Role of H2O2 in the oxidative effects of zinc exposure in human airway epithelial cells

Abstract

Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn2+) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn2+ toxicity is not fully understood. H2O2 and Zn2+ have been shown to mediate signaling leading to adverse cellular responses in the lung and we have previously demonstrated Zn2+ to cause cellular H2O2 production. To determine the role of Zn2+-induced H2O2 production in the human airway epithelial cell response to Zn2+ exposure. BEAS-2B cells expressing the redox-sensitive fluorogenic sensors HyPer (H2O2) or roGFP2 (EGSH) in the cytosol or mitochondria were exposed to 50 µM Zn2+ for 5 min in the presence of 1 µM of the zinc ionophore pyrithione. Intracellular H2O2 levels were modulated using catalase expression either targeted to the cytosol or ectopically to the mitochondria. HO-1 mRNA expression was measured as a downstream marker of response to oxidative stress induced by Zn2+ exposure. Both cytosolic catalase overexpression and ectopic catalase expression in mitochondria were effective in ablating Zn2+-induced elevations in H2O2. Compartment-directed catalase expression blunted Zn2+-induced elevations in cytosolic EGSH and the increased expression of HO-1 mRNA levels. Zn2+ leads to multiple oxidative effects that are exerted through H2O2-dependent and independent mechanisms.

Live-cell imaging approaches for the investigation of xenobiotic-induced oxidant stress

“…HyPer has been chimerically linked to the growth factor receptors EGFR and PDGFR in order to create proteins positioned to sense localized increases in H 2 O 2 production [198]. Studies using HyPer have demonstrated that xenobiotic induction of H 2 O 2 is specific to subcellular compartments [114,195,199]. The group that developed HyPer recently reported an improved version, called HyPer-3, that contains point mutations that increase the sensor dynamic range [197].…”

“…This can be done using pH specific sensors such as such as pHluorin or pHluorin2 [200], albeit these sensors also emit in the green channel, requiring separate experiments or spectral unmixing [137]. pHRed is an alternative pH sensor that emits in the red channel and can therefore be co-expressed and monitored concurrently with HyPer in the same cells [199,201]. However, the ideal pH sensor to use as a control for HyPer is SypHer, which is a version of HyPer that contains a single point mutation (C199S) that renders it unable to sense H 2 O 2 [202].…”

“…Our experiments showed that glucose deprivation effectively sensitized the cells to increases in E GSH induced by a subsequent exposure to O 3 [212], without a detectable alteration in basal E GSH . Depletion of intracellular levels of GSH using an inhibitor of glutathione synthesis, such as buthionine sulfoximine (BSO), is also an effective tool that can be brought to bear in studies aimed at scrutinizing the integrity of the roGFP redox relay in cells exposed to xenobiotics [199]. …”

“…This approach has been used to support data obtained using HyPer in human respiratory cells exposed to the ambient air electrophile 1.2-naphthoquinone [114,213]. A subsequent study investigated the role of H 2 O 2 in Zn 2+ -induced oxidative responses using cytosolic as well as ectopic mitochondrial expression of catalase in airway epithelial cells co-expressing HyPer in either compartment, thus providing independent spatial information to confirm the imaging data [199]. This approach could also be used to validate responses reported by small molecule sensors.…”

Mutagenicity and oxidative damage induced by an organic extract of the particulate emissions from a simulation of the deepwater horizon surface oil burns

“…We assessed the effect of the extract on extracellular flux of mitochondrial respiration in cultured human airway epithelial cells (BEAS‐2B), as well as on expression of the heme oxygenase ( HMOX1 ) gene. We have shown previously that HMOX1 expression, a marker of oxidative stress in mammalian cells, is induced by a wide variety of air pollutants [Silbajoris et al, ; Cheng et al, ; Wages et al, ].…”