ABSTRACT
We have previously shown that PM10 exposure causes oxidative stress and reduces Nrf2 protein levels, and SKQ1 pre-treatment protects against this damage in human corneal epithelial cells (HCE-2). The current study focuses on uncovering the mechanisms underlying acute PM10 toxicity and SKQ1-mediated protection. HCE-2 were pre-treated with SKQ1 and then exposed to 100 µg/mL PM10. Cell viability, oxidative stress markers, programmed cell death, DNA damage, senescence markers, and pro-inflammatory cytokines were analyzed. Nrf2 cellular location and its transcriptional activity were determined. Effects of the Nrf2 inhibitor ML385 were similarly evaluated. Data showed that PM10 decreased cell viability, Nrf2 transcriptional activity, and mRNA levels of antioxidant enzymes, but increased p-PI3K, p-NFκB, COX-2, and iNOS proteins levels. Additionally, PM10 exposure significantly increased DNA damage, phosphor-p53, p16 and p21 protein levels, and ß-galactosidase (ß-gal) staining, which confirmed the senescence. SKQ1 pre-treatment reversed these effects. ML385 lowered the Nrf2 protein levels and mRNA levels of its downstream targets. ML385 also abrogated the protective effects of SKQ1 against PM10 toxicity by preventing the restoration of cell viability and reduced oxidative stress. In conclusion, PM10 induces inflammation, reduces Nrf2 transcriptional activity, and causes DNA damage, leading to a senescence-like phenotype, which is prevented by SKQ1.
Subject(s)
Cornea , NF-E2-Related Factor 2 , Oxidative Stress , Particulate Matter , Humans , Cornea/drug effects , Cornea/metabolism , NF-E2-Related Factor 2/genetics , RNA, Messenger/genetics , Particulate Matter/toxicityABSTRACT
Urinary oxalate is a biomarker for calcium oxalate kidney stone disease; however, its assay is insensitive and nonspecific. Calcium oxalate monohydrate (COM) binding protein (45 kDa) is a promoter of calcium oxalate kidney disease, which is markedly upregulated by oxalate induced oxidative stress. The current study was carried out to evaluate whether COM binding protein can serve as a diagnostic marker for calcium oxalate kidney stone formers. COM binding protein was isolated, purified and antibody was raised against it in rabbits. Urine samples (24 h) were collected from patients suffering from various kidney diseases such as acute nephritis, chronic nephritis, nephrotic syndrome, calcium oxalate (CaOx) stone formers, uric acid stone formers, struvite stone formers and calcium phosphate stone formers. This COM binding protein was quantified by an in house ELISA method and the excretion was found to lie between 2 and 3 mg in control samples, while in CaOx stone formers it was detected between 11 and 19 mg. Urinary risk factors were assayed. We conclude that COM binding protein can serve as a diagnostic marker for CaOx stone formers.
