Paracetamol-induced Liver Injury in An Experimental Rat Model: Noninvasive Assessment using DKI and the Effect of Gadoxetate on DKI Parameters.

PURPOSE: To explore the potential of diffusion kurtosis imaging (DKI) for assessing the degree of liver injury in a paracetamol-induced rat model and to simultaneously investigate the effect of intravenous gadoxetate on DKI parameters. METHODS: Paracetamol was used to induce hepatoxicity in 39 rats. The rats were pathologically classified into 3 groups: normal (n=11), mild necrosis (n=18), and moderate necrosis (n=10). DKI was performed before and, 15 min, 25 min, and 45 min after gadoxetate administration. Repeated-measures ANOVA with Tukey's multiple comparison test was used to investigate the effect of gadoxetate on mean diffusivity (MD) and mean diffusion kurtosis (MK) and to assess the differences in MD and MK among the three groups. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic accuracy of the MD values when discriminating between the necrotic groups. RESULTS: Gadoxetate had no significant effect on either the MD or the MK, and the effect size was small. The MD in the moderate necrosis group was significantly lower than that in the other two groups (F = 13.502, p < 0.001; η2 = 0.428 [95% CI: 0.082-0.637]), while the MK did not significantly differ among the three groups (F = 2.702, p = 0.081; η2 = 0.131 [95% CI: 0.001-0.4003]). The AUCs of MD for discriminating the moderate necrosis or normal group from the other groups were 0.921 (95% CI: 0.832-1.000) and 0.831 (95% CI: 0.701-0.961), respectively. CONCLUSION: It would be better to measure the MD and MK before gadoxetate injection. MD showed potential for assessing the degree of liver necrosis in a paracetamol-induced liver injury rat model.

Development of a lung-liver in vitro coculture model for inhalation-like toxicity assessment.

Animal models are considered prime study models for inhalation-like toxicity assessment. However, in light of animal experimentation reduction (3Rs), we developed and investigated an alternative in vitro method to study systemic-like responses to inhalation-like exposures. A coculture platform was established to emulate inter-organ crosstalks between a pulmonary barrier, which constitutes the route of entry of inhaled compounds, and the liver, which plays a major role in xenobiotic metabolism. Both compartments (Calu-3 insert and HepG2/C3A biochip) were jointly cultured in a dynamically-stimulated environment for 72 h. The present model was characterized using acetaminophen (APAP), a well-documented hepatotoxicant, to visibly assess the passage and circulation of a xenobiotic through the device. Based on viability and functionality parameters the coculture model showed that the bronchial barrier and the liver biochip can successfully be maintained viable and function in a dynamic coculture setting for 3 days. In a stress-induced environment, present results reported that the coculture model emulated active and functional in vitro crosstalk that seemingly was responsive to xenobiotic exposure doses. The hepatic and bronchial cellular responses to xenobiotic exposure were modified in the coculture setting as they displayed earlier and stronger detoxification processes, highlighting active and functional organ crosstalk between both compartments.

Potential of paracetamol for reproductive disruption: molecular interaction, dynamics, and MM-PBSA based in-silico assessment.

Paracetamol is generally recommended for pain and fever. However, as per experimental and epidemiological data, widespread and irrational or long-term use of paracetamol may be harmful to human endocrine homeostasis, especially during pregnancy. Some researchers suggest that prenatal exposure to paracetamol might alter fetal development and also enhance the risk of reproductive disorders. An imbalance in the levels of these hormones may play a significant role in the emergence of various diseases, including infertility. Therefore, in this study, the interaction mechanism of paracetamol with reproductive hormone receptors was investigated by molecular docking, molecular dynamics (MD) simulations, and Poisson-Boltzmann surface area (MM-PBSA) for assessing paracetamol's potency to disrupt reproductive hormones. The results indicate that paracetamol has the ability to interact with reproductive hormone receptors (estrogen 1XP9; 1QKM with binding energy of -5.61 kcal/mol; -5.77 kcal/mol; androgen 5CJ6 - 5.63 kcal/mol; and progesterone 4OAR -5.60 kcal/mol) by hydrogen bonds as well as hydrophobic and van der Waals interactions to maintain its stability. In addition, the results of the MD simulations and MM-PBSA confirm that paracetamol and reproductive receptor complexes are stable. This research provides a molecular and atomic level understanding of how paracetamols disrupt reproductive hormone synthesis. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), Radius of Gyration and hydrogen bonding exhibited that paracetamol mimic at various attribute to bisphenol and native ligand.

Assessment of the cellular integrity and expression of melatonin receptor (MTNR1A) in the retina assaulted by ethanol and acetaminophen.

Ethanol exposures have been reported to disrupt the development of the retina and optic nerve which can be considered as part of underlying mechanisms of visual pathway impairments. This study aims to investigate the cellular integrity of the retina and the expression of melatonin receptor (MTNR1A) in the retina when assaulted chronically and simultaneously by ethanol and acetaminophen. Animals were randomly grouped into five groups. Control (normal saline), Alcohol group (25% alcohol in 2% sucrose solution), Acetaminophen group, (100 mg/kg BW for 14 days), Acetaminophen + Alcohol group (25% alcohol in 2% sucrose solution + 100 mg/kg BW of paracetamol). Withdrawal group (25% alcohol in 2% sucrose solution + 100 mg/kg BW of paracetamol). The body weight and rectal temperature of the animals were taking every 2 days and a post mortem study was conducted by quantitatively assessing the markers of oxidative stress. Melatonin level was quantified in the retina tissue and Immunohistochemistry was done via MTNR1A to study the expression of melatonin receptor type 1A in the retina. These results demonstrate that alcohol and acetaminophen significantly reduced the activity of retina rat melatonin (MTNR1A) levels, lowers the SOD and MDA activity. Expression of MTNR1A was reduced in the ganglionic cell layer of Alcohol and acetaminophen group as compared to the control and withdrawal group. It can be inferred that chronic simultaneous intake/consumption of alcohol and acetaminophen altered the melatonin level in the retina and this may implicate the circadian clock and melatonin in Wistar rat visual system.

Liver Acinus Dynamic Chip for Assessment of Drug-Induced Zonal Hepatotoxicity.

Zonation along the liver acinus is considered a key feature of liver physiology. Here, we developed a liver acinus dynamic (LADY) chip that recapitulates a key functional structure of the liver acinus and hepatic zonation. Corresponding to the blood flow from portal triads to the central vein in vivo, gradual flow of oxygen and glucose-carrying culture medium into the HepG2 cell chamber of the LADY chip generated zonal protein expression patterns in periportal (PP) zone 1 and perivenous (PV) zone 3. Higher levels of albumin secretion and urea production were obtained in a HepG2/HUVECs co-culture LADY chip than in HepG2 mono-culture one. Zonal expression of PEPCK as a PP marker and CYP2E1 as a PV marker was successfully generated. Cell death rate of the PV cells was higher than that of the PP cells since zonal factors responsible for metabolic activation of acetaminophen (APAP) were highly expressed in the PV region. We also found the co-culture enhanced metabolic capacity to process APAP, thus improving resistance to APAP toxicity, in comparison with HepG2 mono-culture. These results indicate that our LADY chip successfully represents liver zonation and could be useful in drug development studies as a drug-induced zonal hepatotoxicity testing platform.

The human hepatocyte TXG-MAPr: gene co-expression network modules to support mechanism-based risk assessment.

Mechanism-based risk assessment is urged to advance and fully permeate into current safety assessment practices, possibly at early phases of drug safety testing. Toxicogenomics is a promising source of mechanisms-revealing data, but interpretative analysis tools specific for the testing systems (e.g. hepatocytes) are lacking. In this study, we present the TXG-MAPr webtool (available at https://txg-mapr.eu/WGCNA_PHH/TGGATEs_PHH/ ), an R-Shiny-based implementation of weighted gene co-expression network analysis (WGCNA) obtained from the Primary Human Hepatocytes (PHH) TG-GATEs dataset. The 398 gene co-expression networks (modules) were annotated with functional information (pathway enrichment, transcription factor) to reveal their mechanistic interpretation. Several well-known stress response pathways were captured in the modules, were perturbed by specific stressors and showed preservation in rat systems (rat primary hepatocytes and rat in vivo liver), with the exception of DNA damage and oxidative stress responses. A subset of 87 well-annotated and preserved modules was used to evaluate mechanisms of toxicity of endoplasmic reticulum (ER) stress and oxidative stress inducers, including cyclosporine A, tunicamycin and acetaminophen. In addition, module responses can be calculated from external datasets obtained with different hepatocyte cells and platforms, including targeted RNA-seq data, therefore, imputing biological responses from a limited gene set. As another application, donors' sensitivity towards tunicamycin was investigated with the TXG-MAPr, identifying higher basal level of intrinsic immune response in donors with pre-existing liver pathology. In conclusion, we demonstrated that gene co-expression analysis coupled to an interactive visualization environment, the TXG-MAPr, is a promising approach to achieve mechanistic relevant, cross-species and cross-platform evaluation of toxicogenomic data.

Acetaminophen degradation by hydroxyl and organic radicals in the peracetic acid-based advanced oxidation processes: Theoretical calculation and toxicity assessment.

The research on the mechanisms and kinetics of radical oxidation in peracetic acid-based advanced oxidation processes was relatively limited. In this work, HO• and organic radicals mediated reactions of acetaminophen (ACT) were investigated, and the reactivities of important organic radicals (CH3COO• and CH3COOO•) were calculated. The results showed that initiated reaction rate constants of ACT are in the order: CH3COO• (5.44 × 1010 M-1 s-1) > HO• (7.07 × 109 M-1 s-1) > CH3O• (1.57 × 107 M-1 s-1) > CH3COOO• (3.65 × 105 M-1 s-1) >> •CH3 (5.17 × 102 M-1 s-1) > CH3C•O (1.17 × 102 M-1 s-1) > CH3OO• (11.80 M-1 s-1). HO•, CH3COO• and CH3COOO• play important roles in ACT degradation. CH3COO• is another important radical in the hydroxylation of aromatic compounds in addition to HO•. Reaction rate constants of CH3COO• and aromatic compounds are 1.40 × 106 - 6.25 × 1010 M-1 s-1 with addition as the dominant pathway. CH3COOO• has high reactivity to phenolate and aniline only among the studied aromatic compounds, and it was more selective than CH3COO•. CH3COO•-mediated hydroxylation of aromatic compounds could produce their hydroxylated products with higher toxicity.

Assessment of the biochemical pathways for acetaminophen toxicity: Implications for its carcinogenic hazard potential.

In 2019 California's Office of Environmental Health Hazard Assessment (OEHHA) initiated a review of the carcinogenic hazard potential of acetaminophen. In parallel with this review, herein we evaluated the mechanistic data related to the steps and timing of cellular events following therapeutic recommended (≤4 g/day) and higher doses of acetaminophen that may cause hepatotoxicity to evaluate whether these changes indicate that acetaminophen is a carcinogenic hazard. At therapeutic recommended doses, acetaminophen forms limited amounts of N-acetyl-p-benzoquinone-imine (NAPQI) without adverse cellular effects. Following overdoses of acetaminophen, there is potential for more extensive formation of NAPQI and depletion of glutathione, which may result in mitochondrial dysfunction and DNA damage, but only at doses that result in cell death - thus making it implausible for acetaminophen to induce the kind of stable, genetic damage in the nucleus indicative of a genotoxic or carcinogenic hazard in humans. The collective data demonstrate a lack of a plausible mechanism related to carcinogenicity and are consistent with rodent cancer bioassays, epidemiological results reviewed in companion manuscripts in this issue, as well as conclusions of multiple international health authorities.

Acute effects of acetaminophen on the developmental, swimming performance and cardiovascular activities of the African catfish embryos/larvae (Clarias gariepinus).

Acetaminophen is a widely used analgesic that has been detected in many water bodies with few reports concerning its potential toxicity to fish. This study sought to assess the developmental, swimming performance and cardiovascular activities of embryo/larvae catfish (Clarias gariepinus) exposed to acetaminophen. The Organization for Economic Development (OECD) Fish Embryo Acute Toxicity Test (OECD 236) was employed. Fertilized embryo were exposed to different concentrations of acetaminophen (0, 0.5, 1, 10 µg/L) for 96 h. Hatching rates of the embryo were observed to decrease with increasing concentrations of acetaminophen. Fish embryo exposed to acetaminophen displayed varying levels of teratogenic effects at different levels of development in a dose-dependent manner. The results also showed a significant (p < 0.05) dose-dependent increase in swimming speed and movement patterns in fish larvae exposed to acetaminophen, with distance travelled in larvae exposed to the highest concentration of acetaminophen (10 µg/L) about eight (8) times the distance travelled by the control larvae, indicating that acetaminophen-induced erratic swimming behaviour in the catfish species. Cardiotoxicity was evident, with a significant reduction in heartbeat rate with increasing concentrations of acetaminophen. The results showed that exposure to acetaminophen resulted in teratogenic, neurotoxic and cardiotoxic effects in embryo/larvae of Clarias gariepinus. The findings suggest that acetaminophen which has recently been detected in many water bodies could potentially impact on survival of aquatic life, especially catfish.

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 µM and 2 µM respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

Assessment of antioxidant traits and protective action of Egyptian acacia pods extracts against paracetamol-induced liver toxicity in rats.

This study investigates the protective effect of Egyptian acacia pod extracts against overdose of paracetamol-induced liver damage. Egyptian acacia green and brown pods were extracted by mixture of ethanol 80%: HCl (6 M) (99:1 v/v). In extracts of green and brown pods, total phenolic content in hydrolyzed ethyl acetate fraction (HEF) at pH 4, was 649.89 and 712.14 mg GAE/g while antioxidant activity was 95.55% and 97.35%, both being the highest than any fraction. HEF (pH 4) in brown pods was analyzed by HPLC, there were 22 phenolic compounds rich in ethyl vanillin about 227 mg/g and 11 flavonoids rich in catechin 48.70 mg/g. A biological experiment was conducted using HEF (pH4) in brown pods against overdose of paracetamol in albino rats induced to hepatotoxicity. Thirty rats were divided into five groups; a control group, a paracetamol group, and the other three received paracetamol plus silymarin or two doses of HEF. Animals were received paracetamol and treated with either silymarin or HEF showed reduced levels of liver (ALT, AST, and ALP) and kidney (urea, creatinine, and uric acid) markers compared with the control group as well as reduction of oxidative stress and increment antioxidant enzyme activity in liver tissue when compared with the paracetamol group. It could be concluded that both HEF and silymarin are considerably high hepatoprotector against paracetamol-induced hepatotoxicity in rats due to their strong antioxidant activity. PRACTICAL APPLICATIONS: Both HEF and silymarin improved liver functions and exerted strong antioxidant activities. This antioxidant activity would have a positive effect against oxidative liver damage caused by parcetamol. Thus, it may be concluded that the liver plasma membranes were protected and the regenerative and reparative capacity of liver by phenolic compound in HEF treatment. The study demonstrated the HEF hepatoprotective activity and recommends using Egyptian acacia pods for treatment of liver disorders.

Application of tail vein serial microsampling for plasma or dried plasma spots in toxicokinetic assessment in rats using acetaminophen as the model compound.

In the current study, two groups of rats (five per group) were administered a single oral dose of 500 mg/kg acetaminophen. For toxicokinetic assessment, the Group 1 animals were bled via conventional sparse (two animals/time point) sublingual vein bleeding (~0.5 ml) with anesthesia, while the Group 2 animals were bled via serial tail vein microsampling (~0.075 ml) without anesthesia. All collected blood was processed for plasma. Each Group 2 plasma sample (~30 µl) was divided into 'wet' and 'dried' (dried plasma spots). All plasma samples were analyzed by LC-MS/MS for acetaminophen and its major metabolites acetaminophen glucuronide and acetaminophen sulfate. In addition, plasma and urine samples were collected for analysis of corticosterone and creatinine to assess stress levels. Comparable plasma exposure to acetaminophen and its two metabolites was observed in the plasma obtained via conventional sparse sublingual vein bleeding and serial tail vein microsampling and between the 'wet' and 'dried' plasma obtained by the latter. Furthermore, comparable corticosterone levels or corticosterone/creatinine ratios between the two groups suggested that serial microsampling without anesthesia did not increase the levels of stress as compared with conventional sampling with anesthesia, confirming the utility of microsampling for plasma or dried plasma spots in rodent toxicokinetic assessment.

Contrasting model mechanisms of alanine aminotransferase (ALT) release from damaged and necrotic hepatocytes as an example of general biomarker mechanisms.

Interpretations of elevated blood levels of alanine aminotransferase (ALT) for drug-induced liver injury often assume that the biomarker is released passively from dying cells. However, the mechanisms driving that release have not been explored experimentally. The usefulness of ALT and related biomarkers will improve by developing mechanism-based explanations of elevated levels that can be expanded and elaborated incrementally. We provide the means to challenge the ability of closely related model mechanisms to generate patterns of simulated hepatic injury and ALT release that scale (or not) to be quantitatively similar to the wet-lab validation targets, which are elevated plasma ALT values following acetaminophen (APAP) exposure in mice. We build on a published model mechanism that helps explain the generation of characteristic spatiotemporal features of APAP hepatotoxicity within hepatic lobules. Discrete event and agent-oriented software methods are most prominent. We instantiate and leverage a small constellation of concrete model mechanisms. Their details during execution help bring into focus ways in which particular sources of uncertainty become entangled with cause-effect details within and across several levels. We scale ALT amounts in virtual mice directly to target plasma ALT values in individual mice. A virtual experiment comprises a set of Monte Carlo simulations. We challenge the sufficiency of four potentially explanatory theories for ALT release. The first of the tested model theories failed to achieve the initial validation target, but each of the three others succeeded. Results for one of the three model mechanisms matched all target ALT values quantitatively. It explains how ALT externalization is the combined consequence of lobular-location-dependent drug-induced cellular damage and hepatocyte death. Falsification of one (or more) of the model mechanisms provides new knowledge and incrementally shrinks the constellation of model mechanisms. The modularity and biomimicry of our explanatory models enable seamless transition from mice to humans.

Assessment of oxidative stress of paracetamol to Daphnia magna via determination of Nrf1 and genes related to antioxidant system.

Paracetamol (APAP) is one of the most widely used anti-inflammatory and analgesic drugs in human being health care and has been universally detected in various aquatic environments. However, its potential adverse effects and toxic mechanisms on freshwater invertebrates still remain unclear. In the present study, the effects of APAP on the expressions of Nrf1 and the antioxidant related genes including GCLC, GST, GPX, CAT, TRX, TrxR and Prx1 in Daphnia magna (D. magna) were evaluated after 24, 48 and 96 h, and the changes of GPX, GST and CAT enzyme activities, as well as the GSH and MDA content under APAP exposure for 48 h were also determined. Results showed that paracetamol affected the expressions of Nrf1 and antioxidant related genes in D. magna, which were related to the exposure time and concentration of APAP. Nrf1 was inhibited at 48 h, but induced at 96 h under the APAP exposure, being about two fold of the control in 5.0 µg/L. CAT were significantly induced in all treatments. But Prx decreased in an concentration-dependent manner in all treatments. In comparison with the mRNA expression, antioxidant enzymes activity displayed less changes in D. magna. Overall, APAP exposure altered the expression of Nrf1 and genes related to antioxidant system and disturbed the redox homeostasis of D. magna.

Dynamic and accurate assessment of acetaminophen-induced hepatotoxicity by integrated photoacoustic imaging and mechanistic biomarkers in vivo.

The prediction and understanding of acetaminophen (APAP)-induced liver injury (APAP-ILI) and the response to therapeutic interventions is complex. This is due in part to sensitivity and specificity limitations of currently used assessment techniques. Here we sought to determine the utility of integrating translational non-invasive photoacoustic imaging of liver function with mechanistic circulating biomarkers of hepatotoxicity with histological assessment to facilitate the more accurate and precise characterization of APAP-ILI and the efficacy of therapeutic intervention. Perturbation of liver function and cellular viability was assessed in C57BL/6J male mice by Indocyanine green (ICG) clearance (Multispectral Optoacoustic Tomography (MSOT)) and by measurement of mechanistic (miR-122, HMGB1) and established (ALT, bilirubin) circulating biomarkers in response to the acetaminophen and its treatment with acetylcysteine (NAC) in vivo. We utilised a 60% partial hepatectomy model as a situation of defined hepatic functional mass loss to compared acetaminophen-induced changes to. Integration of these mechanistic markers correlated with histological features of APAP hepatotoxicity in a time-dependent manner. They accurately reflected the onset and recovery from hepatotoxicity compared to traditional biomarkers and also reported the efficacy of NAC with high sensitivity. ICG clearance kinetics correlated with histological scores for acute liver damage for APAP (i.e. 3h timepoint; r=0.90, P<0.0001) and elevations in both of the mechanistic biomarkers, miR-122 (e.g. 6h timepoint; r=0.70, P=0.005) and HMGB1 (e.g. 6h timepoint; r=0.56, P=0.04). For the first time we report the utility of this non-invasive longitudinal imaging approach to provide direct visualisation of the liver function coupled with mechanistic biomarkers, in the same animal, allowing the investigation of the toxicological and pharmacological aspects of APAP-ILI and hepatic regeneration.

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

In vitro assessment of hepatoprotective agents against damage induced by acetaminophen and CCl4.

BACKGROUND: In vitro bioassays are important in the evaluation of plants with possible hepatoprotective effects. The aims of this study were to evaluate the pretreatment of HepG2 cells with hepatoprotective agents against the damage induced by carbon tetrachloride (CCl4) and paracetamol (APAP). METHODS: Antioxidative activity was measured using an assay to measure 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging. The in vitro hepatotoxicity of CCl4 and APAP, and the cytotoxic and hepatoprotective properties of silymarin (SLM), silybinin (SLB), and silyphos (SLP) were evaluated by measuring cell viability; activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH); total antioxidant capacity (TAOxC); and reduced glutathione (GSH), superoxide dismutase (SOD), and lipid peroxidation (malondialdehyde (MDA) levels). RESULTS: Only SLB and SLM showed strong antioxidative activity in the DPPH assay (39.71 ± 0.85 µg/mL and 14.14 ± 0.65 µg/mL, respectively). CCl4 induced time- and concentration-dependent changes. CCl4 had significant effects on cell viability, enzyme activities, lipid peroxidation, TAOxC, and SOD and GSH levels. These differences remained significant up to an exposure time of 3 h. APAP induced a variety of dose- and time-dependent responses up to 72 h of exposure. SLM, SLB, and SLP were not cytotoxic. Only SLB at a concentration of 100 µg/mL or 150 µg/mL significantly decreased the enzyme activities and MDA level, and prevented depletion of total antioxidants compared with CCl4. CONCLUSIONS: CCl4 was more consistent than APAP in inducing cell injury. Only SLB provided hepatoprotection. AST, LDH, and MDA levels were good markers of liver damage.

Occurrence and preliminarily environmental risk assessment of selected pharmaceuticals in the urban rivers, China.

Twelve selected pharmaceuticals including antibiotics, analgesics, antiepileptics and lipid regulators were analysed and detected in water samples collected from 18 sampling sections along the three main urban rivers in Yangpu District of Shanghai, China during four sampling campaigns. Besides, algal growth inhibition test was conducted to preliminarily assess the eco-toxicology induced by the target pharmaceuticals in the rivers. Mean levels for most of target compounds were generally below 100 ng/L at sampling sections, with the exception of caffeine and paracetamol presenting considerably high concentration. The detected pharmaceuticals in the urban rivers ranged from

Leading articles in medical journals in 1966.

The British Journal of Hospital Medicine is 50 years old. This article takes a look back at articles published during the year of its inception from the British Medical Journal, the Lancet and the Journal of the American Medical Association.

Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.