A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H(2)O(2). After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H(2)O(2) concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.     

Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission

The present study describes the development and characterization of a novel technique, the alkaline-halo assay, for the assessment of DNA single strand breakage in mammalian cells. This technique allows the measurement of DNA lesions at the single cell level and presents the additional advantages of being rapid, sensitive, virtually costless and environmentally friendly, because it does not require the use of isotopes. The alkaline halo assay involves a series of sequential steps in which the cells are first treated, then embedded in melted agarose and spread onto microscope slides that are incubated for 2 min at ice-bath temperature to allow complete geling. The slides are then incubated for 20 min in a high salt alkaline lysis solution, for an additional 15 min in a hypotonic alkaline solution and, finally, for 10 min in ethidium bromide. Under these conditions, single-stranded DNA fragments spread radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nucleus remnants. The area of the halos increased at increasing levels of DNA fragmentation and this process was associated with a progressive reduction of areas of the nuclear remnants. These events were conveniently monitored with a fluorescence microscope and quantified by image processing analysis. The sensitivity of the alkaline-halo assay, which is based on the osmotically driven radial diffusion of single-stranded DNA fragments through agarose pores, is remarkably similar to that of the widely used alkaline elution and comet assays.     

A comparison of four assays detecting oxidizing species

Quin2, a fluorescent calcium probe, has a low affinity for calcium in comparison to its affinities for transition metal ions. Chelation of ferric ion with quin2 strongly enhanced the formation of oxidizing species in the presence of bolus H₂O₂ as detected with four assays, electron spin resonance with the spin-trap DMPO, the deoxyribose assay, the DMSO assay, and plasmid DNA strand breakage. In comparison, Fe(III)-EDTA reacted with bolus H₂O₂ only as detected with electron spin resonance and the deoxyribose assay, but not as detected with the two latter assays. The addition of reductants, like ascorbate or superoxide generated by hypoxanthine/xanthine oxidase, to Fe(III)-EDTA in the presence of H₂O₂ produced plasmid DNA strand breakage and strong reactivity in both the DMSO and the deoxyribose assays. Our findings suggest that the main oxidizing species produced in Fenton-type reactions is hydroxyl radical. However, the reaction between Fe(III)-EDTA and bolus H₂O₂ appears to be exceptional and dominated by a nonhydroxyl radical species.     

Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay

Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to ?80 °C—a laborious process. A recent publication (Al‐Salmani et al. Free Rad Biol Med 2011; 51: 719–725) describes a simple method in which small volumes of whole blood are frozen to ?20 or ?80 °C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H₂O₂, and so a common test of antioxidant status (resistance to strand breakage by H₂O₂) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H₂O₂. In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H₂O₂ sensitivity and endogenous damage—both reflecting the antioxidant status of the cells—correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory. Copyright © 2013 John Wiley & Sons, Ltd.     

Hydrogen peroxide induced by modulated electromagnetic radiation protects the cells from DNA damage

It is believed that non-ionizing electromagnetic radiation (EMR) and low-level hydrogen peroxide (H₂O₂) may change nonspecific resistance and modify DNA damage caused by ionizing radiation. To check this assumption, the combined effects of extremely high-frequency EMR (EHF EMR) and X-rays on induction of DNA damage in mouse whole blood leukocytes were studied. The cells were exposed to X-rays with or without preliminary treatment with EHF EMR or low-level H₂O₂. With the use of enhanced chemiluminescence, it was shown for the first time that pulse-modulated EHF EMR (42.2 GHz, incident power density of 0.1 mW/cm², exposure duration of 20 min, modulation frequency of 1 Hz) induced H₂O₂ at a concentration of 4.6 ± 0.3 nM L^?1 in physiological saline. With the use of an alkaline comet assay, it was found that the exposure of cells to the pulse-modulated EHF EMR, 25 min prior to treatment with X-rays at a dose of 4 Gy reduced the level of ionizing radiation-induced DNA damage. Continuous EHF EMR was inefficient. In turn, it was shown that low-level H₂O₂ (30–500 nM L^?1) protected the cells against X-irradiation. Thus, the mechanisms of radiation protective effect of EHF EMR are connected with the induction of the adaptive response by nanomolar concentrations of reactive oxygen species formed by pulse-modulated EHF EMR.     

A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage

The molecular role of poly (ADP-ribose) polymerase-1 in DNA repair is unclear. Here, we show that the single-strand break repair protein XRCC1 is rapidly assembled into discrete nuclear foci after oxidative DNA damage at sites of poly (ADP-ribose) synthesis. Poly (ADP-ribose) synthesis peaks during a 10 min treatment with H₂O₂ and the appearance of XRCC1 foci peaks shortly afterwards. Both sites of poly (ADP-ribose) and XRCC1 foci decrease to background levels during subsequent incubation in drug-free medium, consistent with the rapidity of the single-strand break repair process. The formation of XRCC1 foci at sites of poly (ADP-ribose) was greatly reduced by mutation of the XRCC1 BRCT I domain that physically interacts with PARP-1. Moreover, we failed to detect XRCC1 foci in Adprt1^–/– MEFs after treatment with H₂O₂. These data demonstrate that PARP-1 is required for the assembly or stability of XRCC1 nuclear foci after oxidative DNA damage and suggest that the formation of these foci is mediated via interaction with poly (ADP-ribose). These results support a model in which the rapid activation of PARP-1 at sites of DNA strand breakage facilitates DNA repair by recruiting the molecular scaffold protein, XRCC1.     

Cellular protection of morin against the oxidative stress induced by hydrogen peroxide

Flavonoids are a class of secondary metabolites abundantly found in fruits and vegetables. In addition, flavonoids have been reported as potent antioxidants with beneficial effects against oxidative stress-related diseases such as cancer, aging, and diabetes. The present study was carried out to investigate the cytoprotective effects of morin (2′,3,4′,5,7-pentahydroxyflavone), a member of the flavonoid group, against hydrogen peroxide (H₂O₂)-induced DNA and lipid damage. Morin was found to prevent the cellular DNA damage induced by H₂O₂ treatment, which is shown by the inhibition of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation (a modified form of DNA base), inhibition of comet tail (a form of DNA strand breakage), and decrease of nuclear phospho histone H2A.X expression (a marker for DNA strand breakage). In addition, morin inhibited membrane lipid peroxidation, which is detected by inhibition of thiobarbituric acid reactive substance (TBARS) formation. Morin was found to scavenge the intracellular reactive oxygen species (ROS) generated by H₂O₂ treatment in cells, which is detected by a spectrofluorometer, flow cytometry, and confocal microscopy after staining of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Morin also induces an increase in the activity of catalase and protein expression. The results of this study suggest that morin protects cells from H₂O₂-induced damage by inhibiting ROS generation and by inducing catalase activation.     

Apoptotic signaling induced by H2O2-mediated oxidative stress in differentiated C2C12 myotubes

AimsApoptotic signaling proteins were evaluated in postmitotic skeletal myotubes to test the hypothesis that oxidative stress induced by H₂O₂ activates both caspase-dependent and caspase-independent apoptotic proteins in differentiated C2C12 myotubes. We hypothesized that oxidative stress would decrease anti-apoptotic protein levels in C2C12 myotubes.Main methodsApoptotic regulatory factors and apoptosis-associated proteins including Bcl-2, Bax, Apaf-1, XIAP, ARC, cleaved PARP, p53, p21^Cip1/Waf1, c-Myc, HSP70, CuZnSOD, and MnSOD protein content were measured by immunoblots.Key findingsH₂O₂ induced apoptosis in myotubes as shown by DNA laddering and an elevation of apoptotic DNA fragmentation. Cell death ELISA showed increase in the extent of apoptotic DNA fragmentation following treatment with H₂O₂. Treatment with 4 mM of H₂O₂ for 24 or 96 h caused increase in Bax (56%, 227%), cytochrome c (282%, 701%), Smac/DIABLO (155%, 260%), caspase-3 protease activity (51%, 141%), and nuclear and cytosolic p53 (719%, 1581%) levels in the myotubes. As an estimate of the mitochondrial AIF release to the cytosol, AIF protein content measured in the mitochondria-free cytosolic fraction was elevated by 65% after 96 h treatment with 4 mM of H₂O₂. AIF measured in the nuclear protein fraction increased by 74% and 352% following treatment with 4 mM of H₂O₂ for 24 and 96 h, respectively. Bcl-2 declined in myotubes by 61% and 69% after 24 or 96 h of treatment in 4 mM H₂O₂, respectively.SignificanceThese findings indicate that both caspase-dependent and caspase-independent mechanisms are involved in coordinating the activation of apoptosis induced by H₂O₂ in differentiated myotubes.     

A Highly Sensitive Method for Quantitative Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Ferric-Xylenol Orange Formation

L-amino acid oxidase (LAAO) has important biological roles in many organisms, thus attracting great attention from researchers to establish its detection methods. In this study, a new quantitative in-gel determination of LAAO activity based on ferric-xylenol orange (Fe^IIIXO) formation was established. This method showed that due to the conversion of Fe^II to Fe^III by H₂O₂ and subsequent formation of Fe^IIIXO complex halo in agar medium, the logarithm of H₂O₂ concentration from 5 to 160 µM was linearly correlated to the diameter of purplish red Fe^IIIXO halo. By extracting the LAAO-generated H₂O₂ concentration, the LAAO activity can be quantitatively determined. This Fe^IIIXO agar assay is highly sensitive to detect H₂O₂ down to micromolar range. More importantly, it is easy to handle, cheap, reproducible, convenient and accurate. Coupled with SDS-PAGE, it can directly be used to determine the number and approximate molecular weight of LAAO in one assay. All these features make this in-gel Fe^IIIXO assay useful and convenient as a general procedure for following enzyme purification, assaying fractions from a column, or observing changes in activity resulting from enzyme modifications, hence endowing this method with broad applications.     

Similarity in the acute cytotoxic response of mammalian cells to mercury (II) and X-rays: DNA damage and glutathione depletion

Pronounced strand breakage of DNA analyzed by alkaline elution techniques was produced in intact Chinese hamster ovary cells by 25 μM HgCl₂ within 1 hr or 100 μM HgCl₂ within 15 min. HgCl₂-induced strand breakage was directly proportional to concentration up to 100 μM and to time within 1 hr. Levels of reduced glutathione decreased following HgCl₂ in parallel with the induction of DNA strand breakage. Evidence is presented that this rapid and pronounced induction of DNA strand breaks and other cytotoxic responses following acute exposure to HgCl₂ resembles the cellular effects of X-rays.     

Timely termination of repair DNA synthesis by ATAD5 is important in oxidative DNA damage-induced single-strand break repair

Reactive oxygen species (ROS) generate oxidized bases and single-strand breaks (SSBs), which are fixed by base excision repair (BER) and SSB repair (SSBR), respectively. Although excision and repair of damaged bases have been extensively studied, the function of the sliding clamp, proliferating cell nuclear antigen (PCNA), including loading/unloading, remains unclear. We report that, in addition to PCNA loading by replication factor complex C (RFC), timely PCNA unloading by the ATPase family AAA domain-containing protein 5 (ATAD5)-RFC–like complex is important for the repair of ROS-induced SSBs. We found that PCNA was loaded at hydrogen peroxide (H₂O₂)-generated direct SSBs after the 3′-terminus was converted to the hydroxyl moiety by end-processing enzymes. However, PCNA loading rarely occurred during BER of oxidized or alkylated bases. ATAD5-depleted cells were sensitive to acute H₂O₂ treatment but not methyl methanesulfonate treatment. Unexpectedly, when PCNA remained on DNA as a result of ATAD5 depletion, H₂O₂-induced repair DNA synthesis increased in cancerous and normal cells. Based on higher H₂O₂-induced DNA breakage and SSBR protein enrichment by ATAD5 depletion, we propose that extended repair DNA synthesis increases the likelihood of DNA polymerase stalling, shown by increased PCNA monoubiquitination, and consequently, harmful nick structures are more frequent.     

Strand scission in DNA by Gossypol and Cu(II): Role of Cu(I) and oxygen-free radicals

Gossypol, a polyphenolic binaphthyl dialdehyde found in cotton seeds, is a dietary mutagen and a potential male contraceptive. In the presence of Cu(II), gossypol caused breakage of supercoiled plasmid pBR322 DNA. The products were relaxed circles or a mixture of these and linear molecules. Other metal ions tested [Ni(II), Co(II), Mn(II), and Fe(II)] were ineffective or less effective in the DNA breakage reaction. In the case of gossypol-Cu(II) mediated cleavage, (Cu(I) was shown to be an essential intermediate by using the Cud) sequestering reagent bathocuproine. By using job plots, it was established that in the absence of DNA, eight Cu(II) ions can be reduced by one gossypol molecule. The involvement of active oxygen species, such as singlet oxygen and H₂O₂, was established by the inhibition of DNA breakage by catalase and by sodium azide. It was further shown that gossypol is capable of directly producing H₂O₂.     

Genoprotection and genotoxicity of green tea (Camellia sinensis): Are they two sides of the same redox coin?

Abstract

Objectives

Regular intake of green tea associates with lower DNA damage and increased resistance of DNA to oxidant challenge. However, in vitro pro-oxidant effects of green tea have been reported. Both effects could be mediated by hydrogen peroxide (H₂O₂) which is generated by autoxidation of tea catechins. In large amounts, H₂O₂ is genotoxic, but low concentrations could activate the redox-sensitive antioxidant response element (ARE) via the Keap-1/Nrf2 redox switch, inducing genoprotective adaptations. Our objective was to test this hypothesis.

Methods

Peripheral lymphocytes from healthy volunteers were incubated for 30 minutes at 37°C in freshly prepared tea solutions (0.005, 0.01, 0.05%w/v (7, 14, 71 µmol/l total catechins) in phosphate buffered saline (PBS), with PBS as control) in the presence and absence of catalase (CAT). H₂O₂ in tea was measured colorimetrically. Oxidation-induced DNA lesions were measured by the Fpg-assisted comet assay.

Results

H₂O₂ concentrations in 0.005, 0.01, and 0.05% green tea after 30 minutes at 37°C were, respectively, ～3, ～7, and ～52 µmol/l. Cells incubated in 0.005 and 0.01% tea showed less (P < 0.001) DNA damage compared to control cells. Cells treated with 0.05% green tea showed ～50% (P < 0.001) more DNA damage. The presence of CAT prevented this damage, but did not remove the genoprotective effects of low-dose tea. No significant changes in expression of ARE-associated genes (HMOX1, NRF2, KEAP1, BACH1, and hOGG1) were seen in cells treated with tea or tea + CAT.

Conclusion

Genoprotection by low-dose green tea could be due to direct antioxidant protection by green tea polyphenols, or to H₂O₂-independent signalling pathways.     

DNA strand breakage induced by CuII and NiII, in the presence of peptide models of histone H2B

In the present study we used the plasmid relaxation assay, a very sensitive method for detection of DNA strand breaks in vitro, in order to evaluate the role of peptide fragments of histone H2B in DNA strand breakage induced by copper and nickel. We have found that in the presence of peptides modeling the histone fold domain (H2B_32-62 and H2B_63-93) as well as the N-terminal tail (H2B_1-31) of histone H2B there is an increased DNA damage by Cu²⁺/H₂O₂ and Ni²⁺/H₂O₂ reaction mixtures. On the contrary, the C-terminal tail (H2B_94-125) seems to have a protective role on the attack of ROS species to DNA. We have rendered our findings to the interactions of the peptides with DNA, as well as with the metal.     

Content of iron and copper in the nuclei and induction of pH 9-labile lesions in L5178Y sublines inversely cross-sensitive to H2O2 and x-rays

Cells from the L5178Y murine lymphoma subline LY-R are twice as resistent to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by H₂O₂, the effect being more pronounced at 37 °C than 0 °C. Initial DNA damage after H₂O₂ treatment (both temperatures, 5 min) has been estimated by the comet assay (single-cell gel electrophoresis) and fluorescent halo technique. According to both methods, the initial damage is significantly higher in LY-R cells, particularly that inflicted at O °C. Differences between DNA unwinding and rewinding abilities at pH 9 and 6.9 (estimated by the fluorescent halo technique) point to a considerable difference in pH-9-labile damage between the sublines, as observed previously for x-irradiated cells (Kapiszewska et al. 1992). In contrast to findings with x-irradiated cells, however, after H₂O₂ treatment this damage is more extensive in LY-R cells than in LY-S cells. Thus, the initial pH-9-labile damage corresponds to the pattern of sensitivity to H₂O₂ and x-rays. We suggest that this is caused by different proportions of cuprous and ferric ions found in the nuclei of LY sublines and by the different ability of these ions to react with H₂O₂ and water radiolysis products. The copper/iron ratio in the nucleus is 1.31 in LY-R cells and 4.84 in LY-S cells.     

Optimization of the alkaline comet assay for easy repair capacity quantification of oxidative DNA damage in PBMC from human volunteers using aphidicolin block

Assessment of DNA repair capacity (DRC) upon ex vivo challenge of peripheral blood mononuclear cells (PBMC) with oxidative damage inducing agents, as evaluated by the comet assay, is widely used as biomarker to assess the antioxidant status in human studies. Here, the alkaline comet assay was now optimized for easy and time saving detection of repair capacity upon oxidative stress-induced DNA damage using the DNA polymerase inhibitor aphidicolin (APC) to block repair of hydrogen peroxide (H₂O₂) induced DNA damage. Addition of a DMSO-containing DNA damage stop solution was found suitable to replace washing steps for H₂O₂ removal before APC block. Cell treatment with APC at 6 μM did not impact baseline DNA damage but could reliably block DNA repair after H₂O₂ challenge in both fresh and cryopreserved samples thus omitting the use of a starting time point control. Under the conditions used, frozen cells, with or without an additional 4 h rest, showed the same repair capacity as their fresh counterpart. The intra assay coefficient of variation (CV) was 3.3%. To provide proof of principle, the modified assay was applied to cryopreserved PBMC from 19 participants of a short-term Brassica diet intervention study investigating potential health promoting effects of the food intervention. Then, a 33% increase in DRC (p ≤ 0.01) could be shown in samples after intervention (mean ± SD: 5.82 ± 1) as compared to baseline (mean ± SD: 4.38 ± 1.21). Individual samples from baseline and intervention showed an inter-individual CV of 27.65% (baseline) and 17.26% (intervention). Taken together this modified comet assay protocol allows the facilitated detection of DNA repair in fresh or cryopreserved human PBMC samples with a good sensitivity and reliability and could be useful in human studies addressing the antioxidant status and repair capacity of PBMC.     

Opposing effects of nitroxide free radicals in Escherichia coli mutants deficient in DNA repair

Nitroxide free radicals have been previously shown to function as superoxide dismutase (SOD) mimics and to protect bacterial and mammalian cells against oxidative damage, particularly from superoxide and hydrogen peroxide. Although nitroxides are generally considered to be non-toxic nor mutagenic, there is no agreement regarding their potential adverse effect. Some toxic effects were observed upon using high concentration of six-membered ring derivatives. Conflicting evidence has also been reported regarding the mutagenic activity of nitroxides toward Salmonella typhimurium. It was also demonstrated that nitroxides exert two opposing effects on exonuclease III deficient cells of Escherichia coli upon exposure to naphthoquinones. The attempts to use nitroxides as contrast agents in nuclear magnetic resonance imaging (MRI) and as a new class of anti-oxidants underscore the need to examine their potential adverse effects. Since nitroxides protected xthA cells from DNA scission caused by H₂O₂, it was anticipated that they would provide even greater t protection for recA DNA repair-deficient cells of E. coli, which are more sensitive to H₂O₂-induced oxidative stress. The results of the present study showed that: (1) nitroxides exert bactericidal and bacteriostatic effects on recA but not on xthA or wild-type E. coli K12 cells, (b) nitroxides and H₂O₂ act synergistically on recA cells, both under aerobic and hypoxic conditions; (c) the nitroxide-induced toxicity in recA cells and the synergistic effect with H₂O₂ were not accompanied by a decrease in the cellular level of reduced glutathione; (d) TEMPAMINE protected against DNA scission induced by H₂O₂ and 1,10-o-phenanthroline chelate of Cu(II) in xthA cells, but potentiated DNA double-strand breakage in recA cells.     

Effects of light-emitting diodes on thermally-induced oxidative stress in the bay scallop Argopecten irradians

ABSTRACT

Water temperature is an important stressor that affects the physiological and biochemical responses of scallops. In this study, we investigated the effect of different light-emitting diodes (LEDs; red, green and blue) on oxidative stress in Argopecten irradians. PCR revealed MnSOD mRNA expression in the digestive diverticula, gill, adductor muscle and eye. CAT and HSP70 mRNA were expressed in the digestive diverticula, gill and adductor muscle. Additionally, we measured the changes in the expression of HSP70, MnSOD and CAT as well as H₂O₂ levels during thermal/laboratory stress. In the digestive diverticula, gill and adductor muscle, the mRNA expressions and activities and H₂O₂ levels significantly increased in response to thermal changes. The gene expressions and activities and H₂O₂ levels were significantly lower in scallops that received green LED light than in those that received no mitigating treatment. A comet assay revealed that thermal change groups had increased rates of nuclear DNA damage; however, treatment with green LED reduced the frequency of damage. The results indicated that low or high water temperature conditions induced oxidative stress in A. irradians but that green LED significantly reduced this stress.     

Hydrogen peroxide enhanced Ca(2+)-activated BK currents and promoted cell injury in human dermal fibroblasts

AimsRecent studies have shown that dermal fibroblasts possess multiple types of voltage-dependent K⁺ channels, and the activation of these channels induces apoptosis. In the present study, we aimed to investigate whether hydrogen peroxide (H₂O₂), an oxidative stress inducer, could modulate these channels or induce human dermal fibroblasts injury.Main methodsThe effects of H₂O₂ on K⁺ currents were studied using a whole-cell recording. Intracellular PKC levels were measured with a direct human PKC enzyme immunoassay kit. Cell viability was assessed using PI staining and apoptotic nuclei were detected with TdT-mediated digoxigenin-dUTP nick-end labelling assay (TUNEL) assay.Key findingsTreatment of cells with 100 μM H₂O₂ resulted in a partially reversible increase in non-inactivating outward K⁺ currents and an alteration in the steady-state activation property of the channels. The H₂O₂-induced increase in K⁺ currents was mimicked by a PKC activator, and was blocked by the PKC inhibitor or the large conductance Ca²⁺-activited K⁺ (BK) channel blockers. The intracellular PKC levels were significantly enhanced by H₂O₂ treatment in a concentration-dependent manner. After exposure to H₂O₂, evaluation of fibroblasts survival rate and damaged cell number with TUNEL-positive nuclei revealed an increased cell injury. Blocking the K⁺ channels with blockers significantly decreased the H₂O₂-induced human dermal fibroblasts injury.SignificanceOur results revealed that H₂O₂ could enhance BK currents by PKC pathway. Increased K⁺ currents might be related to H₂O₂-induced human dermal fibroblasts injury. The results reported here contribute to our understanding of the mechanism underlying H₂O₂-induced human dermal fibroblasts injury.