Intramuscular Exposure to a Lethal Dose of Ricin Toxin Leads to Endothelial Glycocalyx Shedding and Microvascular Flow Abnormality in Mice and Swine.

Ricin toxin isolated from the castor bean (Ricinus communis) is one of the most potent and lethal molecules known. While the pathophysiology and clinical consequences of ricin poisoning by the parenteral route, i.e., intramuscular penetration, have been described recently in various animal models, the preceding mechanism underlying the clinical manifestations of systemic ricin poisoning has not been completely defined. Here, we show that following intramuscular administration, ricin bound preferentially to the vasculature in both mice and swine, leading to coagulopathy and widespread hemorrhages. Increased levels of circulating VEGF and decreased expression of vascular VE-cadherin caused blood vessel impairment, thereby promoting hyperpermeability in various organs. Elevated levels of soluble heparan sulfate, hyaluronic acid and syndecan-1 were measured in blood samples following ricin intoxication, indicating that the vascular glycocalyx of both mice and swine underwent extensive damage. Finally, by using side-stream dark field intravital microscopy imaging, we determined that ricin poisoning leads to microvasculature malfunctioning, as manifested by aberrant blood flow and a significant decrease in the number of diffused microvessels. These findings, which suggest that glycocalyx shedding and microcirculation dysfunction play a major role in the pathology of systemic ricin poisoning, may serve for the formulation of specifically tailored therapies for treating parenteral ricin intoxication.

Constructing a Tandem Mass Spectral Library for Forensic Ricin Identification.

Ricin, a protein found in castor seeds, is a lethal toxin that is designated as a category 2 select agent, and cases of attempted ricin poisoning are relatively common. Many methods to detect protein toxins such as ricin use targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify toxin peptides, usually tryptic peptides. The successful use of untargeted methods has also been reported. However, the use of untargeted proteomics methods, including database search, for peptide and protein identification is less common in forensic practice and may be unfamiliar to forensic science practitioners. Here, we propose a method to create spectral libraries of tryptic ricin peptides and use these libraries for ricin identification by spectral library search, which may be more familiar to forensic scientists because of the use of spectral libraries in small molecule identification. Peptide spectral libraries offer a direct comparison to an authentic standard, a key element of forensic analysis, but have not previously been used in a forensic context. To construct these spectral libraries, two pure ricin samples (one from a proposed standard reference material) were digested with trypsin and analyzed using a standard shotgun LC-MS/MS protocol. Spectral libraries were created from resulting tryptic peptides identified from filtered search results from four database search tools. The library was then used in a search using SpectraST on forensically realistic castor seed extracts. These castor seed samples were made using the crude methods commonly encountered in real-world ricin cases. Analysis showed that the spectral library search resulted in more peptides identified from crude castor seed samples compared to MS-GF+ and Sequest plus Percolator database searches. These results, the first published use of spectral library search to detect protein toxins in forensically relevant samples, suggest that computational comparison of putative ricin peptide spectra to library spectra can be an effective method to detect ricin in an unknown sample. Data are available via ProteomeXchange with identifier PXD013711.

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies.

In this report we investigated, within a group of closely related single domain camelid antibodies (VH Hs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast-acting toxin and biothreat agent. The V1C7-like VH Hs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin-neutralizing activities. Using the X-ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta-based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1R29G mutant was largely devoid of toxin-neutralizing activity (TNA). However, the TNA of the V1C7G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen-deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.

Multichannel Open Tubular Enzyme Reactor Online Coupled with Mass Spectrometry for Detecting Ricin.

For counterterrorism purposes, a selective nano liquid chromatography-mass spectrometry (nanoLC-MS) platform was developed for detecting the highly lethal protein ricin from castor bean extract. Manual sample preparation steps were omitted by implementing a trypsin/Lys-C enzyme-immobilized multichannel reactor (MCR) consisting of 126 channels (8 µm inner diameter in all channels) that performed online digestion of proteins (5 min reaction time, instead of 4-16 h in previous in-solution methods). Reduction and alkylation steps were not required. The MCR allowed identification of ricin by signature peptides in all targeted mode injections performed, with a complete absence of carry-over in blank injections. The MCRs (interior volume ≈ 1 µL) have very low backpressure, allowing for trivial online coupling with commercial nanoLC-MS systems. The open tubular nature of the MCRs allowed for repeatable within/between-reactor preparation and performance.

Review - Ricinus cmmunis - Ethnomedicinal uses and pharmacological activities.

Ricinus cmmunis L. (Castor oil plant) is an important medicinal plant belonging to family Euphorbiaceae. Its phytochemistry, biological and pharmacological activities, and ethnomedicinal uses have been reviewed in the present study. The reported chemical constituents showed the presence of flavonoids, phenolic compounds, fatty acids, amino acids, terpenoids, phytosterol etc. The compounds have been reported to exhibit anticonceptive, antidiabetic, antifertility, anti-inflammatory, antimicrobial, antioxidant, hepatoprotective, insecticidal and wound-healing activities. They also showed free radical scavenging and Hg scavenging activities, and repellent properties. Various parts of R. communis have been widely used in traditional medicine such as abdominal disorders, arthritis, backache, muscle aches, bilharziasis, chronic backache and sciatica, chronic headache, constipation, expulsion of placenta, gallbladder pain, period pain, menstrual cramps, rheumatism, sleeplessness, and insomnia. Castor oil plant has also revealed toxic effects due to the presence of ricin (protein) and ricinine (alkaloid). Comparatively, ricin is more toxic. But still there is need of more research to be conducted with reference to its medicinal importance (particularly exploring of medicinal recipes) and active compounds responsible for various activities.

An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies.

Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL) technique, Staphylococcus protein A (SPA) functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb) as magnetic capturing probes, and Ru(bpy)3(2+)-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD) was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy)3(2+)-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy)3(2+)-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Inhibition of protein synthesis leading to unfolded protein response is the major event in abrin-mediated apoptosis.

Abrin obtained from the plant Abrus precatorius inhibits protein synthesis and also triggers apoptosis in cells. Previous studies from our laboratory suggested a link between these two events. Using an active site mutant of abrin A-chain which exhibits 225-fold lower protein synthesis inhibitory activity than the wild-type abrin A-chain, we demonstrate in this study that inhibition of protein synthesis induced by abrin is the major factor triggering unfolded protein response leading to apoptosis. Since abrin A-chain requires the B-chain for internalization into cells, the wild-type and mutant recombinant abrin A-chains were conjugated to native ricin B-chain to generate hybrid toxins, and the toxic effects of the two conjugates were compared. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild-type ricin B-rABRA conjugate as expected. The mutant conjugate activated p38MAPK and caspase-3 similar to its wild-type counterpart although at later time points. Overall, these results confirm that inhibition of protein synthesis is the major event contributing to abrin-mediated apoptosis.

Immunomodulatory activity of recombinant Ricin toxin binding Subunit B (RTB).

Ricin toxin binding subunit B (RTB) is one of the subunits of the ricin protein. RTB has been used as adjuvant, but little is known about its mechanism. In this study, we found that RTB increased not only nitric oxide (NO) release, but also tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in mouse macrophage cell line RAW264.7 cells. They subsequently exhibited enhanced ConA-induced T-cell and LPS-induced B-cell proliferative responses. We also examined the cytokines that were produced from splenocytes following in vitro RTB administration. Increased levels of IL-2, interferon (IFN)-γ and TNF-α were observed, while IL-4 and IL-5 were unaffected. These results demonstrate that recombinant RTB can act on the immune system and activate T-cells by introducing a Th1 immune response. Th1 cells might be the primary cellular target affected by RTB. Our results suggest that the recombinant RTB can promote the activation of macrophages and has a beneficial effect on immunomodulatory activity.

Integration of gas chromatography mass spectrometry methods for differentiating ricin preparation methods.

The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of Ricinus communis, commonly known as the castor plant. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatography-mass spectrometry (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid, as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods, starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid, or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method, independent of the seed source. In particular, the abundance of mannose, arabinose, fucose, ricinoleic acid, and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation than would be possible using a single analytical method.

CNL, a ricin B-like lectin from mushroom Clitocybe nebularis, induces maturation and activation of dendritic cells via the toll-like receptor 4 pathway.

A novel lectin, isolated from the basidiomycete mushroom Clitocybe nebularis and termed C. nebularis lectin (CNL), exhibits an immunostimulatory effect on the most potent antigen-presenting cells, the dendritic cells (DCs). Treatment of human monocyte-derived DCs with CNL in doses from 1 to 10 µg/ml resulted in a dose-dependent induction of overall DC maturation characteristics. Exposure of DCs to CNL for 48 hr resulted in extensive up-regulation of co-stimulatory molecules CD80 and CD86, as well as of the maturation marker CD83 and HLA-DR molecules. Such CNL-matured DCs (CNL-DCs) were capable of inducing a T helper type 1-polarized response in naive CD4+ CD45RA+ T cells in 5-day allogeneic co-cultures. The allostimulatory potential of CNL-DCs was significantly increased relative to untreated controls, as was their capacity to produce several pro-inflammatory cytokines such as interleukin-6, interleukin-8 and tumour necrosis factor-α. By using a specific Toll-like receptor 4 (TLR4) signalling inhibitor, CLI-095, as well as Myd88 inhibitory peptide, we have shown that DC activation by CNL is completely dependent on the TLR4 activation pathway. Furthermore, activation of TLR4 by CNL was confirmed via TLR4 reporter assay. Measurement of p65 nuclear factor-κB and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CNL stimulation of DCs revealed primarily an increase in nuclear factor-κB activity, with less effect on the induction of p38 MAPK signalling than of lipopolysaccharide-matured DCs. The CNL had the ability to activate human DCs in such a way as to subsequently direct T helper type 1 T-cell responses. Our results encourage the use of mushroom-derived lectins for use in therapeutic strategies with aims such as to strengthen anti-tumour immune responses.

Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars.

Ricin, a highly toxic protein from Ricinus communis, is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.

Identification of ricin in crude and purified extracts from castor beans using on-target tryptic digestion and MALDI mass spectrometry.

Ricin is a toxic protein produced in the seeds of the castor bean plant. The toxicity of the protein and the ease in which it can be extracted from the seeds makes it a potential biological warfare agent. There has been extensive work in the development of analytical techniques that can identify the protein robustly and rapidly. On-target tryptic digestion and MALDI MS was used to distinguish ricin from bovine serum albumin and three other type 2 ribsome-inactivating proteins (RIPs), abrin, agglutinin (RCA(120)), and viscumin, using the peptide mass fingerprint. The sequence coverage obtained was enhanced using methanol-assisted tryptic digestion and was particularly useful for the detection of these toxins in complex matrixes. When used in conjunction with intact protein MALDI mass measurement, a positive identification of ricin (or any of the other RIPs) was achieved including confirmation of the integrity of the disulfide bond between the A and B chains. This applicability of this methodology was demonstrated by the identification of ricin in a typical "crude white powder" that may be illicitly produced in a clandestine lab. The analysis on the solubilized sample using this method can be undertaken in around an hour with minimal sample preparation.

Development of an integrated microfluidic instrument for unattended water-monitoring applications.

Field-deployable detection technologies in the nation's water supplies have become a high priority in recent years. The unattended water sensor is presented which employs microfluidic chip-based gel electrophoresis for monitoring proteinaceous analytes in a small integrated sensor platform. The instrument collects samples directly from a domestic water flow. The sample is then processed in an automated microfluidic module using in-house designed fittings, microfluidic pumps and valves prior to analysis via Sandia's microChemLab module, which couples chip-based electrophoresis separations with sensitive LIF detection. The system is controlled using LabVIEW software to analyze water samples about every 12 min. The sample preparation, detection and data analysis has all been fully automated. Pressure transducers and a positive control verify correct operation of the system, remotely. A two-color LIF detector with internal standards allows corrections to migration time to account for ambient temperature changes. The initial unattended water sensor prototype is configured to detect protein biotoxins such as ricin as a first step toward a total bioanalysis capability based on protein profiling. The system has undergone significant testing at two water utilities. The design and optimization of the sample preparation train is presented with results from both laboratory and field testing.

Characterization of native and denatured ricin using MALDI-TOF/MS.

Ricin is a toxic protein present in the seeds of castor bean plant. It can be inactivated by heat; therefore characterization of denatured ricin is essential to differentiate it from native ricin and to avoid any ambiguity in its identification. In this study, potential of mass spectrometry using MALDI—TOF/MS has been exploited to investigate the effects of heat treatment on ricin and spiked food matrices. The molecular weights of ricin, ricin A (A1 and A2) and B chain were found to be 62.8 kDa, 31.2 kDa, 32.5 kDa and 32 kDa respectively. The mass spectrum revealed a polypeptide chain of 11.1 kDa for denatured ricin. The peptide mass fingerprinting showed 24 peptides, six were common both in native and denatured ricin. The differentiating peptide at position 294—318 (m/z 934.533) was observed only in denatured ricin. The three selected marker peptides m/z 1013.6, 1310.7, 1728.9 are chosen for identification of ricin inactivated by heat in spiked apple juice and milk samples by immunocapture analysis. There is always a probability of denatured non— toxic ricin being confused with native (toxic) ricin to create unnecessary panic. Keeping this probability in mind, our study will be of immense value in minimising such risk.

Development of a novel immuno-PCR assay for detection of ricin in ground beef, liquid chicken egg, and milk.

Reliable, sensitive, and high-throughput methods are essential for food defense, to detect foodborne contaminants and to facilitate remediation and recovery from potential toxin-related incidents. Ricin is a protein toxin that has been used for intentional contamination of foods in the past. In this study, we developed procedures for quantification of ricin in foods using immuno-PCR (IPCR). The direct adsorption of ricin onto the wells of a microtitration plate was compared with indirect immobilization via a capture antibody (sandwich IPCR). The latter procedure provided much greater sensitivity. We also compared a protocol with the immunoassay and PCR conducted in a single plate to a two-step procedure in which the PCR was conducted in a second plate, following release and transfer of the DNA marker. The two-step procedure proved 1,000-fold more sensitive for ricin detection, so this format was used to detect ricin in spiked samples of ground beef, chicken egg, and milk, and the results were compared with those obtained from enzyme-linked immunosorbent assay (ELISA). The IPCR had a limit of detection of 10 pg/ml in chicken egg and milk samples and 100 pg/ml in ground beef extracts. Comparable ELISA results were in the 1 to 10 ng/ml range. Thus, IPCR affords sensitivity that is 10-fold greater in the ground beef matrix, 100-fold greater in the milk, and 1,000-fold greater in the egg matrix than the sensitivity obtained by ELISA. Further optimization of the sandwich IPCR was performed by comparing various antibody combinations. Among the four formats investigated, the pAb-pAb combination yielded the lowest limit of detection (10 fg/ml).

Establishment of a Novel Oral Murine Model of Ricin Intoxication and Efficacy Assessment of Ovine Ricin Antitoxins.

Ricin, produced from the castor beans of Ricinus communis, is a cytotoxin that exerts its action by inactivating ribosomes and causing cell death. Accidental (e.g., ingestion of castor beans) and/or intentional (e.g., suicide) exposure to ricin through the oral route is an area of concern from a public health perspective and no current licensed medical interventions exist to protect from the action of the toxin. Therefore, we examined the oral toxicity of ricin in Balb/C mice and developed a robust food deprivation model of ricin oral intoxication that has enabled the assessment of potential antitoxin treatments. A lethal oral dose was identified and mice were found to succumb to the toxin within 48 h of exposure. We then examined whether a despeciated ovine F(ab')2 antibody fragment, that had previously been demonstrated to protect mice from exposure to aerosolised ricin, could also protect against oral intoxication. Mice were challenged orally with an LD99 of ricin, and 89 and 44% of mice exposed to this otherwise lethal exposure survived after receiving either the parent anti-ricin IgG or F(ab')2, respectively. Combined with our previous work, these results further highlight the benefit of ovine-derived polyclonal antibody antitoxin in providing post-exposure protection against ricin intoxication.

Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli.

BACKGROUND: Gag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB) when fused to p24. RESULTS: A fusion between ricin toxin B subunit and p24 HIV (RTB/p24) was expressed in E. coli. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin. CONCLUSION: In this work, we report the expression in E. coli of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when conjugated to an HIV structural protein. This is the first report in which a eukaryotic toxin produced in E. coli is employed as an adjuvant to elicit immune responses to p24 HIV core antigen.

Ricin and Ricinus communis in pharmacology and toxicology-from ancient use and "Papyrus Ebers" to modern perspectives and "poisonous plant of the year 2018".

While probably originating from Africa, the plant Ricinus communis is found nowadays around the world, grown for industrial use as a source of castor oil production, wildly sprouting in many regions, or used as ornamental plant. As regards its pharmacological utility, a variety of medical purposes of selected parts of the plant, e.g., as a laxative, an anti-infective, or an anti-inflammatory drug, have been described already in the sixteenth century BC in the famous Papyrus Ebers (treasured in the Library of the University of Leipzig). Quite in contrast, on the toxicological side, the native plant has become the "poisonous plant 2018" in Germany. As of today, a number of isolated components of the plant/seeds have been characterized, including, e.g., castor oil, ricin, Ricinus communis agglutinin, ricinin, nudiflorin, and several allergenic compounds. This review mainly focuses on the most toxic protein, ricin D, classified as a type 2 ribosome-inactivating protein (RIP2). Ricin is one of the most potent and lethal substances known. It has been considered as an important bioweapon (categorized as a Category B agent (second-highest priority)) and an attractive agent for bioterroristic activities. On the other hand, ricin presents great potential, e.g., as an anti-cancer agent or in cell-based research, and is even explored in the context of nanoparticle formulations in tumor therapy. This review provides a comprehensive overview of the pharmacology and toxicology-related body of knowledge on ricin. Toxicokinetic/toxicodynamic aspects of ricin poisoning and possibilities for analytical detection and therapeutic use are summarized as well.

Antiproliferative potential from aqueous Viscum album L. preparations and their main constituents in comparison with ricin and purothionin on human cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Mistletoe has been used since ancient times in Europe mostly for medicinal purposes. Since 1917, mistletoe preparations have been applied in cancer therapy and today are the most frequently used complementary medicine in tumor treatment. The main cytotoxic constituents of Viscum album are lectins and viscotoxins. AIM OF THE STUDY: The aim of this in vitro study was to investigate the antiproliferative potential of Viscum album preparations from different host trees and to assess the impact of mistletoe lectin 1 (ML-1) and viscotoxin A (VT-A) in comparison to a structurally similar lectin and thionin. MATERIALS AND METHODS: By means of widely accepted 2D Alamar Blue Assay, based on population counting of living cells using a fluorescent cell viability dye, the potential impact to inhibit tumor cell of the mistletoe preparations (Iscucin®) and their single compounds (ML-1 and VT-A) on the cell growth of six human cancer cell lines were evaluated. Also the mixture of ML-1 and VT-A corresponding to the contents in the specific mistletoe preparations were monitored. Ricin and purothionin were used as reference lectin and reference thionin, respectively. RESULTS: The lung carcinoma cell line HCC827 was very sensitive to the Iscucin® preparations. Very strong antiproliferative effects were found with Iscucin®Salicis and Tiliae and a strong with Iscucin®Crataegi, Mali and Populi. The IC50 concentrations of the Iscucin® preparations correlated with their respective ML-1 contents, but the ML-1 levels were much lower than the IC50 concentration of isolated ML-1 (1 ng/ml - 56 ng/ml). ML-1 was much more effective than ricin. Iscucin® preparations, ML-1 and ricin showed antiproliferative activity on human tumor cells. VT-A and purothionin had no effect on cell viability in the concentration ranges tested. CONCLUSION: The complete mistletoe extract is more potent to inhibit tumor cell proliferation than isolated ML-1 at an equivalent concentration level. Phenolic compounds found in all Iscucin® preparations might contribute to uphold the cytotoxic activity of ML-1 by antioxidative action. However, further studies are necessary to evaluate the role of VT-A and possible synergistic actions to the antiproliferative effect of aqueous mistletoe extracts.