Elsevier

Toxicology

Volume 223, Issues 1–2, 1 June 2006, Pages 61-70
Toxicology

Retrospective identification of ricin in animal tissues following administration by pulmonary and oral routes

https://doi.org/10.1016/j.tox.2006.03.010Get rights and content

Abstract

A previously characterised amplified ELISA for ricin (sensitivity limit approximately 200 pg mL−1) has been employed to quantify ricin following a novel recovery method from selected tissues. Tissue samples from rats dosed by pulmonary instillation or orally with ricin were homogenised and treated with an elution buffer to extract ricin. This is the first time that ex vivo recovery of ricin post exposure following pulmonary or oral challenge has been achieved using clinically acceptable sampling methods, with promise in terms of diagnosis for the timely implementation of therapy.

The toxin was detected and quantified using the ELISA in conjunction with pure ricin standards. Extracts from tissues sampled, including lung, blood, liver and spleen tested positive for ricin with maximum yield in lung associated fractions for pulmonary dosing and liver tissue for oral administration. This indicates the potential of lavage and blood sampling for timely diagnosis of ricin poisoning by pulmonary and oral routes, respectively.

Time course analysis at 24 and 48 h also indicated the progression of ricin from surfaces of the lung into the lung tissue. Inter-subject variation was observed in the case of oral dosing, with data for ricin-treated and vehicle control tissues not statistically different in all samples. In addition the oral toxicity of the crude ricin administered was found to be higher than expected in the rat, based upon published information and an unpublished in house murine study.

Introduction

Ricin is a major protein produced in the seeds of the castor oil plant, Ricinus communis and represents about 2% of the total weight of a seed. The greatest component by weight of Ricinus seeds is the lipid, castor oil, which is the reason why large quantities of the plant are cultivated in tropical and sub-tropical areas of the world. In fact castor oil represents about 60% by weight of each seed.

Ricin is a potent toxin, which kills eukaryotic cells by inhibiting protein synthesis. It is one of a class of toxins known as ribosome-inactivating proteins (RIPs; Barbieri et al., 1993). These proteins interact with a discrete part of the 60 S subunit of cellular ribosomal RNA and cleave one nucleotide base, adenine 4324. This action prevents the binding of elongation factor 2 and arrests protein synthesis (Jiminez and Vasquez, 1985). RIPs exist as type 1 or type 2, based upon their structure, being either monomeric or dimeric, respectively. Ricin is an example of a type 2 RIP. Others include abrin, modeccin, volkensin and viscum album toxin. (Stirpe et al., 1992).

Type 1 RIPs are produced by many plants and consist of an A chain which has N-glycosidase activity, capable of cleaving ribosomal RNA. Type 2 RIPs consist of the A chain but additionally, this is bound through a disulphide bond to a B chain. The B chain has lectin properties, binding galactose or N-acetyl-galactosamine sugars (Olsnes and Pihl, 1982). By virtue of this lectin domain, type 2 RIPs are able to bind to appropriately-glycosylated cell surface proteins or lipids and become internalised by endocytosis. During this process the toxin becomes dissociated from the membrane and the A and B chains become separated through hydrolysis of the connecting disulphide bond when the endocytic vesicle is acidified. A proportion of the internalised ribotoxin is channelled via the trans-Golgi to the cytoplasm, where it gains access to the ribosomes. A more detailed description of this process and the subsequent cleavage of adenine is given in the review by Lord et al. (1994).

It is very easy to prepare a toxic extract rich in ricin from castor beans using a simple, low technology approach. Since a simple crude preparation of ricin is considered to be a realistic terrorist chemical weapon, emergency medical service providers need information on the properties of the toxin (Bradberry et al., 2003). Furthermore, it is important to be able to interrogate tissue samples in order to determine whether exposure has occurred. Consequently, there is a requirement for a reliable method to positively determine whether or not an individual has been exposed to this toxin. This might be used for therapeutic benefit or retrospective identification of poisoning for medicolegal reasons.

In view of the potency of ricin, the method must be very sensitive. An enzyme-linked immunoassay (ELISA) was developed (Leith et al., 1988), which could detect ricin in body tissues to a limit sensitivity of about 200 pg mL−1 following intramuscular injection. The detection limit is partly a function of the quality and properties of the antibodies used in the system but also of the efficacy with which ricin is extracted from tissue samples and made available to the assay. The present study describes a current version of this tissue treatment and ELISA developed at Dstl, Porton Down. Despite employing different capture and reporter antibodies, the procedure was found to have the same limit of detection as the original assay. This adapted ELISA was applied to tissue samples following intoxication of a small number of rodents with ricin by pulmonary and oral routes. While information on inhalational exposure must come from animal studies, there have been many accidental or intentional poisonings by oral administration of ricin, examples of which are given (Smith et al., 1985, Aplin and Eliseo, 1998).

The methods discussed enabled both the novel recovery and identification of ricin in several tissue samples with the quantification of the toxin in representative samples, in conjunction with a standard curve using purified ricin. The method offers scope for early screening of potential victims of ricin exposure by oral or pulmonary routes, using appropriate, acceptable clinical sampling techniques, with a view to implementation of therapeutic strategies.

Section snippets

Animals

Rats used in this study were male Porton Wistar rats of bodyweight approximately 250 g. Animals were habituated to the experimental animal unit for 1 week prior to use in the study. All work was conducted in accordance with the Animal (Scientific Procedures) Act, 1986.

Ricin

Pure ricin toxin was prepared in-house from seeds of Ricinis communis var. zanzibariensis as described previously (Griffiths et al., 1995). Briefly, following homogenisation of the seeds, ricin toxin was extracted from the

Analysis of crude ricin preparation

Crude ricin was found to have a total protein concentration of 10 mg mL−1 by BCA assay. Using SDS-PAGE at least five protein bands were contained within the centrifuged crude ricin preparation of which the major component was ricin (RCA60) which was found by densitometry to represent about 50% of the total protein (Fig. 1). Thus the crude ricin preparation was, in this way, determined to contain 5 mg ricin per mL solution. The position of ricin was inferred by running a sample of pure ricin in the

Discussion

The method of extraction of ricin from tissues and the ELISA was originally developed to detect and quantify ricin in various tissues following intramuscular dosing with the toxin (Leith et al., 1988). This present study has shown that it is feasible to use this approach to detect the presence of ricin in realistic samples taken from living victims of poisoning, after moderate doses of this toxin, by pulmonary or oral routes.

The method of exposure by the pulmonary route was by intratracheal

References (33)

  • P.K. Smith et al.

    Measurement of protein using bicinchoninic acid

    Anal. Biochem.

    (1985)
  • P.J. Aplin et al.

    Ingestion of castor oil plant seeds (comment)

    Comments Med. J. Aust.

    (1998)
  • A. Bingen et al.

    The Kupffer cell is the first target in ricin-induced hepatitis

    J. Submic. Cyto.

    (1987)
  • S.M. Bradberry et al.

    Ricin poisoning

    Toxicol. Rev.

    (2003)
  • A. Brech et al.

    Receptor-mediated endocytosis of ricin in rat liver endothelial cells. An immunocytochemical study

    Eur. J. Cell Biol.

    (1993)
  • R.F.R. Brown et al.

    Ultrastructure of rat lung following inhalation of ricin aerosol

    Int. J. Exp. Path.

    (1997)
  • Cited by (41)

    • Ricin

      2018, Comprehensive Toxicology: Third Edition
    • Safety assessment of foods from genetically modified crops in countries with developing economies

      2015, Food and Chemical Toxicology
      Citation Excerpt :

      In addition to allergenicity, some proteins are known to exist in nature that are capable of causing adverse effects when consumed. Though many are found in venomous snakes and insects or are produced by pathogenic bacteria, there are some that are found in plants such as kidney bean lectin and ricin (Rossi et al., 1984; Lafont et al., 1988; Weinman et al., 1989; Ishiguro et al., 1992a and 1992b; Cook et al., 2006). Accordingly, proteins used in GM crops have also been assessed for potential to cause adverse effects if for no other reason that they too are proteins.

    • Characteristics and safety assessment of intractable proteins in genetically modified crops

      2014, Regulatory Toxicology and Pharmacology
      Citation Excerpt :

      Most dietary proteins do not cause adverse effects to humans because they are metabolized into amino acids and small peptides in the gut that are readily absorbed for nutritive purposes (Delaney et al., 2008). Conversely, proteins that are known to be toxic to humans and other animals following oral exposure are often resistant to digestion by the target organism such that they have either toxicity primarily directed toward intestinal cells (e.g., kidney bean lectin [Lafont et al., 1988; Rossi et al., 1984; Weinman et al., 1989]) or can also be accompanied by systemic toxicity following absorption from the GI system (e.g., ricin [Cook et al., 2006; Ishiguro et al., 1992a, 1992b]). Furthermore, the potential toxicity of proteins introduced into GM crops has been evaluated in mice or other laboratory animals by exposure to purified proteins, though to date, none have demonstrated any evidence of adverse effects (for example, see Hérouet et al., 2005; Juberg et al., 2009; Mathesius et al., 2009; Rice et al., 2008).

    • Ricin

      2010, Comprehensive Toxicology, Second Edition
    • Pest Control Agents from Natural Products

      2010, Hayes' Handbook of Pesticide Toxicology
    View all citing articles on Scopus
    View full text