ROLE OF GLUTATHIONE IN FORMALDEHYDE METABOLISM AND TOXICITY

Glutathione (GSH) is involved in the detoxication of numerous chemicals exogenously exposed or endogenously generated. Exposure to these agents cause depletion of cellular GSH rendering these cells more susceptible to the toxic action of these same agents. Formaldehyde (CH(,2)O) was found to deplete cellular GSH, presumably by the formation of the GSH-CH(,2)O complex, S-hydroxymethylglutathione, and its rapid extrusion into the extracellular medium.^ The metabolism and toxicity of CH(,2)O were determined to be dependent upon cellular GSH in vitro and in vivo. The rate of CH(,2)O oxidation decreased and the extent of toxicity increased when isolated rat hepatocytes or strain A/J mice were pretreated with the GSH-depleting agent, diethyl maleate (DEM). Additional experiments were designed to further study the role GSH plays in detoxication using isolated rat hepatocytes.^ L-Methionine protected against the extent of lipid peroxidation and leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), caused by CH(,2)O in DEM-pretreated hepatocytes, further supporting the protective role of GSH against cellular toxicity. The antioxidants, ascorbate, butylated hydroxytoluene, and (alpha)-tocopherol, were all protective against the extent of lipid peroxidation and leakage of LDH in isolated rat hepatocytes. Whereas L-methionine may be protective by increasing the cellular concentration of GSH which is used to detoxify free radicals or by facilitating the rate of CH(,2)O oxidation, the antioxidant, ascorbate, was protective without altering the rate of CH(,2)O oxidation or increasing cellular GSH levels. These results suggest that the free radical-mediated toxicity caused by CH(,2)O in DEM-pretreated hepatocytes is due to the further depletion of GSH by CH(,2)O and not to increased CH(,2)O persistence. How this further depletion in GSH by CH(,2)O in DEM-pretreated hepatocytes results in lipid peroxidation and cell death was further investigated.^ The further decrease in GSH caused by CH(,2)O in DEM-pretreated hepatocytes, suspected of stimulating lipid peroxidation and cell death, was found not to be due to depletion of mitochondrial GSH but to depletion of protein sulfhydryl groups. In addition, cellular toxicity appears more closely correlated with depletion of protein sulfhydryl groups than with an increase in cytosolic free Ca('2+). The combination of CH(,2)O and DEM may be a useful tool in identifying these critical sulfhydryl-protein(s) and to further understand the role GSH plays in detoxication. ^

An in vitro toxicity evaluation of gold-, PLLA- and PCL-coated silica nanoparticles in neuronal cells for nanoparticle-assisted laser-tissue soldering.

The uptake of silica (Si) and gold (Au) nanoparticles (NPs) engineered for laser-tissue soldering in the brain was investigated using microglial cells and undifferentiated and differentiated SH-SY5Y cells. It is not known what effects NPs elicit once entering the brain. Cellular uptake, cytotoxicity, apoptosis, and the potential induction of oxidative stress by means of depletion of glutathione levels were determined after NP exposure at concentrations of 10(3) and 10(9)NPs/ml. Au-, silica poly (ε-caprolactone) (Si-PCL-) and silica poly-L-lactide (Si-PLLA)-NPs were taken up by all cells investigated. Aggregates and single NPs were found in membrane-surrounded vacuoles and the cytoplasm, but not in the nucleus. Both NP concentrations investigated did not result in cytotoxicity or apoptosis, but reduced glutathione (GSH) levels predominantly at 6 and 24h, but not after 12 h of NP exposure in the microglial cells. NP exposure-induced GSH depletion was concentration-dependent in both cell lines. Si-PCL-NPs induced the strongest effect of GSH depletion followed by Si-PLLA-NPs and Au-NPs. NP size seems to be an important characteristic for this effect. Overall, Au-NPs are most promising for laser-assisted vascular soldering in the brain. Further studies are necessary to further evaluate possible effects of these NPs in neuronal cells.

Aluminum toxicity to tropical montane forest tree seedlings in southern Ecuador: response of biomass and plant morphology to elevated Al concentrations

In acid tropical forest soils (pH < 5.5) increased mobility of aluminum might limit aboveground productivity. Therefore, we evaluated Al phytotoxicity of three native tree species of tropical montane forests in southern Ecuador. An hydroponic dose-response experiment was conducted. Seedlings of Cedrela odorata L., Heliocarpus americanus L., and Tabebuia chrysantha (Jacq.) G. Nicholson were treated with 0, 300, 600, 1200, and 2400 mu M Al and an organic layer leachate. Dose-response curves were generated for root and shoot morphologic properties to determine effective concentrations (EC). Shoot biomass and healthy leaf area decreased by 44 % to 83 % at 2400 mu M Al, root biomass did not respond (C. odorata), declined by 51 % (H. americanus), or was stimulated at low Al concentrations of 300 mu M (T. chrysantha). EC10 (i.e. reduction by 10 %) values of Al for total biomass were 315 mu M (C. odorata), 219 mu M (H. americanus), and 368 mu M (T. chrysantha). Helicarpus americanus, a fast growing pioneer tree species, was most sensitive to Al toxicity. Negative effects were strongest if plants grew in organic layer leachate, indicating limitation of plant growth by nutrient scarcity rather than Al toxicity. Al toxicity occurred at Al concentrations far above those in native organic layer leachate.

Late toxicity and five year outcomes after high-dose-rate brachytherapy as a monotherapy for localized prostate cancer

BACKGROUND To determine the 5-year outcome after high-dose-rate brachytherapy (HDR-BT) as a monotherapy. METHODS Between 10/2003 and 06/2006, 36 patients with low (28) and intermediate (8) risk prostate cancer were treated by HDR-BT monotherapy. All patients received one implant and 4 fractions of 9.5 Gy within 48 hours for a total prescribed dose (PD) of 38 Gy. Five patients received concomitant androgen deprivation therapy (ADT). Toxicity was scored according to the common terminology criteria for adverse events from the National Cancer Institute (CTCAE) version 3.0. Biochemical recurrence was defined according to the Phoenix criteria and analyzed using the Kaplan Meier method. Predictors for late grade 3 GU toxicity were analyzed using univariate and multivariate Cox regression analyses. RESULTS The median follow-up was 6.9 years (range, 1.5-8.0 years). Late grade 2 and 3 genitourinary (GU) toxicity was observed in 10 (28%) and 7 (19%) patients, respectively. The actuarial proportion of patients with late grade 3 GU toxicity at 5 years was 17.7%. Late grade 2 and 3 gastrointestinal (GI) toxicities were not observed. The crude erectile function preservation rate in patients without ADT was 75%. The 5 year biochemical recurrence-free survival (bRFS) rate was 97%. Late grade 3 GU toxicity was associated with the urethral volume (p = 0.001) and the urethral V120 (urethral volume receiving ≥120% of the PD; p = 0.0005) after multivariate Cox regression. CONCLUSIONS After HDR-BT monotherapy late grade 3 GU was observed relatively frequently and was associated with the urethral V120. GI toxicity was negligible, the erectile function preservation rate and the bRFS rate was excellent.

Biological, diagnostic and therapeutic relevance of the MET receptor signaling in head and neck cancer.

Head and neck cancer constitutes the 6th most common malignancy worldwide and affects the crucial anatomical structures and physiological functions of the upper aerodigestive tract. Classical therapeutic strategies such as surgery and radiotherapy carry substantial toxicity and functional impairment. Moreover, the loco-regional control rates as well as overall survival still need to be improved in subgroups of patients. The scatter-factor/hepatocyte growth factor receptor tyrosine kinase MET is an established effector in the promotion, maintenance and progression of malignant transformation in a wide range of human malignancies, and has been gaining considerable interest in head and neck cancer over the last 15 years. Aberrant MET activation due to overexpression, mutations, tumor-stroma paracrine loops, and cooperative/redundant signaling has been shown to play prominent roles in epithelial-to-mesenchymal transition, angiogenesis, and responses to anti-cancer therapeutic modalities. Accumulating preclinical and translational evidence highly supports the increasing interest of MET as a biomarker for lymph node and distant metastases, as well as a potential marker of stratification for responses to ionizing radiation. The relevance of MET as a therapeutic molecular target in head and neck cancer described in preclinical studies remains largely under-evaluated in clinical trials, and therefore inconclusive. Also in the context of anti-cancer targeted therapy, a large body of preclinical data suggests a central role for MET in treatment resistance towards multiple therapeutic modalities in malignancies of the head and neck region. These findings, as well as the potential use of combination therapies including MET inhibitors in these tumors, need to be further explored.

Aluminum toxicity in a tropical montane forest ecosystem in southern Ecuador

Aluminum phytotoxicity frequently occurs in acid soils (pH < 5.5) and was therefore discussed to affect ecosystem functioning of tropical montane forests. The susceptibility to Al toxicity depends on the sensitivity of the plant species and the Al speciation in soil solution, which can vary highly depending e.g., on pH, ionic strength, and dissolved organic matter. An acidification of the ecosystem and periodic base metal deposition from Saharan dust may control plant available Al concentrations in the soil solutions of tropical montane rainforests in south Ecuador. The overall objective of my study was to assess a potential Al phytotoxicity in the tropical montane forests in south Ecuador. For this purpose, I exposed three native Al non-accumulating tree species (Cedrela odorata L., Heliocarpus americanus L., and Tabebuia chrysantha (Jacq.) G. Nicholson) to increased Al concentrations (0 – 2400 μM Al) in a hydroponic experiment, I established dose-response curves to estimate the sensitivity of the tree species to increased Al concentrations, and I investigated the mechanisms behind the observed effects induced by elevated Al concentrations. Furthermore, the response of Al concentrations and the speciation in soil solution to Ca amendment in the study area were determined. In a final step, I assessed all major Al fluxes, drivers of Al concentrations in ecosystem solutions, and indicators of Al toxicity in the tropical montane rainforest in Ecuador in order to test for indications of Al toxicity. In the hydroponic experiment, a 10 % reduction in aboveground biomass production occurred at 126 to 376 μM Al (EC10 values), probably attributable to decreased Mg concentrations in leaves and reduced potosynthesis. At 300 μM Al, increased root biomass production of T. chrysantha was observed. Phosphorus concentrations in roots of C. odorata and T. chrysantha were significantly highest in the treatment with 300 μM Al and correlated significantly with root biomass, being a likely reason for stimulated root biomass production. The degree of organic complexation of Al in the organic layer leachate, which is central to plant nutrition because of the high root density, and soil solution from the study area was very high (mean > 99 %). The resulting low free Al concentrations are not likely to affect plant growth, although the concentrations of potentially toxic Al3+ increased with soil depth due to higher total Al and lower dissolved organic matter concentrations in soil solutions. The Ca additions caused an increase of Al in the organic layer leachate, probably because Al3+ was exchanged against the added Ca2+ ions while pH remained constant. The free ion molar ratios of Ca2+:Al3+ (mean ratio ca. 400) were far above the threshold (≤ 1) for Al toxicity, because of a much higher degree of organo-complexation of Al than Ca. High Al fluxes in litterfall (8.8 – 14.2 kg ha−1 yr−1) indicate a high Al circulation through the ecosystem. The Al concentrations in the organic layer leachate were driven by the acidification of the ecosystem and increased significantly between 1999 and 2008. However, the Ca:Al molar ratios in organic layer leachate and all aboveground ecosystem solutions were above the threshold for Al toxicity. Except for two Al accumulating and one non-accumulating tree species, the Ca:Al molar ratios in tree leaves from the study area were above the Al toxicity threshold of 12.5. I conclude that toxic effects in the hydroponic experiment occurred at Al concentrations far above those in native organic layer leachate, shoot biomass production was likely inhibited by reduced Mg uptake, impairing photosynthesis, and the stimulation of root growth at low Al concentrations can be possibly attributed to improved P uptake. Dissolved organic matter in soil solutions detoxifies Al in acidic tropical forest soils and a wide distribution of Al accumulating tree species and high Al fluxes in the ecosystem do not necessarily imply a general Al phytotoxicity.

The secretome of induced pluripotent stem cells reduces lung fibrosis in part by hepatocyte growth factor.

INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic lung disease, resulting in respiratory insufficiency and reduced survival. Pulmonary fibrosis is a result of repeated alveolar epithelial microinjuries, followed by abnormal regeneration and repair processes in the lung. Recently, stem cells and their secretome have been investigated as a novel therapeutic approach in pulmonary fibrosis. We evaluated the potential of induced pluripotent stem cells (iPSC) conditioned media (iPSC-cm) to regenerate and repair the alveolar epithelium in vitro and improve bleomycin induced lung injury in vivo. METHODS IPSC-cm was collected from cultured iPSC derived from human foreskin fibroblasts and its biological effects on alveolar epithelial wound repair was studied in an alveolar wound healing assay in vitro. Furthermore, iPSC-cm was intratracheally instilled 7 days after bleomycin induced injury in the rat lungs and histologically and biochemically assessed 7 days after instillation. RESULTS iPSC-cm increased alveolar epithelial wound repair in vitro compared with medium control. Intratracheal instillation of iPSC-cm in bleomycin-injured lungs reduced the collagen content and improved lung fibrosis in the rat lung in vivo. Profibrotic TGFbeta1 and alpha-smooth muscle actin (alpha-sma) expression were markedly reduced in the iPSC-cm treated group compared with control. Antifibrotic hepatocyte growth factor (HGF) was detected in iPSC-cm in biologically relevant levels, and specific inhibition of HGF in iPSC-cm attenuated the antifibrotic effect of iPSC-cm, indicating a central role of HGF in iPSC-cm. CONCLUSION iPSC-cm increased alveolar epithelial wound repair in vitro and attenuated bleomycin induced fibrosis in vivo, partially due to the presence of HGF and may represent a promising novel, cell free therapeutic option against lung injury and fibrosis.

Intra- and interlaboratory reliability of a cryopreserved trout hepatocyte assay for the prediction of chemical bioaccumulation potential

Measured rates of intrinsic clearance determined using cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of improving modeled bioaccumulation predictions for fish. To date, however, the intra- and interlaboratory reliability of this procedure has not been determined. In the present study, three laboratories determined in vitro intrinsic clearance of six reference compounds (benzo[a]pyrene, 4-nonylphenol, di-tert-butyl phenol, fenthion, methoxychlor and o-terphenyl) by conducting substrate depletion experiments with cryopreserved trout hepatocytes from a single source. O-terphenyl was excluded from the final analysis due to nonfirst-order depletion kinetics and significant loss from denatured controls. For the other five compounds, intralaboratory variability (% CV) in measured in vitro intrinsic clearance values ranged from 4.1 to 30%, while interlaboratory variability ranged from 27 to 61%. Predicted bioconcentration factors based on in vitro clearance values exhibited a reduced level of interlaboratory variability (5.3-38% CV). The results of this study demonstrate that cryopreserved trout hepatocytes can be used to reliably obtain in vitro intrinsic clearance of xenobiotics, which provides support for the application of this in vitro method in a weight-of-evidence approach to chemical bioaccumulation assessment.

Clinical importance of risk variants in the dihydropyrimidine dehydrogenase gene for the prediction of early-onset fluoropyrimidine toxicity.

We investigated the clinical relevance of dihydropyrimidine dehydrogenase gene (DPYD) variants to predict severe early-onset fluoropyrimidine (FP) toxicity, in particular of a recently discovered haplotype hapB3 and a linked deep intronic splice site mutation c.1129-5923C>G. Selected regions of DPYD were sequenced in prospectively collected germline DNA of 500 patients receiving FP-based chemotherapy. Associations of DPYD variants and haplotypes with hematologic, gastrointestinal, infectious, and dermatologic toxicity in therapy cycles 1-2 and resulting FP-dose interventions (dose reduction, therapy delay or cessation) were analyzed accounting for clinical and demographic covariates. Fifteen additional cases with toxicity-related therapy delay or cessation were retrospectively examined for risk variants. The association of c.1129-5923C>G/hapB3 (4.6% carrier frequency) with severe toxicity was replicated in an independent prospective cohort. Overall, c.1129-5923G/hapB3 carriers showed a relative risk of 3.74 (RR, 95% CI = 2.30-6.09, p = 2 × 10(-5)) for severe toxicity (grades 3-5). Of 31 risk variant carriers (c.1129-5923C>G/hapB3, c.1679T>G, c.1905+1G>A or c.2846A>T), 11 (all with c.1129-5923C>G/hapB3) experienced severe toxicity (15% of 72 cases, RR = 2.73, 95% CI = 1.61-4.63, p = 5 × 10(-6)), and 16 carriers (55%) required FP-dose interventions. Seven of the 15 (47%) retrospective cases carried a risk variant. The c.1129-5923C>G/hapB3 variant is a major contributor to severe early-onset FP toxicity in Caucasian patients. This variant may substantially improve the identification of patients at risk of FP toxicity compared to established DPYD risk variants (c.1905+1G>A, c.1679T>G and c.2846A>T). Pre-therapeutic DPYD testing may prevent 20-30% of life-threatening or lethal episodes of FP toxicity in Caucasian patients.

Nuclear Factor I-C acts as a regulator of hepatocyte proliferation at the onset of liver regeneration

BACKGROUND & AIMS: Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed abnormal skin wound healing and growth of its appendages, suggesting a role in controlling cell proliferation in adult regenerative processes. Liver regeneration following partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes lead to their rapid and phased proliferation. Although NFI-C is highly expressed in the liver, no hepatic function was yet established for this transcription factor. This study aimed to determine whether NFI-C may play a role in hepatocyte proliferation and liver regeneration. METHODS: Liver regeneration and cell proliferation pathways following two-thirds PH were investigated in NFI-C knockout (ko) and wild-type (wt) mice. RESULTS: We show that the absence of NFI-C impaired hepatocyte proliferation because of plasminogen activator I (PAI-1) overexpression and the subsequent suppression of urokinase plasminogen activator (uPA) activity and hepatocyte growth factor (HGF) signalling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wt mice. The subsequent transient down regulation of NFI-C, as can be explained by a self-regulatory feedback loop with transforming growth factor beta 1 (TGF-ß1), may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. CONCLUSION: NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration.

Optimizing the aquatic toxicity assessment under REACH through an integrated testing strategy (ITS).

To satisfy REACH requirements a high number of data on chemical of interest should be supplied to the European Chemicals Agency. To organize the various kinds of information and help the registrants to choose the best strategy to obtain the needed information limiting at the minimum the use of animal testing, integrated testing strategies (ITSs) schemes can be used. The present work deals with regulatory data requirements for assessing the hazards of chemicals to the aquatic pelagic environment. We present an ITS scheme for organizing and using the complex existing data available for aquatic toxicity assessment. An ITS to optimize the choice of the correct prediction strategy for aquatic pelagic toxicity is described. All existing information (like physico-chemical information), and all the alternative methods (like in silico, in vitro or the acute-to-chronic ratio) are considered. Moreover the weight of evidence approach to combine the available data is included.

Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis.

BACKGROUND & AIMS Unhealthy lifestyles predispose to non-alcoholic steatohepatitis (NASH), which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity, it is unknown whether regular exercise reduces the risk of developing HCC. Therefore, we studied the effect of regular exercise on the development of HCC in male hepatocyte-specific PTEN-deficient mice (AlbCrePten(flox/flox)), which develop steatohepatitis and HCC spontaneously. METHODS Mice were fed a standardized 10% fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 minutes/day, 5 days/week during 32 weeks. RESULTS After 32 weeks of regular exercise, 71% of exercised mice developed nodules larger than 15 mm(3) vs 100% of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise, as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the Non-alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically, exercise stimulated the phosphorylation of AMPK and its substrate raptor, which decreased the kinase activity of mTOR. CONCLUSIONS These data show a benefit of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC.