Effects of cellular uptake of flavonoids against peroxynitrite-mediated cell cytotoxicity.

Objective: Flavonoids-derived antioxidant properties have been proposed to be beneficial to various pathologies associated with reactive species including peroxynitrite. It is thus crucial to investigate cellular uptake of flavonoids which are responsible for biological effects in target cells. The objectives of this study were to evaluate the uptake of flavonoids in human colon cancer (HT-29) and chondrosarcoma (SW1353) cells by assessing intracellular phenolic contents and effects against peroxynitrite-mediated SW1353 cytotoxicity. Methods: The radical scavenging activities of flavonoids (catechin, epicatechin or quercetin) were determined using Thin layer chromatorgraphy-1, 1-diphenyl-2-picrylhydrazyl (TLC-DPPH) assay. HT-29 and SW1353 cells were treated with the flavonoids and removed after 18-h incubation before the assay. Flavonoid uptake in HT-29 and SW1353 cells was demonstrated by assessing phenolic contents in the cell lysates using the Folin-Ciocalteau method. Additionally, their effects in inhibiting peroxynitrite-mediated SW1353 cytotoxicity were studied by using 4 cytotoxicity models: crystal violet-staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction, cellular glutathione (GSH) assay and intracellular oxidant formation. Results: DPPH assay showed that quercetin exhibited the greatest radical scavenging activity. Phenolic contents detected in both HT-29 and SW1353 cell lines pre-incubated with quercetin were also greater in HT-29 cell lysates. SW1353 cytotoxicity by peroxynitrite was also inhibited by pre-incubation with quercetin. Additionally, phenolic contents and cytoprotective effects were not found in cells pre-incubated with catechin or epicatechin. Conclusion: There appears to be an uptake of quercetin but neither catechin nor epicatechin in HT-29 and SW1353 cells. Moreover, accumulation of quercetin, the powerful free radical scavenger, in SW1353 cells may contribute to its cytoprotective effect against peroxynitrite-induced cytotoxicity.

Clinical and molecular characterization of an extended family with Fabry disease.

OBJECTIVE: To characterize clinical manifestations, biochemical changes, mutation of alpha-Galactosidase (alpha-Gal A) gene A (GLA), and functional capability of mutant protein. MATERIAL AND METHOD: Seventeen subjects from a family with a newly diagnosed patient with Fabry disease were enrolled in the present study. In each individual, clinical history, physical examination, leukocyte enzyme activity of alpha-Gal A, and mutation analysis were performed. Those with a mutation were further investigated by ophthalmological and audiological evaluations, electrocardiography, echocardiogram, urinalysis, and blood tests to determine renal insufficiency. Expression study of the mutant protein was performed using a Pichia pastoris expression system. RESULTS: Four affected males and five symptomatic female carriers were identified. Clinical manifestations included severe neuropathic pain, acroparesthesia, hypo-/hyper-hidrosis, frequent syncope, ischemic stroke, cardiac hypertrophy, corneal dystrophy and cart-wheel cataract, high frequency sensorineural hearing loss, periorbital edema and subcutaneous edema over hands and interphalangeal joints. None had angiokeratoma or renal symptoms. The authors identified a novel mutation, p.L106R, in the GLA gene. Recombinant expression of the mutant protein gave little or no enzyme activity compared to the normal protein. CONCLUSION: There were intrafamilial clinical variabilities, but consistent findings of the absence of angiokeratoma and renal symptoms, which could represent a unique feature of this particular mutation.