Copper Helps Control Deadly Prion Protein Infection

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<HR class=title width="100%" SIZE=1></TD></TR><TR><TD>October 2006
Copper Helps Control Deadly Prion Protein Infection
By William H. Dresher, Ph.D., P. E
Introduction | Background | Definitions | Relative Resistance | Role of Copper | Conclusions | References
Introduction
In a previous paper<SUP>1</SUP>, we discussed the role of copper in maintaining hospital cleanliness in the fight against hospital-acquired (or nosocomial) infections. In this paper we will discuss the use of a copper salt and hydrogen peroxide to sterilize surgical instruments against deadly prion protein contamination and transmission. Recent studies have shown that the combination of a copper salt with hydrogen peroxide is an effective way of destroying prion proteins on surgical instruments without running the danger of incapacitating the instrument. This is expected to be particularly useful in such instruments as endoscopes that cannot withstand the currently recommended procedure for prion inactivation.
Background
Prion (pronounced ‘PREE-on’) proteins are natural complex amino acid molecules that were first identified in 1982 by Dr. Stanley B. Prusiner at the University of California–San Francisco (UCSF)<SUP>2</SUP>. While the scientific community was initially somewhat skeptical about Prusiner's finding, he was subsequently awarded a Nobel Prize for his discovery. Prion proteins occur in the brain and other parts of the central nervous system and, in their normal forms, are believed to act as antioxidants that protect brain cells. As an example, scientists at Case Western Reserve University and elsewhere have genetically engineered strains of mice that lack prion proteins<SUP>3</SUP>. These “prion knock-out” mice exhibit increased levels of oxidative stress in their brains compared with normal mice.
Prions have a dark side, however, which results in their classification as proteinaceous infectious particles. They are infectious agents in that they are capable of self-replication. They contain no nucleic acid (DNA or RNA) response. They invoke no immune response. They can change into an abnormal form that causes “prion diseases.” The mechanism proposed for the occurrence of these conditions is that the normal prion protein (shorthand: PrP for protease-resistant protein) is converted into an abnormal or “rogue” form, PrPsc, simply by changing its shape — a Dr. Jekyll and Mr. Hyde of the biological world, so to speak. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein. Rogue proteins accumulate in the brain, disrupting or destroying neurons in large numbers, which inevitably leads to the death of the person or animal.
Prion proteins have been associated with a number of diseases in both humans and animals: Creutzfeldt-Jakob Disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE, or "mad cow disease") in cattle, chronic wasting disease (CWD) in deer and elk, and feline spongiform encephalopathy (FSE) in cats. Collectively, these afflictions are known as transmissible spongiform encephalopathy diseases (TSEs). Spongiform refers to the porous nature of respective characteristic microscopic plaques that form in the brains of afflicted animals or humans. The appearance of these plaques differs between the animal and the human disease; however, both have sponge-like aspects.
CJD in humans was first identified in 1921 and occurs at a frequency of about one in a thousand. CJD, while traditionally a disease among the elderly, was diagnosed in a number of people in their 20s and 30s in the U. K. in 1996, suggesting that a new variant of the disorder, variant CJD (vCJD), has now crossed the species barrier from cattle to humans. BSE, a disease in cattle similar to scrapie in sheep, was first identified in the U. K. in 1986. Prior to that, it had never been identified anywhere in the world. It is the concern for the transmission of BSE to humans in the form of vCJD that brought the incidence of BSE in the U. K. to newspaper headlines and TV screens throughout the world in the 1990s. The role of copper in BSE has been discussed previously in this Innovations series<SUP>4</SUP>.
Although prion diseases are not transmitted by casual contact, they are transmitted perorally and parenterally. A striking feature of prion proteins is their extraordinary resistance to conventional sterilization procedures and their capacity to bind to surfaces of both metal and plastic without losing infectivity<SUP>5</SUP>. Almost 300 cases of transmission of CJD have been reported<SUP>6</SUP>. These have been the result of medical treatments, such as human-pituitary-derived growth hormone injectants, dura mater and corneal tissue transplantation and brain surgery involving contaminated instruments. At least four incidents of CJD have been reported after neurosurgical intervention using instruments previously used on CJD patients<SUP>7</SUP>.
It is already common practice to dispose of instruments used on known CJD patients. However, this is not done on cases where the symptoms have yet to appear and, as yet, there is no diagnostic test available for CJD. Also, some instruments, such as endoscopes, are too expensive for one-time use. Current methods for sterilization and decontamination can render instruments damaged and unusable. The World Health Organization (WHO) recommends, for economic reasons, that instruments used on patients suspected of carrying CJD be quarantined until final lab results are available.
Definitions<SUP>8</SUP>

• Sterilization – the destruction of all microbial life, including bacterial endospores.
• Disinfection – the elimination of virtually all pathogenic microorganisms on inanimate surfaces with the exception of large numbers of bacterial endospores, reducing the level of microbial contamination to an acceptably safe level.
• Decontamination – all of the above. Decontamination is any activity that reduces the microbial load to prevent inadvertent contamination or infection. The appropriateness of a decontamination procedure is situation-dependent. For example, surgical instruments must be sterile, but this level of microbial killing is unnecessary for environmental surfaces such as floors and walls.

Relative Resistance of Different Types of Microorganisms to Sterilization and Disinfection
Microorganisms differ widely in their resistance to sterilization and disinfection. In general, bacterial endospores and prions are the hardest to destroy, and lipid viruses are the easiest to destroy. The following are listed in decreasing order of resistance of microorganisms to sterilization and disinfection<SUP>9</SUP>:

• Prions — may require extended, multiple sterilization cycles.
• Bacterial endospores — destroyed by sterilization or high-level disinfection.
• Mycobacteria — (example: tuberculosis) destroyed by sterilization, high-level disinfection, intermediate level disinfection.
• Non-lipid or small viruses — (example: polio virus) destroyed by sterilization, high level disinfection, intermediate level disinfection.
• Fungi (molds and yeasts) (example: Candida albicans) — destroyed by sterilization, high level disinfection, intermediate level disinfection. Some fungi are also destroyed by low-level disinfection.
• Vegetative bacteria (example, pseudomas aeruginosa) — destroyed by sterilization, high-level disinfection, intermediate-level disinfection, low-level disinfection.
• Lipid or medium-sized viruses (example, hepatitis B virus) — destroyed by sterilization, high-level disinfection, intermediate-level disinfection, low-level disinfection.

Role of Copper in Sterilization of Prion Proteins and Bacterial Endospores
It has been previously shown that copper surfaces and copper ions are effective in the deactivation of bacteria and viruses<SUP>10</SUP>. Recently, Dr. Sylvain Lehmann and his team at the Institute of Human Genetics (IGH) of the French Centre National de la Recherche Scientifique (CNRS) have demonstrated copper systems for the deactivation of prion proteins<SUP>11</SUP>. The Lehmann team had been studying TSEs, the role of abnormal prions in triggering these pathologies and, in particular, the role of metal ions in prion diseases<SUP>12</SUP>. They found that in scrapie-infected mouse brain homogenates even low concentrations of cupric ion, ranging from 0.1–10 mmol/l, are sufficient to convert the scrapie (bad) form of a prion protein (PrPsc) to the normal (good) form (PrP). However, they found that a formulation of copper and hydrogen peroxide can completely deactivate the prion proteins. They ascribe this to a modified Fenton Reaction with cupric ion substituted for ferric ion.

The Fenton reaction is named for H.J.H. Fenton who first studied the catalytic decomposition of hydrogen peroxide by a transition metal ion in 1894. Subsequently, it was determined that the hydroxyl radical (HO-) was responsible for the reagent’s high oxidation efficiency. The iron-catalyzed reaction has been called “Fenton’s Reaction.” The basic mechanism is:

Fe<SUP>3+</SUP> + H<SUB>2</SUB>O<SUB>2 </SUB>——> Fe<SUP>2+</SUP> + HOO* + H<SUP>+</SUP>​

Fe<SUP>2+</SUP> + H<SUB>2</SUB>O<SUB>2 </SUB>——> Fe<SUP>3+</SUP> + HO* + OH<SUP>-</SUP>​

In this reaction, the transition metal (in this case iron) is cycled through its oxidation states to form free radicals. As such, the net reaction is:

Fe<SUP>3+</SUP>​

2H<SUB>2</SUB>O<SUB>2 </SUB>——> HO* + H<SUB>2</SUB>O + HOO*​

Thus, the metal ion serves as a liquid catalytic agent for forming hydroxyl ion and other reactive free radicals. Free radical formation in the copper analog would be written:

Cu<SUP>1+</SUP> + H<SUB>2</SUB>O<SUB>2 </SUB>= HO* + OH<SUP>-</SUP> + Cu<SUP>2+</SUP>​

The team notes that using medical instruments contaminated by CJD or other prion diseases is a problem. The very harsh treatment normally required for sterilization, such as use of sodium hydroxide or steam, is not compatible with delicate equipment. They found that their formulation “dramatically reduced” the level of prion protein in homogenates of samples from prion-infected brains, including those from mice infected with scrape strains and humans with CJD. According to Dr. Lehmann, the new decontamination approach is currently under commercial development and will be particularly adapted to the reduction of the risk of physician-induced prion transmission with medical instruments, such as endoscopes that cannot withstand the currently recommended prion inactivation procedures.
Earlier, researchers at the U.S. Food and Drug Administration (FDA) found that a mixture of cupric chloride and hydrogen peroxide is a powerful viruscide<SUP>13</SUP>. And, subsequent work at Los Alamos National Laboratory applied a modified Fenton Reagent by the use of copper/ascorbic acid and sodium chloride to fight the spread of tuberculosis by killing Bacillus spores<SUP>14</SUP>. Work at the University of Connecticut studied the use of this system on the killing of Bacillus subtilis spores<SUP>15</SUP>. Thus, it appears both hydrogen peroxide and ascorbic acid are capable of forming deadly free radicals in the presence of copper salts.
These last two efforts suggest the possibility of using the copper-modified Fenton Reagent to sterilize objects and surfaces against the spread of anthrax — Bacillus anthracis.
Conclusions
It has been shown by a number of investigators that copper salts together with hydrogen peroxide or ascorbic acid in an aqueous medium represents a powerful sterilization agent capable of sanitizing against the most resistant of microbiological material. The reagent is sufficiently mild to treat even the most sensitive of medical equipment.
Copper's bactericidal, fungicidal and even viriscidal properties have been recognized for many years. The fact that copper, in combination with hydrogen peroxide, is also useful in protecting against the spread of the most resistant — and most dreaded — prion proteins is gratifying. References
1. Dresher, W. H., “Copper Helps Control Infection in Healthcare Facilities,” Copper Development Association Inc., August 2004.
2. Prusiner, S. B., “Prions.” Nobel Lecture, Proceedings of the National Academy of Science Vol 95, No. 23, November 10, 1998, pp 13363-13383.
3. Wong, B. S., T. Liu, R. Li, T. Pan, R. B. Petersen, M. A. Smith, P. Gannett, G. Perry, J. C. Manson, D. R. Brown and M. S. Sy, “Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein,” J. Neurochemistry Vol. 76, 2001, pp 565-572.
4. Dresher, W. H., G. Greetham, and B. J. Harrison, “The Case for the Role of Copper Deficiency in “Mad-Cow” Disease and Human Creutzfeldt-Jakob Disease,” Copper Development Association Inc., December 2001.
5. C. Weissmann, M. Enari, P. C. Llöhn, D. Rossi, and E. Flechsig, “Transmission of Prions,” Proceedings of the National Academy of Sciences, 99 (4) December 10, 2005.
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7. Brown, P., in Prion Diseases, eds. H. Baker and R. M. Ridley, 1996, Humana, Totowa, N.J., pp 139-154.
8. Argonne National Laboratory, Institutional Biosafety Committee “Decontamination and Disinfection Fact Sheet.
9. Muscarella, L. F., “Are Quaternary Ammonium Disinfectants Adequate?” Infection Control Today, May 2005.
10. Michels, H. T., S. A. Wilks, J. O. Noyce, and C. W. Keevil “Copper Alloys for Human Infectious Disease Control,” presented at Materials Science and Technology Conference, September 25-28, 2005, Pittsburgh, PA, Copper for the 21st Century Symposium.
11. Solassol J., M. Pastore., C. Crozet, V. Perrier and S. Lehmann, “A novel copper – hydrogen peroxide formulation for prion decontamination,” J. Infect. Dis. 2006; 194, pp 865-869.
12. Lehmann, S., Metal Ions and Prion Diseases, Curr. Opin. Chem. Biol. 6, 2002, pp 187-92.
13. Sagripanti, J-L, L. B. Routson and C. D. Lytle, “Virus Inactivation by Copper or Iron Ions Alone and in the presence of Peroxide,” Applied and Environmental Microbiology, December 1993, pp 4374-4676.
14. Cross, J. B., R. P. Currier, D. J. Torraco, L. A. Vandenberg, G. L. Wagner, and P. D. Gaden, “Killing of Bacillus Spores by Aqueous Dissolved Oxygen, Ascorbic Acid and Copper Ions.,” Applied and Environmental Microbiology, April 2003, pp 2245 – 2252.
15. Shapiro, M. P., B. Setlow, and P. Setlow, Killing of Bacillus subtilis Spores by a Modified Fenton Reagent Containing CuCl2 and Ascorbic Acid, Applied and Environmental Microbiology, April 2004, pp 2535-2539.</TD></TR></TBODY></TABLE>