ascorbic-acid and 2--7--dichlorodihydrofluorescein-diacetate

Endothelial injury and damage as well as accumulated reactive oxygen species (ROS) in aging play a significant role in the development of cardiovascular disease (CVD). Recent studies show an association of high citrus fruit intake with a lower risk of CVD and stroke but the mechanisms involved are not fully understood. This study investigated the effects of pummelo (Citrus maxima Merr. var. Tubtim Siam, CM) fruit extract on human umbilical vein endothelial cell (HUVECs) migration and aging. The freeze-dried powder of fruit extract was characterized for antioxidant capacity (FRAP assay) and certain natural antioxidants, including ascorbic acid, gallic acid, hesperidin, and naringin (HPLC). Short-term (48 h) co-cultivation of HUVECs with CM enhanced cell migration as evaluated by a scratch wound assay and Boyden chamber assay. A long-term treatment with CM for 35 days significantly increased HUVEC proliferation capability as indicated by population doubling level (PDL). CM also delayed the onset of aging phenotype shown by senescence-associated β-galactosidase (SA-β-gal) staining. Furthermore, CM was able to attenuate increased ROS levels in aged cells when determined by 2',7'-dichlorodihydrofluorescein diacetate (DCDHF) while eNOS mRNA expression was increased but the eNOS protein level was not changed. Thus, further in vivo and clinical studies are warranted to support the use of pummelo as a functional fruit for endothelial health and CVD risk reduction.

Topics: Antioxidants; Ascorbic Acid; Cell Movement; Cellular Senescence; Citrus; Flavanones; Fluoresceins; Fruit; Gallic Acid; Hesperidin; Human Umbilical Vein Endothelial Cells; Humans; Nitric Oxide Synthase Type III; Plant Extracts; Reactive Oxygen Species; RNA, Messenger

In the present work, the mode of cell death induced by Pb in Saccharomyces cerevisiae was studied. Yeast cells Pb-exposed, up to 6 h, loss progressively the capacity to proliferate and maintained the membrane integrity evaluated by the fluorescent probes bis(1,3-dibutylbarbituric acid trimethine oxonol) and propidium iodide. Pb-induced death is an active process, requiring the participation of cellular metabolism, since the simultaneous addition of cycloheximide attenuated the loss of cell proliferation capacity. Cells exposed to Pb accumulated intracellularly reactive oxygen species (ROS), evaluated by 2',7'-dichlorodihydrofluorescein diacetate. The addition of ascorbic acid (a ROS scavenger) strongly reduced the oxidative stress and impaired the loss of proliferation capacity in Pb-treated cells. Pb-exposed cells displayed nuclear morphological alterations, like chromatin fragmentation, as revealed by diaminophenylindole staining. Together, the data obtained indicate that yeast cells exposition to 1 mmol/l Pb results in severe oxidative stress which can be the trigger of programmed cell death by apoptosis.

Topics: Apoptosis; Ascorbic Acid; Chromatin; Colony Count, Microbial; Cycloheximide; Fluoresceins; Lead; Microbial Viability; Oxidative Stress; Phenotype; Reactive Oxygen Species; Saccharomyces cerevisiae