LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.

INTRODUCTION: Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs. METHODS: The present study aimed to validate a simple routine LC-HRMS method to quantify cortisol, cortisone, 6ß-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism. RESULTS: The LC-HRMS method involved liquid-liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6ß-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity). CONCLUSION: A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism.

Identification of Dhx15 as a Major Regulator of Liver Development, Regeneration, and Tumor Growth in Zebrafish and Mice.

RNA helicase DHX15 plays a significant role in vasculature development and lung metastasis in vertebrates. In addition, several studies have demonstrated the overexpression of DHX15 in the context of hepatocellular carcinoma. Therefore, we hypothesized that this helicase may play a significant role in liver regeneration, physiology, and pathology. Dhx15 gene deficiency was generated by CRISPR/Cas9 in zebrafish and by TALEN-RNA in mice. AUM Antisense-Oligonucleotides were used to silence Dhx15 in wild-type mice. The hepatocellular carcinoma tumor induction model was generated by subcutaneous injection of Hepa 1-6 cells. Homozygous Dhx15 gene deficiency was lethal in zebrafish and mouse embryos. Dhx15 gene deficiency impaired liver organogenesis in zebrafish embryos and liver regeneration after partial hepatectomy in mice. Also, heterozygous mice presented decreased number and size of liver metastasis after Hepa 1-6 cells injection compared to wild-type mice. Dhx15 gene silencing with AUM Antisense-Oligonucleotides in wild-type mice resulted in 80% reduced expression in the liver and a significant reduction in other major organs. In addition, Dhx15 gene silencing significantly hindered primary tumor growth in the hepatocellular carcinoma experimental model. Regarding the potential use of DHX15 as a diagnostic marker for liver disease, patients with hepatocellular carcinoma showed increased levels of DHX15 in blood samples compared with subjects without hepatic affectation. In conclusion, Dhx15 is a key regulator of liver physiology and organogenesis, is increased in the blood of cirrhotic and hepatocellular carcinoma patients, and plays a key role in controlling hepatocellular carcinoma tumor growth and expansion in experimental models.

Liver-targeted nanoparticles delivering nitric oxide reduce portal hypertension in cirrhotic rats.

Nitric oxide (NO) is a small vasodilator playing a key role in the pathogenesis of portal hypertension. Here, we assessed the potential therapeutic effect of a NO donor targeted to the liver by poly(beta-amino ester) nanoparticles (pBAE NPs) in experimental cirrhosis. Retinol-functionalized NO donor pBAE NPs (Ret pBAE NPs) were synthetized with the aim of actively targeting the liver. Administration of Ret pBAE NPs resulted in uptake and transfection by the liver and spleen. NPs were not found in other organs or the systemic circulation. Treatment with NO donor Ret pBAE NPs (30 mg/ kg body weight) significantly decreased aspartate aminotransferase, lactate dehydrogenase and portal pressure (9.75 ± 0.64 mmHg) compared to control NPs (13.4 ± 0.53 mmHg) in cirrhotic rats. There were no effects on mean arterial pressure and cardiac output. Liver-targeted NO donor NPs reduced collagen fibers and steatosis, activation of hepatic stellate cells and mRNA expression of profibrogenic and proinflammatory genes. Finally, Ret pBAE NPs displayed efficient transfection in human liver slices. Overall, liver-specific NO donor NPs effectively target the liver and mitigated inflammation and portal hypertension in cirrhotic rats. The use of Ret pBAE may prove to be an effective therapeutic strategy to treat advanced liver disease.

Conservation of the enzyme-like activity and biocompatibility of CeO2 nanozymes in simulated body fluids.

Cerium oxide nanozymes (CeO2NZs) are attracting vast attention due to their antioxidant and catalytic properties and mimic the activities of multiple endogenous enzymes. However, as is the case for nanomedicines in general, the success in showing their unique medical applications has not been matched by an understanding of their pharmacokinetics, which is delaying their implementation in clinical settings. Furthermore, the data of their modifications in body fluids and the impact on their activity are scarce. Herein, two types of widely used CeO2NZs, electrostatically stabilized and coated with a mesoporous silica shell, were exposed to simulated saliva and lung, gastric and intestinal fluids, and cell culture media. Their physicochemical modifications and bioactivity were tracked over time up to 15 days combining the data of different characterization techniques and biological assays. The results show that the biocompatibility and antioxidant activity are retained in all cases despite the different evolution behaviors in different fluids, including agglomeration. This work provides an experimental basis from a pharmacokinetic perspective that supports the therapeutic effectiveness of CeO2NZs observed in vivo for the treatment of many conditions related to chronic inflammation and cancer, and suggests that they can be safely administered through different portals of entry including intravenous injection, oral ingestion or inhalation.

Partial vasopressin 1a receptor agonism reduces portal hypertension and hyperaldosteronism and induces a powerful diuretic and natriuretic effect in rats with cirrhosis and ascites.

The vasopressin system has emerged as a therapeutic focus for lowering portal hypertension and reducing splanchnic vasodilation in patients with refractory ascites. Clinically available vasopressin agonists are limited by preferential selectivity for V1 receptors that also have steep concentration-response curves with potential risks of excess vasoconstriction and/or complete antidiuretic effects. OCE-205 is a novel, selective, partial V1a receptor agonist with mixed agonist/antagonist activity and no V2 receptor activation at therapeutic doses. We carried out two studies assessing the in vivo effects of OCE-205 in different rat models of cirrhosis and ascites. In a carbon tetrachloride rat cirrhosis model, OCE-205 administration produced a marked reduction in portal hypertension and hyperaldosteronism, along with robust diuretic and natriuretic effects. These effects were accompanied by marked decreases in ascites volume, with three of five animals experiencing total mobilization of ascites. There was no evidence of fluid overload or sodium or water retention, confirming OCE-205's lack of V2 receptor activity. In a second, corroborative study using a bile duct ligation rat model of ascites, OCE-205 produced significant decreases in ascites volume and body weight and a significant increase in urine volume versus vehicle. Urine sodium excretion increased significantly after the first administration of OCE-205 relative to vehicle; however, repeat administration over 5 days did not lead to hyponatremia. Thus, in separate in vivo models, the mixed agonist/antagonist OCE-205 demonstrated relevant and expected endpoint findings consistent with its known mechanism of action and in vitro pharmacology without apparent unwanted effects or nonspecific toxicities.

Exploring the Long-Term Tissue Accumulation and Excretion of 3 nm Cerium Oxide Nanoparticles after Single Dose Administration.

Nanoparticle (NP) pharmacokinetics significantly differ from traditional small molecule principles. From this emerges the need to create new tools and concepts to harness their full potential and avoid unnecessary risks. Nanoparticle pharmacokinetics strongly depend on size, shape, surface functionalisation, and aggregation state, influencing their biodistribution, accumulation, transformations, and excretion profile, and hence their efficacy and safety. Today, while NP biodistribution and nanoceria biodistribution have been studied often at short times, their long-term accumulation and excretion have rarely been studied. In this work, 3 nm nanoceria at 5.7 mg/kg of body weight was intravenously administrated in a single dose to healthy mice. Biodistribution was measured in the liver, spleen, kidney, lung, brain, lymph nodes, ovary, bone marrow, urine, and faeces at different time points (1, 9, 30, and 100 days). Biodistribution and urinary and faecal excretion were also studied in rats placed in metabolic cages at shorter times. The similarity of results of different NPs in different models is shown as the heterogeneous nanoceria distribution in organs. After the expectable accumulation in the liver and spleen, the concentration of cerium decays exponentially, accounting for about a 50% excretion of cerium from the body in 100 days. Cerium ions, coming from NP dissolution, are most likely excreted via the urinary tract, and ceria nanoparticles accumulated in the liver are most likely excreted via the hepatobiliary route. In addition, nanoceria looks safe and does not damage the target organs. No weight loss or apathy was observed during the course of the experiments.

The Integration of Nanomedicine with Traditional Chinese Medicine: Drug Delivery of Natural Products and Other Opportunities.

The integration of progressive technologies such as nanomedicine with the use of natural products from traditional medicine (TM) provides a unique opportunity for the longed-for harmonization between traditional and modern medicine. Although several actions have been initiated decades ago, a disparity of reasons including some misunderstandings between each other limits the possibilities of a truly complementation. Herein, we analyze some common challenges between nanomedicine and traditional Chinese medicine (TCM). These challenges, if solved in a consensual way, can give a boost to such harmonization. Nanomedicine is a recently born technology, while TCM has been used by the Chinese people for thousands of years. However, for these disciplines, the regulation and standardization of many of the protocols, especially related to the toxicity and safety, regulatory aspects, and manufacturing procedures, are under discussion. Besides, both TCM and nanomedicine still need to achieve a wider social acceptance. Herein, we first briefly discuss the strengths and weaknesses of TCM. This analysis serves to focus afterward on the aspects where TCM and nanomedicine can mutually help to bridge the existing gaps between TCM and Western modern medicine. As discussed, many of these challenges can be applied to TM in general. Finally, recent successful cases in scientific literature that merge TCM and nanomedicine are reviewed as examples of the benefits of this harmonization.

Validation of a Microwave-Assisted Derivatization Gas Chromatography-Mass Spectrometry Method for the Quantification of 2-Hydroxybutyrate in Human Serum as an Early Marker of Diabetes Mellitus.

Circulating levels of 2-hydroxybutyrate (2HB) are highly related to glycemic status in different metabolomic studies. According to recent evidence, 2HB is an early biomarker of the future development of dysglycemia and type 2 diabetes mellitus and may be causally related to the progression of normal subjects to impaired fasting glucose or insulin resistance. In the present study, we developed and validated a simple, specific and sensitive gas chromatography-mass spectrometry (GC-MS) method specifically intended to quantify serum levels of 2HB. Liquid-liquid extraction with ethyl acetate was followed by 2 min of microwave-assisted derivatization. The method presented acceptable accuracy, precision and recovery, and the limit of quantification was 5 µM. Levels of 2HB were found to be stable in serum after three freeze-thaw cycles, and at ambient temperature and at a temperature of 4 °C for up to 24 h. Extracts derivatized under microwave irradiation were stable for up to 96 h. No differences were found in 2HB concentrations measured in serum or plasma EDTA samples. In summary, the method is useful for a rapid, precise and accurate quantification of 2HB in serum samples assessed for the evaluation of dysglycemia and diabetes mellitus.

The pituitary tumour-transforming gene 1/delta-like homologue 1 pathway plays a key role in liver fibrogenesis.

BACKGROUND AND AIMS: PTTG1 is almost undetectable in adult livers but is highly expressed in hepatocarcinoma. While little is known about its involvement in liver fibrosis, PTTG1 expression is associated with DLK1. We assessed the role of the PTTG1/DLK1 pathway in fibrosis progression and the potential therapeutic effect of PTTG1 silencing in fibrosis. METHODS: Pttg1 and Dlk1 were studied in liver and isolated cell populations of control and fibrotic rats and in human liver biopsies. The fibrotic molecular signature was analysed in Pttg1-/- and Pttg1+/+ fibrotic mice. Finally, Pttg1 silencing was evaluated in rats as a novel antifibrotic therapy. RESULTS: Pttg1 and Dlk1 mRNA selectively increased in fibrotic rats paralleling fibrosis progression. Serum DLK1 concentrations correlated with hepatic collagen content and systemic and portal haemodynamics. Human cirrhotic livers showed greater PTTG1 and DLK1 transcript abundance than non-cirrhotic, and reduced collagen was observed in Pttg1 Pttg1-/- mice. The liver fibrotic molecular signature revealed lower expression of genes related to extracellular matrix remodelling including Mmp8 and 9 and Timp4 and greater eotaxin and Mmp13 than fibrotic Pttg1+/+ mice. Finally, interfering Pttg1 resulted in reduced liver fibrotic area, lower α-Sma and decreased portal pressure than fibrotic animals. Furthermore, Pttg1 silencing decreased the transcription of Dlk1, collagens I and III, Pdgfrß, Tgfrß, Timp1, Timp2 and Mmp2. CONCLUSIONS: Pttg1/Dlk1 are selectively overexpressed in the cirrhotic liver and participate in ECM turnover regulation. Pttg1 disruption decreases Dlk1 transcription and attenuates collagen deposition. PTTG1/DLK1 signalling is a novel pathway for targeting the progression of liver fibrosis.

Scalable synthesis of multicomponent multifunctional inorganic core@mesoporous silica shell nanocomposites.

Integrating multiple materials with different functionalities in a single nanostructure enables advances in many scientific and technological applications. However, such highly sophisticated nanomaterials usually require complex synthesis processes that complicate their preparation in a sustainable and industrially feasible manner. Herein, we designed a simple general method to grow a mesoporous silica shell onto any combination of hydrophilic nanoparticle cores. The synthetic strategy, based on the adjustment of the key parameters of the sol-gel process for the silica shell formation, allows for the embedment of single, double, and triple inorganic nanoparticles within the same shell, as well as the size-control of the obtained nanocomposites. No additional interfacial adhesive layer is required on the nanoparticle surfaces for the embedding process. Adopting this approach, electrostatically stabilized, small-sized (from 4 to 15 nm) CeO2, Fe3O4, Gd2O3, NaYF4, Au, and Ag cores were used to test the methodology. The mean diameter of the resulting nanocomposites could be as low as 55 nm, with high monodispersity. These are very feasible sizes for biological intervention, and we further observed increased nanoparticle stability in physiological environments. As a demonstration of their increased activity as a result of this, the antioxidant activity of CeO2 cores was enhanced when in core-shell form. Remarkably, the method is conducted entirely at room temperature, atmospheric conditions, and in aqueous solvent with the use of ethanol as co-solvent. These facile and even "green" synthesis conditions favor scalability and easy preparation of multicomponent nanocomposite libraries with standard laboratory glassware and simple benchtop chemistry, through this sustainable and cost-effective fabrication process.

Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and ß-Hydroxybutyrate/Acetoacetate in Biological Samples.

The metabolic ratios lactate/pyruvate and ß-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, ß-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency's Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and ß-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

Mesoporous silica coated CeO2 nanozymes with combined lipid-lowering and antioxidant activity induce long-term improvement of the metabolic profile in obese Zucker rats.

Obesity is one of the most important public health problems that is associated with an array of metabolic disorders linked to cardiovascular disease, stroke, type 2 diabetes, and cancer. A sustained therapeutic approach to stop the escalating prevalence of obesity and its associated metabolic comorbidities remains elusive. Herein, we developed a novel nanocomposite based on mesoporous silica coated cerium oxide (CeO2) nanozymes that reduce the circulating levels of fatty acids and remarkably improve the metabolic phenotype in a model of obese Zucker rats five weeks after its administration. Lipidomic and gene expression analyses showed an amelioration of the hyperlipidemia and of the hepatic and adipose metabolic dysregulations, which was associated with a down-regulation of the hepatic PI3K/mTOR/AKT pathway and a reduction of the M1 proinflammatory cytokine TNF-α. In addition, the coating of the CeO2 maximized its cell antioxidant protective effects and minimized non-hepatic biodistribution. The one-pot synthesis method for the nanocomposite fabrication is implemented entirely in aqueous solution, room temperature and open atmosphere conditions, favoring scalability and offering a safe and translatable lipid-lowering and antioxidant nanomedicine to treat metabolic comorbidities associated with obesity. This approach may be further applied to address other metabolic disorders related to hyperlipidemia, low-grade inflammation and oxidative stress.

Cerium Oxide Nanoparticles: A New Therapeutic Tool in Liver Diseases.

Oxidative stress induced by the overproduction of free radicals or reactive oxygen species (ROS) has been considered as a key pathogenic mechanism contributing to the initiation and progression of injury in liver diseases. Consequently, during the last few years antioxidant substances, such as superoxide dismutase (SOD), resveratrol, colchicine, eugenol, and vitamins E and C have received increasing interest as potential therapeutic agents in chronic liver diseases. These substances have demonstrated their efficacy in equilibrating hepatic ROS metabolism and thereby improving liver functionality. However, many of these agents have not successfully passed the scrutiny of clinical trials for the prevention and treatment of various diseases, mainly due to their unspecificity and consequent uncontrolled side effects, since a minimal level of ROS is needed for normal functioning. Recently, cerium oxide nanoparticles (CeO2NPs) have emerged as a new powerful antioxidant agent with therapeutic properties in experimental liver disease. CeO2NPs have been reported to act as a ROS and reactive nitrogen species (RNS) scavenger and to have multi-enzyme mimetic activity, including SOD activity (deprotionation of superoxide anion into oxygen and hydrogen peroxide), catalase activity (conversion of hydrogen peroxide into oxygen and water), and peroxidase activity (reducing hydrogen peroxide into hydroxyl radicals). Consequently, the beneficial effects of CeO2NPs treatment have been reported in many different medical fields other than hepatology, including neurology, ophthalmology, cardiology, and oncology. Unlike other antioxidants, CeO2NPs are only active at pathogenic levels of ROS, being inert and innocuous in healthy cells. In the current article, we review the potential of CeO2NPs in several experimental models of liver disease and their safety as a therapeutic agent in humans as well.

Cerium Oxide Nanoparticles: Advances in Biodistribution, Toxicity, and Preclinical Exploration.

Antioxidant nanoparticles have recently gained tremendous attention for their enormous potential in biomedicine. However, discrepant reports of either medical benefits or toxicity, and lack of reproducibility of many studies, generate uncertainties delaying their effective implementation. Herein, the case of cerium oxide is considered, a well-known catalyst in the petrochemistry industry and one of the first antioxidant nanoparticles proposed for medicine. Like other nanoparticles, it is now described as a promising therapeutic alternative, now as threatening to health. Sources of these discrepancies and how this analysis helps to overcome contradictions found for other nanoparticles are summarized and discussed. For the context of this analysis, what has been reported in the liver is reviewed, where many diseases are related to oxidative stress. Since well-dispersed nanoparticles passively accumulate in liver, it represents a major testing field for the study of new nanomedicines and their clinical translation. Even more, many contradictory works have reported in liver either cerium-oxide-associated toxicity or protection against oxidative stress and inflammation. Based on this, finally, the intention is to propose solutions to design improved nanoparticles that will work more precisely in medicine and safely in society.

Bespoken Nanoceria: An Effective Treatment in Experimental Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Despite the availability of new-generation drugs, hepatocellular carcinoma (HCC) is still the third most frequent cause of cancer-related deaths worldwide. Cerium oxide nanoparticles (CeO2 NPs) have emerged as an antioxidant agent in experimental liver disease because of their antioxidant, anti-inflammatory, and antisteatotic properties. In the present study, we aimed to elucidate the potential of CeO2 NPs as therapeutic agents in HCC. APPROACH AND RESULTS: HCC was induced in 110 Wistar rats by intraperitoneal administration of diethylnitrosamine for 16 weeks. Animals were treated with vehicle or CeO2 NPs at weeks 16 and 17. At the eighteenth week, nanoceria biodistribution was assessed by mass spectrometry (MS). The effect of CeO2 NPs on tumor progression and animal survival was investigated. Hepatic tissue MS-based phosphoproteomics as well as analysis of principal lipid components were performed. The intracellular uptake of CeO2 NPs by human ex vivo perfused livers and human hepatocytes was analyzed. Nanoceria was mainly accumulated in the liver, where it reduced macrophage infiltration and inflammatory gene expression. Nanoceria treatment increased liver apoptotic activity, while proliferation was attenuated. Phosphoproteomic analysis revealed that CeO2 NPs affected the phosphorylation of proteins mainly related to cell adhesion and RNA splicing. CeO2 NPs decreased phosphatidylcholine-derived arachidonic acid and reverted the HCC-induced increase of linoleic acid in several lipid components. Furthermore, CeO2 NPs reduced serum alpha-protein levels and improved the survival of HCC rats. Nanoceria uptake by ex vivo perfused human livers and in vitro human hepatocytes was also demonstrated. CONCLUSIONS: These data indicate that CeO2 NPs partially revert the cellular mechanisms involved in tumor progression and significantly increase survival in HCC rats, suggesting that they could be effective in patients with HCC.

Cerium Oxide Nanoparticles Protect against Oxidant Injury and Interfere with Oxidative Mediated Kinase Signaling in Human-Derived Hepatocytes.

Cerium oxide nanoparticles (CeO2NPs) possess powerful antioxidant properties, thus emerging as a potential therapeutic tool in non-alcoholic fatty liver disease (NAFLD) progression, which is characterized by a high presence of reactive oxygen species (ROS). The aim of this study was to elucidate whether CeO2NPs can prevent or attenuate oxidant injury in the hepatic human cell line HepG2 and to investigate the mechanisms involved in this phenomenon. The effect of CeO2NPs on cell viability and ROS scavenging was determined, the differential expression of pro-inflammatory and oxidative stress-related genes was analyzed, and a proteomic analysis was performed to assess the impact of CeO2NPs on cell phosphorylation in human hepatic cells under oxidative stress conditions. CeO2NPs did not modify HepG2 cell viability in basal conditions but reduced H2O2- and lipopolysaccharide (LPS)-induced cell death and prevented H2O2-induced overexpression of MPO, PTGS1 and iNOS. Phosphoproteomic analysis showed that CeO2NPs reverted the H2O2-mediated increase in the phosphorylation of peptides related to cellular proliferation, stress response, and gene transcription regulation, and interfered with H2O2 effects on mTOR, MAPK/ERK, CK2A1 and PKACA signaling pathways. In conclusion, CeO2NPs protect HepG2 cells from cell-induced oxidative damage, reducing ROS generation and inflammatory gene expression as well as regulation of kinase-driven cell survival pathways.

Cerium oxide nanoparticles improve liver regeneration after acetaminophen-induced liver injury and partial hepatectomy in rats.

BACKGROUND AND AIMS: Cerium oxide nanoparticles are effective scavengers of reactive oxygen species and have been proposed as a treatment for oxidative stress-related diseases. Consequently, we aimed to investigate the effect of these nanoparticles on hepatic regeneration after liver injury by partial hepatectomy and acetaminophen overdose. METHODS: All the in vitro experiments were performed in HepG2 cells. For the acetaminophen and partial hepatectomy experimental models, male Wistar rats were divided into three groups: (1) nanoparticles group, which received 0.1 mg/kg cerium nanoparticles i.v. twice a week for 2 weeks before 1 g/kg acetaminophen treatment, (2) N-acetyl-cysteine group, which received 300 mg/kg of N-acetyl-cysteine i.p. 1 h after APAP treatment and (3) partial hepatectomy group, which received the same nanoparticles treatment before partial hepatectomy. Each group was matched with vehicle-controlled rats. RESULTS: In the partial hepatectomy model, rats treated with cerium oxide nanoparticles showed a significant increase in liver regeneration, compared with control rats. In the acetaminophen experimental model, nanoparticles and N-acetyl-cysteine treatments decreased early liver damage in hepatic tissue. However, only the effect of cerium oxide nanoparticles was associated with a significant increment in hepatocellular proliferation. This treatment also reduced stress markers and increased cell cycle progression in hepatocytes and the activation of the transcription factor NF-κB in vitro and in vivo. CONCLUSIONS: Our results demonstrate that the nanomaterial cerium oxide, besides their known antioxidant capacities, can enhance hepatocellular proliferation in experimental models of liver regeneration and drug-induced hepatotoxicity.

Cerium oxide nanoparticles display antilipogenic effect in rats with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide, ranging from steatosis to non-alcoholic steatohepatitis (NASH). Recently, cerium oxide nanoparticles (CeO2NPs) have emerged as a new antioxidant agent with hepatoprotective properties in experimental liver disease. The aim of the current investigation was to elucidate whether CeO2NPs display beneficial effects in an experimental model of NAFLD.Therefore, fifteen Wistar rats were subjected to a methionine and choline deficient diet (MCDD) for 6 weeks and intravenously treated with CeO2NP or vehicle during the weeks three and four of the diet. The effect of CeO2NPs on serum biochemistry, hepatic steatosis, inflammation, fatty acid content and expression of reactive oxygen species (ROS) and lipid metabolism related genes was assessed. MCDD fed rats showed increased inflammation, enhanced hepatic lipid accumulation of both saturated and unsaturated fatty acids (FAs) and overexpression of genes related to fatty liver and ROS metabolism. Treatment with CeO2NPs was able to reduce the size and content of hepatocyte lipid droplets, the hepatic concentration of triglyceride- and cholesterol ester-derived FAs and the expression of several genes involved in cytokine, adipokine and chemokine signaling pathways. These findings suggest that CeO2NPs could be of beneficial value in NAFLD.

Beyond the Scavenging of Reactive Oxygen Species (ROS): Direct Effect of Cerium Oxide Nanoparticles in Reducing Fatty Acids Content in an In Vitro Model of Hepatocellular Steatosis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic accumulation of lipids. Antisteatotic effects of cerium oxide nanoparticles (CeO2NPs) have recently been shown in animal models of liver disease. However, it is unclear whether the activity of CeO2NPs is related solely to the decrease in oxidative stress or, in addition, they directly decrease liver fatty acid accumulation. To address this question, in this work, we used an in vitro model of hepatocellular steatosis, exposing HepG2 cells to oleic and palmitic acid. Cell uptake of CeO2NPs and their effect on oxidative stress and viability of hepatic cells cultured with H2O2 were also evaluated. Results show that CeO2NPs were uptaken by HepG2 cells and reduced oxidative stress and improved cell viability. Treatment with oleic and palmitic acid increased lipogenesis and the content of different fatty acids. CeO2NPs reduced palmitic and stearic acid and most fatty acids consisting of more than 18 carbon atoms. These effects were associated with significant changes in elongase and desaturase activity. In conclusion, CeO2NPs directly protected HepG2 cells from cell injury in oxidative stress conditions and reduced fatty acid content in steatotic conditions by inducing specific changes in fatty acid metabolism, thus showing potential in the treatment of NAFLD.

Metastatic Tissue Proteomic Profiling Predicts 5-Year Outcomes in Patients with Colorectal Liver Metastases.

Colorectal cancer (CRC) is one of the most common cancers in the developed countries, and nearly 70% of patients with CRC develop colorectal liver metastases (CRLMs). During the last decades, several scores have been proposed to predict recurrence after CRLM resection. However, these risk scoring systems do not accurately reflect the prognosis of these patients. Therefore, this investigation was designed to identify a proteomic profile in human hepatic tumor samples to classify patients with CRLM as "mild" or "severe" based on the 5-year survival. The study was performed on 85 CRLM tumor samples. Firstly, to evaluate any distinct tumor proteomic signatures between mild and severe CRLM patients, a training group of 57 CRLM tumor samples was characterized by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, and a classification and regression tree (CART) analysis was subsequently performed. Finally, 28 CRLM tumor samples were used to confirm and validate the results obtained. Based on all the protein peaks detected in the training group, the CART analysis was generated, and four peaks were considered to be the most relevant to construct a diagnostic algorithm. Indeed, the multivariate model yielded a sensitivity of 85.7% and a specificity of 86.1%, respectively. In addition, the receiver operating characteristic (ROC) curve showed an excellent diagnostic accuracy to discriminate mild from severe CRLM patients (area under the ROC: 0.903). Finally, the validation process yielded a sensitivity and specificity of 68.8% and 83.3%, respectively. We identified a proteomic profile potentially useful to determine the prognosis of CRLM patients based on the 5-year survival.