GRAMD2 + alveolar type I cell plasticity facilitates cell state transitions in organoid culture.

Alveolar epithelial regeneration is critical for normal lung function and becomes dysregulated in disease. While alveolar type 2 (AT2) and club cells are known distal lung epithelial progenitors, determining if alveolar epithelial type 1 (AT1) cells also contribute to alveolar regeneration has been hampered by lack of highly specific mouse models labeling AT1 cells. To address this, the Gramd2 CreERT2 transgenic strain was generated and crossed to Rosa mTmG mice. Extensive cellular characterization, including distal lung immunofluorescence and cytospin staining, confirmed that GRAMD2 + AT1 cells are highly enriched for green fluorescent protein (GFP). Interestingly, Gramd2 CreERT2 GFP + cells were able to form organoids in organoid co-culture with Mlg fibroblasts. Temporal scRNAseq revealed that Gramd2 + AT1 cells transition through numerous intermediate lung epithelial cell states including basal, secretory and AT2 cell in organoids while acquiring proliferative capacity. Our results indicate that Gramd2 + AT1 cells are highly plastic suggesting they may contribute to alveolar regeneration.

Targeting upregulated RNA binding protein RCAN1.1: a promising strategy for neuroprotection in acute ischemic stroke.

AIMS: To explore the expression changes and roles of the RNA-binding protein RCAN1.1 in acute ischemic stroke (AIS), and to preliminarily confirm the medicinal value of the RNA aptamer R1SR13 in AIS by targeting RCAN1.1. METHODS: Two mouse AIS models of middle cerebral artery occlusion (MCAO) and right common carotid artery ligation (R-CCAL) and oxygen glucose deprivation (OGD) model of AIS in primary neurons and SH-SY5Y were performed. The expression pattern of RCAN1.1 was assessed using real-time quantitative PCR (RT-qPCR) and western blotting (WB) in vivo and in vitro. The underlying mechanism for the elevation of RCAN1.1 in the upstream was investigated. Lentiviruses were administrated and the effect of RCAN1.1 in AIS was assessed by ATP level, caspase 3/7 assay, TUNEL and WB. The protective function of R1SR13 in AIS was evaluated both in vivo and in vitro. RESULTS: In two mouse models of AIS, RCAN1.1 mRNA and RCAN1.1 L protein were significantly upregulated in the ischemic brain tissue. The same results were detected in the OGD model of primary neurons and SH-SY5Y. The mechanistic analysis proved that hypoxia-inducible factor-1α (HIF1α) could specifically activate the RCAN1.1 gene promoter through combining with the functional hypoxia-responsive element (HRE) site (-325 to -322 bp). The increased expression of RCAN1.1 L markedly depleted ATP production and aggravated neuronal apoptosis under OGD condition. R1SR13, an antagonizing RNA aptamer of RCAN1.1, was demonstrated to reduce neuronal apoptosis caused by the elevated RCAN1.1 L in the cellular and animal models of AIS. CONCLUSION: RCAN1.1 is a novel target gene of HIF1α and the functional HRE in the RCAN1.1 promoter region is -325 to -322 bp. The marked upregulation of RCAN1.1 in AIS promoted neuronal apoptosis, an effect that could be reversed by its RNA aptamer R1SR13 in vivo and in vitro. Thus, R1SR13 represents a promising strategy for neuroprotection in AIS and our study lays a theoretical foundation for it to become a clinically targeted drug.

Alterations of the Gut Microbiota in Response to Total Sleep Deprivation and Recovery Sleep in Rats.

INTRODUCTION: Accumulating evidence suggests that both sleep loss and gut dysbiosis can lead to metabolic disorders. However, less is known about the impact of total sleep deprivation (SD) and sleep recovery on the composition, function, and metabolic dynamics of the gut microbiota. METHODS: Specific-pathogen free Sprague-Dawley rats were subjected to 48 h of SD with gentle handling and then allowed to recover for 1 week. Taxonomic profiles of fecal microbiota were obtained at baseline, 24 h of SD, 48 h of SD, and 1 week of recovery. We used 16S rRNA gene sequencing to analyze the gut microbial composition and function and further characterize microbiota-derived metabolites in rats. RESULTS: The microbiota composition analysis revealed that gut microbial composition and metabolites did not change in the rats after 24 h of SD but were significantly altered after 48 h of SD. These changes were reversible after 1 week of sleep recovery. A functional analysis was performed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, indicating that 19 KEGG pathways were significantly altered in the gut microbiota in SD rats. These functional changes occurred within 24 h of SD, were more apparent after 48 h of SD, and did not fully recover after 1 week of sleep recovery. CONCLUSION: These results indicate that acute total SD leads to significant compositional and functional changes in the gut microbiota, and these changes are reversible.

Gut microbiota modulates the inflammatory response and cognitive impairment induced by sleep deprivation.

Sleep deprivation (SD) is increasingly common in modern society, which can lead to the dysregulation of inflammatory responses and cognitive impairment, but the mechanisms remain unclear. Emerging evidence suggests that gut microbiota plays a critical role in the pathogenesis and development of inflammatory and psychiatric diseases, possibly via gut microbiota-brain interactions and neuroinflammation. The present study investigated the impact of SD on gut microbiota composition and explored whether alterations of the gut microbiota play a causal role in chronic inflammatory states and cognitive impairment that are induced by SD. We found that SD-induced gut dysbiosis, inflammatory responses, and cognitive impairment in humans. Moreover, the absence of the gut microbiota suppressed inflammatory response and cognitive impairment induced by SD in germ-free (GF) mice. Transplantation of the "SD microbiota" into GF mice activated the Toll-like receptor 4/nuclear factor-κB signaling pathway and impaired cognitive function in the recipient mice. Mice that harbored "SD microbiota" also exhibited increases in neuroinflammation and microglial activity in the hippocampus and medial prefrontal cortex. These findings indicate that gut dysbiosis contributes to both peripheral and central inflammatory processes and cognitive deficits that are induced by SD, which may open avenues for potential interventions that can relieve the detrimental consequences of sleep loss.

Diurnal rhythm disruptions induced by chronic unpredictable stress relate to depression-like behaviors in rats.

The relationship between circadian rhythms and mood disorders has been established. Circadian dysregulations are believed to exacerbate the severity of mood disorders and vice versa. Although many studies on diurnal changes of clock genes in animal model of depression have been performed from the RNA level, only a few studies have been carried out from the protein level. In this study, we investigated the diurnal changes induced by chronic unpredictable stress (CUS) using free-running wheel test and Western Blotting (WB). Besides, we examined the depression-like behaviors of rats by sucrose preference test (SPT) and forced swim test (FST). We found that CUS induced significant reductions in the quantity of free-running wheel activity and rhythmic disruptions of clock proteins in hippocampus. Furthermore, we found that the amplitude of PER1 in CA1 was positively related to the severity of depression-like behaviors. These results suggest that CUS results in both changes in diurnal rhythms and in depression-like behaviors and that it is suggested that these changes are related.

Retraction notice to "Diurnal rhythm disruptions induced by chronic unpredictable stress relate to depression-like behaviors in rats" [Pharmacol. Biochem. Behav. 194 (2020) 172939].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor because of serious doubts regarding the data on melatonin levels. The authors used a melatonin ELISA kit that was not fit for purpose, resulting in data showing peak secretion of this hormone occurring in the middle of the light period, which does not make any physiological sense since melatonin is only produced during darkness.

Integrated Single-Cell RNA-Sequencing Analysis of Aquaporin 5-Expressing Mouse Lung Epithelial Cells Identifies GPRC5A as a Novel Validated Type I Cell Surface Marker.

Molecular and functional characterization of alveolar epithelial type I (AT1) cells has been challenging due to difficulty in isolating sufficient numbers of viable cells. Here we performed single-cell RNA-sequencing (scRNA-seq) of tdTomato+ cells from lungs of AT1 cell-specific Aqp5-Cre-IRES-DsRed (ACID);R26tdTomato reporter mice. Following enzymatic digestion, CD31-CD45-E-cadherin+tdTomato+ cells were subjected to fluorescence-activated cell sorting (FACS) followed by scRNA-seq. Cell identity was confirmed by immunofluorescence using cell type-specific antibodies. After quality control, 92 cells were analyzed. Most cells expressed 'conventional' AT1 cell markers (Aqp5, Pdpn, Hopx, Ager), with heterogeneous expression within this population. The remaining cells expressed AT2, club, basal or ciliated cell markers. Integration with public datasets identified three robust AT1 cell- and lung-enriched genes, Ager, Rtkn2 and Gprc5a, that were conserved across species. GPRC5A co-localized with HOPX and was not expressed in AT2 or airway cells in mouse, rat and human lung. GPRC5A co-localized with AQP5 but not pro-SPC or CC10 in mouse lung epithelial cell cytospins. We enriched mouse AT1 cells to perform molecular phenotyping using scRNA-seq. Further characterization of putative AT1 cell-enriched genes revealed GPRC5A as a conserved AT1 cell surface marker that may be useful for AT1 cell isolation.

Network Pharmacology Combined with Transcriptional Analysis to Unveil the Biological Basis of Astaxanthin in Reducing the Oxidative Stress Induced by Diabetes Mellitus.

PURPOSE: Astaxanthin (Ast) has been reported to reduce oxidative stress induced by diabetes mellitus (DM). The aim of this research was to give a systematic overview of the biological basis for this process. METHODS: Ast-targeted proteins were collected from the BATMAN database, Comparative Toxicogenomics Database, and STITCH database. Putative DM-related protein targets were collected from the GeneCards database. A DM-rat model was then built with streptozotocin (STZ) combined with a high-sugar, high-fat diet for 30 days. Total cholesterol (TC), triglycerides (TGs), and insulin levels were examined using whole tail-vein blood from overnight-fasted rats. SOD, GSH, and MDA activy was detected in liver tissue (p<0.05). In addition, we used RNA-sequencing analysis to detect gene-transcription level in liver tissue of rats and GO biological process analysis to show all the log2FC≥2 genes in the Ast-fed DM rats compared with the DM group using the STRING database. Ast-intersecting targets were collected with Venn analysis. Docking analysis between Ast and targeted proteins was down with the SwissDock server. Ast targets-pathway networks were built using Cytoscape 3.7.2 software. RESULTS: A total of 120 Ast-targeted proteins and 13,784 DM-related targets were collected. Ast functioned in reducing TC, TG, and MDA levels, promoting SOD activity and GSH expression, and alleviating islet-cell injury in Ast-fed DM rats compared with DM control rats. Furthermore, genes involved in MAPK, TNF, AMPK, and FOXO signaling pathways were differently expressed in Ast-treated DM rats compared with DM rats. The differentially expressed genes were enriched in euchromatin, thyroid cancer, and metaphase-plate congression. Three Ast-intersecting targets - Col5A1, Nqo1, and Notch2 - were then identified. We found possible binding patterns of Ast with Nqo1 and Notch2, respectively. Ast targets-pathway networks were finally built to show a systematic overview of how Ast works in multiple pathways to reduce oxidative stress. Taken together, Ast is predicted to target Col5A1, Nqo1, and Notch2 to form a network of systemic pharmacological effects to: 1) promote insulin-releasing balance and relieve insulin resistance, 2) reduce testicular cell apoptosis, and 3) maintain normal size in marginal-zone B cells and inhibit autoimmune DM, all of which contribute to the balance of lipid metabolism and reduction of oxidative stress in DM patients. CONCLUSION: Ast functions in reducing oxidative stress in DM rats by regulating a variety of targets to form a comprehensive antioxidative network.

Diurnal rhythm disruptions induced by chronic unpredictable stress relate to depression-like behaviors in rats.

The relationship between circadian rhythms and mood disorders has been established, circadian dysregulations are believed to exacerbate the severity of mood disorders and vice versa. Although many studies on diurnal changes of clock genes in animal model of depression have been performed from the RNA level, only a few studies have been carried out from the protein level. In this study, we investigated the diurnal changes induced by chronic unpredictable stress (CUS) using various methods, including free-running wheel test, enzyme-linked immunosorbent assay (ELISA) and Western Blotting (WB). Besides, we examined the depression-like behaviors of rats by sucrose preference test (SPT) and forced swim test (FST). We found that CUS induced significant reductions in the quantity of free-running wheel activity and the amplitude of melatonin secretion rhythm. We also found that CUS induced rhythmic disruptions of clock proteins in hippocampus. Furthermore, we found that the amplitude of PER1 in CA1 was positively related to the severity of depression-like behaviors. These results suggest that stress results in both changes in circadian rhythms and in depression-like behaviors and that it is suggested that these changes are related.

Comparison of three common nutritional screening tools with the new European Society for Clinical Nutrition and Metabolism (ESPEN) criteria for malnutrition among patients with geriatric gastrointestinal cancer: a prospective study in China.

OBJECTIVE: The aim of this study was to evaluate and compare three common nutritional screening tools with the new European Society for Clinical Nutrition and Metabolism (ESPEN) diagnostic criteria for malnutrition among elderly patients with gastrointestinal cancer. RESEARCH METHODSANDPROCEDURES: Nutritional screening tools, including the Nutritional Risk Screening 2002 (NRS 2002), the Malnutrition Universal Screening Tool (MUST) and the Short Form of Mini Nutritional Assessment (MNA-SF), were applied to 255 patients with gastrointestinal cancer. We compared the diagnostic values of these tools for malnutrition, using the new ESPEN diagnostic criteria for malnutrition as the 'gold standards'. RESULTS: According to the new ESPEN diagnostic criteria for malnutrition, 20% of the patients were diagnosed as malnourished. With the use of NRS 2002, 52.2% of the patients were found to be at high risk of malnutrition; with the use of MUST, 37.6% of the patients were found to be at moderate/high risk of malnutrition; and according to MNA-SF, 47.8% of the patients were found to be at nutritional risk. MUST was best correlated with the ESPEN diagnostic criteria (К=0.530, p<0.001) compared with NRS 2002 (К=0.312, p<0.001) and MNA-SF (К=0.380, p<0.001). The receiver operating characteristic curve of MUST had the highest area under the curve (AUC) compared with NRS 2002 and MNA-SF. CONCLUSIONS: Among the tools, MUST was found to perform the best in identifyingmalnourished elderly patients with gastrointestinal cancer distinguished by the new ESPEN diagnostic criteria for malnutrition. Nevertheless, further studies are needed to verify our findings. TRIAL REGISTRATION NUMBER: ChiCTR-RRC-16009831; Pre-results.

Effect of nutritional risk on short-term outcomes after laparoscopic-assisted gastrectomy: a prospective study from two centers.

BACKGROUND/AIM: The aim of this prospective double-center study was to explore the effect of nutritional risk on short-term outcomes in the patients who had gastric cancer and underwent a laparoscopic-assisted gastrectomy. PATIENTS AND METHODS: We conducted a study of patients who underwent laparoscopic-assisted gastrectomy in two large centers between June 2014 and April 2017. Patients' demographic and clinical characteristics and postoperative short-term outcomes were prospectively analyzed. Patients were divided into two groups depend on the preoperative presence of nutritional risk. Clinical variables were compared. Univariate analyses and multivariate logistic regression analyses evaluating the risk factors for postoperative complications were performed. RESULTS: A total of 256 patients, comprising 187 males and 69 females, met the inclusion criteria and were included in this study. The mean age was 61.81 years, the average BMI was 22.44 kg/m, and the average preoperative serum albumin was 39.42 g/l. Older age (P=0.001), higher tumor stage (P=0.047), lower BMI (P<0.001), lower preoperative serum albumin (P=0.005), and lower hemoglobin (P=0.013) were more common in the nutritional risk group. There were no significant differences in the short-term postoperative outcomes between nutritional risk and non-nutritional risk groups. Advanced age (P=0.024) and hypoalbuminemia (P=0.004) were independent risk factors for postoperative complications after laparoscopic-assisted gastrectomy. CONCLUSION: Nutritional risk may not be a clinical predictor of short-term outcomes after laparoscopic-assisted gastrectomy. Advanced age and preoperative hypoalbuminemia were independent risk factors for grade II or more postoperative complications.

Effect of surgery-induced acute muscle wasting on postoperative outcomes and quality of life.

BACKGROUND: Gastrectomy results in a significant loss of body composition in the long term, but the acute skeletal muscle wasting after gastrectomy has been rarely investigated. Moreover, the association between postoperative muscle wasting and quality of life (QOL) has never been reported. In the present study, we aimed to investigate the risk factors for acute muscle wasting after gastric cancer surgery and its effect on QOL and short-term postoperative outcomes. METHODS: We conducted a prospective study of patients who underwent curative gastrectomy for gastric cancer between June 2015 and December 2015. Skeletal muscle mass was measured by computed tomography within 1 month before and 1 week after surgery. QOL was assessed 1, 3, and 6 months postoperatively. Univariate and multivariate analyses were performed to identify the risk factors for clinically relevant muscle wasting (muscle wasting ≥10%). RESULTS: A total of 110 patients were included, in which 35 patients had muscle wasting ≥10% within 1 week after surgery. Age ≥65 years and diabetes were independent risk factors for muscle wasting ≥10%. Patients with muscle wasting ≥10% had a poorer QOL in terms of fatigue and physical functioning at 1 and 3 months postoperatively, as well as a higher incidence of postoperative complications, a higher incidence of handgrip strength reduction ≥10%, longer hospital stays, and higher costs. CONCLUSIONS: Age ≥65 years and diabetes were independently associated with clinically relevant muscle wasting within 1 week after gastric cancer surgery. Clinically relevant muscle wasting was associated with a poorer QOL and short-term outcomes after surgery.

Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance.

Active ion transport by basolateral Na-K-ATPase (Na pump) creates an Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and ß1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the ß1 subunit and the relative contributions of alveolar epithelial type II (AT2) versus type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which the ß1 subunit was knocked out in either AT1 cells or both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing, we believe for the first time, that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung weight ratios in knockout lungs relative to control lungs. Knockout mice showed increases in Na pump ß3 subunit expression and ß2-adrenergic receptor expression. These results demonstrate a crucial role for the Na pump ß1 subunit in alveolar ion and fluid transport and indicate that both AT1 and AT2 cells make major contributions to these processes and to AFC. Furthermore, they support the feasibility of a general approach to altering alveolar epithelial function in a cell-specific manner that allows direct insights into AT1 versus AT2 cell-specific roles in the lung.

Knockout mice reveal key roles for claudin 18 in alveolar barrier properties and fluid homeostasis.

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased ß-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase ß1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.

In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide.

The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum, and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions.

Cyclotides, a novel ultrastable polypeptide scaffold for drug discovery.

Cyclotides are a unique and growing family of backbone cyclized peptides that also contain a cystine knot motif built from six conserved cysteine residues. This unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Aside from the conserved residues forming the cystine knot, cyclotides have been shown to have high variability in their sequences. Consisting of over 160 known members, cyclotides have many biological activities, ranging from anti-HIV, antimicrobial, hemolytic, and uterotonic capabilities; additionally, some cyclotides have been shown to have cell penetrating properties. Originally discovered and isolated from plants, cyclotides can also be produced synthetically and recombinantly. The high sequence variability, stability, and cell penetrating properties of cyclotides make them potential scaffolds to be used to graft known active peptides or engineer peptide-based drug design. The present review reports recent findings in the biological diversity and therapeutic potential of natural and engineered cyclotides.

Role of microsomal glutathione transferase 1 in the mechanism-based biotransformation of glyceryl trinitrate in LLC-PK1 cells.

Although glyceryl trinitrate (GTN) has been used in the treatment of angina for many years, details of its conversion to the proximal activator (presumed to be NO or an NO congener) of soluble guanylyl cyclase (sGC) are still unclear. We reported previously that purified microsomal glutathione transferase 1 (MGST1) mediates the denitration of GTN. In the current study, we investigated in intact cells whether this enzyme also converts GTN to species that activate sGC (mechanism-based biotransformation). We utilized LLC-PK1 cells, a cell line with an intact NO/sGC/cGMP system, and generated a stable cell line that overexpressed MGST1. MGST1 in the stably transfected cells was localized to the endoplasmic reticulum, and microsomes from these cells exhibited markedly increased GST activity. Although incubation of these cells with GTN resulted in a 3-4-fold increase in GTN biotransformation, attributed primarily to an increase in formation of the 1,3-glyceryl dinitrate metabolite, GTN-induced cGMP accumulation in cells overexpressing MGST1 was not different than that observed in wild type cells or in cells stably transfected with empty vector. To determine whether overexpression of NADPH cytochrome P450 reductase might act in concert with MGST1 to generate activators of sGC, we assessed GTN-induced cGMP accumulation in MGST1-overexpressing cells that had been transiently transfected with CPR. In this case, GTN-induced cGMP accumulation was also not different than that observed in wild type cells. We conclude that although MGST1 mediates the biotransformation of GTN in intact cells, this biotransformation does not contribute to the formation of activators of sGC.

Direct mitochondrial dysfunction precedes reactive oxygen species production in amiodarone-induced toxicity in human peripheral lung epithelial HPL1A cells.

Amiodarone (AM), a drug used in the treatment of cardiac dysrrhythmias, can produce severe pulmonary adverse effects, including fibrosis. Although the pathogenesis of AM-induced pulmonary toxicity (AIPT) is not clearly understood, several hypotheses have been advanced, including increased inflammatory mediator release, mitochondrial dysfunction, and free-radical formation. The hypothesis that AM induces formation of reactive oxygen species (ROS) was tested in an in vitro model relevant for AIPT. Human peripheral lung epithelial HPL1A cells, as surrogates for target cells in AIPT, were susceptible to the toxicity of AM and N-desethylamiodarone (DEA), a major AM metabolite. Longer incubations (> or =6 h) of HPL1A cells with 100 microM AM significantly increased ROS formation. In contrast, shorter incubations (2 h) of HPL1A cells with AM resulted in mitochondrial dysfunction and cytoplasmic cytochrome c translocation. Preexposure of HPL1A cells to ubiquinone and alpha-tocopherol was more effective than that with Trolox C or 5,5-dimethylpyrolidine N-oxide (DMPO) at preventing AM cytotoxicity. These data suggest that mitochondrial dysfunction, rather than ROS overproduction, represents an early event in AM-induced toxicity in peripheral lung epithelial cells that may be relevant for triggering AIPT, and antioxidants that target mitochondria may potentially have beneficial effects in AIPT.

Effectiveness of novel imidazole-dioxolane heme oxygenase inhibitors in renal proximal tubule epithelial cells.

To enhance our understanding of the physiological roles of heme oxygenase (HO) isozymes, HO-1 (inducible) and HO-2 (constitutive), we developed novel imidazole-based HO inhibitors. Unlike the metalloporphyrins, these imidazole-dioxolane compounds are selective for the in vitro inhibition of HO with minimal effects on other heme-dependent enzymes such as nitric oxide synthase and soluble guanylyl cyclase. In the current study, we tested the hypothesis that these novel HO inhibitors are effective in intact cells by extending their application to cultured, renal proximal tubule epithelial cells (LLC-PK1). HO-1 and HO-2 protein expression was enhanced by pretreatment of cells with hemin, transduction with adenovirus encoding human HO-1, and transfection with cDNA for HO-2, respectively. Total HO activity was measured by determining the formation of carbon monoxide (CO), whereas cell viability and apoptosis were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the expression of activated caspase-3. Gliotoxin/tumor necrosis factor-alpha (TNF-alpha) produced cytotoxicity in wild-type LLC-PK1 cells (P < 0.05) but not in HO-1 and HO-2 overexpressing or wild type cells pretreated with hemin (10 microM). The presence of imidazole-dioxolane HO inhibitors (2-25 microM) decreased cell viability (P < 0.05). A CO-releasing molecule reversed, in a dose-dependent manner, the cytotoxic effects and caspase-3 activation induced by the combination of gliotoxin/TNF-alpha and the HO inhibitors, suggesting an important role for CO in protection against renal toxicity. These data demonstrate a protective role of both HO-1 and HO-2 against gliotoxin/TNF-alpha-induced cytotoxicity in LLC-PK1 cells. The novel imidazole-dioxolane compounds can be used as effective inhibitors of HO activity in cell culture.

Biotransformation of glyceryl trinitrate by rat hepatic microsomal glutathione S-transferase 1.

Although the biotransformation of organic nitrates by the cytosolic glutathione S-transferases (GSTs) is well known, the relative contribution of the microsomal GST (MGST1) to nitrate biotransformation has not been described. We therefore compared the denitration of glyceryl trinitrate (GTN) by purified rat liver MGST1 and cytosolic GSTs. Both MGST1 and cytosolic GSTs catalyzed the denitration of GTN, but the activity of MGST1 toward GTN was 2- to 3-fold higher. To mimic oxidative/nitrosative stress in vitro, we treated enzyme preparations with hydrogen peroxide, S-nitrosoglutathione, and peroxynitrite. Both oxidants and nitrating reagents increased the activity of MGST1 toward the GST substrate, 1-chloro-2,4-dinitrobenzene (CDNB) whereas these treatments inhibited GTN denitration by MGST1. Alkylation of the sole cysteine residue of MGST1 by N-ethylmaleimide markedly increased enzyme activity with CDNB as substrate but decreased the rate of GTN denitration. In aortic microsomes from GTN-tolerant animals, there was a decreased abundance of MGST1 dimers and trimers. In hepatic microsomes from GTN-tolerant animals, GTN biotransformation was unaltered whereas the rate of CDNB conjugation was doubled, suggesting that chronic GTN exposure causes structural modifications to the enzyme, resulting in increased activity to certain substrates. Collectively, these data indicate that MGST1 contributes significantly to the biotransformation of GTN and that chemical modification of the microsomal enzyme has differential effects on the catalytic activity toward different substrates.