The mouse Gene Expression Database (GXD): 2019 update.

The mouse Gene Expression Database (GXD) is an extensive, well-curated community resource freely available at www.informatics.jax.org/expression.shtml. Covering all developmental stages, GXD includes data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot and western blot experiments in wild-type and mutant mice. GXD's gene expression information is integrated with the other data in Mouse Genome Informatics and interconnected with other databases, placing these data in the larger biological and biomedical context. Since the last report, the ability of GXD to provide insights into the molecular mechanisms of development and disease has been greatly enhanced by the addition of new data and by the implementation of new web features. These include: improvements to the Differential Gene Expression Data Search, facilitating searches for genes that have been shown to be exclusively expressed in a specified structure and/or developmental stage; an enhanced anatomy browser that now provides access to expression data and phenotype data for a given anatomical structure; direct access to the wild-type gene expression data for the tissues affected in a specific mutant; and a comparison matrix that juxtaposes tissues where a gene is normally expressed against tissues, where mutations in that gene cause abnormalities.

The mouse Gene Expression Database (GXD): 2017 update.

The Gene Expression Database (GXD; www.informatics.jax.org/expression.shtml) is an extensive and well-curated community resource of mouse developmental expression information. Through curation of the scientific literature and by collaborations with large-scale expression projects, GXD collects and integrates data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot and western blot experiments. Expression data from both wild-type and mutant mice are included. The expression data are combined with genetic and phenotypic data in Mouse Genome Informatics (MGI) and made readily accessible to many types of database searches. At present, GXD includes over 1.5 million expression results and more than 300 000 images, all annotated with detailed and standardized metadata. Since our last report in 2014, we have added a large amount of data, we have enhanced data and database infrastructure, and we have implemented many new search and display features. Interface enhancements include: a new Mouse Developmental Anatomy Browser; interactive tissue-by-developmental stage and tissue-by-gene matrix views; capabilities to filter and sort expression data summaries; a batch search utility; gene-based expression overviews; and links to expression data from other species.

Periodontitis and diabetes mellitus co-morbidity: A molecular dialogue.

BACKGROUND: Type 2 diabetes mellitus (T2DM) and periodontitis are two biologically linked diseases that often coexist in complex interaction. While periodontitis may lead to insulin receptor desensitization, diabetes may increase the expression of inflammatory cytokines, such as Tumor Necrosis Factor-α (TNF-α) and Interleukin 6 (IL-6), in the gingival crevicular fluid and activate osteoclasts via Receptor activator of nuclear factor kappa-Β ligand (RANK-L) production, leading to bone resorption. However, the association between the two diseases processes, where one may exacerbate the progression of the other, is unclear. In addition, both diseases have similar mechanistic themes, such as chronic inflammation and oxidative stress. This review aimed to investigate the pathophysiological and molecular mechanisms underlying T2DM and periodontitis. HIGHLIGHT: Uncontrolled diabetes is often associated with severe periodontitis, measured by clinical attachment loss. Alteration in the oral microbiome composition, which may activate the host inflammatory response and lead to irreversible oxidative stress, is a common finding in both diseases. An understanding of the molecular crosstalk between the two disease processes is crucial for developing therapeutic targets that inhibit bone resorption and halt the progression of periodontitis in patients with diabetes. CONCLUSION: The Oral microbiome composition in T2DM and periodontitis shifts toward dysbiosis, favoring bacterial pathogens, such as Fusobacteria and Porphyromonas species. Both conditions are marked by pro-inflammatory immune activity via the activation of Interleukin 17 (IL-17), Interleukin 1 (IL-1), TNF-α, and Nuclear Factor Kappa Beta (NF-κB). Common molecular crosstalk signaling appears to involve advanced glycation end products (AGEs) and oxidative stress. Thus, future drug targets are multifactorial, ranging from modulatory of host inflammatory response to preventing the accumulation of AGEs and oxidative free radicals.

Effect of systemic parathyroid hormone (1-34) and a beta-tricalcium phosphate biomaterial on local bone formation in a critical-size rat calvarial defect model.

OBJECTIVE: The objective of this study was to evaluate local bone formation following systemic administration of parathyroid hormone (1-34) (PTH), a surgically implanted synthetic beta-tricalcium phosphate (beta-TCP) bone biomaterial serving as a matrix to support new bone formation. MATERIALS AND METHODS: Critical-size, 8 mm, calvarial through-and-through osteotomy defects were surgically created in 100 adult male Sprague-Dawley rats. The animals were randomized into five groups of 20 animals each to receive one of the following treatments: PTH (15 microg PTH/kg/day; subcutaneously), PTH/beta-TCP, beta-TCP, or particulate human demineralized freeze-dried bone (DFDB), and sham-surgery controls. Ten animals/group were euthanized at 4 and 8 weeks post-surgery for radiographic and histometric analysis. RESULTS: The histometric analysis showed that systemic PTH significantly enhanced local bone formation, bone fill averaging (+/-SE) 32.2+/-4.0% compared with PTH/beta-TCP (15.7+/-2.4%), beta-TCP (12.5+/-2.3%), DFDB (14.5+/-2.3%), and sham-surgery control (10.0+/-1.5%) at 4 weeks (p<0.014). Systemic PTH showed significantly enhanced bone formation (41.5+/-4.0%) compared with PTH/beta-TCP (22.4+/-3.0%), beta-TCP (21.3+/-4.4%), and with the sham-surgery control (23.8+/-4.2%) at 8 weeks (p<0.025). The DFDB group showed significantly increased bone formation from 4 (14.5+/-2.3%) to 8 weeks (32.0+/-3.2%) (p<0.006). The PTH/beta-TCP and beta-TCP groups both showed limited biomaterials resorption. The radiographic analysis was not diagnostic to distinguish local bone formation from the radiopaque beta-TCP biomaterial. CONCLUSIONS: Systemic administration of PTH significantly stimulates local bone formation. Bone formation was significantly limited by the beta-TCP biomaterial.

Rosmarinic Acid/ Blue Light Combination Treatment Inhibits Head and Neck Squamous Cell Carcinoma In Vitro.

BACKGROUND/AIM: This study investigated a novel combined therapy of rosmarinic acid (RA)/blue light on head and neck squamous cell carcinoma (HNSCC) cell proliferation in vitro. MATERIALS AND METHODS: HNSCC cells were exposed to BL (500 mW/cm2) for 90 s, and incubated with 80 µg/ml RA for 1 hour. Cell viability was determined after 24 h using WST-1 assay. Western blot was used to detect treatment-induced changes in epidermal growth factor receptor (EGFR) activation. Hydrogen peroxide (H2O2) and nitric oxide levels were quantified using CM-H2DCFH-DA assays. Apoptosis was assessed using Annexin V/PI staining and flow cytometry. RESULTS: RA/blue light treatment resulted in a significant reduction in cell viability, EGFR activation and H2O2 levels in all HNSCC cell lines. However, no significant changes in NO production or apoptosis induction were found. CONCLUSION: RA/blue light effectively decreased HNSCC cell proliferation through reduction in EGFR activation and H2O2 production, and not via induction of apoptosis.

In vitro cytotoxic response to lithium disilicate dental ceramics.

OBJECTIVES: The use of lithium disilicate dental ceramics is increasing in dentistry and previous reports have suggested that they may have greater biological risks than previously thought. We tested a hypothesis that composition and processing influence the biological properties of these ceramics. METHODS: The cytotoxicity of two machined and three pressed lithium disilicate materials (n=6) were tested in vitro using mouse fibroblasts in direct contact with the materials for 72h. Cellular response was estimated by mitochondrial succinate dehydrogenase activity (MTT method). Mitochondrial activity was expressed as a percentage of Teflon controls, then compared to Teflon using 2-sided t-tests (alpha=0.05). Polished materials were aged in artificial saliva and tested for cytotoxicity periodically over 6 weeks, then were repolished (320grit SiC paper), aged and tested again for 4 weeks. RESULTS: All materials significantly (50-70%) suppressed cellular mitochondrial activity in the initial week, but suppression decreased by 25-30% over the next 2 weeks. In weeks 4 and 6 some materials exhibited a cytotoxic 'relapse' of 10-20%. The cytotoxic response was no different for machined or pressed materials, but the presence of ZnO had at least an association with longer-term cytotoxicity and relapse. Repolishing to 320grit did not increase cytotoxicity significantly. SIGNIFICANCE: Our results suggest that lithium disilicates are not biologically inert, and that many have a similar cytotoxicity dynamic regardless of small differences in composition or processing.

Effect of mercury(II) on Nrf2, thioredoxin reductase-1 and thioredoxin-1 in human monocytes.

OBJECTIVES: Human blood levels of mercury are commonly 10nM, but may transiently reach 50-75nM after dental amalgam placement or removal. Controversy persists about the use of mercury because the effects of these 'trace' levels of mercury are not clear. Concentrations of mercury > or =5000nM unequivocally alter redox balance in blood cells including monocytes. In the current study, we tested a hypothesis that concentrations of mercury <100nM altered levels and activities of key proteins that maintain monocytic redox balance. METHODS: Human THP1 monocytes were exposed to 10-75nM of Hg(II) for 6-72h, with or without activation by lipopolysaccharide (LPS). The redox management proteins Nrf2 and thioredoxin-1 (Trx1) were separated by electrophoresis, then quantified by immunoblotting. The activity of the seleno-enzyme thioredoxin reductase (TrxR1), important in maintaining Trx1 redox balance, was measured by cell-free and cell-dependent assays. RESULTS: Concentrations of Hg(II) between 10-75nM increased Nrf2 levels (3.5-4.5 fold) and decreased Trx1 levels (2-3 fold), but these changes persisted <24h. Hg(II) potently inhibited (at concentrations of 5-50nM) TrxR1 activity in both cell-free and intracellular assays. Furthermore, Hg(II) transiently amplified LPS-induced Nrf2 levels by 2-3 fold and limited LPS-induced decreases in Trx1. All effects of Hg(II) were mitigated by pre-adding N-acetyl-cysteine (NAC) or sodium selenide (Na2SeO3), supplements of cellular thiols and selenols, respectively. SIGNIFICANCE: Our results suggest that nanomolar concentrations of Hg(II) transiently alter cellular redox balance in monocytes that trigger changes in Nrf2 and Trx1 levels. These changes indicate that monocytes have a capacity to adapt to trace concentrations of Hg(II) that are introduced into the bloodstream after dental amalgam procedures or fish consumption. The ability of monocytes to adapt suggests that low levels of mercury exposure from dental amalgam may not overtly compromise monocyte function.

Biological effects of Ni(II) on monocytes and macrophages in normal and hyperglycemic environments.

Corrosion and release of nickel ions from biomedical alloys are well documented, but little is still known about the effects of released nickel ions on cellular function with recurrent inflammatory challenges. Evidence suggests Ni(II) ions amplify LPS-induced secretion of several pro-inflammatory cytokines from monocytes. Exacerbating the inflammatory response, hyperglycemic conditions also affect monocytic function. This study investigated how Ni(II) and hyperglycemic conditions, both singly and in combination, alter monocyte proliferation, mitochondrial activity, inflammatory responses, and differentiation. Results showed that Ni(II) did not affect proliferation, but decreased mitochondrial activity in monocytic-cells and macrophages under normal conditions. However, hyperglycemic conditions negated the toxicity seen with Ni(II) exposure. Cytokine secretion in response to LPS was variable, with little effect on IL6 secretion, but significantly increased secretion of IL1ß at intermediate Ni(II) concentrations. Hyperglycemic conditions did not alter these results significantly. Finally, exposure to eluants from nickel-based commercial alloys caused enhanced IL1ß secretion from PMA-treated cells. These data suggest that corrosion products from nickel-containing dental alloys increased Ni(II)-induced changes in cytokine secretion by monocytes and macrophages. By better defining the effects of Ni(II) at these lower, biomedically relevant concentrations, we improve understanding of the biomedical alloy risk in the context of dental inflammation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2433-2439, 2018.

Downregulation of retinoblastoma protein is involved in the enhanced cytotoxicity of 4-hydroxytamoxifen plus mifepristone combination therapy versus antiestrogen monotherapy of human breast cancer.

In this study, human MCF-7 breast cancer cells, which express functional estrogen and progesterone receptors, were used to compare the efficacy of combined antiestrogen plus antiprogestin therapy to antiestrogen monotherapy. Cells were treated with the antiestrogen 4-hydroxytamoxifen (4-OHT) and/or the antiprogestin mifepristone (MIF) and effects on cell proliferation (cytostatic action), cell cycle phase, the phosphorylation state of the tumor suppressor retinoblastoma protein (Rb), and induction of active cell death (cytotoxic action) were determined. Combination hormonal therapy showed both increased cytostatic and cytotoxic activity as compared to either monotherapy. The increased cytostatic action was mediated by Rb activation; whereas, the cytotoxic (pro-apoptotic) action of combined hormonal therapy correlated to a significant reduction in Rb protein levels. To test the apparent role of Rb protein loss in the pro-apoptotic action of combined hormonal therapy, Rb was downregulated in MCF-7 cells using siRNA-targeting. The siRNA-mediated knockdown of Rb combined with 4-OHT therapy resulted in a pro-apoptotic action similar to that resulting from 4-OHT and MIF combination treatment, which included increased cell detachment from the monolayer, high-molecular-weight genomic DNA fragmentation, and cleavage of poly ADP-ribose polymerase (PARP) and lamin A. From these studies, we conclude that Rb protein downregulation is required for 4-OHT-treated, estrogen receptor positive (ER+) breast cancer cells to undergo active cell death. We discuss the potential of using an antiprogestin such as MIF plus antiestrogen treatment to more effectively downregulate Rb in ER+ breast cancer cells to increase the overall cytotoxic action of hormonal therapy.

Ni(II) activates the Nrf2 signaling pathway in human monocytic cells.

Nickel is a component of biomedical alloys that is released during corrosion and causes inflammation in tissues by as yet unknown mechanisms. Recent data show that Ni(II) at concentrations of 10-50 microM amplifies lipopolysaccharide-triggered, NFkappaB-mediated cytokine secretion from monocytes. In the current study, we tested the hypothesis that Ni(II) amplifies cytokine secretion by activating the Nrf2 antioxidant pathway rather than by enhancing activity of the NFkappaB signaling pathway. Human THP1 monocytes were exposed to Ni(II) concentrations of 10-30 microM for 6-72 h, then immunoblots of whole-cell lysates or cytosolic and nuclear proteins were used to detect changes in Nrf2 or NFkappaB signaling. Our results show that Ni(II) increased (by 1-2 fold) whole-cell Nrf2 levels and nuclear translocation of Nrf2, and amplified lipopolysaccharide (LPS)-induction of Nrf2 (by 3-5 fold), but had no detectable effect on the initial activation or nuclear translocation of NFkappaB. Because Nrf2 target gene products are known regulators of NFkappaB nuclear activity, our results suggest that Ni(II) may affect cytokine secretion indirectly via modulation of the Nrf2 pathway.

Attachment of human epithelial cells and periodontal ligament fibroblasts to tooth dentin.

A goal of treatment in periodontal therapy is to regenerate a new fibroblastic attachment rather than to repair lost attachment with a long junctional epithelium. To date, there is no evidence that fibroblastic attachment formed during regeneration is stronger or less susceptible to periodontal breakdown than a long junctional epithelial attachment. We measured the rate and strength of attachment of epithelial cells (NHEK) and periodontal ligament fibroblasts (PDLF) cultured individually and cocultured to dentin surfaces to determine which cell type has a faster attachment rate and greater adhesive strength to human dentin, and whether the cell types attach independently. Longitudinal dentin slices were seeded with either PDLF or NHEK for 2 or 24 h. The specimens were placed into a parallel plate flow chamber and defined laminar shear stresses were applied. Shear stress was created by step increases in fluid flow rate. Effluent fluid was collected and cell numbers (detached) were counted using a hemocytometer. Cocultures of PDLF and NHEK at three seeding ratios (10:1, 1:1, 1:10) were also tested. Each cell type attached equally well to polystyrene or dentin. PDLF showed a stronger attachment to polystyrene and dentin at 24 versus 2 h. NHEK attached to polystyrene or dentin equally well at 2 and 24 h. NHEK were more strongly attached after 2 h when compared to PDLF. PDLF were more strongly attached after 24 h versus NHEK. When NHEK and PDLF were seeded together on dentin at a 1:1 ratio, PDLF appeared to be more strongly attached than NHEK at 2 but not 24 h. At a ratio of 10 PDLF:1 NHEK, PDLF appeared to be more strongly attached at 2 and 24 h. At a ratio of 1 PDLF:10 NHEK, NHEK appeared to be more strongly attached at 2 h, but PDLF showed a trend of stronger attachment at 24 h. We conclude that epithelial cells attach more quickly to dentin surfaces than PDLF, but do not demonstrate increased attachment strength over time (PDLF do show increased attachment strength overtime). The purported advantages of periodontal regeneration over periodontal repair are supported by our results. Furthermore, our results support the concept of guided tissue regeneration. On the basis of on cellular competition experiments, epithelial cells and PDLF do not act independently, because epithelial cells enhanced the attachment rate of PDLF.

Gold-induced reactive oxygen species (ROS) do not mediate suppression of monocytic mitochondrial or secretory function.

UNLABELLED: The toxicity of anti-rheumatic gold compounds has limited their use and development, yet both the toxicological and therapeutic actions of these compounds remain unclear. In the current study, we tested the hypothesis that intracellular reactive oxygen species (ROS) induced by Au(I) or Au(III) compounds mediate their ability to suppress mitochondrial activity. METHODS: Human THP1 monocytes were exposed to HAuCl(4) x 3H(2)O (Au(III)), or the anti-rheumatic compounds auranofin (AF) or gold sodium thiomalate (GSTM) for 6-72 h, after which mitochondrial activity (succinate dehydrogenase) was measured. To assess the role of cellular redox status as a mediator of mitochondrial suppression, monocytes were pre-treated with a pro-oxidant (t-butyl hydroquinone, t-BHQ) or antioxidant (N-acetyl cysteine, NAC ). ROS levels were measured 0-24h post-gold addition to determine their role as mediators of mitochondrial activity suppression. RESULTS: AF was the most potent inhibitor of mitochondrial activity, followed by Au(III) and GSTM. Only Au(III) induced intracellular ROS; no ROS formation was observed in response to AF or GSTM exposure. Although anti- and pro-oxidants had some effects on mitochondrial suppression of Au compounds, collectively the data do not support redox effects or ROS formation as major mediators of Au-compound mitochondrial suppression. CONCLUSIONS: Our results do not indicate that ROS and redox effects play major roles in mediating the cytotoxicity of AF, GSTM or Au(III).

Sublethal concentrations of diverse gold compounds inhibit mammalian cytosolic thioredoxin reductase (TrxR1).

UNLABELLED: Thioredoxin reductase (TrxR) reduces thioredoxin (Trx), thereby contributing to cellular redox balance, facilitating the synthesis of deoxy-ribose sugars for DNA synthesis, and regulating redox-sensitive gene expression. Auranofin is a gold compound that potently inhibits TrxR. This inhibition is one suspected mechanism of auranofin's therapeutic benefit in the treatment of rheumatoid arthritis. The use of other gold compounds to treat cancer or inflammatory disease may rely on their ability to inhibit TrxR. In the current study, we tested the hypothesis that a variety of gold compounds may inhibit TrxR. METHODS: We exposed rat-TrxR1 to auranofin, gold sodium thiomalate, sodium aurothiosulfate, triphenyl phosphine gold chloride, or gold acetate, and measured TrxR activity ex vivo. We then compared TrxR1 inhibitory levels of gold compounds to those that inhibited mitochondrial activity of THP1 monocytes and OSC2 epithelial cells, estimated by succinate dehydrogenase activity. RESULTS: All gold compounds inhibited TrxR1 at concentrations ranging from 5 to 4000 nM (50% inhibitory concentration). The oxidation state of gold did not correlate with inhibitory potency, but ligand configuration was important. Au(I)-phosphine compounds (triphenyl phosphine gold chloride and auranofin) were the most potent inhibitors of TrxR. All TrxR1 inhibitory concentrations were sublethal to mitochondrial activity in both THP1 and OSC2 cells. CONCLUSIONS: Diverse types of gold compounds may be effective inhibitors of TrxR1 at concentrations that do not suppress cellular mitochondrial function. Inhibition may be optimized to some degree by altering the ligand configuration of the compounds. These results support future study of a variety of Au compounds for therapeutic development as inhibitors of TrxR1.

Extracellular environment as one mediator of blue light-induced mitochondrial suppression.

OBJECTIVES: The current study tested the hypothesis that the extracellular environment mediates mitochondrial suppression of oral epithelial cells and fibroblasts by blue light. METHODS: We exposed Balb fibroblasts (Balb), normal human epidermal keratinocytes (NHEK), and oral squamous carcinoma cells (OSC2) to blue light (30-120J/cm2) in different cell-culture media and in phosphate buffered saline (PBS). Mitochondrial activity (MTT method) was used to assess cellular response 72 h post-light exposure. Cell-culture media were replaced or supplemented before or after light exposure to assess the variables of exposure time and medium degradation as mediators of blue light-induced effects. RESULTS: Mitochondrial activity of NHEK was not suppressed by exposure to blue light regardless of extracellular conditions. The mitochondrial activity of OSC2 and Balb cells was suppressed most when cells were exposed to light in cell-culture medium (versus PBS). Blue light suppressed mitochondrial activity more when irradiated medium remained in contact with the cells at least 1h, indicating a time-dependence of the medium effects. Neither a replacement nor a supplementation of medium components reduced blue light-induced mitochondrial suppression. SIGNIFICANCE: Our results suggest that tissue environments influence cellular responses to blue light and that these environments should be considered when assessing any biological effects of blue light during the photopolymerization of restorative resins.

Parameters in the prediction of the sensitivity of parathyroid scanning.

OBJECTIVE: Although parathyroid scintigraphy with technetium-99m sestamibi has been shown to be a sensitive and specific test for the detection and localization of parathyroid adenomas in patients with primary hyperparathyroidism, false-negative studies occur. Our goal was to determine whether the presurgical parathyroid hormone level could be used to predict whether a scan would be positive or negative. MATERIALS AND METHODS: A retrospective review of parathyroid scans was performed. Analysis included patients with surgical confirmation of a parathyroid adenoma and a documented parathyroid hormone (PTH) level obtained within 6 months of the scan. Patients with secondary hyperparathyroidism were excluded. A true-positive study was defined by the surgical finding of an adenoma on the side indicated by the scan. A scan was false-negative if it did not detect the adenoma found at surgery. A scan was false-positive if an adenoma was identified in the wrong side of the neck or if an adenoma was called but not found. RESULTS: Of 166 scans, 83 met the criteria for inclusion. There were 56 true-positives, 21 false-negatives, and 6 false-positives. The mean PTH in patients with true-positive scans was 367 pg/mL (range, 46-3231 pg/mL) and with false-positive and false-negative scans was 148 pg/mL (range, 46-390 pg/mL). The difference was statistically significant. CONCLUSION: There is a correlation between the sensitivity of parathyroid scintigraphy and presurgical PTH. The overlap, however, of parathyroid hormone levels in positive and negative scans does not allow one to confidently preselect candidates for preoperative scanning.

Inherent physical characteristics and gene expression differences between alveolar and basal bones.

OBJECTIVES: The objective of this study was to evaluate the baseline differences between alveolar and basal areas of the rat mandible. STUDY DESIGN: Rat mandibular alveolar and basal bones were evaluated using histology and micro-computed tomography to compare osteocyte number as well as bone density and architecture and polymerase chain reaction to measure gene expression levels. RESULTS: Micro-computed tomography data indicated that basal bone is denser and less porous than alveolar bone. Histologic analysis showed that alveolar bone has more osteocytes per unit area compared with basal bone. Real-time polymerase chain reaction results showed higher levels of expression of the following genes in basal bone than in alveolar bone: SOST, E-11, DMP-1, and MEPE. CONCLUSIONS: Three of these gene products are associated with mature osteocytes, and this suggests that basal bone has more mature osteocyte phenotypes compared with alveolar bone. These findings are suggestive of fewer bone mineralization units and therefore a slower remodeling rate.

Role of p21WAF1 in green tea polyphenol-induced growth arrest and apoptosis of oral carcinoma cells.

The cyclin-dependent kinase inhibitor p21WAF1 participates in cell growth, differentiation, and apoptosis. p21WAF1 can be induced by green tea polyphenol EGCG in several cancer cell types, but its role in the oral cancer cell response to EGCG is not known. We found that EGCG upregulates p21WAF1 in an oral carcinoma cell line, OSC2, by cDNA microarray. The current study determined the impact of siRNA-suppressed p21WAF1 and its response to EGCG on cell growth, DNA synthesis and apoptosis by RT-PCR, Western blot, BrdU incorporation, MTT and caspase 3 activity assays. Suppression of p21WAF1 by siRNA resulted in an accelerated cell growth and DNA synthesis, and increased cell viability. However, caspase 3 activity was not significantly inhibited. The evidence showed that p21WAF1 is involved in EGCG-induced growth arrest of OSC2 cells, which may facilitate caspase 3-mediated apoptosis. Thus, expression of functional p21WAF1 may promote phytochemical-mediated growth arrest and apoptosis in oral carcinoma cells.

Mercury (II) alters mitochondrial activity of monocytes at sublethal doses via oxidative stress mechanisms.

The perennial controversy about the safety of mercury in dental amalgams has adversely affected the availability and the quality of dental care. Chronic Hg(II) blood concentrations above 300 nM are known to alter function of the nervous system and the kidney. However, the effects of blood concentrations of 10 to 75 nM, far more common in the general population, are not clear and mechanisms of any effects are not known. The monocyte is an important potential target of Hg(II) because of its critical role in directing inflammatory and immune responses. In the current study we tested the hypothesis that concentrations of Hg(II) of 10 to 300 nM alter monocyte activity via a redox-dependent mechanism. Mitochondrial activity was used to establish inhibitory concentrations of Hg(II) following 6 to 72 h of exposures to THP1 human monocytic cells. Then subinhibitory concentrations were applied, and total glutathione levels and reactive oxygen species (ROS) were measured. Antioxidants [N-acetyl cysteine, (NAC); Na2SeO3, (Se)] and a pro-oxidant (tert-butylhydroquinone, tBHQ) were used to support the hypothesis that Hg(II) effects were redox-mediated. After 72 h of exposure, 20 microM of Hg(II) inhibited monocytic mitochondrial activity by 50%. NAC mitigated Hg(II)-induced mitochondrial suppression only at concentrations of greater than 10 microM, but Se had few effects on Hg-induced mitochondrial responses. tBHQ significantly enhanced mitochondrial suppression at higher Hg(II) concentrations. Hg(II) concentrations of 75 and 300 nM (0.075 and 0.30 microM, respectively) significantly increased total glutathione levels, and NAC mitigated these increases. Se plus Hg(II) significantly elevated Hg-induced total cellular glutathione levels. Increased ROS levels were not detected in monocytes exposed to mercury. Hg(II) acts in monocytic cells, at least in part, through redox-mediated mechanisms at concentrations below those commonly associated with chronic mercury toxicity, but commonly occurring in the blood of some dental patients.

Blue light differentially alters cellular redox properties.

Blue light (lambda = 380-500 nm) historically has been used to initiate polymerization of biomaterials and recently has been proposed as a therapeutic agent. New evidence suggests that cell-type-specific responses result from redox changes induced by exposure to blue light. Cultured cells were exposed to defined doses of blue light, equivalent to exposure times of 10 s and 2 min, to achieve energies of 5 J/cm2 and 60 J/cm2, respectively, after which (a) viable cell number, (b) cellular protein profiles, (c) mitochondrial succinate dehydrogenase (SDH) activity, (d) total reactive oxygen species (ROS), and (e) induction of apoptosis were compared to that of nonexposed control cultures. Results showed that blue-light exposure arrested monocyte cell growth and increased levels of peroxiredoxins. SDH activity of normal epidermal keratinocytes (NHEK) was slightly enhanced by blue light, whereas identical treatment of OSC2 oral tumor cells resulted in significant suppression of SDH activity. Blue-light exposure generally induced higher levels of total ROS in OSC2 cells than in NHEK. Finally, only OSC2 cells exhibited signs of apoptosis via Annexin V staining following exposure to blue light. These data support the central hypothesis that blue light induces an oxidative stress response in cultured cells resulting in cell-type-specific survival outcomes. The identification of oxidative stress as a mediator of the effects of blue light is a critical first step in defining its biological risks and therapeutic opportunities.

Blue light generates reactive oxygen species (ROS) differentially in tumor vs. normal epithelial cells.

OBJECTIVES: Blue light of high intensity is commonly used in dentistry to activate polymerization of resin restorative materials. Other than its effects on the retina, the biological effects of blue light (380-500nm wavelengths) are poorly studied. Limited evidence suggests that blue light acts by forming intracellular reactive oxygen species (ROS) that then affect critical cell functions. If the biological effects of blue light are redox-mediated, antioxidants might be used to mitigate unwanted side effects of blue light during clinical use, or blue light might be used therapeutically to modulate redox-sensitive cell signaling responses. METHODS: Intracellular ROS were estimated using HFLUOR-DA (dihydrofluorescein diacetate), a vital fluorescein-based, redox-sensitive dye. ROS were measured in normal (NHEK) and oral squamous carcinoma (OSC2) epithelial cells, shown previously to respond differentially to blue light irradiation. Two-hour cumulative levels of ROS and approximate ROS lifetimes were measured after irradiation doses of 5-30 J/cm(2). The blue light-induced generation of ROS was further tested by the ability of the antioxidants N-acetylcysteine (NAC) and vitamin E to mitigate intracellular ROS levels. RESULTS: Dose-dependent ROS levels were generated in both NHEK and OSC2 cells, but cumulative levels were higher and persisted longer in the OSC2 cells. Both vitamin E and NAC significantly reduced blue-light-induced levels of ROS, but were more effective in the OSC2 cells. SIGNIFICANCE: The induction of intracellular ROS by blue light implies that redox effects may mediate cellular responses to blue light. This result suggests the opportunity to mitigate any effects of direct or coincident exposure during dental treatment via antioxidants, and the opportunity to exploit differences in redox processing among cells for possible treatment of epithelial cancer or wound healing.