Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.

Effect of cytochrome c peroxidase on the corneal epithelial healing process after excimer laser photo-ablation in transgenic mice.

AIM: To evaluate the role of commercially prepared cytochrome c peroxidase eye drops in corneal epithelial healing of transgenic B6(A)-Rpe65rd12/J mice after excimer laser photo-ablation. MATERIALS AND METHODS: In our prospective animal series, 72 eyes of 36 mice had uneventful bilateral excimer laser photo-ablation. In each mouse, one eye received standard topical postoperative therapy with tobramycin, diclofenac, and dexamethasone eye drops plus cytochrome c peroxidase eye drops (two drops three times a day for 1 week or until corneal re-epithelialization was complete, corresponding to 15,000 IU/day). The fellow eye served as the control and received standard postoperative therapy plus placebo. The mice were monitored daily, commencing on the day after surgery, for 7 days to evaluate the corneal re-epithelialization rate using a video slit lamp camera with cobalt blue light. The mean diameter of the corneal wounds was measured. Videotaped images were recorded and analyzed by computer planimetry. RESULTS: All eyes treated with cytochrome c peroxidase eye drops healed completely before day 5 after surgery, with a mean re-epithelialization time of 92 +/- (SD) 10 h; the mean re-epithelialization time was 121 +/- 8 h in the eyes receiving placebo (p < 0.05). There were no statistically significant differences between the two groups in corneal haze presentation during the follow-up period (p = 0.70), perhaps because the observation period was too short (7 days). However, the corneal clarity, on slit lamp biomicroscopy, in the study group was higher than that in the control group. No side effects or toxic effects were observed. CONCLUSIONS: These data suggest that cytochrome c peroxidase significantly accelerates epithelial healing after phototherapeutic keratectomy. Further clinical studies should be performed to prove the results obtained in this study and the long-term efficacy of cytochrome c peroxidase to prevent corneal haze.

Secreted products of a nonmucoid Pseudomonas aeruginosa strain induce two modes of macrophage killing: external-ATP-dependent, P2Z-receptor-mediated necrosis and ATP-independent, caspase-mediated apoptosis.

A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium. These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium. Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5'-nucleotidase (ATPase and/or phosphatase), were detected in the medium. These ATP-utilizing enzymes are believed to convert external ATP, presumably effluxed from macrophages, to various adenine nucleotides, which then activate purinergic receptors such as P2Z, leading to enhanced macrophage cell death. Pretreatment of macrophages with periodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z receptor activation, prevented subsequent ATP-induced macrophage cell death. A second type of cytotoxic factor(s) operated in an ATP-independent manner such that it triggered activation of apoptotic processes in macrophages, leading to proteolytic conversion of procaspase-3 to active caspase-3. This cytotoxic factor(s) did not appear to act on procaspase-3 present in macrophage cytosolic extracts. Intact macrophages, when exposed to the cytotoxic factor(s) for 6-16 h, underwent apoptosis and demonstrated the presence of active caspase-3 in their cytosolic extracts. Interestingly, two redox proteins, azurin and cytochrome c(551), were detected in the cytotoxic preparation. When cell-line-derived or peritoneal macrophages or mast cells were incubated overnight with Q-Sepharose column flow-through fraction or with a mixture of azurin and cytochrome c(551), they underwent extensive cell death due to induction of apoptosis.