A Novel Copper–Hydrogen Peroxide Formulation for Prion Decontamination

Acquired human prion diseases are generally connected to a specific history of exposure to prions through medical procedures (iatrogenic Creutzfeldt-Jakob disease [CJD]) or cannibalism (kuru). However, in 1996, a novel form of human prion disease, termed “variant CJD” (vCJD) [1], that was linked to bovine spongiform encephalopathy (BSE) was identified in the United Kingdom. The link to BSE was suggested by epidemiological studies and was strongly supported initially by molecular strain typing and subsequently by experimental transmission experiments. Only a limited number of patients with vCJD have been described to date [2], and recent figures suggest that a first peak in the epidemic has been reached. However, for reasons of public health, there is still great concern about the length of the incubation period, the prevalence of the disease in the population, and the risk of iatrogenic transmission. Moreover, in patients with vCJD, prion protein (PrP)^Sc (the scrapie isoform of PrP) is detected not only in the central nervous system, as in patients with sporadic CJD, but also in lymphoid organs (such as the tonsils, appendix, and spleen [3]), where it can be detected more than a year before the appearance of the first symptoms. In addition, blood donor–related vCJD cases have been reported, and PrP^Sc has been detected in the spleens and muscles of patients with sporadic CJD [4]. All these data suggest that there is an important risk of iatrogenic transmission through human-derived products, medical equipment, and such instruments as endoscopes [5]

Over the past 5 years, a growing interest has emerged in the role played by metal ions in transmissible spongiform encephalophathies (TSEs). The amino-terminus of the cellular isoform of PrP, PrP^C, indeed contains a series of octapeptide repeats, which are among the most conserved regions of mammalian PrPs and have been implicated in the binding of divalent metal ions, particularly copper [6]. Knowledge of whether this binding is of structural or functional significance remains elusive. Copper is an essential redox transition element that is able to induce ion-mediated damage to proteins. This process has been described as “the Fenton reaction” and consists of the reduction of Cu²⁺ by an electron donor and generation of a hydroxyl radical through the reduction of hydrogen peroxide (i.e., H₂O₂) by the reduced metal. We have previously demonstrated that PrP^C undergoes a site-specific cleavage of the octapeptide-repeat region on exposure to Cu²⁺ and H₂O₂ [7]. In view of this, we investigated their effect on PrP^Sc, and we demonstrate here justification for the interest in a novel Cu²⁺-H₂O₂ formulation for prion decontamination

MethodsProteinase K (PK) and Pefabloc were purchased from Roche Diagnostics. Secondary antibodies were purchased from Jackson ImmunoResearch. All other reagents were purchased from Sigma. Scrapie-associated fibril (SAF) 60, SAF69, and SAF70, monoclonal antibodies that recognize the 142–160-aa peptide epitope of hamster PrP (provided by J. Grassi, Centre d’Étude Atomique, Saclay, France), were used for Western blot analysis. Brain homogenates (10% in PBS) were prepared using terminally ill C57BL/6 mice infected with scrapie strain RML or 22L. Human brain samples were provided by J. Ironside (CJD Surveillance Unit, Edinburgh, United Kingdom)

For in vitro assays, diluted brain homogenates corresponding to 150 μg of brain tissue were mixed, at room temperature for 2 h, with a PBS solution containing various concentrations of metal ions (CuSO₄ [Cu²⁺], CaCl₂ [Ca²⁺], ZnSO₄ [Zn²⁺], AlSO₄ [Al²⁺], MnCl₂ [Mn²⁺], and FeCl₂ [Fe²⁺]) and H₂O₂, as indicated in Results. This solution was then mixed with an equal volume of 2× Triton-DOC lysis buffer (150 mmol/L NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, and 50 mmol/L Tris-HCl) and digested with PK (1 μg of PK/150 μg of tissue) for 30 min at 37°C. Digestion was terminated by the addition of Pefabloc to a final concentration of 1 mmol/L. Samples were mixed with an equal volume of 2× Laemmli buffer and boiled for 5 min before Western blot analysis using a mixture of SAF60, SAF69, and SAF70 antibodies [8]

For the animal bioassay, C57BL/6 mice were injected intracerebrally with the equivalent of 20 μL of 2% 22L brain homogenate, which had been treated as described above and then dialyzed in Slide-A-Lyzer cassettes (10-kDa molecular weight cutoff; Pierce) to eliminate any chemical that might harm the mice. The mice were observed once a day, to detect the onset and progression of clinical signs. Behavioral analysis included evaluation for posture, balance, coordination, and the presence of tremors. Mice were killed by cervical dislocation at a predefined clinical end point

ResultsTo investigate whether copper ions can affect the protease resistance of PrP^Sc in vitro, 22L brain homogenates were incubated with concentrations of Cu²⁺ ranging from 0.01 to 10 mmol/L, as described in Methods. PrP^Sc detection (figure 1A, insert) revealed that incubation with Cu²⁺ resulted in a dose-dependent decrease in PrP^Sc level that was significant starting at a concentration of 0.1 mmol/L (figure 1A). These Western blot results were confirmed using a filter retention assay (data not shown) [8]

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Figure 1

Cu²⁺ and H₂O₂ specifically influence prion protein (PrP) levels in brain homogenates. In panel A, 22L brain homogenates were incubated with PBS and increasing concentrations of Cu²⁺ (0.01, 0.1, 0.5, 1.0, and 10.0 mmol/L) for 2 h at room temperature. Samples were lysed and digested with proteinase K (PK), and PrP^Sc (the scrapie isoform of PrP) was detected by Western blot analysis (insert). PrP^Sc bands from 3 separate experiments were quantified by densitometry and normalized to those of the control sample (PBS only) (graph). Bars represent mean±SD results. *P<.01 and **P<.001, compared with the control (Student’s t test). In panel B, 22L brain homogenates were treated (as in panel A) with 0.01 (lanes 2–4) 0.1 (lanes 5–7) or 0.5 (lanes 8–10) mmol/L Cu²⁺, either alone (lanes 2, 5 and 8) or with 50 (lanes 3, 6 and 9) or 100 (lanes 4, 7 and 10) mmol/L H₂O₂. After lysis, samples were treated (top) or not treated (bottom) with PK, and PrP was detected by Western blot analysis. In panel C, 22L brain homogenates were incubated with PBS alone (control) or with PBS supplemented with one of the following metal ions: Ca²⁺, Zn²⁺, Cu²⁺, Al²⁺, Mn²⁺, and Fe²⁺ (10 mmol/L). PrP^Sc was detected as described for panel A. Molecular mass markers (in kilodaltons) are indicated on the left of the blots

To examine the effect of the combination of H₂O₂ and Cu²⁺ [7], 22L brain homogenates were incubated with 50 or 100 mmol/L H₂O₂ in the presence of 0.01, 0.1, or 0.5 mmol/L Cu²⁺. Although the presence of H₂O₂ alone did not reduce the PrP^Sc signal (data not shown), the addition of H₂O₂ to Cu²⁺ enhanced the disappearance of PrP^Sc signal to the point of undetecability, whereas Cu²⁺ alone did not (figure 1B, top [compare lane 8 with lanes 9 and 10]). This result is comparable with the effect that autoclave has on PrP^Sc, which we demonstrated in a previous study [8]. In addition, Western blot analysis of non–PK-digested PrP (figure 1B, bottom) showed a decrease of the signal in a dose-dependant manner, indicating that not only PrP^Sc but also PrP^C—and, to some extent, all proteins—are affected by high concentrations of Cu²⁺ and H₂O₂. Finally, when additional heavy metal ions (Ca²⁺, Zn²⁺, Al²⁺, Mn²⁺, and Fe²⁺) were tested along with Cu²⁺ on brain homogenates at a high concentration (10 mmol/L), it was found that only Cu²⁺ significantly affected PrP^Sc level (figure 1C). Combination of these additional metal ions with 100 mmol/L H₂O₂ did not have a significant synergistic effect on PrP^Sc level (data not shown), as was observed for Cu²⁺

To test whether the effect of Cu²⁺ and H₂O₂ was limited to a particular brain homogenate, species, or prion strain, we tested the effect of the formulation on additional brain homogenate. Following the same experimental protocol as for the 22L brain homogenate but with an incubation time of only 30 min (instead of 2 h), PrP^Sc levels in mouse RML brain homogenates and in homogenates of brain samples from persons with sporadic CJD or vCJD were also dramatically reduced after treatment with 50 mmol/L H₂O₂ plus 0.5 mmol/L of Cu²⁺ (figure 2A). Similar results were observed when genuine BSE homogenate was used (data not shown)

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Figure 2

Cu²⁺ and H₂O₂ reduce prion infectivity of infectious brain homogenates in a mouse bioassay. In panel A, mouse RML brain homogenates and homogenates of brain samples from humans with sporadic Creutzfeldt-Jakob disease (sCJD; M/M type 1) or variant CJD (vCJD) were either not treated or treated with 0.5 mmol/L Cu²⁺ plus 50 mmol/L H₂O₂ for 30 min at room temperature, and prion protein (PrP)^Sc (the scrapie isoform of PrP) was detected by Western blot analysis. The results shown are representative of several independent experiments. In panel B, C57BL/6 mice were inoculated intracerebrally with 20 μL of a 2% 22L brain homogenate that had not been subjected to any decontamination procedure and were either not treated or treated with 0.5 mmol/L Cu²⁺, 1 mmol/L Cu²⁺, or 0.5 mmol/L Cu²⁺ plus 100 mmol/L H₂O₂ (as described in Methods). All groups were observed daily for the onset of clinical symptoms. The Kaplan-Meier survival curves show the percentage of surviving mice versus time. The Western blot in panel C demonstrates that PrP^Sc could be detected in the brains of mice from each bioassay group (lane 1 no treatment; lane 2 0.5 mmol/L Cu²⁺; lane 3 1 mmol/L Cu²⁺; lane 4 0.5 mmol/L Cu²⁺ plus 100 mmol/L H₂O₂). Molecular mass markers (in kilodaltons) are indicated on the left of the blots

To examine whether the biochemical disappearance of PrP^Sc reported above was associated with a decrease in prion infectivity, treated and dialyzed 22L brain homogenates were used as starting infectious materials. After an initial series of convincing experiments using cell culture were conducted as described elsewhere [8] (data not shown), an animal bioassay was performed using C57BL/6 mice (see Methods). 22L brain homogenates were treated as described above with either unsupplemented PBS or PBS supplemented with 0.5 mmol/L Cu²⁺, 1 mmol/L Cu²⁺, or 0.5 mmol/L Cu²⁺ plus 100 mmol/L H₂O₂; were dialyzed extensively against PBS; and then were inoculated into mice. Cu²⁺ alone significantly delayed the onset of clinical signs and prolonged the survival time of the mice, all of which eventually developed clinically and biochemically confirmed scrapie (figure 2B and 2C). The magnitude of the effect depended on the Cu²⁺ concentration and whether H₂O₂ was included. The mean±SD survival times of the mice were as follows: for the mice (n=5) that received brain homogenate treated only with PBS (the control mice), 167±0.5 days after infection; for the mice (n=5) that received brain homogenate treated with 0.5 mmol/L Cu²⁺, 200.2±0.5 days after infection (P=.0003); for the mice (n=5) that received brain homogenate treated with 1 mmol/L Cu²⁺, 217.2±13.4 days after infection (P=.0001); and for the mice (n=5) that received brain homogenate treated with 0.5 mmol/L Cu²⁺ plus 100 mmol/L H₂O₂, 266.4±15.6 days after infection (P<.0001) (figure 2B). The reduction in infectivity, expressed as a logarithmic reduction factor, was estimated to be 2.7 log for 0.5 mmol/L Cu²⁺, 4.2 log for 1 mmol/L Cu²⁺, and >5.2 log for 0.5 mmol/L Cu²⁺ plus 100 mmol/L H₂O₂, on the basis of the incubation time obtained from an incubation time/dilution of 22L homogenate curve provided by S. Priola (Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health)

DiscussionIn the present study, several concentrations of heavy metal ions (Cu²⁺, Zn²⁺, Fe²⁺, Mn²⁺, Ca²⁺, and Al²⁺), in the presence or absence of H₂O₂, were assessed for their ability to reduce PrP^Sc levels in human and mouse brain homogenates and to decrease prion infectivity, as tested in an animal bioassay. A major effect was observed using a formulation of Cu²⁺ at 0.5 mmol/L (80 mg/L CuSO₄) in combination with 100 mmol/L (0.34%) H₂O₂. Our observation that Cu²⁺ decreases PrP^Sc level is in apparent discrepancy with the results of previous studies [9–11]. However, in all these previous works the experimental conditions were significantly different—detergent was present during the incubation with copper, and the concentrations of metal ions were lower by 1 or 2 logs of magnitude than those in the present study. In addition, although we observed an effect with Cu²⁺ alone, a much more efficient and reproducible PrP^Sc-degradation effect was obtained when Cu²⁺ was used in combination with H₂O₂. These results with copper may be explained by the fact that PrP molecules are able to bind up to 5 Cu²⁺ molecules with a femtomolar affinity, whereas other metal ions have lower affinities for PrP^C by at least 3 orders of magnitude [6]. According to the Fenton reaction, Cu²⁺ generates, through the breakdown of H₂O₂, a hydroxyl radical, which is a highly reactive oxygen species that causes direct damage to nucleic acids and proteins—a property already used for inactivation of conventional pathogens [12]. A Cu²⁺-H₂O₂ combination, therefore, preferentially cleaves Cu²⁺-loaded PrP, as we demonstrated in a previous study [7]. However, it has been suggested that PrP^Sc is preferentially associated with Zn²⁺ or Mn²⁺ rather than with Cu²⁺ [13]. In the present study, we used concentrations of Cu²⁺ to affect PrP^Sc that were much higher than what we had used earlier for PrP^C [7]. This might be indicative of a 2-step mechanism. In the first step, high concentrations of copper could, through an exchange mechanism, modify the metal ion content of PrP^Sc (from Zn²⁺ or Mn²⁺ to Cu²⁺). This might affect the conformation of the molecule [14] and, in the second step, render it sensitive to PK [11] and to high levels of hydroxyl radicals arising from H₂O₂ and Cu²⁺

It has been well documented that the prion agent has a number of unusual characteristics, including a remarkable resistance to such physicochemical inactivation procedures as heat, ionization, UV light, microwaves, radiation, and conventional disinfectants (e.g., detergents, alcohol, glutaraldehyde, and formalin) [15–17]. Prion decontamination, therefore, requires very harsh treatment in most cases, and recommended methods include sodium hydroxide, sodium hypochlorite, or steam sterilization. However, these methods are not adapted to sensitive medical equipment and instruments and/or present serious handling risks, whereas our novel Cu²⁺-H₂O₂ formulation is expected to have a low corrosive and biotoxicity impact. In conclusion, our study strongly supports the idea that high levels of hydroxyl radicals arising from H₂O₂ and Cu²⁺ cause oxidative damage to PrP^Sc, eventually leading to a decrease in prion infectivity. This effect was observed not only for prion-infected mouse brain homogenates but also for genuine human samples. Studies are now in progress to validate this novel Cu²⁺-H₂O₂ formulation as an effective yet instrument-friendly and routinely applicable reprocessing procedure for prion decontamination

• Received January 3, 2006.
• Accepted May 8, 2006.

• © 2006 by the Infectious Diseases Society of America