Shaopeng Chen

Constitutive nitric oxide acting as a possible intercellular signaling molecule in the initiation of radiation-induced DNA double strand breaks in non-irradiated bystander cells

The initiation and propagation of the early processes of bystander signaling induced by low-dose α-particle irradiation are very important for understanding the underlying mechanism of the bystander process. Our previous investigation showed that the medium collected from cell culture exposed to low-dose α-particle rapidly induced phosphorylated form of H2AX protein foci formation among the non-irradiated medium receptor cells in a time-dependent manner. Using NG-methyl-L-arginine, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate and Nω-nitro-L-arginine (L-NNA) treatment before exposure to 1 cGy α-particle, we showed in the present study that nitric oxide (NO•) produced in the irradiated cells was important and necessary for the DNA double strand break inducing activity (DIA) of conditioned medium and the generation of NO• in irradiated confluent AG1522 cells is in a time-dependent manner and that almost all NO• was generated within 15 min post-irradiation. Concurrently, the kinetics of NO• production in the medium of irradiated cells after irradiation was rapid and in a time-dependent manner as well, with a maximum yield observed at 10 min after irradiation with electron spin resonance analysis. Furthermore, our results that 7-Nitroindazole and L-NNA, but not aminoguanidine hemisulfate, treatment before exposure to 1 cGy α-particle significantly decrease the DIA of the conditioned medium suggested that constitutive NO• from the irradiated cells possibly acted as an intercellular signaling molecule to initiate and activate the early process (⩽30 min) of bystander response after low-dose irradiation.

Up-regulation of ROS by mitochondria-dependent bystander signaling contributes to genotoxicity of bystander effects

Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander gamma-H2AX induction. In this paper, we used normal (rho(+)) and mtDNA-depleted (rho(0)) human-hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with gamma-H2AX, we found that the fraction of gamma-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated rho(+) cells at 10 min post-irradiation (rho(+) ICCM(10 min)) caused larger increases of bystander gamma-H2AX induction comparing to rho(0) ICCM(10 min), which only caused a slight increase of bystander gamma-H2AX induction. The rho(+) ICCM(10 min) could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with rho(0) ICCM(10 min). We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched gamma-H2AX induction by rho(+) ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59(-) gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of rho(+) ICCM-treated bystander cells. In conclusion, the work presented here suggested that up-regulation of the mitochondria-dependent ROS might be very important in mediating genotoxicity of bystander effects.

Nitric oxide mediated DNA double strand breaks induced in proliferating bystander cells after alpha-particle irradiation

Low-dose alpha-particle exposures comprise 55% of the environmental dose to the human population and have been shown to induce bystander responses. Previous studies showed that bystander effect could induce stimulated cell growth or genotoxicity, such as excessive DNA double strand breaks (DSBs), micronuclei (MN), mutation and decreased cell viability, in the bystander cell population. In the present study, the stimulated cell growth, detected with flow cytometry (FCM), and the increased MN and DSB, detected with p53 binding protein 1 (53BP1) immunofluorescence, were observed simultaneously in the bystander cell population, which were co-cultured with cells irradiated by low-dose alpha-particles (1-10 cGy) in a mixed system. Further studies indicated that nitric oxide (NO) and transforming growth factor beta1 (TGF-beta1) played very important roles in mediating cell proliferation and inducing MN and DSB in the bystander population through treatments with NO scavenger and TGF-beta1 antibody. Low-concentrations of NO, generated by spermidine, were proved to induce cell proliferation, DSB and MN simultaneously. The proliferation or shortened cell cycle in bystander cells gave them insufficient time to repair DSBs. The increased cell division might increase the probability of carcinogenesis in bystander cells since cell proliferation increased the probability of mutation from the mis-repaired or un-repaired DSBs.

Exogenous carbon monoxide protects the bystander Chinese hamster ovary cells in mixed coculture system after alpha-particle irradiation

In the present work, the inhibitory effect of carbon monoxide (CO), generated by tricarbonyldichlororuthenium (II) dimer [CO-releasing molecule (CORM-2)], on the toxicity of radiation-induced bystander effect (RIBE) after alpha-particle irradiation was studied in a mixed coculture system. CO (CORM-2) treatment showed a significant inhibitory effect to the formation of p53 binding protein 1 (BP1) and micronuclei (MN) induced by RIBE in a concentration-dependent manner, but in the directly irradiated cell population no distinct decreases of BP1 and MN formation were observed. In this mixed coculture system, nitric oxide (NO) or superoxide anion (O2(*-)) was also proved to mediate the transduction of RIBE by using a NO synthase inhibitor or NADPH-oxidase-specific inhibitor treatment. The elevated O2(*-) was attenuated by CO (CORM-2) treatment in the bystander cells as measured by hydroethidine staining and fluorescence assessment. The exogenous NO (sper) or O2(*-) (H2O2) was used to mimic NO/O(2)-mediated RIBE, and CO (CORM-2) treatment also showed a protective effect to cells against the toxicity of these exogenous factors. Considering the inhibitory effect of CO on RIBE and the wide use of CO in therapy of diseases, it is hoped that a low concentration of CO can protect normal tissues against RIBE during radiotherapy.

Cellular ATP content was decreased by a homogeneous 8.5 T static magnetic field exposure: Role of reactive oxygen species

The literature on the impact of strong static magnetic fields (SMF) on human health is vast and contradictory. The present study focused on the cellular effects of strong homogeneous SMF in human-hamster hybrid (A(L) ) cells, mitochondria-deficient (ρ(0) A(L) ) cells, and double-strand break (DSB) repair-deficient (XRS-5) cells. Adenosine triphosphate (ATP) content was significantly decreased in A(L) cells exposed to 8.5 Tesla (T) but not 1 or 4 T SMF for either 3 or 5 h. In addition, ATP content significantly decreased in the two deficient cell lines exposed to 8.5 T SMF for 3 h. With further incubation of 12 or 24 h without SMF exposure, ATP content could retrieve to the control level in the A(L) cells but not ρ(0) A(L) and XRS-5 cells. Under a fluorescence reader, the levels of reactive oxygen species (ROS) in the three cell lines were significantly increased by exposure to 8.5 T SMF for 3 h. Concurrent treatment with ROS inhibitor, DMSO, dramatically suppressed the ATP content in exposed A(L) cells. However, the CD59 mutation frequency and the cell cycle distribution were not significantly affected by exposure to 8.5 T SMF for 3 h. Our results indicated that the cellular ATP content was reduced by 8.5 T SMF for 3 h exposure, which was partially mediated by mitochondria and the DNA DSB repair process. Moreover, ROS were involved in the process of the cellular perturbations from the SMF.

Mitochondrial dysfunction resulting from loss of cytochrome c impairs radiation-induced bystander effect

Cytochrome c is a pivotal protein that resides in mitochondria as component of mitochondria respiration and apoptosis initiator. Using murine cells lacking cytochrome c, we showed here that cytochrome c-deficient cells had attenuated reactive oxygen species/nitric oxide and micronuclei induction to radiation-induced bystander signals, indicating cytochrome c is essential for the bystander effect.

PFOS-induced apoptosis through mitochondrion-dependent pathway in human–hamster hybrid cells

Perfluorooctane sulfonate (PFOS) was listed as one of the persistent organic pollutants (POPs) in Stockholm Convention in 2009. Recent evidence showed that PFOS could induce apoptosis both in vivo and in vitro. However, the apoptotic mechanisms induced by PFOS as well as the possible relationship between apoptosis and other PFOS-induced endpoints, remain unclear. In the present study, normal human-hamster hybrid (AL) cells and mtDNA-depleted (ρ(0) AL) cells were exposed to PFOS, and assayed for cytotoxicity, mutagenicity, and apoptosis (caspase-3/7, caspase-9 activities). Our results showed that PFOS decreased cell viability in a time- and concentration-dependent manner in AL cells, but not in ρ(0) AL cells. However, long-term exposure to PFOS failed to induce the mutagenic effects at the CD59 locus in AL cells. Exposure to 200 μM PFOS significantly increased the activities of caspase-3/7 and caspase-9 in AL cells, but the activities of these caspases were not affected in ρ(0) AL cells. In addition, PFOS increased the levels of reactive oxygen species (ROS), superoxide anion (O2(-)), as well as nitric oxide (NO), and decreased mitochondrial membrane potential (MMP) at the concentrations of 100 and 200μM in AL cells. On the other hand, exposure to PFOS had no effect on intracellular ROS, O2(-), and NO production in ρ(0) AL cells. Caspase-3/7 activity, which was increased by 200 μM PFOS, could be suppressed by ROS/O2(-) scavengers and nitric oxide synthases (NOSs) inhibitors in AL cells. These results implicate that PFOS-induced apoptosis and oxidative stress is mediated by a mitochondrion-dependent pathway and that the induction of apoptosis might be a protective function against mutagenesis in AL cells exposed to PFOS.

Mutagenicity of PFOA in Mammalian Cells: Role of Mitochondria-Dependent Reactive Oxygen Species

Mutagenicity is often a prerequisite to the development of malignancy. Evidences have shown that exposure to perfluorooctanoic acid (PFOA) results in various cancer inductions. However, whether any mutagenic base exists is still puzzling. In the present study, we exposed exponentially growing AL cells to PFOA and assayed the cells for survival, mutation induction, and caspase-3/7, -9 activities. Mitochondrial-DNA deficient human-hamster hybrid (ρ(0) AL) cells and reactive oxygen species (ROS) inhibitor were used to elucidate the possible mechanism. Our results showed that treatment of AL cells with PFOA for 16 days induced significant mutagenic effects together with the increment of ROS, superoxide anions (O2(.-)), and nitrogen oxide (NO) levels, while treatment of ρ(0) AL cells did not have much change. Concurrent treatment of AL cells with ROS inhibitor significantly decreased the mutagenic potential of PFOA. In addition, caspase activities in AL cells were increased by PFOA exposure and suppressed by ROS/RNS (reactive oxygen/nitrogen species) inhibitors. Our results suggest that exposure to PFOA lead to mutagenicity induction in AL cells, and mitochondria-dependent ROS plays an important role in this process. This provides a direct base for PFOA mediated cancer induction.

Graphene Oxide Attenuates the Cytotoxicity and Mutagenicity of PCB 52 via Activation of Genuine Autophagy

Graphene oxide (GO), owing to its large surface area and abundance of oxygen-containing functional groups, is emerging as a potential adsorbent for polychlorinated biphenyls (PCBs), which accumulate over time and are harmful to both natural ecosystems and human health. However, the effect of GO against PCB-induced toxicity remains largely unexplored. The present study aimed to investigate the protective effect of GO against PCB 52 induced cytotoxic and genotoxic response in mammalian cells at various exposure conditions and clarify the protective role of autophagy. Pretreatment with GO dramatically decreased PCB 52 induced cytotoxicity and CD59 gene mutation in human-hamster hybrid (AL) cells. The toxic response in cells either pretreated with PCB 52 and then treated with GO or concurrently treated with GO and PCB 52 did not differ significantly from the toxic response in the cells treated with PCB 52 alone. Using autophagy inhibitors (3-methyladenine and wortmannin) and inducers (trehalose and rapamycin), we found that genuine autophagy induced by GO was involved in decreasing PCB 52 induced toxicity. These findings suggested that GO has an antagonistic effect against the toxicity of PCB 52 mainly by triggering a genuine autophagic process, which might provide new insights into the potential application of GO in PCB disposal and environmental and health risk assessment.

Activated toxicity of diesel particulate extract by ultraviolet a radiation in mammalian cells: role of singlet oxygen.

Background Diesel exhaust [diesel exhaust particles (DEPs) and their extracts (DPE)] and ultraviolet A radiation (UVA) are two ubiquitous environmental factors that have been identified as essential risk factors for various benign or malignant human diseases, either alone or in combination with other agents. Objectives We aimed to investigate the synergistic effects of DPE and UVA at low-dose exposures in human–hamster hybrid (AL) cells and their underlying mechanisms. Methods We exposed exponentially growing AL cells to DPE and/or UVA radiation with or without reactive oxygen species (ROS) quenchers and then assayed the cells for survival, mutation induction, apoptosis, and micronucleus generation. In addition, using a singlet oxygen (1O2) trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron paramagnetic resonance spectroscopy, we determined the production of 1O2. Results Treatment of AL cells with DPE + UVA induced significant cytotoxic and genotoxic damage. In contrast, we found no significant damage in cells treated with either UVA or DPE alone at the same doses. Mutation spectra of CD59− mutants showed that treatment with DPE + UVA easily induces multilocus deletions. Sodium azide significantly inhibited both cellular and DNA damage induced by DPE + UVA treatment, whereas other ROS inhibitors had little protecting effect. Furthermore, we found a significant increase of 1O2 in the cells that received DPE + UVA treatment. Conclusion These findings suggest that UVA activated the genotoxicity and cytotoxicity of DPE in mammalian cells and that 1O2 played an important role in these processes.