MicroRNA as biomarkers of mitochondrial toxicity.

Mitochondrial toxicity can be difficult to detect as most cells can tolerate reduced activity as long as minimal capacity for function is maintained. However, once minimal capacity is lost, apoptosis or necrosis occurs quickly. Identification of more sensitive, early markers of mitochondrial toxicity was the objective of this work. Rotenone, a mitochondrial complex I inhibitor, and 3-nitropropionic acid (3-NP), a mitochondrial complex II inhibitor, were administered daily to male Sprague-Dawley rats at subcutaneous doses of 0.1 or 0.3mg/kg/day and intraperitoneal doses of 5 or 10mg/kg/day, respectively, for 1week. Samples of kidney, skeletal muscle (quadriceps femoris), and serum were collected for analysis of mitochondrial DNA (mtDNA) copy number and microRNA (miRNA) expression patterns. MtDNA was significantly decreased with administration of rotenone at 0.3mg/kg/day and 3-NP at 5 and 10mg/kg/day in the quadriceps femoris and with 3-NP at 10mg/kg/day in the kidney. Additionally, rotenone and 3-NP treatment produced changes to miRNA expression that were similar in direction (i.e. upregulation, downregulation) to those previously linked to mitochondrial functions, such as mitochondrial damage and biogenesis (miR-122, miR-202-3p); regulation of ATP synthesis, abolished oxidative phosphorylation, and loss of membrane potential due to increased reactive oxygen species (ROS) production (miR-338-5p, miR-546, miR-34c); and mitochondrial DNA damage and depletion (miR-546). These results suggest that miRNAs may be sensitive biomarkers for early detection of mitochondrial toxicity.

D-α-tocopheryl polyethylene glycol 1000 succinate-containing vehicles provide no detectable chemoprotection from oxidative damage.

The objective of this study was to evaluate potential protective effects of vehicles containing d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), which may impact nonclinical safety assessments of oxidative processes. This was achieved by evaluating plasma, liver and adrenal gland concentrations of d-α-tocopheryl succinate (TS) and d-α-tocopherol as well as oxidative status of plasma following oral dosing of TPGS-containing vehicles, intraperitoneal (IP) dosing of TS or ex vivo treatment of blood with H2O2. Male and female rats were dosed orally with formulations containing 5% or 40% TPGS (70 or 550 mg kg(-1) day(-1) TS, respectively) for 1 week. A control group was dosed orally with polyethylene glycol-400 (PEG-400; no vitamin E) and positive control animals received a single 100 mg kg(-1) day(-1) IP injection of TS. Whole blood from untreated animals was treated ex vivo with 5 or 50 mm H(2)O(2), with or without TS (0.5, 5, 50 or 500 µm) or ascorbate (1 mm), for 1 h. Oral TPGS treatments did not affect d-α-tocopherol concentrations in plasma or adrenal glands and caused only transient increases in liver. Concentrations of TS in plasma, liver and adrenal glands were undetectable in control animals, but increased in all other groups. Oral administration of TPGS did not reduce plasma lipid peroxidation in vivo. Substantially greater TS concentrations used ex vivo (100× greater than in vivo) were also unable to reduce lipid peroxidation in H2O2 -treated whole blood. These results provide evidence that administration of oral TPGS vehicles is unlikely to impact nonclinical safety assessments of pharmaceuticals.

Allogeneic murine pregnancy models for assessing the developmental effects of immune-stimulating antibodies: Challenges in reproducibility.

Literature suggests that murine allogeneic pregnancy models are an alternative approach for evaluating the developmental toxicity of immune-stimulating agents. In this study, multiple syngeneic and allogeneic murine pregnancy models were used to assess the potential embryo-fetal effects of four different murine antibodies (IgG1 or IgG2 ) that activate the immune system by binding to T-cell receptors (PD-L1, LAG-3, and GITR). The pregnancy models were generated by within and between matings of five different inbred strains of mice (CBA/CaJ, DBA/2J, BALB/c, C57BL/6, and CBA/J). The antibodies were administered every 2-3 days by intraperitoneal injection (n = 12-29/group) during gestation days 6 to 14. There were no differences in embryo-fetal endpoints between the allogeneic and syngeneic pregnancies. Additionally, treatment with the antibodies had no effect on mean postimplantation loss in either the syngeneic or allogeneic pregnancies despite confirmation of pharmacologically-relevant systemic exposures. These results suggest that allogeneic murine pregnancy models need further validation and testing before they can be reliably used as an alternative approach for assessing the developmental effects of agents that stimulate the immune system.

Effects of BMS-986094, a Guanosine Nucleotide Analogue, on Mitochondrial DNA Synthesis and Function.

BMS-986094, the prodrug of a guanosine nucleotide analogue (2'-C-methylguanosine), was withdrawn from clinical trials due to serious safety issues. Nonclinical investigative studies were conducted as a follow up to evaluate the potential for BMS-986094-related mitochondrial-toxicity. In vitro, BMS-986094 was applied to human hepatoma cells (HepG2 and Huh-7) or cardiomyocytes (hiPSCM) up to 19 days to assess mitochondrial DNA content and specific gene expression. There were no mitochondrial DNA changes at concentrations ≤10 µM. Transcriptional effects, such as reductions in Huh-7 MT-ND1 and MT-ND5 mRNA content and hiPSCM MT-ND1, MT-COXII, and POLRMT protein expression levels, occurred only at cytotoxic concentrations (≥10 µM) suggesting these transcriptional effects were a consequence of the observed toxicity. Additionally, BMS-986094 has a selective weak affinity for inhibition of RNA polymerases as opposed to DNA polymerases. In vivo, BMS-986094 was given orally to cynomolgus monkeys for 3 weeks or 1 month at doses of 15 or 30 mg/kg/day. Samples of heart and kidney were collected for assessment of mitochondrial respiration, mitochondrial DNA content, and levels of high energy substrates. Although pronounced cardiac and renal toxicities were observed in some monkeys at 30 mg/kg/day treated for 3-4 weeks, there were no changes in mitochondrial DNA content or ATP/GTP levels. Collectively, these data suggest that BMS-986094 is not a direct mitochondrial toxicant.

Differential display in rat livers treated for 13 weeks with phenobarbital implicates a role for metabolic and oxidative stress in nongenotoxic carcinogenicity.

Hepatic enzyme inducers such as phenobarbital are often nongenotoxic rodent hepatocarcinogens. Currently, nongenotoxic hepatocarcinogens can only be definitively identified through costly and extensive long-term, repeat-dose studies (e.g., 2-year rodent carcinogenicity assays). Although liver tumors caused by these compounds are often not found to be relevant to human health, the mechanism(s) by which they cause carcinogenesis are not well understood. Toxicogenomic technologies represent a new approach to understanding the molecular bases of toxicological liabilities such asnongenotoxic carcinogenicity early in the drug discovery/development process. Microarrays have been used to identify mechanistic molecular markers of nongenotoxic rodent hepatocarcinogenesis in short-term, repeat-dose preclinical safety studies. However, the initial "noise" of early adaptive changes may confound mechanistic interpretation of transcription profiling data from short-term studies, and the molecular processes triggered by treatment with a xenobiotic agent are likely to change over the course of long-term treatment. Here, we describe the use of a differential display technology to understand the molecular mechanisms related to 13 weeks of dosing with the prototype rodent nongenotoxic hepatocarcinogen, phenobarbital. These findings implicate a continuing role for oxidative stress in nongenotoxic carcinogenicity.An Excel data file containing raw data is available in full at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. Click on the issue link for 33(1), then select this article. A download option appears at the bottom of this abstract. The file contains raw data for all gene changes detected by AFLP, including novel genes and genes of unknown function; sequences of detected genes; and animal body and liver weight ratios. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Transcription profiling distinguishes dose-dependent effects in the livers of rats treated with clofibrate.

Peroxisome proliferators such as the fibrates act via the peroxisome proliferator activated receptor (PPAR)-alpha as hypolipidemic agents. Many peroxisome proliferators are also nongenotoxic hepatic carcinogens and hepatotoxicants in rodents. We performed transcription profiling using cDNA microarrays on livers of rats treated for 5 days with 3 doses of the peroxisome proliferator clofibrate. All 3 doses had hepatic effects as assessed by liver to body weight ratio, alanine aminotransferase (ALT) increases and histopathology examination. Analysis of the transcription profiling data identified changes in the expression of many genes within several mechanistic pathways that support existing hypotheses regarding peroxisome proliferator mediated carcinogenicity. Additionally, the transcription profiling, histopathology, and clinical chemistry results suggested a biphasic response to clofibrate. These findings provide insight into the pathogenesis of toxic and carcinogenic effects of clofibrate in rodents and demonstrate the ability of cDNA microarrays to provide information regarding mechanisms of toxicity identified during the drug development process.

Acute molecular markers of rodent hepatic carcinogenesis identified by transcription profiling.

Currently, the only way to identify nongenotoxic hepatocarcinogens is through long-term repeat dose studies such as the 2 year rodent carcinogenicity assay. Such assays are both time consuming and expensive and require large amounts of active pharmaceutical or chemical ingredients. Thus, the results of the 2 year assay are not known until very late in the discovery and development process for new pharmaceutical entities. Although in many cases nongenotoxic carcinogenicity in rodents is considered to be irrelevant for humans, a positive finding in a 2 year carcinogenicity assay may increase the number of studies to demonstrate the lack of relevance to humans, delay final submission and subsequent registration of a product, and may result in a "black box" carcinogenicity warning on the label. To develop early identifiers of carcinogenicity, we applied transcription profiling using several prototype rodent genotoxic and nongenotoxic carcinogens, as well as two noncarcinogenic hepatotoxicants, in a 5 day repeat dose in vivo toxicology study. Fluorescent-labeled probes generated from liver mRNA prepared from male Sprague-Dawley rats treated with one of three dose levels of bemitradine, clofibrate, doxylamine, methapyrilene, phenobarbital, tamoxifen, 2-acetylaminofluorene, 4-acetylaminofluorene, or isoniazid were hybridized against rat cDNA microarrays. Correlation of the resulting data with an estimated carcinogenic potential of each compound and dose level identified several candidate molecular markers of rodent nongenotoxic carcinogenicity, including transforming growth factor-beta stimulated clone 22 and NAD(P)H cytochrome P450 oxidoreductase.