Lycium barbarum polysaccharides attenuate nonylphenol and octylphenol-induced oxidative stress and neurotransmitter disorders in PC-12 cells.

Nonylphenol (NP) and octylphenol (OP) are environmental contaminants with potential endocrine disrupting effects. However, there is limited research on the mechanisms and intervention of combined NP and OP exposure-induced neurotoxicity. This study aims to explore the cytotoxicity of combined NP and OP exposure and evaluate the potential of Lycium barbarum polysaccharides (LBP) in mitigating the aforementioned toxicity. In present study, LBP (62.5, 125 and 250 µg/mL) were applied to intervene rat adrenal pheochromocytoma (PC-12) cells treated with combined NP and OP (NP: OP = 4:1, w/w; 1, 2, 4 and 8 µg/mL). The results showed that NP and OP induced oxidative stress, disrupted the 5-hydroxytryptamine (5-HT) and cholinergic systems in PC-12 cells. Additionally, they activated the p38 protein kinase (p38) and suppressed the expression of silent information regulation type 1 (SIRT1), monoamine oxidase A (MAOA), phosphorylated cyclic-AMP response binding protein (p-CREB), brain-derived neurotrophic factor (BDNF) and phosphorylated tropomyosin-related kinase receptor type B (p-TrkB). However, N-acetyl-L-cysteine (NAC) treatment counteracted the changes of signalling molecule p38, SIRT1/MAOA and CREB/BDNF/TrkB pathways-related proteins induced by NP and OP. LBP pretreatment ameliorated combined NP and OP exposure-induced oxidative stress and neurotransmitter imbalances. Furthermore, the application of LBP and administration of a p38 inhibitor both reversed the alterations in the signaling molecule p38, as well as the proteins associated to the SIRT1/MAOA and CREB/BDNF/TrkB pathways. These results implied that LBP may have neuroprotective effects via p38-mediated SIRT1/MAOA and CREB/BDNF/TrkB pathways.

EGLN1: A Biomarker of Poor Prognosis of Cervical Cancer and a Target of Treatment.

Objective: To conduct bioinformatics analysis on the prognostic effect, mechanism of action, and drug sensitivity of Egl-9 family hypoxia-inducible factor 1 (EGLN1) expression on cervical cancer. Methods: Bioinformatics were obtained from Gene Expression Profiling Interactive Analysis (GEPIA), Tumor Immune Estimation Resource (TIMER), and the human cancer metastasis database (HCMDB), and the effect of EGLN1 expression level on the prognosis of cervical cancer was comprehensively analyzed. The protein-protein interaction network was constructed by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), and the possible mechanism of EGLN1 affecting the prognosis of cervical cancer was discussed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In addition, Gene Set Cancer Analysis (GSCALite) was used to predict sensitive drugs online. Results: The higher the expression level of EGLN1, the shorter the tumor-free survival time and overall survival time of cervical cancer. The higher the stage of cervical cancer, the higher the expression level of EGLN1. The expression of EGLN1 affects the degree of immune infiltration, the variation of somatic copy number, and the level of N6-methyladenosine (m6A) modification in cervical cancer. COX regression model suggested that EGLN1 was an independent prognostic factor of cervical cancer. Conclusions: The high expression of EGLN1 in cervical cancer is an independent risk factor for the prognosis of cervical cancer, which affects the prognosis of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) through different signal pathways. It is expected to be used to predict the sensitive anticancer drugs for the treatment of cervical cancer.

Lycium barbarum polysaccharides attenuate oxidative stress and mitochondrial toxicity induced by mixed plasticizers in HepG2 cells through activation of Nrf2.

AIMS: In daily life, it is common for humans to be exposed to multiple phthalate esters (PAEs). However, there is limited research on the mechanisms and intervention of combined PAEs toxicity. This study aims to explore the cytotoxicity of combined PAEs and evaluate the potential of Lycium barbarum polysaccharides (LBP) in mitigating the aforementioned toxicity. MAIN METHODS: LBP (62.5, 125 and 250 µg/mL) were applied to intervene HepG2 cells treated with DEHP and DBP mixtures (50, 100, 200, 400 and 800 µg/mL). Western Blot and different kits were mainly performed in our study. KEY FINDINGS: DEHP and DBP mixtures suppressed the expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and activated MAPK pathway by increasing ROS. Combined DEHP and DBP exposure reduced ATP content and inhibited the mitochondrial biogenesis pathway in HepG2 cells through oxidative stress, which in turn caused cytotoxicity. LBP reduced oxidative stress and cell death induced by mixed plasticizers, upregulated Nrf2 levels and mitochondrial biogenesis pathway levels and inhibited MAPK pathway activation. Notably, after treating HepG2 cells with Nrf2-specific inhibitor (ML385, 0.5 µM), we found that the activation of Nrf2 played a crucial role on LBP intervention of DEHP and DBP induced HepG2 cytotoxicity. SIGNIFICANCE: This study not only enhances our understanding of the toxicological effects caused by combined PAEs exposure, but also has significant implications in devising strategies to mitigate the toxicological consequences of combined exposure to exogenous chemicals through the investigation of the role of LBP.

The feasibility of using the compound kushen injection to treat cervical cancer based on network pharmacology and transcriptomics.

BACKGROUND: To investigate the molecular targets and mechanisms of compound kushen injection (CKI) in the prevention and treatment of cervical cancer based on network pharmacology and transcriptomics. METHODS: In this study, we used network pharmacology methods to screen for effective compounds, integrated the results of network pharmacology and RNA-seq to comprehensively screen and predict target genes, analyze the biological functions and signaling pathways of target genes, and construct a PPI network to screen for hub genes. The results were further verified by biological experiments, molecular docking, RT-PCR, and western blot analysis. RESULTS: The results showed that the hub genes CXCL2, anti-vascular endothelial growth factor, hexokinase 2 are therapeutic targets of CKI for the treatment of Cervical Cancer. These targets were significantly enriched in pathways mainly including pathways in cancer, cell cycle, MAPK signaling pathways, etc. In vitro cell experiments showed that CKI could effectively inhibit the proliferation of cancer cells, promote apoptosis, and induce cell cycle arrest. RT-PCR and western blot experiments showed that the expression of hub genes was significantly decreased. The compounds have good binding activity to hub genes. CONCLUSION: CKI, based on its active ingredients and through multiple targets and multiple pathways, can stop the growth of cervical cancer cells at a certain phase of the cell cycle and cause apoptosis, which proved CKI's effect in treating cervical cancer.

DBP induced autophagy and necrotic apoptosis in HepG2 cells via the mitochondrial damage pathway.

Phthalate esters (PAEs) are widely present in human tissues and pose significant health risks. In this study, HepG2 cells were treated with 0.0625, 0.125, 0.25, 0.5 and 1 mM Dibutyl phthalate (DBP) for 48 h to investigate mitochondrial toxicity. The results showed that DBP caused mitochondrial damage, autophagy, apoptosis and necroptosis; Transcriptomics analysis identified that MAPK and PI3K were significant factors in the cytotoxic changes induced by DBP; N-Acetyl-L-cysteine (NAC), SIRT1 activator, ERK inhibitor, p38 inhibitor and ERK siRNA treatments counteracted the changes of SIRT1/PGC-1α and Nrf2 pathway-related proteins, autophagy and necroptotic apoptosis proteins induced by DBP. While PI3K and Nrf2 inhibitors exacerbated the changes in SIRT1/PGC-1α, Nrf2-associated proteins and autophagy and necroptosis proteins induced by DBP. In addition, the autophagy inhibitor 3-MA alleviated the increase in DBP-induced necroptosis proteins. These results suggested that DBP-induced oxidative stress activated the MAPK pathway, inhibited the PI3K pathway, which in turn inhibited the SIRT1/PGC-1α pathway and Nrf2 pathway, thereby causing cell autophagy and necroptosis.

Effects of Neurod1 Expression on Mouse and Human Schwannoma Cells.

OBJECTIVES: The objective was to explore the effect of the proneuronal transcription factor neurogenic differentiation 1 (Neurod1, ND1) on Schwann cells (SC) and schwannoma cell proliferation. METHODS: Using a variety of transgenic mouse lines, we investigated how expression of Neurod1 effects medulloblastoma (MB) growth, schwannoma tumor progression, vestibular function, and SC cell proliferation. Primary human vestibular schwannoma (VS) cell cultures were transduced with adenoviral vectors expressing Neurod1. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) uptake. STUDY DESIGN: Basic science investigation. RESULTS: Expression of Neurod1 reduced the growth of slow-growing but not fast-growing MB models. Gene transfer of Neurod1 in human schwannoma cultures significantly reduced cell proliferation in dose-dependent way. Deletion of the neurofibromatosis type 2 (Nf2) tumor-suppressor gene via Cre expression in SCs led to increased intraganglionic SC proliferation and mildly reduced vestibular sensory-evoked potentials (VsEP) responses compared to age-matched wild-type littermates. The effect of Neurod1-induced expression on intraganglionic SC proliferation in animals lacking Nf2 was mild and highly variable. Sciatic nerve axotomy significantly increased SC proliferation in wild-type and Nf2-null animals, and expression of Neurod1 reduced the proliferative capacity of both wild-type and Nf2-null SCs following nerve injury. CONCLUSION: Expression of Neurod1 reduces slow-growing MB progression and reduces human SC proliferation in primary VS cultures. In a genetic mouse model of schwannomas, we find some effects of Neurod1 expression; however, the high variability indicates that more tightly regulated Neurod1 expression levels that mimic our in vitro data are needed to fully validate Neurod1 effects on schwannoma progression. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E259-E270, 2021.

Antifouling Photograftable Zwitterionic Coatings on PDMS Substrates.

The foreign body response (FBR) to implantable materials can negatively impact performance of medical devices such as the cochlear implant. Engineering surfaces that resist the FBR could lead to enhanced functionality including potentially improving outcomes for cochlear implant recipients through reduction in fibrosis. In this work, we coat poly(dimethylsiloxane) (PDMS) surfaces with two zwitterionic polymers, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA), using a simultaneous photografting/photo-cross-linking process to produce a robust grafted zwitterionic hydrogel. reduce nonspecific protein adsorption, the first step of the FBR. The coating process uses benzophenone, a photografting agent and type II photoinitiator, to covalently link the cross-linked zwitterionic thin film to the PDMS surface. As the concentration of benzophenone on the surface increases, the adhesive strength of the zwitterionic thin films to PDMS surfaces increases as determined by shear adhesion. Additionally, with increased concentration of the adsorbed benzophenone, failure of the system changes from adhesive delamination to cohesive failure within the hydrogel, demonstrating that durable adhesive bonds are formed from the photografting process. Interestingly, antifouling properties of the zwitterionic polymers are preserved with significantly lower levels of nonspecific protein adsorption on zwitterion hydrogel-coated samples compared to uncoated controls. Fibroblast adhesion is also dramatically reduced on coated substrates. These results show that cross-linked pSBMA and pCBMA hydrogels can be readily photografted to PDMS substrates and show promise in potentially changing the fibrotic response to implanted biomaterials.

Intracellular calcium and cyclic nucleotide levels modulate neurite guidance by microtopographical substrate features.

Micro- and nanoscale surface features have emerged as potential tools to direct neurite growth into close proximity with next generation neural prosthesis electrodes. However, the signaling events underlying the ability of growth cones to respond to topographical features remain largely unknown. Accordingly, this study probes the influence of [Ca(2+) ]i and cyclic nucleotide levels on the ability of neurites from spiral ganglion neurons (SGNs) to precisely track topographical micropatterns. Photopolymerization and photomasking were used to generate micropatterned methacrylate polymer substrates. Dissociated SGN cultures were plated on the micropatterned surfaces. Calcium influx and release from internal stores were manipulated by elevating extracellular K(+) , maintenance in calcium-free media, or bath application of various calcium channel blockers. Cyclic nucleotide activity was increased by application of cpt-cAMP or 8-Br-cGMP. Elevation of [Ca(2+) ]i by treatment of cultures with elevated potassium reduced neurite alignment to physical microfeatures. Maintenance of cultures in Ca(2+) -free medium or treatment with the non-selective voltage-gated calcium channel blocker cadmium or L-type Ca(2+) channel blocker nifedipine did not signficantly alter SGN neurite alignment. By contrast, ryanodine or xestospongin C, which block release of internal calcium stores via ryanodine-sensitive channels or inositol-1,4,5-trisphosphate receptors respectively, each significantly decreased neurite alignment. Cpt-cAMP significantly reduced neurite alignment while 8-Br-cGMP significantly enhanced neurite alignment. Manipulation of [Ca(2+) ]i or cAMP levels significantly disrupts neurite guidance while elevation of cGMP levels increases neurite alignment. The results suggest intracellular signaling pathways similar to those recruited by chemotactic cues are involved in neurite guidance by topographical features. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2037-2048, 2016.

Merlin status regulates p75(NTR) expression and apoptotic signaling in Schwann cells following nerve injury.

After nerve injury, Schwann cells (SCs) dedifferentiate, proliferate, and support axon regrowth. If axons fail to regenerate, denervated SCs eventually undergo apoptosis due, in part, to increased expression of the low-affinity neurotrophin receptor, p75(NTR). Merlin is the protein product of the NF2 tumor suppressor gene implicated in SC tumorigenesis. Here we explore the contribution of merlin to SC responses to nerve injury. We find that merlin becomes phosphorylated (growth permissive) in SCs following acute axotomy and following gradual neural degeneration in a deafness model, temporally correlated with increased p75(NTR) expression. p75(NTR) levels are elevated in P0SchΔ39-121 transgenic mice that harbor an Nf2 mutation in SCs relative to wild-type mice before axotomy and remain elevated for a longer period of time following injury. Replacement of wild-type, but not phospho-mimetic (S518D), merlin isoforms suppresses p75(NTR) expression in primary human schwannoma cultures which otherwise lack functional merlin. Despite elevated levels of p75(NTR), SC apoptosis following axotomy is blunted in P0SchΔ39-121 mice relative to wild-type mice suggesting that loss of functional merlin contributes to SC resistance to apoptosis. Further, cultured SCs from mice with a tamoxifen-inducible knock-out of Nf2 confirm that SCs lacking functional merlin are less sensitive to p75(NTR)-mediated cell death. Taken together these results point to a model whereby loss of axonal contact following nerve injury results in merlin phosphorylation leading to increased p75(NTR) expression. Further, they demonstrate that merlin facilitates p75(NTR)-mediated apoptosis in SCs helping to explain how neoplastic SCs that lack functional merlin survive long-term in the absence of axonal contact.

Influence of cAMP and protein kinase A on neurite length from spiral ganglion neurons.

Regrowth of peripheral spiral ganglion neuron (SGN) fibers is a primary objective in efforts to improve cochlear implant outcomes and to potentially reinnervate regenerated hair cells. Cyclic adenosine monophosphate (cAMP) regulates neurite growth and guidance via activation of protein kinase A (PKA) and Exchange Protein directly Activated by Cylic AMP (Epac). Here we explored the effects of cAMP signaling on SGN neurite length in vitro. We find that the cAMP analog, cpt-cAMP, exerts a biphasic effect on neurite length; increasing length at lower concentrations and reducing length at higher concentrations. This biphasic response occurs in cultures plated on laminin, fibronectin, or tenascin C suggesting that it is not substrate dependent. cpt-cAMP also reduces SGN neurite branching. The Epac-specific agonist, 8-pCPT-2'-O-Me-cAMP, does not alter SGN neurite length. Constitutively active PKA isoforms strongly inhibit SGN neurite length similar to higher levels of cAMP. Chronic membrane depolarization activates PKA in SGNs and also inhibits SGN neurite length. However, inhibition of PKA fails to rescue neurite length in depolarized cultures implying that activation of PKA is not necessary for the inhibition of SGN neurite length by chronic depolarization. Expression of constitutively active phosphatidylinositol 3-kinase, but not c-Jun N-terminal kinase, isoforms partially rescues SGN neurite length in the presence of activated PKA. Taken together, these results suggest that activation of cAMP/PKA represents a potential strategy to enhance SGN fiber elongation following deafness; however such therapies will likely require careful titration so as to promote rather than inhibit nerve fiber regeneration.

Cystitis increases colorectal afferent sensitivity in the mouse.

Studies in humans and rodents suggest that colon inflammation promotes urinary bladder hypersensitivity and, conversely, that cystitis contributes to colon hypersensitivity, events referred to as cross-organ sensitization. To investigate a potential peripheral mechanism, we examined whether cystitis alters the sensitivity of pelvic nerve colorectal afferents. Male C57BL/6 mice were treated with cyclophosphamide (CYP) or saline, and the mechanosensitive properties of single afferent fibers innervating the colorectum were studied with an in vitro preparation. In addition, mechanosensitive receptive endings were exposed to an inflammatory soup (IS) to study sensitization. Urinary bladder mechanosensitive afferents were also tested. We found that baseline responses of stretch-sensitive colorectal afferents did not differ between treatment groups. Whereas IS excited a proportion of colorectal afferents CYP treatment did not alter the magnitude of this response. However, the number of stretch-sensitive fibers excited by IS was increased relative to saline-treated mice. Responses to IS were not altered by CYP treatment, but the proportion of IS-responsive fibers was increased relative to saline-treated mice. In bladder, IS application increased responses of muscular afferents to stretch, although no differences were detected between saline- and CYP-treated mice. In contrast, their chemosensitivity to IS was decreased in the CYP-treated group. Histological examination revealed no changes in colorectum and modest edema and infiltration in the urinary bladder of CYP-treated mice. In conclusion, CYP treatment increased mechanical sensitivity of colorectal muscular afferents and increased the proportion of chemosensitive colorectal afferents. These data support a peripheral contribution to cross-organ sensitization of pelvic organs.

Characterization of mouse lumbar splanchnic and pelvic nerve urinary bladder mechanosensory afferents.

Sensory information from the urinary bladder is conveyed via lumbar splanchnic (LSN) and sacral pelvic (PN) nerves to the spinal cord. In the present report we compared the mechanosensitive properties of single afferent fibers in these two pathways using an in vitro mouse bladder preparation. Mechanosensitive primary afferents were recorded from the LSN or PN and distinguished based on their response to receptive field stimulation with different mechanical stimuli: probing (160 mg to 2 g), stretch (1-25 g), and stroking of the urothelium (10-1,000 mg). Four different classes of afferent were recorded from the LSN and PN: serosal, muscular, muscular/urothelial, and urothelial. The LSN contained principally serosal and muscular afferents (97% of the total sample), whereas all four afferent classes of afferent were present in the PN (63% of which were muscular afferents). In addition, the respective proportions and receptive field distributions differed between the two pathways. Both low- and high-threshold stretch-sensitive muscular afferents were present in both pathways, and muscular afferents in the PN were shown to sensitize after exposure to an inflammatory soup cocktail. The LSN and PN pathways contain different populations of mechanosensitive afferents capable of detecting a range of mechanical stimuli and individually tuned to detect the type, magnitude, and duration of the stimulus. This knowledge broadens our understanding of the potential roles these two pathways play in conveying mechanical information from the bladder to the spinal cord.

Differential chemosensory function and receptor expression of splanchnic and pelvic colonic afferents in mice.

Lumbar splanchnic (LSN) and sacral pelvic (PN) nerves convey different mechanosensory information from the colon to the spinal cord. Here we determined whether these pathways also differ in their chemosensitivity and receptor expression. Using an in vitro mouse colon preparation, individual primary afferents were tested with selective P2X and transient receptor potential vanilloid receptor 1 (TRPV1) receptor ligands. Afferent cell bodies in thoracolumbar and lumbosacral dorsal root ganglia (DRG) were retrogradely labelled from the colon and analysed for P2X3- and TRPV1-like immunoreactivity (LI). Forty per cent of LSN afferents responded to alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP; 1 mm), an effect that was concentration dependent and reversed by the P2X antagonist pyridoxyl5-phosphate 6-azophenyl-2',4'-disulphonic acid (PPADS) (100 microm). Significantly fewer PN afferents (7%) responded to alpha,beta-meATP. Correspondingly, 36% of colonic thoracolumbar DRG neurones exhibited P2X3-LI compared with only 19% of colonic lumbosacral neurones. Capsaicin (3 microm) excited 61% of LSN afferents and 47% of PN afferents; 82% of thoracolumbar and 50% of lumbosacral colonic DRG neurones displayed TRPV1-LI. Mechanically insensitive afferents were recruited by alpha,beta-meATP or capsaicin, and were almost exclusive to the LSN. Capsaicin-responsive LSN afferents displayed marked mechanical desensitization after responding to capsaicin, which did not occur in capsaicin-responsive PN afferents. Therefore, colonic LSN and PN pathways differ in their chemosensitivity to known noxious stimuli and their corresponding receptor expression. As these pathways relay information that may relate to symptoms in functional gastrointestinal disease, these results may have implications for the efficacy of therapies targeting receptor modulation.

Redox modulation of the liver with chronic antioxidant enzyme mimetic treatment prevents age-related oxidative damage associated with environmental stress.

A reduction in stress tolerance is a hallmark of the aging process, and the lowered functional capacity observed in aged organisms is associated with an increased rate of oxidative stress and a greater susceptibility of aged tissues to oxidative injury. In this report, we show that chronic systemic administration of a superoxide dismutase (SOD)/catalase mimetic (EUK-189), delivered over a 1 month period via osmotic pump, prevents heat stress-induced liver injury by dramatically decreasing oxidative damage in aged animals. Widespread liver injury was present in old but not young vehicle-treated rats in response to a 2 day heating protocol. However, SOD/catalase mimetic treatment markedly decreased the hyperthermia-induced liver injury associated in old animals. The reversal of damage with EUK-189 was associated with an improvement in intracellular redox status and a striking reduction in hepatocellular lipid peroxidation. EUK-189 treatment also blocked the activation of activator protein-1 (AP-1), which is a redox-sensitive early response transcription factor involved in the regulation of cellular stress responses. These results demonstrate that oxidative stress plays a unique role in age-related hyperthermic injury and suggest that therapeutic strategies aimed at improving redox potential, such as chronic SOD/catalase mimetic treatment, can prevent the oxidative-mediated damage associated with environmental stress.

Inhibition of oral cancer cell growth by adenovirusMnSOD plus BCNU treatment.

We hypothesized that inhibitors of peroxide removal, such as BCNU, an indirect inhibitor of glutathione peroxidase (GPx), and 3-amino-1,2,4-triazole (AT), a direct inhibitor of catalase (CAT), should cause toxicity to cancer cells after manganese superoxide dismutase (MnSOD) overexpression due to elevated peroxide levels. In vitro, hamster cheek pouch carcinoma cells (HCPC-1) and human oral squamous carcinoma cells (SCC-25) were infected with various combinations of adenovirus containing MnSOD cDNA (AdMnSOD). Cells were then treated with or without BCNU and assayed for viability using Annexin/PI staining and flow cytometry. In AdMnSOD plus BCNU-treated SCC-25 and HCPC-1 cells, a 30-60% decrease in cell viability was observed compared to BCNU alone. In vivo, HCPC-1 and SCC-25 xenografts were allowed to grow to approximately 70 mm(3) and 10(9) plaque forming units (pfu) of AdMnSOD were injected directly into the tumors. Two days later, 15 or 30 mg/kg BCNU was injected intratumorally. Tumor growth was greatly inhibited (4- to 20-fold) by this combined treatment, as well as increasing animal survival. Tumor volume could be decreased further by giving multiple doses of AdMnSOD or inhibiting catalase activity with AT. These results suggest that, by using these combination therapies, a significant decrease in tumor mass can be achieved.

Redox regulation of adenovirus-induced AP-1 activation by overexpression of manganese-containing superoxide dismutase.

Adenovirus gene therapy is a promising tool in the clinical treatment of many genetic and acquired diseases. However, it has also caused pathogenic effects in organs such as the liver. The redox-sensitive transcription factors AP-1 and NF-kappaB have been implicated in these effects. To study the mechanisms of adenovirus-mediated AP-1 and NF-kappaB activation and the possible involvement of oxidative stress in adenovirus transduction, rats were injected with either replication-defective recombinant adenovirus with DNA containing the cytomegalovirus promoter region only (AdCMV), adenovirus containing human manganese-containing superoxide dismutase (MnSOD) cDNA (AdMnSOD), or vehicle. Compared to vehicle and AdCMV transduction, MnSOD gene transfer yielded a fivefold increase in liver MnSOD activity 7 days postinjection. Gel shift assay showed that AdCMV transduction induced DNA binding activity for AP-1 but not NF-kappaB. MnSOD overexpression abolished this activation. Western blotting analysis of c-Fos and c-Jun suggested that up-regulation of c-fos and c-jun gene expression does not directly contribute to the induction of AP-1 activation. Glutathione/glutathione disulfide ratios were decreased by adenovirus transduction and restored by MnSOD overexpression. The AP-1 binding activity that was induced by AdCMV was decreased by immunoprecipitation of Ref-1 protein. Ref-1 involvement was confirmed by restoration of AP-1 binding activity after the immunoprecipitated Ref-1 protein had been added back. AP-1 DNA binding activity was also elevated in control and AdMnSOD-injected rats after addition of the immunoprecipitated Ref-1 protein. These data indicate that cellular transduction by recombinant adenovirus stimulates AP-1 DNA binding activity. Furthermore, our results suggest that MnSOD overexpression decreases AP-1 DNA binding activity by regulating intracellular redox status, with the possible involvement of Ref-1 in this redox-sensitive pathway.