Hepatotoxicity induced by nanomaterials: mechanisms and in vitro models.

The unique physicochemical properties of materials at nanoscale have opened a plethora of opportunities for applications in the pharmaceutical and medical field, but also in consumer products from food and cosmetics industries. As a consequence, daily human exposure to nanomaterials through distinct routes is considerable and, therefore, may raise health concerns. Many nanomaterials have been described to accumulate and induce adversity in the liver. Among these, silica and some types of metallic nanoparticles are the most broadly used in consumer products and, therefore, the most studied and reported. The reviewed literature was collected from PubMed.gov during the month of March 2020 using the search words "nanomaterials induced hepatotoxicity", which yielded 181 papers. This present paper reviews the hepatotoxic effects of nanomaterials described in in vitro and in vivo studies, with emphasis on the underlying mechanisms. The induction of oxidative stress and inflammation are the manifestations of toxicity most frequently reported following exposure of cells or animal models to different nanomaterials. Furthermore, the available in vitro models for the evaluation of the hepatotoxic effects of nanomaterials are discussed, highlighting the continuous interest in the development of more advanced and reliable in vitro models for nanotoxicology.

Primary Human Hepatocyte Spheroids as Tools to Study the Hepatotoxic Potential of Non-Pharmaceutical Chemicals.

Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro.

Robustness testing and optimization of an adverse outcome pathway on cholestatic liver injury.

Adverse outcome pathways (AOPs) have been recently introduced as tools to map the mechanisms underlying toxic events relevant for chemical risk assessment. AOPs particularly depict the linkage between a molecular initiating event and an adverse outcome through a number of intermediate key events. An AOP has been previously introduced for cholestatic liver injury. The objective of this study was to test the robustness of this AOP for different types of cholestatic insult and the in vitro to in vivo extrapolation. For this purpose, in vitro samples from human hepatoma HepaRG cell cultures were exposed to cholestatic drugs (i.e. intrahepatic cholestasis), while in vivo samples were obtained from livers of cholestatic mice (i.e. extrahepatic cholestasis). The occurrence of cholestasis in vitro was confirmed through analysis of bile transporter functionality and bile acid analysis. Transcriptomic analysis revealed inflammation and oxidative stress as key events in both types of cholestatic liver injury. Major transcriptional differences between intrahepatic and extrahepatic cholestatic liver insults were observed at the level of cell death and metabolism. Novel key events identified by pathway analysis included endoplasmic reticulum stress in intrahepatic cholestasis, and autophagy and necroptosis in both intrahepatic as extrahepatic cholestasis. This study demonstrates that AOPs constitute dynamic tools that should be frequently updated with new input information.

Magnetic Hyperthermia for Cancer Treatment: Main Parameters Affecting the Outcome of In Vitro and In Vivo Studies.

Magnetic hyperthermia (MHT) is being investigated as a cancer treatment since the 1950s. Recent advancements in the field of nanotechnology have resulted in a notable increase in the number of MHT studies. Most of these studies explore MHT as a stand-alone treatment or as an adjuvant therapy in a preclinical context. However, despite all the scientific effort, only a minority of the MHT-devoted nanomaterials and approaches made it to clinical context. The outcome of an MHT experiment is largely influenced by a number of variables that should be considered when setting up new MHT studies. This review highlights and discusses the main parameters affecting the outcome of preclinical MHT, aiming to provide adequate assistance in the design of new, more efficient MHT studies.

Industrial, Biocide, and Cosmetic Chemical Inducers of Cholestasis.

A frequent side effect of many drugs includes the occurrence of cholestatic liver toxicity. Over the past couple of decades, drug-induced cholestasis has gained considerable attention, resulting in a plethora of data regarding its prevalence and mechanistic basis. Likewise, several food additives and dietary supplements have been reported to cause cholestatic liver insults in the past few years. The induction of cholestatic hepatotoxicity by other types of chemicals, in particular synthetic compounds, such as industrial chemicals, biocides, and cosmetic ingredients, has been much less documented. Such information can be found in occasional clinical case reports of accidental intake or suicide attempts as well as in basic and translational study reports on mechanisms or testing of new therapeutics in cholestatic animal models. This paper focuses on such nonpharmaceutical and nondietary synthetic chemical inducers of cholestatic liver injury, in particular alpha-naphthylisocyanate, 3,5-diethoxycarbonyl-1,4-dihydrocollidine, methylenedianiline, paraquat, tartrazine, triclosan, 2-octynoic acid, and 2-nonynoic acid. Most of these cholestatic compounds act by similar mechanisms. This could open perspectives for the prediction of cholestatic potential of chemicals.

Mechanisms and in vitro models of drug-induced cholestasis.

Cholestasis underlies one of the major manifestations of drug-induced liver injury. Drug-induced cholestatic liver toxicity is a complex process, as it can be triggered by a variety of factors that induce 2 types of biological responses, namely a deteriorative response, caused by bile acid accumulation, and an adaptive response, aimed at removing the accumulated bile acids. Several key events in both types of responses have been characterized in the past few years. In parallel, many efforts have focused on the development and further optimization of experimental cell culture models to predict the occurrence of drug-induced cholestatic liver toxicity in vivo. In this paper, a state-of-the-art overview of mechanisms and in vitro models of drug-induced cholestatic liver injury is provided.

The mixture of "ecstasy" and its metabolites is toxic to human SH-SY5Y differentiated cells at in vivo relevant concentrations.

The neurotoxicity of "ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) is thought to involve hepatic metabolism, though its real contribution is not completely understood. Most in vitro neurotoxicity studies concern isolated exposures of MDMA or its metabolites, at high concentrations, not considering their mixture, as expected in vivo. Therefore, our postulate is that combined deleterious effects of MDMA and its metabolites, at low micromolar concentrations that may be attained into the brain, may elicit neurotoxicity. Using human SH-SY5Y differentiated cells as dopaminergic neuronal model, we studied the neurotoxicity of MDMA and its MDMA metabolites α-methyldopamine and N-methyl-α-methyldopamine and their correspondent glutathione and N-acetylcysteine monoconjugates, under isolated exposure and as a mixture, at normothermic or hyperthermic conditions. The results showed that the mixture of MDMA and its metabolites was toxic to SH-SY5Y differentiated cells, an effect potentiated by hyperthermia and prevented by N-acetylcysteine. As a mixture, MDMA and its metabolites presented a different toxicity profile, compared to each compound alone, even at equimolar concentrations. Caspase 3 activation, increased reactive oxygen species production, and intracellular Ca(2+) raises were implicated in the toxic effect. The mixture increased intracellular glutathione levels by increasing its de novo synthesis. In conclusion, this study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at low micromolar concentrations, which represents a more realistic approach of the in vivo scenario, elicited toxicity to human SH-SY5Y differentiated cells, thus constituting a new insight into the context of MDMA-related neurotoxicity.

Particulate matter-induced oxidative stress - Mechanistic insights and antioxidant approaches reported in in vitro studies.

Inhaled particulate matter (PM) is a key factor in millions of yearly air pollution-related deaths worldwide. The oxidative potential of PM indicates its ability to promote an oxidative environment. Excessive reactive oxygen species (ROS) can cause cell damage via oxidative stress, leading to inflammation, endoplasmic reticulum stress, airway remodeling, and various cell death modes (apoptosis, ferroptosis, pyroptosis). ROS can also interact with macromolecules, inducing DNA damage and epigenetic modifications, disrupting homeostasis. These effects have been studied extensively in vitro and confirmed in vivo. This review explores the oxidative potential of airborne particles and PM-induced ROS-mediated cellular damage observed in vitro, highlighting the link between oxidative stress, inflammation, and cell death modes described in the latest literature. The review also analyzes the effects of ROS on DNA damage, repair, carcinogenicity, and epigenetics. Additionally, the latest developments on the potential of antioxidants to prevent ROS's harmful effects are described, providing future perspectives on the topic.

Differential impact of diesel exhaust particles on glutamatergic and dopaminergic neurons in Caenorhabditis elegans: A neurodegenerative perspective.

The growing body of evidence links exposure to particulate matter pollutants with an increased risk of neurodegenerative diseases. In the present study, we investigated whether diesel exhaust particles can induce neurobehavioral alterations associated with neurodegenerative effects on glutamatergic and dopaminergic neurons in Caenorhabditis elegans (C. elegans). Exposure to DEP at concentrations of 0.167 µg/cm2 and 1.67 µg/cm2 resulted in significant developmental delays and altered locomotion behaviour. These effects were accompanied by discernible alterations in the expressions of antioxidant genes sod-3 and gst-4 observed in transgenic strains. Behaviour analysis demonstrated a significant reduction in average speed (p < 0.001), altered paths, and decreased swimming activities (p < 0.01), particularly at mid and high doses. Subsequent assessment of neurodegeneration markers in glutamatergic (DA1240) and dopaminergic (BZ555) transgenic worms revealed notable glutamatergic neuron degeneration at 0.167 µg/cm2 (∼30 % moderate, ∼20 % advanced) and 1.67 µg/cm2 (∼28 % moderate, ∼24 % advanced, p < 0.0001), while dopaminergic neurons exhibited structural deformities (∼16 %) without significant degeneration in terms of blebs and breaks. Furthermore, in silico docking simulations suggest the presence of an antagonistic competitive inhibition induced by DEP in the evaluated neuro-targets, stronger for the glutamatergic transporter than for the dopaminergic receptor from the comparative binding affinity point of view. The results underscore DEP's distinctive neurodegenerative effects and suggest a link between locomotion defects and glutamatergic neurodegeneration in C. elegans, providing insights into environmental health risks assessment.

Cocaine-induced kidney toxicity: an in vitro study using primary cultured human proximal tubular epithelial cells.

Renal failure resulting from cocaine abuse has been well documented, although the underlying mechanisms remain to be investigated. In the present study, primary cultured human proximal tubular epithelial cells (HPTECs) of the kidney were used to investigate its ability to metabolize cocaine, as well as the cytotoxicity induced by cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME) and norcocaine (NCOC). Gas chromatography/ion trap-mass spectrometry (GC/IT-MS) analysis of HPTECs exposed to cocaine (1 mM) for 72 h confirmed its metabolism into EME and NCOC, but not BE. EME levels increased along the exposure time to cocaine, while NCOC concentration diminished after reaching a maximum at 6 h, indicating a possible secondary metabolism for this metabolite. Cocaine promoted a concentration-dependent loss of cell viability, whereas BE and EME were found to be non-toxic to HPTECs at the tested conditions. In contrast, NCOC revealed to have higher intrinsic nephrotoxicity than the parent compound. Moreover, cocaine-induced cell death was partially reversed in the presence of ketoconazole (KTZ), a potent CYP3A inhibitor, supporting the hypothesis that NCOC may play a role in cocaine-induced nephrotoxicity. Cocaine-induced cytotoxicity was found to involve intracellular glutathione depletion at low concentrations and to induce mitochondrial damage at higher concentrations. Under the present experimental conditions, HPTECs death pathway followed an apoptotic pattern, which was evident for concentrations as low as 0.1 mM.

Adverse Outcome Pathways as Versatile Tools in Liver Toxicity Testing.

Adverse outcome pathways (AOPs) are tools to capture and visualize mechanisms driving toxicological effects. They share a common structure consisting of a molecular initiating event, a series of key events connected by key event relationships and an adverse outcome. Development and evaluation of AOPs ideally comply with guidelines issued by the Organization for Economic Cooperation and Development. AOPs have been introduced for major types of hepatotoxicity, which is not a surprise, as the liver is a frequent target for systemic adversity. Various applications for AOPs have been proposed in the areas of toxicology and chemical risk assessment, in particular in relation to the establishment of quantitative structure-activity relationships, the elaboration of prioritization strategies, and the development of novel in vitro toxicity screening tests and testing strategies.

Expression of connexins and pannexins in diseased human liver.

Connexin proteins can form hexameric hemichannels and gap junctions that mediate paracrine and direct intercellular communication, respectively. Gap junction activity is crucial for the maintenance of hepatic homeostasis, while connexin hemichannels become particularly active in liver disease, such as hepatitis, fibrosis, cholestasis or even hepatocellular carcinoma. Channels consisting of connexin-like proteins named pannexins have been directly linked to liver inflammation and cell death. The goal of the present study was to characterize the expression and subcellular localization of connexins and pannexins in liver of patients suffering from various chronic and neoplastic liver diseases. Specifically, real-time quantitative reverse transcription polymerase chain reaction, immunoblotting and immunohistochemistry analyses were performed on human liver biopsies. It was found that pannexin1 and pannexin2 gene expression are correlated to a certain degree, as is pannexin1 protein expression with connexin32 and connexin43 protein expression. Furthermore, this study is the first to detect pannexin3 in human patient liver biopsies via both immunoblot and immunohistochemistry.

Nanosafety: An Evolving Concept to Bring the Safest Possible Nanomaterials to Society and Environment.

The use of nanomaterials has been increasing in recent times, and they are widely used in industries such as cosmetics, drugs, food, water treatment, and agriculture. The rapid development of new nanomaterials demands a set of approaches to evaluate the potential toxicity and risks related to them. In this regard, nanosafety has been using and adapting already existing methods (toxicological approach), but the unique characteristics of nanomaterials demand new approaches (nanotoxicology) to fully understand the potential toxicity, immunotoxicity, and (epi)genotoxicity. In addition, new technologies, such as organs-on-chips and sophisticated sensors, are under development and/or adaptation. All the information generated is used to develop new in silico approaches trying to predict the potential effects of newly developed materials. The overall evaluation of nanomaterials from their production to their final disposal chain is completed using the life cycle assessment (LCA), which is becoming an important element of nanosafety considering sustainability and environmental impact. In this review, we give an overview of all these elements of nanosafety.

P-glycoprotein activity in human Caucasian male lymphocytes does not follow its increased expression during aging.

P-glycoprotein (P-gp) is a transmembrane protein that mediates the efflux of innumerous structurally unrelated compounds. It was initially found over-expressed in tumor cells, associated to a multidrug resistance phenotype (MDR). Then, P-gp was found constitutively expressed in excretory cells/tissues and in circulating cells, such as lymphocytes. Considering the importance of this transporter in the establishment of therapeutic protocols and the existence of contradictory results, this study aimed at evaluating the influence of aging in the expression and function of P-gp in human lymphocytes, comparing two different methodologies to assess both parameters. P-gp activity and expression were evaluated in lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, divided into two groups according to age (group 1: under 30-years old; group 2: above 60-years old). P-gp expression was assessed using the anti-P-gp monoclonal antibody, UIC2, in the presence and in absence of vinblastine (Vbl). P-gp activity was evaluated measuring the efflux rate of the fluorescent P-gp substrate rhodamine 123 (Rho 123) and also using UIC2 shift assay. Flow cytometric analysis was performed to assess all the proceedings. Furthermore, P-gp expression and each of the P-gp activity determination methods were compared, through correlation analysis and linear regression models. We observed a significant age-dependent increase in mean P-gp expression (p = 0.029), which was not reflected in the transporter's activity (p > 0.050). Statistical analysis allowed selection of UIC2 shift assay over Rho 123 efflux assay as a more selective method to assess P-gp activity. Despite the significant correlation between P-gp expression and P-gp activity found in lymphocytes (Gp1(group 1)-r = 0.609, p < 0.001; Gp2-r = 0.461, p = 0.012), using UIC2 shift assay, these data reinforce the need for P-gp activity assessment, rather than P-gp expression determination alone, when starting new therapeutic regimens with P-gp substrates, especially in men older than 60 years of age.

Cholestatic liver injury induced by food additives, dietary supplements and parenteral nutrition.

Cholestasis refers to the accumulation of toxic levels of bile acids in the liver due to defective bile secretion. This pathological situation can be triggered by drugs, but also by ingredients contained in food, food supplements and parenteral nutrition. This paper provides an overview of the current knowledge on cholestatic injury associated with such ingredients, with particular emphasis on the underlying mechanisms of toxicity.

Biocompatibility and Bioimaging Potential of Fruit-Based Carbon Dots.

Photo-luminescent carbon dots (CD) have become promising nanomaterials and their synthesis from natural products has attracted attention by the possibility of making the most of affordable, sustainable and, readily-available carbon sources. Here, we report on the synthesis, characterization and bioimaging potential of CDs produced from diverse extensively produced fruits: kiwi, avocado and pear. The in vitro cytotoxicity and anticancer potential of those CDs were assessed by comparing human epithelial cells from normal adult kidney and colorectal adenocarcinoma cells. In vivo toxicity was evaluated using zebrafish embryos given their peculiar embryogenesis, with transparent embryos developing ex-utero, allowing a real-time analysis. In vitro and in vivo experiments revealed that the synthesized CD presented toxicity only at concentrations of ≥1.5 mg mL-1. Kiwi CD exhibited the highest toxicity to both cells lines and zebrafish embryos, presenting lower LD50 values. Interestingly, despite inducing lower cytotoxicity in normal cells than the other CDs, black pepper CDs resulted in higher toxicity in vivo. The bio-distribution of CD in zebrafish embryos upon uptake was investigated using fluorescence microscopy. We observed a higher accumulation of CD in the eye and yolk sac, avocado CD being the ones more retained, indicating their potential usefulness in bio-imaging applications. This study shows the action of fruit-based CDs from kiwi, avocado and pear. However the compounds present in these fruit-based CDs and their mechanism of action as a bioimaging agent need to be further explored.

Primary hepatocytes and their cultures for the testing of drug-induced liver injury.

Drug-induced liver injury is a major reason for discontinuation of drug development and withdrawal of drugs from the market. Intensive efforts in the last decades have focused on the establishment and finetuning of liver-based in vitro models for reliable prediction of hepatotoxicity triggered by drug candidates. Of those, primary hepatocytes and their cultures still are considered the gold standard, as they provide an acceptable reflection of the hepatic in vivo situation. Nevertheless, these in vitro systems cope with gradual deterioration of the differentiated morphological and functional phenotype. The present paper gives an overview of traditional and more recently introduced strategies to counteract this dedifferentiation process in an attempt to set up culture models that can be used for long-term testing purposes. The relevance and applicability of such optimized cultures of primary hepatocytes for the testing of drug-induced cholestatic liver injury is demonstrated.

Portable sensing system based on electrochemical impedance spectroscopy for the simultaneous quantification of free and total microcystin-LR in freshwaters.

Microcystins are the most worldwide extended and common toxins produced by cyanobacteria in freshwater. Microcystin-leucine arginine (MC-LR), associated with the most toxic incidents involving microcystins, are within the cyanobacteria (intracellular) until released into the surrounding waters (extracellular) during cell lysis. Therefore, the relationship between intracellular and extracellular cyanotoxins will allow a comprehensive risk of cyanobacteria-containing waters, preventing disease and improving human safety. In this work, we present the development of a novel portable microfluidic sensing platform for the simultaneous detection of free (extracellular) and total MC-LR (intracellular and extracellular). The integrated system contains the sample processing and detection modules capable of performing the chemical lysis, filtration, sample mixing with antibodies, and electrochemical detection of MC-LR based on an indirect strategy. The performance of the immunosensors was evaluated by electrochemical impedance spectroscopy, showing a linear dynamic range between 3.3 × 10-4 and 10-7 g L-1 and a limit of detection of 5.7 × 10-10 g L-1. The results demonstrate the potential of the developed portable biosensor platform and its suitable application for the analysis of MC-LR at regulated levels for drinking water. Finally, the integrated system was able to simultaneously detect the free and total MC-LR on a Microcystis aeruginosa culture. To the best of our knowledge this is the first described system that can differentiate between intracellular and extracellular concentration of MC-LR. This novel electrochemical sensing platform avoids the multiple processing steps typically needed for standard MC-LR analysis in the laboratory and provides an early warning system for MC-LR remote monitoring in water.

Increased Expression of Adherens Junction Components in Mouse Liver following Bile Duct Ligation.

Adherens junctions, consisting of cadherins and catenins, are a group of cell-to-cell junctions that mediate mechanistic linkage between neighboring cells. By doing so, adherens junctions ensure direct intercellular contact and play an indispensable role in maintaining tissue architecture. Considering these critical functions, it is not surprising that adherens junctions are frequently involved in disease. In the present study, the effects of bile duct ligation-a surgical procedure to experimentally induce cholestatic and fibrotic liver pathology-on hepatic adherens junctions were investigated in mice. In essence, it was found that liver mRNA and protein levels of E-cadherin, ß-catenin and γ-catenin drastically increase following bile duct ligation. These results could suggest a cytoprotective role for hepatic adherens junctions following bile duct ligation.

Green synthesis of fluorescent carbon dots from spices for in vitro imaging and tumour cell growth inhibition.

Carbon dots have demonstrated great potential as luminescent nanoparticles in bioapplications. Although such nanoparticles appear to exhibit low toxicity compared to other metal luminescent nanomaterials, today we know that the toxicity of carbon dots (C-dots) strongly depends on the protocol of fabrication. In this work, aqueous fluorescent C-dots have been synthesized from cinnamon, red chilli, turmeric and black pepper, by a one-pot green hydrothermal method. The synthesized C-dots were firstly characterized by means of UV-vis, fluorescence, Fourier transform infrared and Raman spectroscopy, dynamic light scattering and transmission electron microscopy. The optical performance showed an outstanding ability for imaging purposes, with quantum yields up to 43.6%. Thus, the cytotoxicity of the above mentioned spice-derived C-dots was evaluated in vitro in human glioblastoma cells (LN-229 cancer cell line) and in human kidney cells (HK-2 non-cancerous cell line). Bioimaging and viability studies were performed with different C-dot concentrations from 0.1 to 2 mg·mL-1, exhibiting a higher uptake of C-dots in the cancer cultures compared to the non-cancerous cells. Results showed that the spice-derived C-dots inhibited cell viability dose-dependently after a 24 h incubation period, displaying a higher toxicity in LN-229, than in HK-2 cells. As a control, C-dots synthesized from citric acid did not show any significant toxicity in either cancerous or non-cancerous cells, implying that the tumour cell growth inhibition properties observed in the spice-derived C-dots can be attributed to the starting material employed for their fabrication. These results evidence that functional groups in the surface of the C-dots might be responsible for the selective cytotoxicity, as suggested by the presence of piperine in the surface of black pepper C-dots analysed by ESI-QTOF-MS.