optical parameters such as phase-shift, optical thickness, and effective-calculated volume were observed. These effects were completely inhibited by specific neutralizing antibodies. An enhanced intoxication effect was observed for preadherent compared to adherent cells, as was detected in early morphology changes and confirmed by annexin V/propidium iodide (PI) apoptosis assay. Detection of the dynamic changes in cell morphology at initial stages of cell intoxication by DHM emphasizes the highly sensitive and rapid nature of this method, allowing the early detection of active toxins.

Real-time and in-situ monitoring of Abrin induced cell apoptosis by using SERS spectroscopy.

Abrin is a cytotoxic protein isolated from seeds of leguminous plants and has become a potential bioterrorism weapon for its high toxity and difficult detection. In the early stage of poisoning, Arbin can damage cells and induce apoptosis. Raman spectroscopy is a molecular fingerprint that can identify and compare various intracellular substances. In this work, thiolated polyethylene glycol (mPEG-SH) and cell-penetrating peptide (TAT) modified 70-80 nm gold nanostars (AuNSs) have been developed as label-free Raman enhancement substrates to realize real-time and in-situ monitoring of toxin-induced adherent cell apoptosis. The changes for the surface-enhanced Raman scattering (SERS) spectra of cells before and after the damage (0 h, 2 h, 4 h, 8 h, 12 h and 24 h) of Abrin can be characterized via SERS spectroscopy. The intracellular substances at different time can be compared by using differential spectrum analysis and the cells in different states can be identified and distinguished by means of principal component analysis (PCA). The abundant spectral features in SERS spectra can also reveal the molecular dynamics during apoptosis. Results show that SERS spectroscopy provides a platform for in-situ monitoring of substance changes in adherent cells except detecting cell apoptosis induced by toxin in real-time, which achieves a more detailed and comprehensive understanding of the pathogenesis of toxins in molecular biology and provides a new idea for toxicology experiments.

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.

Influence of Food Matrices on the Stability and Bioavailability of Abrin.

Abrin, a highly toxic plant toxin, is a potential bioterror weapon. Work from our laboratory and others have shown that abrin is highly resistant to both thermal and pH inactivation methods. We sought to evaluate the effectiveness of selected food processing thermal inactivation conditions against abrin in economically important food matrices (whole milk, non-fat milk, liquid egg, and ground beef). The effectiveness of toxin inactivation was measured via three different assays: (1) In vitro cell free translation (CFT) assay, (2) Vero cell culture cytotoxicity; and the in vivo mouse intraperitoneal (ip) bioassay. For both whole and non-fat milk, complete inactivation was achieved at temperatures of ≥ 80 °C for 3 min or 134 °C for 60 s, which were higher than the normal vat/batch pasteurization or the high temperature short time pasteurization (HTST). Toxin inactivation in liquid egg required temperatures of ≥ 74 °C for 3 min higher than suggested temperatures for scrambled eggs (22% solids) and plain whole egg. Additionally, the ground beef (80:20%) matrix was found to be inhibitory for full toxin activity in the mouse bioassay while retaining some activity in both the cell free translation assay and Vero cell culture cytotoxicity assay.

Novel Phage Display-Derived Anti-Abrin Antibodies Confer Post-Exposure Protection against Abrin Intoxication.

Abrin toxin is a type 2 ribosome inactivating glycoprotein isolated from the seeds of Abrus precatorius (jequirity pea). Owing to its high toxicity, relative ease of purification and accessibility, it is considered a biological threat agent. To date, there is no effective post-exposure treatment for abrin poisoning and passive immunization remains the most effective therapy. However, the effectiveness of anti-abrin monoclonal antibodies for post-exposure therapy following abrin intoxication has not been demonstrated. The aim of this study was to isolate high affinity anti-abrin antibodies that possess potent toxin-neutralization capabilities. An immune scFv phage-display library was constructed from an abrin-immunized rabbit and a panel of antibodies (six directed against the A subunit of abrin and four against the B subunit) was isolated and expressed as scFv-Fc antibodies. By pair-wise analysis, we found that these antibodies target five distinct epitopes on the surface of abrin and that antibodies against all these sites can bind the toxin simultaneously. Several of these antibodies (namely, RB9, RB10, RB28 and RB30) conferred high protection against pulmonary intoxication of mice, when administered six hours post exposure to a lethal dose of abrin. The data presented in this study demonstrate for the first time the efficacy of monoclonal antibodies in treatment of mice after pulmonary intoxication with abrin and promote the use of these antibodies, one or several, for post-exposure treatment of abrin intoxication.

Abrin Toxicity and Bioavailability after Temperature and pH Treatment.

Abrin, one of most potent toxins known to man, is derived from the rosary pea (jequirity pea), Abrus precatorius and is a potential bioterror weapon. The temperature and pH stability of abrin was evaluated with an in vitro cell free translation (CFT) assay, a Vero cell culture cytotoxicity assay, and an in vivo mouse bioassay. pH treatment of abrin had no detrimental effect on its stability and toxicity as seen either in vitro or in vivo. Abrin exposure to increasing temperatures did not completely abrogate protein translation. In both the cell culture cytotoxicity model and the mouse bioassay, abrin's toxic effects were completely abrogated if the toxin was exposed to temperatures of 74 °C or higher. In the cell culture model, 63 °C-treated abrin had a 30% reduction in cytotoxicity which was validated in the in vivo mouse bioassay with all mice dying but with a slight time-to-death delay as compared to the non-treated abrin control. Since temperature inactivation did not affect abrin's ability to inhibit protein synthesis (A-chain), we hypothesize that high temperature treatment affected abrin's ability to bind to cellular receptors (affecting B-chain). Our results confirm the absolute need to validate in vitro cytotoxicity assays with in vivo mouse bioassays.

Evaluation of abrin induced nephrotoxicity by using novel renal injury markers.

Abrin is a potent plant toxin analogous to ricin that is derived from the seeds of Abrus precatorius plant. It belongs to the family of type II ribosome-inactivating proteins and causes cell death by irreversibly inactivating ribosomes through site-specific depurination. In this study we examined the in vivo nephrotoxicity potential of abrin toxin in terms of oxidative stress, inflammation, histopathological changes and biomarkers of kidney injury. Animals were exposed to 0.5 and 1.0 LD50 dose of abrin by intraperitoneal route and observed for 1, 3, and 7 day post-toxin exposure. Depletion of reduced glutathione and increased lipid peroxidation levels were observed in abrin treated mice. In addition, abrin also induced inflammation in the kidneys as observed through expression of MMP-9 and MMP-9/NGAL complex in abrin treated groups by using zymography method. Nephrotoxicity was also evaluated by western blot analysis of kidney injury biomarkers including Clusterin, Cystatin C and NGAL, and their results indicate severity of kidney injury in abrin treated groups. Kidney histology confirmed inflammatory changes due to abrin. The data generated in the present study clearly prove the nephrotoxicity potential of abrin.