Hepatic insulin sensitivity is improved in high-fat diet-fed Park2 knockout mice in association with increased hepatic AMPK activation and reduced steatosis.

Park2 is an E3 ubiquitin ligase known for its role in mitochondrial quality control via the mitophagy pathway. Park2 KO mice are protected from diet-induced obesity and hepatic insulin sensitivity is improved in high-fat diet (HFD)-fed Park2 KO mice even under body weight-matched conditions. In order to better understand the cellular mechanism by which Park2 KO mice are protected from diet-induced hepatic insulin resistance, we determined changes in multiple pathways commonly associated with the pathogenesis of insulin resistance, namely levels of bioactive lipid species, activation of the endoplasmic reticulum (ER) stress response and changes in cytokine levels and signaling. We report for the first time that whole-body insulin sensitivity is unchanged in regular chow (RC)-fed Park2 KO mice, and that liver diacylglycerol levels are reduced and very-long-chain ceramides are increased in Park2 KO mice fed HFD for 1 week. Hepatic transcriptional markers of the ER stress response were reduced and plasma tumor necrosis factor-α (TNFα), interleukin-6 and -10 (IL6, IL10) were significantly increased in HFD-fed Park2 KO mice; however, there were no detectable differences in hepatic inflammatory signaling pathways between groups. Interestingly, hepatic adenylate charge was reduced in HFD-fed Park2 KO liver and was associated increased activation of AMPK. These data suggest that negative energy balance that contributed to protection from obesity during chronic HFD manifested at the level of the hepatocyte during short-term HFD feeding and contributed to the improved hepatic insulin sensitivity.

GDF15 is an epithelial-derived biomarker of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and devastating of the interstitial lung diseases. Epithelial dysfunction is thought to play a prominent role in disease pathology, and we sought to characterize secreted signals that may contribute to disease pathology. Transcriptional profiling of senescent type II alveolar epithelial cells from mice with epithelial-specific telomere dysfunction identified the transforming growth factor-ß family member, growth and differentiation factor 15 (Gdf15), as the most significantly upregulated secreted protein. Gdf15 expression is induced in response to telomere dysfunction and bleomycin challenge in mice. Gdf15 mRNA is expressed by lung epithelial cells, and protein can be detected in peripheral blood and bronchoalveolar lavage following bleomycin challenge in mice. In patients with IPF, GDF15 mRNA expression in lung tissue is significantly increased and correlates with pulmonary function. Single-cell RNA sequencing of human lungs identifies epithelial cells as the primary source of GDF15, and circulating concentrations of GDF15 are markedly elevated and correlate with disease severity and survival in multiple independent cohorts. Our findings suggest that GDF15 is an epithelial-derived secreted protein that may be a useful biomarker of epithelial stress and identifies IPF patients with poor outcomes.

Petite Integration Factor 1 (PIF1) helicase deficiency increases weight gain in Western diet-fed female mice without increased inflammatory markers or decreased glucose clearance.

Petite Integration Factor 1 (PIF1) is a multifunctional helicase present in nuclei and mitochondria. PIF1 knock out (KO) mice exhibit accelerated weight gain and decreased wheel running on a normal chow diet. In the current study, we investigated whether Pif1 ablation alters whole body metabolism in response to weight gain. PIF1 KO and wild type (WT) C57BL/6J mice were fed a Western diet (WD) rich in fat and carbohydrates before evaluation of their metabolic phenotype. Compared with weight gain-resistant WT female mice, WD-fed PIF1 KO females, but not males, showed accelerated adipose deposition, decreased locomotor activity, and reduced whole-body energy expenditure without increased dietary intake. Surprisingly, PIF1 KO females did not show obesity-induced alterations in fasting blood glucose and glucose clearance. WD-fed PIF1 KO females developed mild hepatic steatosis and associated changes in liver gene expression that were absent in weight-matched, WD-fed female controls, linking hepatic steatosis to Pif1 ablation rather than increased body weight. WD-fed PIF1 KO females also showed decreased expression of inflammation-associated genes in adipose tissue. Collectively, these data separated weight gain from inflammation and impaired glucose homeostasis. They also support a role for Pif1 in weight gain resistance and liver metabolic dysregulation during nutrient stress.

Association of genetic variation in COMT gene with pain related to sickle cell disease in patients from the walk-PHaSST study.

BACKGROUND: Vaso-occlusive pain episodes (VOEs) are the hallmark of sickle cell disease (SCD), and our current understanding of disease biology, treatment, and psychological covariates does not adequately explain the variability of pain in SCD. Functional variants in catechol-O-methyltransferase (COMT) gene contribute to variability in pain perception, but their impact on pain perception in African American SCD patients is not well known. METHODS: We studied COMT single-nucleotide polymorphisms (SNPs) rs6269, rs4633, rs4818, rs4680, and rs165599 to determine their relationship to patient self-reported pain, the number of acute VOEs, and their impact on daily life and health care utilization in 438 hemoglobin SS patients who participated in the walk-PHaSST study. RESULTS: In women, two risk SNPs (rs4633 and rs165599) and the corresponding haplotype (ATCAA) were associated with increased frequency of pain-related emergency room visit. CONCLUSION: COMT functional variants may predispose SCD patients to worse acute pain in women. The association of COMT variants with the intensity of self-reported acute pain warrants further genetic study of pain perception in SCD.

Variable Susceptibility to Cigarette Smoke-Induced Emphysema in 34 Inbred Strains of Mice Implicates Abi3bp in Emphysema Susceptibility.

Chronic obstructive pulmonary disease (COPD) is caused by a complex interaction of environmental exposures, most commonly cigarette smoke, and genetic factors. Chronic cigarette smoke exposure in the mouse is a commonly used animal model of COPD. We aimed to expand our knowledge about the variable susceptibility of inbred strains to this model and test for genetic variants associated with this trait. To that end, we sought to measure differential susceptibility to cigarette smoke-induced emphysema in the mouse, identify genetic loci associated with this quantitative trait, and find homologous human genes associated with COPD. Alveolar chord length (CL) in 34 inbred strains of mice was measured after 6 months of exposure to cigarette smoke. After testing for association, we connected a murine candidate locus to a published meta-analysis of moderate-to-severe COPD. We identified deleterious mutations in a candidate gene in silico and measured gene expression in extreme strains. A/J was the most susceptible strain in our survey (Δ CL 7.0 ± 2.2 µm) and CBA/J was the least susceptible (Δ CL -0.3 ± 1.2 µm). By integrating mouse and human genome-wide scans, we identified the candidate gene Abi3bp. CBA/J mice harbor predicted deleterious variants in Abi3bp, and expression of the gene differs significantly between CBA/J and A/J mice. This is the first report of susceptibility to cigarette smoke-induced emphysema in 34 inbred strains of mice, and Abi3bp is identified as a potential contributor to this phenotype.

Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow-derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury.

Transcriptional Regulation of Frizzled-1 in Human Osteoblasts by Sp1.

The wingless pathway has a powerful influence on bone metabolism and is a therapeutic target in skeletal disorders. Wingless signaling is mediated in part through the Frizzled (FZD) receptor family. FZD transcriptional regulation is poorly understood. Herein we tested the hypothesis that Sp1 plays an important role in the transcriptional regulation of FZD1 expression in osteoblasts and osteoblast mineralization. To test this hypothesis, we conducted FZD1 promoter assays in Saos2 cells with and without Sp1 overexpression. We found that Sp1 significantly up-regulates FZD1 promoter activity in Saos2 cells. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assays identified a novel and functional Sp1 binding site at -44 to -40 from the translation start site in the FZD1 promoter. The Sp1-dependent activation of the FZD1 promoter was abolished by mithramycin A (MMA), an antibiotic affecting both Sp1 binding and Sp1 protein levels in Saos2 cells. Similarly, down-regulation of Sp1 in hFOB cells resulted in less FZD1 expression and lower alkaline phosphatase activity. Moreover, over-expression of Sp1 increased FZD1 expression and Saos2 cell mineralization while MMA decreased Sp1 and FZD1 expression and Saos2 cell mineralization. Knockdown of FZD1 prior to Sp1 overexpression partially abolished Sp1 stimulation of osteoblast differentiation markers. Taken together, our results suggest that Sp1 plays a role in human osteoblast differentiation and mineralization, which is at least partially mediated by Sp1-dependent transactivation of FZD1.

Expression of RXFP1 Is Decreased in Idiopathic Pulmonary Fibrosis. Implications for Relaxin-based Therapies.

RATIONALE: Relaxin is a hormone that has been considered as a potential therapy for patients with fibrotic diseases. OBJECTIVES: To gauge the potential efficacy of relaxin-based therapies in idiopathic pulmonary fibrosis (IPF), we studied gene expression for relaxin/insulin-like family peptide receptor 1 (RXFP1) in IPF lungs and controls. METHODS: We analyzed gene expression data obtained from the Lung Tissue Research Consortium and correlated RXFP1 gene expression data with cross-sectional clinical and demographic data. We also employed ex vivo donor and IPF lung fibroblasts to test RXFP1 expression in vitro. We tested CGEN25009, a relaxin-like peptide, in lung fibroblasts and in bleomycin injury. MEASUREMENTS AND MAIN RESULTS: We found that RXFP1 is significantly decreased in IPF. In patients with IPF, the magnitude of RXFP1 gene expression correlated directly with diffusing capacity of the lung for carbon monoxide (P < 0.0001). Significantly less RXFP1 was detected in vitro in IPF fibroblasts than in donor controls. Transforming growth factor-ß decreased RXFP1 in both donor and IPF lung fibroblasts. CGEN25009 was effective at decreasing bleomycin-induced, acid-soluble collagen deposition in vivo. The relaxin-like actions of CGEN25009 were abrogated by RXFP1 silencing in vitro, and, in comparison with donor lung fibroblasts, IPF lung fibroblasts exhibited decreased sensitivity to the relaxin-like effects of CGEN25009. CONCLUSIONS: IPF is characterized by the loss of RXFP1 expression. RXFP1 expression is directly associated with pulmonary function in patients with IPF. The relaxin-like effects of CGEN25009 in vitro are dependent on expression of RXFP1. Our data suggest that patients with IPF with the highest RXFP1 expression would be predicted to be most sensitive to relaxin-based therapies.

Elevated NT-pro-brain natriuretic peptide level is independently associated with all-cause mortality in HIV-infected women in the early and recent HAART eras in the Women's Interagency HIV Study cohort.

BACKGROUND: HIV-infected individuals are at increased risk of right and left heart dysfunction. N-terminal-pro-brain natriuretic peptide (NT-proBNP), a marker of cardiac ventricular strain and systolic dysfunction, may be associated with all-cause mortality in HIV-infected women. The aim of this study was to determine if elevated levels of NT-proBNP is associated with increased mortality in HIV-infected women. DESIGN: Prospective cohort study. METHODS AND RESULTS: We measured NT-proBNP in 936 HIV-infected and 387 age-matched HIV-uninfected women early (10/11/94 to 7/17/97) and 1082 HIV-infected and 448 HIV-uninfected women late (4/1/08 to 10/7/08) in the highly active antiretroviral therapy (HAART) periods in the Women's Interagency HIV Study. An NT-proBNP >75th percentile was more likely in HIV-infected persons, but only statistically significant in the late period (27% vs. 21%, unadjusted p = 0.03). In HIV-infected participants, NT-proBNP>75th percentile was independently associated with worse 5-year survival in the early HAART period (HR 1.8, 95% CI 1.3-2.4, p<0.001) and remained a predictor of mortality in the late HAART period (HR 2.8, 95% CI 1.4-5.5, p = 0.002) independent of other established risk covariates (age, race/ethnicity, body mass index, smoking, hepatitis C serostatus, hypertension, renal function, and hemoglobin). NT-proBNP level was not associated with mortality in HIV-uninfected women. CONCLUSION: NT-proBNP is a novel independent marker of mortality in HIV-infected women both when HAART was first introduced and currently. As NT-proBNP is often associated with both pulmonary hypertension and left ventricular dysfunction, these findings suggest that these conditions may contribute significantly to adverse outcomes in this population, requiring further definition of causes and treatments of elevated NT-proBNP in HIV-infected women.

AP2 suppresses osteoblast differentiation and mineralization through down-regulation of Frizzled-1.

Transcription factor activating protein 2 (AP2) plays an important role in cellular differentiation. Although profound craniofacial and long bone developmental abnormalities have been observed in AP2-knockout mice, the molecular effects of AP2 on osteoblasts are poorly defined. We demonstrated that AP2 regulates the expression of human Frizzled 1 (FZD1), a co-receptor for the Wnt signalling pathway, in human osteoblast cell lines and primary bone marrow stromal cells (BMSCs). We also identified a putative AP2-binding site in the FZD1 proximal promoter in silico and characterized this binding element further in Saos2 in vitro by ChIP, electrophoretic mobility shift and promoter reporter assays. The transcriptional repression of the FZD1 promoter by AP2 was confirmed in normal human fetal osteoblasts (hFOB). Furthermore, overexpression of AP2 resulted in a significant reduction in both differentiation and mineralization of Saos2 cells. Knockdown of FZD1 expression before AP2 up-regulation diminished the AP2-dependent inhibition of Saos2 cell differentiation and mineralization. Similarly, overexpressing FZD1 before AP2 treatment in both Saos2 and BMSCs diminished the inhibitory effect of AP2 on osteoblast differentiation and mineralization. Taken together, these results demonstrate that AP2 is a negative regulator of osteoblast differentiation and mineralization, and its inhibitory effect may be mediated in part through down-regulation of FZD1 expression.

Diagnostic value of 18F-FDG-PET or PET-CT in recurrent cervical cancer: a systematic review and meta-analysis.

OBJECTIVES: Accurate detection of recurrent cervical cancer remains a clinical difficulty. This study aims to assess the diagnostic value of PET or PET-computed tomography (PET-CT) using F-fluorodeoxyglucose (F-FDG) in recurrent cervical cancer using a meta-analysis. STUDY DESIGN: All published studies in English evaluating the diagnostic value of PET or PET-CT in detecting recurrent cervical cancer were collected. The methodological quality of the included studies was evaluated. Pooled sensitivity, specificity, diagnostic odds ratio, and summary receiver-operating characteristic curves were obtained using statistical software. Twenty studies were included in the meta-analysis. RESULTS: The meta-analysis showed that the pooled sensitivity and specificity of PET and PET-CT to detect distant metastasis in recurrent cervical cancer were 0.87 [95% confidence interval (CI): 0.80-0.92] and 0.97 (95% CI: 0.96-0.98), respectively. The pooled sensitivity and specificity for local regional recurrence were 0.82 (95% CI: 0.72-0.90) and 0.98 (95% CI: 0.96-0.99), respectively. CONCLUSION: F-FDG-PET and PET-CT are valuable methods for the assessment of recurrent cervical cancer.

MicroRNA-590 promotes cervical cancer cell growth and invasion by targeting CHL1.

MicroRNAs (miRNAs) may function as oncogenes or tumor suppressors. Here, we identified that miR-590-5p was up-regulated in human cervical cancer. Over-expression of miR-590-5p promoted cervical cancer cell growth, cell cycle and invasion via Growth curve, Colony formation, FACS and Transwell assays in HeLa and C33A cell lines. Subsequently, CHL1 was identified as a potential miR-590-5p target by bioinformatics analysis. Moreover, we showed that CHL1 was negatively regulated by miR-590-5p at the posttranscriptional level, via a specific target site within the 3'UTR by luciferase reporter assay. Furthermore, the mRNA and protein levels of CHL1 in cervical cancer cells were downregulated by miR-590-5p. And we identified the cell phenotype altered by miR-590-5p can be rescued by over-expression of CHL1. Therefore, our findings suggest that miR-590-5p acts as an oncogene by targeting the CHL1 gene and promotes cervical cancer proliferation. The findings of this study contribute to current understanding of the functions of miR-590-5p in cervical cancer.

Cartilage oligomeric matrix protein in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5-3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-ß1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-ß1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-ß1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-ß1 signaling should be persuaded.

E2F1 effects on osteoblast differentiation and mineralization are mediated through up-regulation of frizzled-1.

Frizzled homolog 1 (FZD1) is a transmembrane receptor that mediates Wnt signaling. The transcriptional regulation of FZD1 and the role of FZD1 in osteoblast biology are not well understood. We examined the role of E2F1 in FZD1 promoter activation and osteoblast differentiation and mineralization. A putative E2F1 binding site in the FZD1 promoter region was initially identified in silico and characterized further in Saos2 cells in vitro by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift (EMSA) and promoter reporter assays. Over-expression of E2F1 transactivated the FZD1 promoter and increased endogenous FZD1 mRNA and protein levels in Saos2 cells. Over-expression of E2F1 in Saos2 cells up-regulated osteoblast differentiation markers alkaline phosphatase (ALP), type I collagen α (COL1A), and osteocalcin (OCN). Furthermore, E2F1 over-expression enhanced mineralization of differentiated Saos2 cells, whereas siRNA knockdown of FZD1 diminished the effects of E2F1 on osteoblast mineralization. The effects of E2F1 on FZD1 expression and osteoblast mineralization were further confirmed in normal human FOB osteoblasts. Taken together, our experiments demonstrate a role of E2F1 in osteoblast differentiation and mineralization and suggest that FZD1 is required, in part, for E2F1 regulation of osteoblast mineralization.

[Effects of verapamil on the proliferation and migration of cervical cancer cells].

OBJECTIVE: To investigate the effects of Verapamil, a L-type calcium channel blocker, on the proliferation and migration of human cervical cancer cells. METHODS: Human cervical cancer cell lines (HeLa cells) were incubated with serum-free media for 24 hours. These cells were then treated with Verapamil for different time points. Cell proliferation was evaluated by MTT assay and EdU fluorescent assay. The migration of cells was measured by the wound healing assay. Results MTT assay demonstrated that, Verapamil (1, 5, 10 and 20 microg/mL concentrations) could inhibit the proliferation of HeLa cells after 48 or 72 hours treatment. The inhibition rate for 48 h-treatment was 20.87% +/- 1.71%, 23.55% +/- 4.46%, 28.65% +/- 1.32%, 27.37% +/- 3.05% respectively (P < 0.05). After 5, 10 and 20 microg/mL concentrations of verapamil treatment for 24h, the proportion of HeLa cells in the stage of DNA synthesis was 15.7% +/- 1.2%, 11.7% +/- 0.3%, 2.8% +/- 0.5% as evaluated with EdU fluorescent assay, which was significantly lower than that of control (27.34% +/- 2.1%, P < 0.05). With the increase of drug concentrations, Verapamil could significantly suppressed the DNA synthesis in HeLa cell. Cell migration assay revealed that, treatment with Verapamil (5, 10 and 20 microg/mL) for 24 hours significantly inhibited cells migration distance (P < 0.05). CONCLUSION: Calcium channel blocker Verapamil could inhibit proliferation and migration of the cervical cancer HeLa cells.

Systematic comparison of radical vaginal trachelectomy and radical hysterectomy in the treatment of early-stage cervical cancer.

OBJECTIVE: To review the effects of radical vaginal trachelectomy (RVT) and radical hysterectomy (RH) on overall progression-free survival rate, and intraoperative and postoperative complications in patients with cervical cancer (FIGO stage IA-IB1). METHODS: Electronic searches for studies of RVT and RH in the treatment of cervical cancer between 1994 and January 2010 were made on MEDLINE, the Cochrane Library, the China National Knowledge Infrastructure, and the Wan Fang dissertation database. RESULTS: No significant differences were found between RVT and RH in 5-year overall survival rate (relative risk [RR] 0.97; 95% confidence interval [CI], 0.93-1.02); 5-year progression-free survival rate (RR 0.99; 95% CI, 0.95-1.02); intraoperative complications (RR 1.99; 95% CI, 0.61-6.52)]; and postoperative complications (RR 0.36; 95% CI, 0.10-1.27). There were fewer blood transfusions (RR 0.33; 95% CI, 0.12-0.90), less blood loss, and shorter hospital stays in patients undergoing RVT. CONCLUSION: Radical vaginal trachelectomy should be considered as a viable treatment option for young patients with early cervical cancer (FIGO stage IA-IB1) who wish to preserve their fertility.

[Relationship between polymorphism of hypoxia inducible factor-1alpha and cervical cancer in Han population in Sichuan Province of China].

OBJECTIVE: To investigate the relationship between single nucleotide polymorphism (SNP) of hypoxia inducible factor-1alpha (HIF-1alpha) C1772T and genetic susceptibility to and clinical-pathological features of cervical cancers in Han population in Sichuan province of China. METHODS: A case control study was undertaken in Sichuan province of China, with 97 patients with uterine cervical cancer as case group and 117 negative for intraepithelial lesion or malignancy (NILM) patients as control group. Their gene types in HIF-1alpha C1772T were identified with a combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The distribution of the frequencies of T/T, T/C and C/C genotypes in the two groups differed significantly (P < 0.01); T allele frequency in the patients with cervical cancer was much higher than that in the controls (P < 0.01). There were no significant differences in the distributions of T/T, T/C and C/C genotypes among cervical cancer patients at different FIGO stages, pathological grading, stromal invasive depth, lymph node metastasis and vascular invasions (P > 0.05). CONCLUSION: T/C and T/T genotypes of HIF-1alpha C1772T are genetic susceptibility factors for cervical cancer in Han population in Sichuan province of China. HIF-1alpha C1772T SNP probably has no relationship with clinical-pathological features of cervical cancer.

Pyrrolidine dithiocarbamate (PDTC) blocks apoptosis and promotes ionizing radiation-induced necrosis of freshly-isolated normal mouse spleen cells.

Ionizing radiation (IR) is a pro-oxidant that kills cells by both apoptotic and necrotic mechanisms. Pyrrolidine dithiocarbamate (PDTC) is a thiol-containing compound that may act either as a pro- or anti-oxidant depending on the experimental conditions. This study was designed to determine whether PDTC would reduce or enhance IR-induced cell death of freshly-isolated normal mouse B6/129 spleen cells (NMSC). We determined the effect of increasing doses of IR, PDTC alone and PDTC followed by IR on the viability of NMSC. Annexin V and propidium iodide (Annexin V/PI) staining demonstrated a dose and time-dependent relationship in which PDTC enhanced the percentage of IR-induced apoptotic/necrotic NMSC. Trypan blue dye inclusion confirmed that a loss of membrane integrity was occurring 1 h after incubation with PDTC plus IR. Reduction in the glutathione (GSH)/glutathione disulfide (GSSG) ratio and GSH demonstrated that both IR (8.5 Gy) and PDTC acted as pro-oxidants, but their mechanisms of action differed: In contrast to IR, which promoted p53 activation and caspase 3/7-mediated apoptosis, PDTC inhibited IR-induced p53 and caspase 3/7 activity. However, PDTC increased H(2)O(2) formation and necrosis, resulting in an overall increase in IR-induced cell death. Catalase prevented the PDTC-induced increase in IR cytotoxicity implicating the generation of H(2)O(2) as a major factor in this mechanism. These results demonstrate that in NMSC PDTC acts as pro-oxidant and enhances IR-induced cell cytotoxicity by increasing H(2)O(2)formation and thiol oxidation. As such, they strongly suggest that the use of PDTC as an adjunct to reduce radiation toxicity should be avoided.

The manganese superoxide dismutase mimetic, M40403, protects adult mice from lethal total body irradiation.

Over-expression of manganese superoxide dismutase (MnSOD) protects tissues from radiation. M40403 is a stable non-peptidyl mimetic of MnSOD that crosses cell membranes and is effective in reducing experimental inflammation. Male BALB/c mice were injected intraperitoneally (i.p.) and subcutaneously (s.c.) with M40403, 30 min before 6.5, 7.5 and 8.5 Gy total body irradiation (TBI). Whereas all control injected mice died after receiving 8.5 Gy TBI by day 17, 30 day survival of mice pre-treated i.p. with 40, 30, 20 or 10 mg/kg was 100%, 90%, 81% and 25%, respectively. The Dose Reduction Factor 50/30 for animals treated with 30 mg M40403 s.c. 30 min prior to TBI was 1.41. Decreased apoptosis of the large and particularly the small bowel and marked recovery of both lymphoid and hematopoietic tissues occurred in the M40403 pre-treated animals. M40403 is effective in reducing TBI-induced tissue destruction and has potential as a new radioprotective agent.