Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder disease. Ten percent of the ALS patients are congenital (familial ALS), and the other 90% are sporadic ALS (SALS). It has been shown that mutations found in the Cu,Zn‐SOD cause 20% of the familial ALS due to its low enzyme activity. We hypothesized that heavy metals may interfere the structure of Cu,Zn‐SOD protein to suppress its activity in some of the SALS. In this study, we expressed and characterized the recombinant human Cu,Zn‐SOD under various concentrations of Cu²⁺, Zn²⁺, and Cd²⁺. By atomic absorption spectrophotometry, we demonstrated that adding of cadmium significantly increased the content of cadmium ion, but reduced its Zn²⁺ content and enzyme activity of the Cu,Zn‐SOD protein. The data of circular dichroism spectra demonstrated that the secondary structure of Cu,Zn‐SOD/Cd is different from Cu,Zn‐SOD, but close to apo‐SOD. In addition to the effect of cadmium on Cu,Zn‐SOD, cadmium was also shown to induce neural cell apoptosis. To further investigate the mechanism of neural cell apoptosis induced by cadmium, we used proteomics to analyze the altered protein expressions in neural cells treated with cadmium. The altered proteins include cellular structural proteins, stress‐related and chaperone proteins, proteins involved in reactive oxygen species (ROS), enzyme proteins, and proteins that mediated cell death and survival signaling. Taken together, in this paper, we demonstrate that cadmium decreases the content of Zn²⁺, changes the conformation of Cu,Zn‐SOD protein to decrease its enzyme activity, and causes oxidative stress‐induced neural cell apoptosis. J. Cell. Biochem. 98: 577–589, 2006.

The antioxidant properties of isovitexin and related flavonoids were studied. Isovitexin inhibited xanthine oxidase with an IC₅₀ value of = 15.2 μM. The flavonoid analogues, apigenin, kaempferol, quercetin, myricetin, and genistein also inhibited xanthine oxidase with IC₅₀ values of 0.58, 2.18, 1.09, 9.90, and 4.83 μM, respectively. Isovitexin protected DNA from the Fenton reaction-induced breakage in a dose-dependent manner with an IC₅₀ value of 9.52 μM. Isovitexin also protected HL-60 cells from the ROS damage induced by the xanthine/xanthine oxidase reaction. Isovitexin exhibited the lowest cytotoxicity toward HL-60 cells (LD₅₀ >400 μM) compared to the other flavonoids examined. In addition, excess hydrogen peroxide induced by cadmium in A2780 ovarian cells was significantly suppressed by isovitexin. These results suggest that isovitexin in rice may protect cells from oxidative stress.

Isovitexin exhibits potent antioxidant activities. In this study, the activity of nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated RAW264.7 macrophages after incubation with isovitexin was investigated. Isovitexin was able to reduce the production of hydrogen peroxide induced by LPS in mouse macrophage RAW264.7 cells. The cells incubated with isovitexin had markedly reduced LPS-stimulated NO production with an IC₅₀ value of 58.5 μM. The expression of iNOS was also inhibited when the cells were treated with isovitexin. A transient transfection experiment showed that isovitexin suppressed the iNOS promoter and NF-κB-dependent transcriptional activities. It was also found to inhibit IKK kinase activity and prevent the degradation of IκBα in activated RAW264.7 cells. Additionally, Western blotting analysis revealed that isovitexin prevented the translocation of NF-κB from the cytoplasm to the nucleus. Our results indicate that its ROS scavenger and IKK inhibitory activities also contribute to the suppression of ROS-mediated NF-κB activity. These results suggest that isovitexin, a food phytochemical contained in dietary rice products, might have biological significance.