November 2012

Milk offers possible defense against the deadly bioterrorism agent ricin

Photo of Ricinus communis 
Figure 1. Ricinus communis. Photo from Wikipedia. 

What if a simple glass of milk contained the antidote to one of the most deadly toxins known to man? Well, it turns out that this common household beverage, often recognized for its role in promoting strong bones, also may be a strong inhibitor of the highly toxic compound ricin.

What is ricin?
Ricin is found naturally in seeds of ricinus communis, also known as the castor plant (see Fig. 1). The plant is indigenous to the southeastern Mediterranean Basin but is widespread in tropical regions. It produces seeds, or beans (see Fig. 2), that when pressed produce an oil. Castor oil has been used in traditional medicine as a laxative and to stimulate full-term labor. While the plant is known for these health benefits, it is also the source of the lethal poison ricin, which remains in the pulp of the seeds after they are pressed. In other words, the plant can heal you or kill you, depending on how it’s processed. Ricin is a Category B compound, the second-highest-priority agent class designated by the Centers for Disease Control and Prevention. According to Vern Schramm, professor of biochemistry at the Albert Einstein College of Medicine in New York and a prominent researcher of ricin, “one castor bean contains enough ricin to kill thousands of people.”

Photo of castor beans 
Figure 2. Castor beans. Photo from Wikipedia. 

How does ricin work?
Ricin is activated intracellularly by proteolytic cleavage to form the A chain, which has enzymatic activity. The A chain is linked to a B chain by a disulfide bond. The B chain is a lectin that binds carbohydrates such as galactose and galactosamine on the cell surface, facilitating the toxin’s entry into the cell by endocytosis (see Fig. 3 here). Once this happens, ricin is translocated into the cytosol, where the disulfide bond holding the chains together is reduced in the environment of the cell. This releases the enzymatic A chain, which can recognize and cleave a single adenine on the ribosome, the organelle responsible for catalyzing the synthesis of proteins. The enzyme acts like a lawnmower that goes along and cuts one adenine off of every ribosome, leaving inactive ribosomes behind. Within a few hours, the ribosomes in the cell can no longer make protein, and the cell dies. This sequence of molecular events manifests as organ failure and, finally, death of the organism.

Reported cases of ricin poisoning
While much of the research and speculation around the use of the toxin as a terrorist or warfare agent began in the 1940s, it received a great deal of public attention in 1978. In that year, Georgi Markov, a Bulgarian journalist and vocal dissident of the Communist party, was attacked by a man with an umbrella in London while he was on his way to work. The umbrella had been modified to inject a poison ricin pellet under his skin. Markov died three days later from just a 0.2 milligram dose.

Over the past decade, almost a dozen more cases of ricin possession or attempted poisoning have been reported, including an incident in which several U.S. Senate office buildings were shut down when the toxin was found in 2004 in the office suite of the Senate majority leader, Bill Frist. These incidents have created concern among government and public health officials due to the potential for ricin to be used as a biological weapon. At present, symptomatic ricin poisoning is treated by giving supportive medical care to minimize the effects. Symptoms include difficulty breathing, fever, cough, nausea, tightness in the chest, heavy sweating and pulmonary edema. Unfortunately, no antidote for ricin exists. Therefore, there is a great deal of interest in identifying inhibitors for the castor bean compound.

Recent strides to inhibit ricin
Researchers at the U.S., Department of Agriculture, led by Reuven Rasooly, initially set out to create a method to detect ricin in various foods in hopes of eventually inhibiting its biological activity. However, the group had difficulty detecting ricin in milk. Based on this finding, the team hypothesized that the carbohydrate-binding B chain of ricin was interacting with the galactose present in the milk. This led the researchers to examine ricin toxicity in the widely consumed natural food.

In a recent Journal of Biological Chemistry article by the team, the effect of ricin on living cells was visualized and quantified by measuring the changes in the fluorescence intensity level of the green fluorescent protein reporter in African green monkey kidney cells. The more the cells fluoresce, the more protein is being made by the ribosomes. The effect of milk on ricin post-exposure was mimicked by measuring the amount of bound toxin in the presence of milk after 15 minutes of exposure to the castor bean derivative. According to Rasooly, “this is like a treatment, and you need to do it very soon after you know that you have the ricin [exposure], because when the toxin enters the cell it’s too late.”

Upon exposure of the cells to the poison, it was determined, milk not only removed bound ricin but also reduced the attachment of the toxin to its receptor by up to 88 percent. Furthermore, the biological activity of 1 ng/ml ricin was completely neutralized in solutions containing as little as 1 percent milk.

The mechanism of ricin inhibition also was elucidated in the study. It was confirmed that milk inhibits ricin activity by competitively binding the B chain lectin of the toxin, making it unavailable to galactoside receptor sites on the cell surface, thus reducing the number of toxin molecules that enter the cell.

What next?
It is important to note that this research was conducted in an in vitro system and, therefore, the results may not apply to living beings. In addition, the information gleaned from this study is on the basis of ricin ingestion and highlights a therapeutic approach that also requires ingestion. On one hand, inhibiting ricin toxicity by using milk is an appealing therapy because “milk is a food that we can find in any grocery store and it has no side effects unless people have allergies,” says Rasooly. However, according to Schramm “ricin is not very toxic when it is ingested…because most of the protein is degraded [by digestive enzymes] in the stomach and never gets into the [blood] circulation.”

Nevertheless, the finding offers an insightful biological mechanism for how milk acts on the toxin. Schramm goes on to say, “The paper in JBC really shows a way of preventing the action of the native ricin in cases of poisoning. I think the most important part of this paper is that, if you understand the exact carbohydrates that are blocking the action of the ricin B chain on human cells, you might be able to get those into a form that could be injected or inhaled and prevent the toxicity of accidental or intentional bioterrorism releases of ricin.”
 

Shannadora HollisShannadora Hollis (sholl002@umaryland.edu) received her B.S. in chemical engineering from North Carolina State University and is a Ph.D. student in the molecular medicine program at the University of Maryland, Baltimore. Her research focuses on the molecular mechanisms that control salt balance and blood pressure in health and disease. She is a native of Washington, D.C., and in her spare time enjoys cooking, thrift-store shopping and painting.


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  • GOOD JOB.

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