Validation of reference gene stability for APAP hepatotoxicity studies in different in vitro systems and identification of novel potential toxicity biomarkers

Fox, Bridget; Devonshire, Alison S.; Schutte, Maaike E.; Foy, Carole A.; Minguez, Jesus; Maltman, Daniel J.; Bokhari, Maria; Marshall, Damian

doi:10.1016/j.tiv.2010.08.007

Cited by 26 publications

(18 citation statements)

References 44 publications

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“…In this study, several candidate RGs (among them, the conventional GAPDH and ACTB ) showed altered expression in the acute hepatotoxicity models studied. A similar situation was found in different studies evaluating candidate RGs for mRNA normalization: in a cytotoxicity study using AA [49] and in dioxin-treated rats [26], it was observed that about 40 and 50% of the candidate RGs changed their expressions in response to the hepatotoxin, respectively. These findings highlight the importance of testing the alteration of expression of RGs in toxicity studies.…”

Section: Discussionsupporting

confidence: 75%

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Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity

et al. 2012

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Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity.

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Section: Discussionsupporting

confidence: 75%

“…Despite this, only few previous reports have evaluated this possibility [26], [38], [47]–[49]. In this study, several candidate RGs (among them, the conventional GAPDH and ACTB ) showed altered expression in the acute hepatotoxicity models studied.…”

Section: Discussionmentioning

confidence: 72%

Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity

et al. 2012

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“…For testing toxicity of compounds, in vitro screening is cost-effective and time-saving than in vivo assays (Fox et al 2010). The urinary biomarkers such as albumin, β2-microglobulin, clusterin, cystatin C, Kim-1, trefoil factor 3, and total protein acknowledged by the U.S. Food and Drug Administration and European Medicines Agency are excreted in the urine, but do not increase in their mRNAs or proteins after injury except Kim-1 (Dieterle et al 2010;Togashi et al 2012;Vaidya et al 2005Vaidya et al , 2008, which is a limitation for the use in cell-based assays.…”

Section: Discussionmentioning

confidence: 99%

Phlda3, a urine-detectable protein, causes p53 accumulation in renal tubular cells injured by cisplatin

et al. 2015

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Measurable indicators of renal injury are required for the assessment of kidney function after toxicant challenge. In our previous study, pleckstrin homology-like domain, family A, member 3 (Phlda3) was a most greatly up-regulated molecule downstream from p53, culminating with kidney tubular injury. This study investigated the positive feedforward effect of Phlda3 on p53 in an effort to explain the largest increase of Phlda3 in injured tubules and the potential of its urine excretion. qRT-PCR assays confirmed a rapid and substantial increase in Phlda3 messenger RNA (mRNA) in the kidney cortex of mice treated with a single dose of cisplatin. Cisplatin overexpression of Phlda3 was verified by gene set analyses of three different microarray databases. In the immunohistochemistry, Phlda3 staining intensities were augmented in the tubules as kidney injury worsened. Moreover, the urinary content of Phlda3 was increased after cisplatin treatment, as were those of other kidney injury markers (Kim-1 and Timp-1). By contrast, cisplatin failed to increase Phlda3 mRNA in the liver despite hepatocyte necrosis and ensuing increases in serum transaminase activities. In NRK52E tubular cells, siRNA knockdown of Phlda3 enhanced the ability of cisplatin to increase p-Mdm2 presumably via Akt, enforcing the interaction between Mdm2 and p53. Consistently, a deficiency in Phlda3 abrogated p53 increase by cisplatin, indicating that Phlda3 promotes p53 accumulation. Phlda3 overexpression had the opposite effect. In addition, treatment with cyclosporine A or CdCl2, other nephrotoxicants, increased Phlda3 mRNA and protein levels in NRK52E cells, as did cisplatin treatment. Overall, Phlda3 may cause p53 accumulation through a feedforward pathway, facilitating tubular injury and its urine excretion.

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“…Additionally, Fox et al . () have evaluated reference genes for acetaminophen hepatotoxicity studies in different cell types and observed different stable reference genes for each cell type. Plasma miR‐103, which had consistently ranked as the most stably expressed reference gene for acetaminophen hepatotoxicity in this study, was often seen as one of the least stably expressed gene in other previous studies (Song et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Identification of endogenous reference genes for RT‐qPCR analysis of plasma microRNAs levels in rats with acetaminophen‐induced hepatotoxicity

Wang

Tang

Hui

et al. 2013

J of Applied Toxicology

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Circulating microRNA (miRNA) expression profiles have been reported to be promising biomarkers for drug-induced liver injury in preclinical and clinical practice. Proper normalization is critical for accurate miRNAs expression analysis. Herein, using SYBR green quantitative real-time PCR (RT-qPCR), we evaluated the expression stability of six candidate reference genes including two commonly used small RNAs (U6, 5S) and four miRNAs (let-7a, miR-92a, miR-103 and miR-16) in plasma of rats with acetaminophen-induced hepatotoxicity. Data were analysed using geNorm, Normfinder, BestKeeper and comparative delta-Ct statistical models, and the results consistently show that miR-103 is the most stably expressed reference gene. Whereas the commonly used housekeeping genes 5S or U6 are all not suitable normalizers, because 5S exhibits extensive variability in expression and U6 has a low expression level across the plasma samples. Then the effect of reference genes on normalization of plasma miR-122 was assessed; when normalized to the most stable reference gene there were significant differences between the acetaminophen-treated group and the vehicle group. However, when the data were normalized to a less stably expressed gene, miR-16, a biased result was obtained. Therefore, we recommend that miR-103 as suitable reference gene for plasma miRNAs analysis for acetaminophen-induced liver injury. Data presented in this paper are crucial to successful biomarker discovery and validation for the diagnosis of the early stage of acetaminophen hepatotoxicity.

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Validation of reference gene stability for APAP hepatotoxicity studies in different in vitro systems and identification of novel potential toxicity biomarkers

Cited by 26 publications

References 44 publications

Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity

Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity

Phlda3, a urine-detectable protein, causes p53 accumulation in renal tubular cells injured by cisplatin

Identification of endogenous reference genes for RT‐qPCR analysis of plasma microRNAs levels in rats with acetaminophen‐induced hepatotoxicity

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