The use of sensitive biomarkers to monitor skeletal muscle toxicity in preclinical toxicity studies is important for the risk assessment in humans during the development of a novel compound. Skeletal ...muscle toxicity in Sprague Dawley Rats was induced with clofibrate at different dose levels for 7 days to compare standard clinical pathology assays with novel skeletal muscle and cardiac muscle biomarkers, gene expression and histopathological changes. The standard clinical pathology assays aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) enzyme activity were compared to novel biomarkers fatty acid binding protein 3 (Fabp3), myosin light chain 3 (Myl3), muscular isoform of CK immunoreactivity (three isoforms CKBB, CKMM, CKMB), parvalbumin (Prv), skeletal troponin I (sTnI), cardiac troponin T (cTnT), cardiac troponin I (cTnI), CKMM, and myoglobin (Myo). The biomarker elevations were correlated to histopathological findings detected in several muscles and gene expression changes. Clofibrate predominantly induced skeletal muscle toxicity of type I fibers of low magnitude. Useful biomarkers for skeletal muscle toxicity were AST, Fabp3, Myl3, (CKMB) and sTnI. Measurements of CK enzyme activity by a standard clinical assay were not useful for monitoring clofibrate-induced skeletal muscle toxicity in the rat at the doses used in this study.
Abstract Rainfall variability over Australia is revisited from the viewpoint of the atmospheric moisture budgets in three regions: the extratropics, Subtropics, and Tropics. The budgets are ...calculated using three‐hourly European Centre for Medium‐Range Weather Forecasts Reanalysis v5 (ERA5) and ERA5‐Land data between 1979 and 2022. The use of the moisture budget at short time‐scales enables the investigation of the relationship between synoptic weather‐scale processes and the longer term variability of the rainfall climate. The total variability in the vertically integrated moisture flux divergence (VIMD) is significantly larger than the evaporation minus precipitation ( E − P ), to a large extent due to the sub‐daily time‐scales. E − P is related more closely to moisture flux convergence in winter (summer) over south (north) Australia, suggesting a clear seasonality in the relationship between the two budget terms. The E − P –VIMD relationship is nearly in phase in the Tropics, whereas VIMD leads E − P by 9–15 hr with eastward‐propagating signals in the extratropics and Subtropics. Such seasonal and regional discrepancies in the relationship are attributed to the background state of moisture availability and temperature as represented by relative humidity and lifting condensation levels. The variability of the budget imbalance and its seasonality are dominated by the variability in VIMD. The imbalance reduces rapidly with temporal smoothing, with the storage term approaching zero at approximately 20 days, which can be thought of as making a transition time‐scale from high‐frequency weather‐related variability into slow‐varying background conditions. Weather‐related variability (cyclones, fronts, and thunderstorms) dominates the overall E − P variability in the extratropics and Subtropics, whereas slow‐varying background conditions contribute equally to the total variability in the Tropics.
Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is associated with idiosyncratic adverse drug reactions in humans. Previous studies revealed a crucial role for intestine-derived ...bacteria and/or lipopolysaccharide (LPS) in DCLF-induced hepatotoxicity. We further explored this mechanism by conducting gene expression analysis of livers from rats treated with a hepatotoxic dose of DCLF (100 mg/kg) with or without oral antibiotic pretreatment. Genes for which expression was altered by DCLF were divided into two groups: genes with expression altered by antibiotic treatment and those unaffected by antibiotics. The former group of genes represented the ones for which DCLF-induced alterations in expression depended on intestinal bacteria. The expression of the latter group of genes was probably changed by direct effect of DCLF rather than by intestinal bacteria. Functional analysis of genes in the former group revealed LPS-related signaling, further suggesting a role for bacterial endotoxin in the liver injury. Functional analysis of genes in the latter group revealed changes in signaling pathways related to inflammation, hypoxia, oxidative stress, the aryl hydrocarbon receptor, and peroxisome proliferator-activated receptor alpha. Neutrophil depletion failed to protect from DCLF-induced hepatotoxicity, suggesting that intestinal bacteria contribute to liver injury in a neutrophil-independent manner. Hypoxia occurred in the livers of rats treated with DCLF, and hypoxia in vitro rendered hepatocytes sensitive to DCLF-induced cytotoxicity. These results support the hypothesis that intestinal bacteria are required for DCLF-induced hepatotoxicity and suggest that hypoxia plays an important role in the pathogenesis.
Blood is an ideal tissue for the identification of novel genomic biomarkers for toxicity or efficacy. However, using blood for transcriptomic profiling presents significant technical challenges due ...to the transcriptomic changes induced by ex vivo handling and the interference of highly abundant globin mRNA. Most whole blood RNA stabilization and isolation methods also require significant volumes of blood, limiting their effective use in small animal species, such as rodents. To overcome these challenges, a QIAzol-based RNA stabilization and isolation method (QSI) was developed to isolate sufficient amounts of high quality total RNA from 25 to 500 μL of rat whole blood. The method was compared to the standard PAXgene Blood RNA System using blood collected from rats exposed to saline or lipopolysaccharide (LPS). The QSI method yielded an average of 54 ng total RNA per μL of rat whole blood with an average RNA Integrity Number (RIN) of 9, a performance comparable with the standard PAXgene method. Total RNA samples were further processed using the NuGEN Ovation Whole Blood Solution system and cDNA was hybridized to Affymetrix Rat Genome 230 2.0 Arrays. The microarray QC parameters using RNA isolated with the QSI method were within the acceptable range for microarray analysis. The transcriptomic profiles were highly correlated with those using RNA isolated with the PAXgene method and were consistent with expected LPS-induced inflammatory responses. The present study demonstrated that the QSI method coupled with NuGEN Ovation Whole Blood Solution system is cost-effective and particularly suitable for transcriptomic profiling of minimal volumes of whole blood, typical of those obtained with small animal species.
Idiosyncratic drug reactions (IDRs) are poorly understood, unpredictable, and not detected in preclinical studies. Although the cause of these reactions is likely multi-factorial, one hypothesis is ...that an underlying inflammatory state lowers the tolerance to a xenobiotic. Previously used in an inflammation IDR model, bacterial lipopolysaccharide (LPS) is heterogeneous in nature, making development of standardized testing protocols difficult. Here, the use of rat tumor necrosis factor-α (TNFα) to replace LPS as an inflammatory stimulus was investigated. Sprague-Dawley rats were treated with separate preparations of LPS or TNFα, and hepatic transcriptomic effects were compared. TNFα showed enhanced consistency at the transcriptomic level compared to LPS. TNFα and LPS regulated similar biochemical pathways, although LPS was associated with more robust inflammatory signaling than TNFα. Rats were then codosed with TNFα and trovafloxacin (TVX), an IDR-associated drug, and evaluated by liver histopathology, clinical chemistry, and gene expression analysis. TNFα/TVX induced unique gene expression changes that clustered separately from TNFα/levofloxacin, a drug not associated with IDRs. TNFα/TVX cotreatment led to autoinduction of TNFα resulting in potentiation of underlying gene expression stress signals. Comparison of TNFα/TVX and LPS/TVX gene expression profiles revealed similarities in the regulation of biochemical pathways. In conclusion, TNFα could be used in lieu of LPS as an inflammatory stimulus in this model of IDRs.
Idiosyncratic drug reactions (IDRs) of a hepatic origin are a major health concern and a notoriously difficult challenge for the pharmaceutical industry. These types of adverse events are rare, with ...a typical occurrence of 1 in 100 to 1 in 100,000 patients. Typical adverse outcomes are most likely statistically impossible to predict in traditional preclinical safety studies or clinical trials. Unfortunately, these reactions can pose a significant risk to the public health, resulting in devastating consequences such as irreversible liver injury, liver transplantation and fatality. This review provides many examples of experimental efforts that are underway for a better understanding of molecular events that may be responsible for IDRs. A list of existing hypotheses for IDRs is also provided, each with current literature examples or supporting evidence. The possibilities for developing suitable animal models for the prediction and characterisation of IDRs are elaborated, especially for a drug-inflammation interaction rat model of hepatic IDR. The need for predictive biomarkers of IDR is addressed, with the exploration of some possible candidates. Finally, the use of primary human hepatocyte culture systems is explored as an in vitro system, with application for providing an increased mechanistic knowledge of IDR. Several examples of informative studies on the nature of IDRs that employ toxicogenomic and proteomic technologies are summarised.
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Impedance‐based real time cytolethality profiling has become an important lead optimization toxicology tool in pharmaceutical discovery. This technique offers the advantage of ...continuous, non‐destructive monitoring of changes in electrical impedance (reported as cellular index or CI) that correlates to alterations in cell health, morphology, and proliferation of adherent primary cells as well as cell lines. In this study, we used a panel of reference toxicants representing several distinct mechanisms of hepatotoxicity and evaluated their impact on CI profiles of cultured primary human hepatocytes (PHH). The mechanisms of action included: apoptosis (staurosporine; ceramide c2), cytoskeletal disruption (phalloidin; colchicine), mitochondrial inhibition (antimycin A; carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP)), plasma membrane perforation (Triton X‐100), and general cellular detachment (trypsin digestion). PHH donor to donor variability (n= 6 donors) was assessed using chlorpromazine, which acts through two cytotoxicity mechanisms: oxidative stress via quinoneimine formation and changes in mitochondrial membrane potential. Cryopreserved PHH were thawed and cultivated using conventional conditions in Williams E‐based media. Cells were treated with eight concentrations of each toxicant or vehicle (0.1 to 0.5% DMSO) in electrode coated 96‐well plates and monitored with an xCELLigence real‐time cell analyzer (Acea Biosciences) for up to 3 days. Treatment of PHH with surfactant (Triton X‐100) or trypsin resulted in an immediate decline of normalized cellular index (NCI). Mitochondrial function inhibitors caused a rapid decrease of NCI at doses as low as 1.6 and 3.1 μM. Staurosporine and ceramide treatment (apoptosis inducers) decreased NCI (IC
50
=1 to 6 μM), but over an extended time (>10 h) compared to the mitochondrial toxicants. Interestingly, exposure to cytoskeletal disruptors led to a gradual increase in NCI over 3 days, plausibly due to subtle alterations in cell shape. There was a high degree of inter‐individual variability in response to chlorpromazine treatment suggesting that cryopreserved PHH should be characterized using a positive control before an impedance‐based compound evaluation. Altogether, these results are useful to understand differences in impedance profiles based on compound toxicity mechanisms and can be compared to profiles from related liver‐derived primary cells and cell lines.
Support or Funding Information
All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Dibromoacetic acid (DBAA), a by-product formed during disinfection of drinking water, alters spermatogenesis in rats through defective spermiation. The mechanism underlying this toxicity is not fully ...understood. In this study, gene expression data generated with microarrays from testes were used to generate a mechanistic understanding of DBAA-induced testicular toxicity. Testes were collected from male Sprague–Dawley rats dosed orally for 1 and 4 days with DBAA at 250 mg/kg/day. At both time points, DBAA administration induced delayed spermiation in Stage X tubules and regulated the expression of a small number of genes, including a mild but consistent downregulation of cytochrome P450c17α (CYP17) mRNA, an enzyme expressed by Leydig cells and essential for the production of testicular androgens. Downregulation of CYP17 was confirmed at the protein level and its biological significance was substantiated by demonstrating reduced testicular testosterone levels in DBAA-dosed rats. Furthermore, testosterone production by human chorionic gonadotrophin (hCG)-stimulated rat primary Leydig cells was reduced following treatment with 100 μM DBAA. Collectively, these results indicate that DBAA can directly target rat Leydig cells and downregulate testicular CYP17 expression with a resulting decreased testicular testosterone production. This disruption of testicular steroidogenesis is likely to contribute to the mechanism of failed spermiation observed in rats following exposure to DBAA.
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Mitochondrial dysfunction has been frequently implicated in some drug‐induced toxicities. Various methods for the detection of mitochondrial toxicity are available such as an oxygen ...consumption assay to measure mitochondrial basal respiration, assays to measure direct effects of drugs on the five complexes in the mitochondrial electron transport chain (ETC), and assays to detect changes in mitochondrial DNA (mtDNA) copy number or changes in mitochondrial membrane potential. The Seahorse technology platform has emerged as a new technique in assessing mitochondrial respiration in cells, where basal, ATP‐linked, maximal and non‐mitochondrial respiration can be measured in the same experiment. Spare respiratory capacity (SRC) is calculated as the difference between maximal and basal respiration. In this study, Seahorse XF Cell Mito Stress Test was evaluated in the PC‐3 cell line using tool compounds known to directly inhibit the complex or complexes in the ETC. Cells were acutely treated with amiodarone (5–80 μM), simvastatin (5–75 μM), tamoxifen (0.625–20 μM), troglitazone (2.5–50 μM), penicillin (12.5–100 μM, negative control), and vehicle (0.1% DMSO). Exposure of these tool compounds revealed an immediate and pronounced decrease in SRC with a ranking of (greatest to lowest inhibition) troglitazone (IC
50
< 2.5 μM)> amiodarone (IC
50
<5 μM) >tamoxifen (IC
50
= 17.7 μM) >simvastatin (IC
50
= 20.7μM).
Tool compounds known to cause mitochondrial toxicity through an indirect mechanism were also explored in the Seahorse platform. 2′, 3′‐dideoxycytidine (ddC), an inhibitor of mtDNA synthesis, and chloramphenicol (CAM), an inhibitor of mitochondrial protein synthesis, were tested. A 3‐day pre‐treatment of ddC (0.12–30 μM) and CAM (0.04–10 μM) showed a decrease of SRC in PC‐3 cells with IC
50
=0.39 μM and 2.7 μM respectively. The decrease of SRC was further supported by an in‐cell ELISA assay where a set of proteins in the ETC complexes was measured. A decrease of mitochondrial encoded COX‐1 to nuclear encoded SDH‐A protein ratio was observed after a 5‐day treatment of ddC and CAM in PC‐3 cells with IC
50
=0.016 μM and <0.1 μM respectively. In the dose ranges tested, the 3 or 5 day pre‐treatment of ddC and CAM showed minimal cytotoxicity in the PC‐3 cells. In conclusion, the Seahorse XF Cell Mito Stress Test is a sensitive assay to evaluate mitochondrial dysfunction using a set of tool compounds, and this new method combined with traditional methods will be helpful to better understand the mechanism of mitochondrial toxicity in the development of new drugs.
Support or Funding Information
The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Abstract
Introduction: In vitro assays using rat and human hepatocytes are used for hepatotoxicity studies; however, in vitro methods are less established for canine hepatocytes. In particular, ...little is known about the effects of plating and culture on canine hepatocytes. The goal of this study was to conduct a descriptive analysis of an in vitro canine hepatocyte system to evaluate its utility and limitations. The study objectives were to determine if canine hepatocytes shipped in suspension or pre-plated were transcriptomically different from one another and their liver of origin, and to understand temporal transcriptomic changes.
Materials and methods: Frozen canine liver samples were delivered on dry ice; hepatocytes from these livers were delivered in a cell/media suspension (S) or pre-plated (P). Hepatocytes were harvested at arrival and after up to 120 hr of culture in naïve media, or after 48 hr treatment with prototypical enzyme inducing xenobiotics (phenobarbital or rifampin).
Results: A global transcriptomic comparison between liver and hepatocyte preparations indicated that the transcriptome was affected post-plating; transporters and genes involved in xenobiotic metabolism were generally down-regulated. Basal mRNA levels of CYP3A12 and CYP2B11 decreased temporally; after 120 hr CYP3A12 levels decreased by 1000-fold. CYP3A12 and CYP2B11 induction after phenobarbital or rifampin treatment was robust in both cell types but stronger in S cells.
Conclusions: These results indicate that S and P hepatocytes cultured under the current conditions are appropriate for specific in vitro tests. Further characterization of endpoints should be conducted for a thorough understanding of the model's limitations.
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