Induction of systemic disease resistance in Nicotiana benthamiana by the cyclodipeptides cyclo (l‐Pro‐l‐Pro) and cyclo (d‐Pro‐d‐Pro)

Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l ‐Pro‐l ‐Pro) and cyclo (d ‐Pro‐d ‐Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up‐regulated the expression of the plant defence gene PR‐1a and the PR‐1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA‐dependent defence pathway is involved in cyclodipeptide‐mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases.     

Lead‐induced DNA damage and cell apoptosis in human renal proximal tubular epithelial cell: Attenuation via N‐acetyl cysteine and tannic acid

This study investigates the exposure of lead‐induced reactive oxygen species (ROS) generation, DNA damage, and apoptosis and also evaluates the therapeutic intervention using antioxidants in human renal proximal tubular cells (HK‐2 cells). Following treatment of HK‐2 cells with an increasing concentration of lead nitrate (0–50 μM) for 24 h, the intracellular ROS level increased whereas the GSH level decreased significantly in a dose‐dependent manner. Comet assay results revealed that lead nitrate showed the ability to increase the levels of DNA strand breaks in HK‐2 cells. Lead exposure also induced apoptosis through caspase‐3 activation at 30 μg/mL. Pretreatment with N‐acetylcysteine (NAC) and tannic acid showed a significant ameliorating effect on lead‐induced ROS, DNA damage, and apoptosis. In conclusion, lead induces ROS, which may exacerbate the DNA damage and apoptosis via caspase‐3 activation. Additionally, supplementation of antioxidants such as NAC and tannic acid may be used as salvage therapy for lead‐induced DNA damage and apoptosis in an exposed person.     

Gypenosides improve diabetic cardiomyopathy by inhibiting ROS‐mediated NLRP3 inflammasome activation

NLRP3 inflammasome activation plays an important role in diabetic cardiomyopathy (DCM), which may relate to excessive production of reactive oxygen species (ROS). Gypenosides (Gps), the major ingredients of Gynostemma pentaphylla (Thunb.) Makino, have exerted the properties of anti‐hyperglycaemia and anti‐inflammation, but whether Gps improve myocardial damage and the mechanism remains unclear. Here, we found that high glucose (HG) induced myocardial damage by activating the NLRP3 inflammasome and then promoting IL‐1β and IL‐18 secretion in H9C2 cells and NRVMs. Meanwhile, HG elevated the production of ROS, which was vital to NLRP3 inflammasome activation. Moreover, the ROS activated the NLRP3 inflammasome mainly by cytochrome c influx into the cytoplasm and binding to NLRP3. Inhibition of ROS and cytochrome c dramatically down‐regulated NLRP3 inflammasome activation and improved the cardiomyocyte damage induced by HG, which was also detected in cells treated by Gps. Furthermore, Gps also reduced the levels of the C‐reactive proteins (CRPs), IL‐1β and IL‐18, inhibited NLRP3 inflammasome activation and consequently improved myocardial damage in vivo. These findings provide a mechanism that ROS induced by HG activates the NLRP3 inflammasome by cytochrome c binding to NLRP3 and that Gps may be potential and effective drugs for DCM via the inhibition of ROS‐mediated NLRP3 inflammasome activation.     

Ehrlichia type IV secretion effector ECH0825 is translocated to mitochondria and curbs ROS and apoptosis by upregulating host MnSOD

Ehrlichia chaffeensis infects monocytes/macrophages and causes human monocytic ehrlichiosis. To determine the role of type IV secretion (T4S) system in infection, candidates for T4S effectors were identified by bacterial two‐hybrid screening of E. chaffeensis hypothetical proteins with positively charged C‐terminus using E. chaffeensis VirD4 as bait. Of three potential T4S effectors, ECH0825 was highly upregulated early during exponential growth in a human monocytic cell line. ECH0825 was translocated from the bacterium into the host‐cell cytoplasm and localized to mitochondria. Delivery of anti‐ECH0825 into infected host cells significantly reduced bacterial infection. Ectopically expressed ECH0825 also localized to mitochondria and inhibited apoptosis of transfected cells in response to etoposide treatment. In double transformed yeast, ECH0825 localized to mitochondria and inhibited human Bax‐induced apoptosis. Mitochondrial manganese superoxide dismutase (MnSOD) was increased over ninefold in E. chaffeensis‐infected cells, and the amount of reactive oxygen species (ROS) in infected cells was significantly lower than that in uninfected cells. Similarly, MnSOD was upregulated and the ROS level was reduced in ECH0825‐transfected cells. These data suggest that, by upregulating MnSOD, ECH0825 prevents ROS‐induced cellular damage and apoptosis to allow intracellular infection. This is the first example of host ROS levels linked to a bacterial T4S effector.     

Cyclo(d‐Tyr‐d‐Phe): a new antibacterial,anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode

A new microbial cyclic dipeptide (diketopiperazine), cyclo(d ‐Tyr‐d ‐Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d ‐Tyr‐d ‐Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d ‐Tyr‐d ‐Phe) recorded significant antitumor activity against A549 cells (IC₅₀ value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d ‐Tyr‐d ‐Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d ‐Tyr‐d ‐Phe). Flow cytometry analysis showed that the cyclo(d ‐Tyr‐d ‐Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase‐3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d ‐Tyr‐d ‐Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d ‐Tyr‐d ‐Phe) with synthetic cyclo(d ‐Tyr‐d ‐Phe) and cyclo(l ‐Tyr‐l ‐Phe). Natural and synthetic cyclo(d ‐Tyr‐d ‐Phe) recorded similar pattern of activity. Although synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded significant activity than natural and synthetic cyclo(d ‐Tyr‐d ‐Phe). The results of the present study reveals that cyclo(d ‐Tyr‐d ‐Phe) is more bioactive than cyclo(l ‐Tyr‐l ‐Phe). To the best of our knowledge, this is the first time that cyclo(d ‐Tyr‐d ‐Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d ‐Tyr‐d ‐Phe) is also reported for the first time. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Reactive oxygen species and gastric carcinogenesis: The complex interaction between Helicobacter pylori and host

Helicobacter pylori (H. pylori) is a highly successful human pathogen that colonizes stomach in around 50% of the global population. The colonization of bacterium induces an inflammatory response and a substantial rise in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), mostly derived from host neutrophils and gastric epithelial cells, which play a crucial role in combating bacterial infections. However, H. pylori has developed various strategies to quench the deleterious effects of ROS, including the production of antioxidant enzymes, antioxidant proteins as well as blocking the generation of oxidants. The host's inability to eliminate H. pylori infection results in persistent ROS production. Notably, excessive ROS can disrupt the intracellular signal transduction and biological processes of the host, incurring chronic inflammation and cellular damage, such as DNA damage, lipid peroxidation, and protein oxidation. Markedly, the sustained inflammatory response and oxidative stress during H. pylori infection are major risk factor for gastric carcinogenesis. In this context, we summarize the literature on H. pylori infection-induced ROS production, the strategies used by H. pylori to counteract the host response, and subsequent host damage and gastric carcinogenesis.     

Naringin prevents ultraviolet‐B radiation‐induced oxidative damage and inflammation through activation of peroxisome proliferator‐activated receptor γ in mouse embryonic fibroblast (NIH‐3T3) cells

The present study, we investigate the preventive role of naringin, a dietary flavonoid, against ultraviolet‐B (UVB) radiation (280‐320 nm) induced oxidative damage and inflammatory responses in mouse embryonic fibroblast cell lines (NIH‐3T3). In this study, 20 mJ/cm ² of UVB radiation induces cell cytotoxicity, reactive oxygen species (ROS) generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Treatment with naringin (60 µM) prior UVB exposure prevented the cell cytotoxicity, ROS generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Furthermore, naringin prevents UVB‐induced mitogen‐activated protein kinase families and nuclear factor‐κB (NF‐κB)‐mediated activation of inflammatory factors, that is TNF‐α, IL‐6, IL‐10, and COX‐2 in NIH‐3T3 cells. Peroxisome proliferator‐activated receptor γ (PPARγ) is an anti‐inflammatory agent and it suppressed the UVB‐mediated oxidative and inflammatory responses. In this study, naringin activates PPARγ and prevents inflammatory biomarkers in NIH‐3T3 cells. Thus, naringin prevents UVB‐mediated inflammation and oxidative damage in NIH‐3T3 cells probably over controlling NF‐κB expression and activation of PPARγ.     

Mitochondrial DNA damage is sensitive to exogenous H(2)O(2) but independent of cellular ROS production in prostate cancer cells

Intrinsic oxidative stress through enhanced production of reactive oxygen species (ROS) in prostate and other cancers may contribute to cancer progression due to its stimulating effect on cancer growth. In this study, we investigate differential responses to exogenous oxidative stimuli between aggressive prostate cancer and normal cell lines and explore potential mechanisms through interactions between cytotoxicity, cellular ROS production and oxidative DNA damage. The circular, multi-copy mitochondrial DNA (mtDNA) is used as a sensitive surrogate to oxidative DNA damage. We demonstrate that exogenous H(2)O(2) induces preferential cytotoxicity in aggressive prostate cancer than normal cells; a cascade production of cellular ROS, composed mainly of superoxide (O(2)(-)), is shown to be a critical determinant of H(2)O(2)-induced selective toxicity in cancer cells. In contrast, mtDNA damage and copy number depletion, as measured by a novel two-phase strategy of the supercoiling-sensitive qPCR method, are very sensitive to exogenous H(2)O(2) exposure in both cancer and normal cell lines. Moreover, we demonstrate for the first time that the sensitive mtDNA damage response to exogenous H(2)O(2) is independent of secondary cellular ROS production triggered by several ROS modulators regardless of cell phenotypes. These new findings suggest different mechanisms underpinning cytotoxicity and DNA damage induced by oxidative stress and a susceptible phenotype to oxidative injury associated with aggressive prostate cancer cells in vitro.     

Postmitotic neurons develop a p21‐dependent senescence‐like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro‐inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence‐like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL‐6 production, heterochromatinization and senescence‐associated β‐galactosidase activity. Frequencies of these senescence‐like neurons increased with age. Short‐term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late‐generation TERC?/? mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late‐generation TERC?/?CDKN1A?/? mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence‐like phenotype in neurons, as in senescing fibroblasts and other proliferation‐competent cells. We conclude that a senescence‐like phenotype is possibly not restricted to proliferation‐competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence‐like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.     

Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS‐induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild‐type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin‐binding protein F1‐disrupted mutant SAM1‐infected cells. In Bcl‐2‐overexpressing HeLa cells (HBD98‐2‐4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98‐2‐4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS‐infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.     

The Protecting Effect of Deoxyschisandrin and Schisandrin B on HaCaT Cells against UVB-Induced Damage

Schisandra chinensis is a traditional Chinese medicine that has multiple biological activities, including antioxidant, anticancer, tonic, and anti-aging effects. Deoxyschisandrin (SA) and schisandrin B (SB), the two major lignans isolated from S. chinensis, exert high antioxidant activities in vitro and in vivo by scavenging free radicals, such as reactive oxygen species (ROS). Ultraviolet B-ray (UVB) radiation induces the production of ROS and DNA damage, which eventually leads to cell death by apoptosis. However, it is unknown whether SA or SB protects cells against UVB-induced cellular DNA damage. Our study showed that both SA and SB effectively protected HaCaT cells from UVB-induced cell death by antagonizing UVB-mediated production of ROS and induction of DNA damage. Our results showed that both SA and SB significantly prevented UVB-induced loss of cell viability using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assays showed that the production of ROS following UVB exposure was inhibited by treatment with SA and SB. Moreover, SA and SB decreased the UVB-induced DNA damage in HaCaT cells by comet assays. In addition, SA and SB also prevented UVB-induced cell apoptosis and the cleavage of caspase-3, caspase-8 and caspase-9. In a word, our results imply that the antioxidants SA and SB could protect cells from UVB-induced cell damage via scavenging ROS.     

Deoxycholic acid causes DNA damage while inducing apoptotic resistance through NF-κB activation in benign Barrett's epithelial cells

Gastroesophageal reflux is associated with adenocarcinoma in Barrett's esophagus, but the incidence of this tumor is rising, despite widespread use of acid-suppressing medications. This suggests that refluxed material other than acid might contribute to carcinogenesis. We looked for potentially carcinogenetic effects of two bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on Barrett's epithelial cells in vitro and in vivo. We exposed Barrett's (BAR-T) cells to DCA or UDCA and studied the generation of reactive oxygen/nitrogen species (ROS/RNS); expression of phosphorylated H2AX (a marker of DNA damage), phosphorylated IkBα, and phosphorylated p65 (activated NF-κB pathway proteins); and apoptosis. During endoscopy in patients, we took biopsy specimens of Barrett's mucosa before and after esophageal perfusion with DCA or UDCA and assessed DNA damage and NF-κB activation. Exposure to DCA, but not UDCA, resulted in ROS/RNS production, DNA damage, and NF-κB activation but did not increase the rate of apoptosis in BAR-T cells. Pretreatment with N-acetyl-l-cysteine (a ROS scavenger) prevented DNA damage after DCA exposure, and DCA did induce apoptosis in cells treated with NF-κB inhibitors (BAY 11-7085 or AdIκB superrepressor). DNA damage and NF-κB activation were detected in biopsy specimens of Barrett's mucosa taken after esophageal perfusion with DCA, but not UDCA. These data show that, in Barrett's epithelial cells, DCA induces ROS/RNS production, which causes genotoxic injury, and simultaneously induces activation of the NF-κB pathway, which enables cells with DNA damage to resist apoptosis. We have demonstrated molecular mechanisms whereby bile reflux might contribute to carcinogenesis in Barrett's esophagus.     

1,4‐butanediyl‐bismethanethiosulfonate (BMTS) induces apoptosis through reactive oxygen species‐mediated mechanism

Although methane sulfonate compounds are widely used for the protein modification for their selectivity of thiol groups in proteins, their intracellular signaling events have not yet been clearly documented. This study demonstrated the methane sulfonate chemical 1,4‐butanediyl‐bismethanethiosulfonate (BMTS)‐induced cascades of signals that ultimately led to apoptosis of Jurkat cells. BMTS induced apoptosis through fragmentation of DNA, activation of caspase‐9 and caspase‐3, and downregulation of Bcl‐2 protein with reduction of mitochondrial membrane potential. Moreover, BMTS intensely and transiently induced intracellular reactive oxygen species (ROS) production and ROS produced by BMTS was mediated through mitochondria. We also found that a reducing agent dithiothreitol (DTT) and an anti‐oxidant N‐acetyl cysteine (NAC) inhibited BMTS‐mediated caspase‐9 and ‐3 activation, ROS production and induction of Annexin V/propidium iodide double positive cells, suggesting the involvement of ROS in the apoptosis process. Therefore, this study further extends our understanding on the basic mechanism of redox‐linked apoptosis induced by sulfhydryl‐reactive chemicals. J. Cell. Biochem. 108: 1059–1065, 2009. © 2009 Wiley‐Liss, Inc.     

Antimicrobial activity of the indolicidin‐derived novel synthetic peptide In‐58

Natural peptides with antimicrobial activity are extremely diverse, and peptide synthesis technologies make it possible to significantly improve their properties for specific tasks. Here, we investigate the biological properties of the natural peptide indolicidin and the indolicidin‐derived novel synthetic peptide In‐58. In‐58 was generated by replacing all tryptophan residues on phenylalanine in D‐configuration; the α‐amino group in the main chain also was modified by unsaturated fatty acid. Compared with indolicidin, In‐58 is more bactericidal, more resistant to proteinase K, and less toxic to mammalian cells. Using molecular physics approaches, we characterized the action of In‐58 on bacterial cells at the cellular level. Also, we have found that studied peptides damage bacterial membranes. Using the Escherichia coli luminescent biosensor strain MG1655 (pcolD’::lux), we investigated the action of indolicidin and In‐58 at the subcellular level. At subinhibitory concentrations, indolicidin and In‐58 induced an SOS response. Our data suggest that indolicidin damages the DNA, but bacterial membrane perturbation is its principal mode of action. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

A novel andrographolide derivative AL‐1 exerts its cytotoxicity on K562 cells through a ROS‐dependent mechanism

Andrographolide‐lipoic acid conjugate (AL‐1) is a new in‐house synthesized chemical entity, which was derived by covalently linking andrographolide with lipoic acid. However, its anti‐cancer effect and cytotoxic mechanism remains unknown. In this study, we found that AL‐1 could significantly inhibit cell viability of human leukemia K562 cells by inducing G2/M arrest and apoptosis in a dose‐dependent manner. Thirty‐one AL‐1‐regulated protein alterations were identified by proteomics analysis. Gene ontology and ingenuity pathway analysis revealed that a cluster of proteins of oxidative redox state and apoptotic cell death‐related proteins, such as PRDX2, PRDX3, PRDX6, TXNRD1, and GLRX3, were regulated by AL‐1. Functional studies confirmed that AL‐1 induced apoptosis of K562 cells through a ROS‐dependent mechanism, and anti‐oxidant, N‐acetyl‐l ‐cysteine, could completely block AL‐1‐induced cytotoxicity, implicating that ROS generation played a vital role in AL‐1 cytotoxicity. Accumulated ROS resulted in oxidative DNA damage and subsequent G2/M arrest and mitochondrial‐mediated apoptosis. The current work reveals that a novel andrographolide derivative AL‐1 exerts its anticancer cytotoxicity through a ROS‐dependent DNA damage and mitochondrial‐mediated apoptosis mechanism.     

Busulfan-induced senescence is dependent on ROS production upstream of the MAPK pathway

Induction of cellular senescence is a common response of a normal cell to a DNA-damaging agent, which may contribute to cancer chemotherapy- and ionizing radiation-induced normal tissue injury. The induction has been largely attributed to the activation of p53. However, the results from the present study suggest that busulfan (BU), an alkylating agent that causes DNA damage by cross-linking DNAs and DNA and proteins, induces senescence in normal human diploid WI38 fibroblasts through the extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK) cascade independent of the p53-DNA damage pathway. The induction of WI38 cell senescence is initiated by a transient depletion of intracellular glutathione (GSH) and followed by a continuous increase in reactive oxygen species (ROS) production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which leads to the activation of the Erk and p38 MAPK pathway. Incubation of WI38 cells with N-acetylcysteine (NAC) replenishes intracellular GSH, abrogates the increased production of ROS, ameliorates Erk and p38 MAPK activation, and attenuates senescence induction by BU. Thus, inhibition of senescence induction using a potent antioxidant or specific inhibitor of the Erk and p38 MAPK pathway has the potential to be developed as a mechanism-based strategy to ameliorate cancer therapy-induced normal tissue damage.     

The effect of α-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. α-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of γ-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP⁺+NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.