All tags multiplex-mirna-assays Biomarkers for assessing tissue-specific toxicity

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Biomarkers for assessing tissue-specific toxicity

Find out why miRNAs are ideal biomarkers of toxicity, and what tools are available to study them.

Traditionally, biomarker studies have focused on identifying a single factor to indicate the presence of given disease1. However, recent studies have shown that using combined signatures of multiple biomarkers (multiplex biomarker profiling) can better account for patient heterogeneity, and provide a more accurate indication of patient health1.

Proteins in circulation have been used as biomarkers for many years1; however, miRNAs have recently emerged as ideal biomarkers for studying normal cellular processes, pathological conditions, and response to therapeutic intervention2. Their presence in biofluids such as plasma, urine and cerebrospinal fluid (CSF) makes them ideally suited for liquid biopsies in patients3. Further, miRNAs are highly stable in circulation, and their tissue-specific expression patterns have been found to fluctuate in response to inflammation, acute and chronic tissue injury, and in cancer3.

Organ toxicity is one of the leading causes of compound attrition during pre-clinical and clinical trials, therefore identifying biomarkers that can be used in early stages of drug safety assessment is essential4. Here we describe how miRNAs have been used as biomarker for cardiac and liver toxicity.

miRNAs in cardiac injury and toxicity

Studies in rodent and human samples have identified miRNA signatures for monitoring cardiac injury after myocardial infarction (MI), as well as drug-induced cardiac toxicity (DICI)5-7. Expression of miR-1, miR-133a, miR-208a, and miR-499-5p was found to be upregulated in patients following MI, whereas their levels remained low or undetectable in healthy control patients5,6. Interestingly, the induction of miRNA expression after MI was observed in both patient plasma and urine samples6.

Similarly, increased plasma levels of miR-208a was reported in response to exposure to cardiotoxic agents such as isoproterenol7. The finding that miR-208a is upregulated upon both DICI and MI-induced cardiac injury emphasizes the importance of multiplex biomarker screening. In the case of cardiac tissues, multiplexing enables monitoring both the occurrence of tissue injury, as well as potential causes for the tissue injury.

The Firefly® miRNA Cardiology Panel (ab204063) is specifically designed to enable cardiac injury and toxicity screening studies in both human and rodent samples. This panel consists of a literature-curated list of 68 cardiac-specific miRNAs, including key markers of DICI and MI and suggested normalizers. The Firefly miRNA Assays enable multiplex miRNAs profiling directly from 20 µL of crude biofluids without the need for prior RNA isolation, making them the ideal tool for rodent and clinical trials, where limited sample availability is an issue.

Multiplex miRNA Assay Cardiology Panel - Circulating (ab204063)

Figure 1. The Firefly miRNA Assay was used to profile 68 miRNAs in the Firefly Cardiology miRNA Panel (ab204063) from 40 µL pooled human serum and 40 µL pooled human plasma, each in triplicate. The heatmap displays log values of the background-subtracted, un-normalized signal of the mean fluorescent intensity of each target.


miRNAs of liver toxicity

Liver is another common target of drug-induced organ injury and toxicity2, and biomarkers that can sensitively detect early-stage liver injury are of particular importance for pre-clinical and clinical toxicological risk and safety assessment8. Elevated serum levels of liver enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GDH) are often used to assess liver injury8. However, these enzyme markers cannot differentiate between drug-induced and non-drug-induced liver injuries8. As a result, current research is focused on identifying miRNA signatures associated with global liver injury versus those unique to a particular mode of injury.

Extensive studies using an acetaminophen-induced model of liver injury in rodents have determined that elevated plasma levels of several miRNAs, including miR-122, miR-146a, miR-155, and miR-192, are associated with liver injury8. Similar studies have been used to identify miRNA signatures of liver steatosis, cirrhosis, and alcohol-induced liver toxicity.

The Firefly miRNA Liver Toxicity Panel (ab204065) comprises a literature-curated list of 68 miRNAs associated with liver injury and toxicity, including markers of both drug-induced and non-drug induced liver toxicity. This panel can be applied to liver injury and toxicity screening studies in both pre-clinical studies using rodent models, and to clinical studies using patient samples.

Multiplex miRNA Assay Liver Tox Panel - Circulating (ab204065)

Figure 2. The Firefly miRNA Assay was used to profile 68 miRNAs in the Firefly Liver Toxicity miRNA Panel (ab204065) from 40 µL pooled human serum and 40 µL pooled human plasma, each in triplicate. The heatmap displays log values of the background-subtracted, un-normalized signal of the mean fluorescent intensity of each target.

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References

1. Rifai N., Gillette M.A., Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol. 24, 971-82 (2006).

2. Etheridge A., Lee I., Hood L., Galas D., Wang K. Extracellular microRNA: a new source of biomarkers. Mutat Res. 717, 85-90 (2011).

3. Moldovan L., Batte K.E., Trgovcich J., Wisler J., Marsh C.B., Piper M. Methodological challenges in utilizing miRNAs as circulating biomarkers. J Cell Mol Med. 18, 371-90 (2014).

4. Kramer J.A., Sagartz J.E., Morris D.L. The application of discovery toxicology and pathology towards the design of safer pharmaceutical lead candidates. Nat Rev Drug DIscov. 6, 636-49 (2007).

5. Wang G.K., Zhu J.Q., Zhang J.T., Li Q., Li Y., He J., Qin Y.W., Jing Q. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J. 31, 659-66 (2010).

6. Gidlöf O., Andersson P., van der Pals J., Götberg M., Erlinge D. Cardiospecific microRNA plasma levels correlate with troponin and cardiac function in patients with ST elevation myocardial infarction, are selectively dependent on renal elimination, and can be detected in urine samples. Cardiology. 118, 217-26 (2011).

7. Gilneur S.F., De Ron P., Hanon E., Valentin J.P., Dremier S., Noqueira da Costa A. Paving the Route to Plasma miR-208a-3p as an Acute Cardiac Injury Biomarker: Preclinical Rat Data Supports Its Use in Drug Safety Assessment. Toxicol Sci. 149, 89-97 (2016).

8. Osaki M., Kosaka N., Okada F., Ochiya T. Circulating microRNAs in drug safety assessment for hepatic and cardiovascular toxicity: the latest biomarker frontier? Mol Diagn Ther. 18, 121-6 (2014).