Methodological considerations for measuring biofluid-based microRNA biomarkers.

MicroRNAs (miRNAs) are small non-coding RNA that regulate the expression of messenger RNA and are implicated in almost all cellular processes. Importantly, miRNAs can be released extracellularly and are stable in these matrices where they may serve as indicators of organ or cell-specific toxicity, disease, and biological status. There has thus been great enthusiasm for developing miRNAs as biomarkers of adverse outcomes for scientific, regulatory, and clinical purposes. Despite advances in measurement capabilities for miRNAs, miRNAs are still not routinely employed as noninvasive biomarkers. This is in part due to the lack of standard approaches for sample preparation and miRNA measurement and uncertainty in their biological interpretation. Members of the microRNA Biomarkers Workgroup within the Health and Environmental Sciences Institute's (HESI) Committee on Emerging Systems Toxicology for the Assessment of Risk (eSTAR) are a consortium of private- and public-sector scientists dedicated to developing miRNAs as applied biomarkers. Here, we explore major impediments to routine acceptance and use of miRNA biomarkers and case examples of successes and deficiencies in development. Finally, we provide insight on miRNA measurement, collection, and analysis tools to provide solid footing for addressing knowledge gaps toward routine biomarker use.

RATEmiRs: the rat atlas of tissue-specific and enriched miRNAs for discerning baseline expression exclusivity of candidate biomarkers.

MicroRNAs (miRNAs) are small RNAs that regulate mRNA expression and have been targeted as biomarkers of organ damage and disease. To explore the utility of miRNAs to assess injury to specific tissues, a tissue atlas of miRNA abundance was constructed. The Rat Atlas of Tissue-specific and Enriched miRNAs (RATEmiRs) catalogues miRNA sequencing data from 21 and 23 tissues in male and female Sprague-Dawley rats, respectively. RATEmiRs identifies tissue-enriched (TE), tissue-specific (TS), or organ-specific (OS) miRNAs via comparisons of one or more tissue or organ vs others. We provide a brief overview of RATEmiRs and present how to use it to detect miRNA expression abundance of candidate biomarkers as well as to compare the expression of miRNAs between rat and human. The database is available at https://www.niehs.nih.gov/ratemirs/.

A cross-sector call to improve carcinogenicity risk assessment through use of genomic methodologies.

Robust genomic approaches are now available to realize improvements in efficiencies and translational relevance of cancer risk assessments for drugs and chemicals. Mechanistic and pathway data generated via genomics provide opportunities to advance beyond historical reliance on apical endpoints of uncertain human relevance. Published research and regulatory evaluations include many examples for which genomic data have been applied to address cancer risk assessment as a health protection endpoint. The alignment of mature, robust, reproducible, and affordable technologies with increasing demands for reduced animal testing sets the stage for this important transition. We present our shared vision for change from leading scientists from academic, government, nonprofit, and industrial sectors and chemical and pharmaceutical safety applications. This call to action builds upon a 2017 workshop on "Advances and Roadblocks for Use of Genomics in Cancer Risk Assessment." The authors propose a path for implementation of innovative cancer risk assessment including incorporating genomic signatures to assess mechanistic relevance of carcinogenicity and enhanced use of genomics in benchmark dose and point of departure evaluations. Novel opportunities for the chemical and pharmaceutical sectors to combine expertise, resources, and objectives to achieve a common goal of improved human health protection are identified.

A multi-megabase copy number gain causes maternal transmission ratio distortion on mouse chromosome 2.

Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 - 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, R2d) emerged as the strongest candidate for the causative allele. We mapped R2d sequences to two loci within the candidate interval. R2d1 is located near the proximal boundary, and contains a single copy of R2d in all strains tested. R2d2 maps to a 900 kb interval, and the number of R2d copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (R2d2WSBdel1) that eliminates the majority of the R2d2WSB copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for R2d2WSBdel1, the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for R2d2WSBdel1, thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in R2d2WSB heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the R2d2WSB transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system.

A Synopsis of the "Influence of Epigenetics, Genetics, and Immunology" Session Part A at the 35th Annual Society of Toxicologic Pathology Symposium.

The overarching theme of the 2016 Society of Toxicology Pathology's Annual Symposium was "The Basis and Relevance of Variation in Toxicologic Responses." Session 4 focused on genetic variation as a potential source for variability in toxicologic responses within nonclinical toxicity studies and further explored how knowledge of genetic traits might enable targeted prospective and retrospective studies in drug development and human health risk assessment. In this session, the influence of both genetic sequence variation and epigenetic modifications on toxicologic responses and their implications for understanding risk were explored. In this overview, the presentations in this session will be summarized, with a goal of exploring the ramifications of genetic and epigenetic variability within and across species for toxicity studies and disseminating information regarding novel tools to harness this variability to advance understanding of toxicologic responses across populations.

Green tea epigallocatechin gallate binds to and inhibits respiratory complexes in swelling but not normal rat hepatic mitochondria.

Epigallocatechin gallate (EGCG), the major flavonoid in green tea, is consumed via tea products and dietary supplements, and has been tested in clinical trials. However, EGCG can cause hepatotoxicity in humans and animals by unknown mechanisms. Here EGCG effects on rat liver mitochondria were examined. EGCG showed negligible effects on oxidative phosphorylation at 7.5-100µM in normal mitochondria. However, respiratory chain complexes (RCCs) were profoundly inhibited by EGCG in mitochondria undergoing Ca(2+) overload-induced mitochondrial permeability transition (MPT). As RCCs are located in mitochondrial inner membranes (IM) and matrix, it was reasoned that EGCG could not readily pass through IM to affect RCCs in normal mitochondria but may do so when IM integrity is compromised. This speculation was substantiated in three ways. (1) Purified EGCG-bound proteins were barely detectable in normal mitochondria and contained no RCCs as determined by Western blotting, but swelling mitochondria contained about 1.5-fold more EGCG-bound proteins which included four RCC subunits together with cyclophilin D that locates in mitochondrial matrix. (2) Swelling mitochondria consumed more EGCG than normal ones. (3) The MPT blocker cyclosporine A diminished the above-mentioned difference. Among four subunits of RCC II, only SDHA and SDHB which locate in mitochondrial matrix, but not SDHC or SDHD which insert into the IM, were found to be EGCG targets. Interestingly, EGCG promoted Ca(2+) overload-induced MPT only when moderate MPT already commenced. This study identified hepatic RCCs as targets for EGCG in swelling but not normal mitochondria, suggesting EGCG may trigger hepatotoxicity by worsening pre-existing mitochondria abnormalities.

Dysregulation of protein degradation pathways may mediate the liver injury and phospholipidosis associated with a cationic amphiphilic antibiotic drug.

A large number of antibiotics are known to cause drug-induced liver injury in the clinic; however, interpreting clinical risk is not straightforward owing to a lack of predictivity of the toxicity by standard preclinical species and a poor understanding of the mechanisms of toxicity. An example is PF-04287881, a novel ketolide antibiotic that caused elevations in liver function tests in Phase I clinical studies. In this study, a mouse diversity panel (MDP), comprised of 34 genetically diverse, inbred mouse strains, was utilized to model the toxicity observed with PF-04287881 treatment and investigate potential mechanisms that may mediate the liver response. Significant elevations in serum alanine aminotransferase (ALT) levels in PF-04287881-treated animals relative to vehicle-treated controls were observed in the majority (88%) of strains tested following a seven day exposure. The average fold elevation in ALT varied by genetic background and correlated with microscopic findings of hepatocellular hypertrophy, hepatocellular single cell necrosis, and Kupffer cell vacuolation (confirmed as phospholipidosis) in the liver. Global liver mRNA expression was evaluated in a subset of four strains to identify transcript and pathway differences that distinguish susceptible mice from resistant mice in the context of PF-04287881 treatment. The protein ubiquitination pathway was highly enriched among genes associated with PF-04287881-induced hepatocellular necrosis. Expression changes associated with PF-04287881-induced phospholipidosis included genes involved in drug transport, phospholipid metabolism, and lysosomal function. The findings suggest that perturbations in genes involved in protein degradation leading to accumulation of oxidized proteins may mediate the liver injury induced by this drug.

Liver biomarker and in vitro assessment confirm the hepatic origin of aminotransferase elevations lacking histopathological correlate in beagle dogs treated with GABAA receptor antagonist NP260.

NP260 was designed as a first-in-class selective antagonist of α4-subtype GABAA receptors that had promising efficacy in animal models of pain, epilepsy, psychosis, and anxiety. However, development of NP260 was complicated following a 28-day safety study in dogs in which pronounced elevations of serum aminotransferase levels were observed, although there was no accompanying histopathological indication of hepatocellular injury. To further investigate the liver effects of NP260, we assayed stored serum samples from the 28-day dog study for liver specific miRNA (miR-122) as well as enzymatic biomarkers glutamate dehydrogenase and sorbitol dehydrogenase, which indicate liver necrosis. Cytotoxicity assessments were conducted in hepatocytes derived from dog, rat, and human liver samples to address the species specificity of the liver response to NP260. All biomarkers, except ALT, returned toward baseline by Day 29 despite continued drug treatment, suggesting adaptation to the initial injury. In vitro analysis of the toxicity potential of NP260 to primary hepatocytes indicated a relative sensitivity of dog>human>rat, which may explain, in part, why the liver effects were not evident in the rodent safety studies. Taken together, the data indicate that a diagnostic biomarker approach, coupled with sensitive in vitro screening strategies, may facilitate interpretation of toxicity potential when an adaptive event masks the underlying toxicity.

Keratin-18 and microRNA-122 complement alanine aminotransferase as novel safety biomarkers for drug-induced liver injury in two human cohorts.

BACKGROUND & AIMS: There is a demand for more sensitive, specific and predictive biomarkers for drug-induced liver injury (DILI) than the gold standard used today, alanine aminotransferase (ALT). The aim of this study was to qualify novel DILI biomarkers (keratin-18 markers M65/M30, microRNA-122, glutamate dehydrogenase and alpha-foetoprotein) in human DILI. METHODS: Levels of the novel biomarkers were measured by enzyme-linked immunosorbent assay or real-time quantitative reverse-transcription PCR (qRT-PCR) in two human DILI cohorts: a human volunteer study with acetaminophen and a human immunodeficiency virus (HIV)/tuberculosis (TB) study. RESULTS: In the acetaminophen study, serum M65 and microRNA-122 levels were significantly increased at an earlier time point than ALT. Furthermore, the maximal elevation of M65 and microRNA-122 exceeded the increase in ALT. In the HIV/TB study, all the analysed novel biomarkers increased after 1 week of treatment. In contrast to ALT, the novel biomarkers remained stable in a human cohort with exercise-induced muscular injury. CONCLUSIONS: M65 and microRNA-122 are potential biomarkers of DILI superior to ALT with respect to sensitivity and specificity.

Mouse population-guided resequencing reveals that variants in CD44 contribute to acetaminophen-induced liver injury in humans.

Interindividual variability in response to chemicals and drugs is a common regulatory concern. It is assumed that xenobiotic-induced adverse reactions have a strong genetic basis, but many mechanism-based investigations have not been successful in identifying susceptible individuals. While recent advances in pharmacogenetics of adverse drug reactions show promise, the small size of the populations susceptible to important adverse events limits the utility of whole-genome association studies conducted entirely in humans. We present a strategy to identify genetic polymorphisms that may underlie susceptibility to adverse drug reactions. First, in a cohort of healthy adults who received the maximum recommended dose of acetaminophen (4 g/d x 7 d), we confirm that about one third of subjects develop elevations in serum alanine aminotransferase, indicative of liver injury. To identify the genetic basis for this susceptibility, a panel of 36 inbred mouse strains was used to model genetic diversity. Mice were treated with 300 mg/kg or a range of additional acetaminophen doses, and the extent of liver injury was quantified. We then employed whole-genome association analysis and targeted sequencing to determine that polymorphisms in Ly86, Cd44, Cd59a, and Capn8 correlate strongly with liver injury and demonstrated that dose-curves vary with background. Finally, we demonstrated that variation in the orthologous human gene, CD44, is associated with susceptibility to acetaminophen in two independent cohorts. Our results indicate a role for CD44 in modulation of susceptibility to acetaminophen hepatotoxicity. These studies demonstrate that a diverse mouse population can be used to understand and predict adverse toxicity in heterogeneous human populations through guided resequencing.

An analysis of N-acetylcysteine treatment for acetaminophen overdose using a systems model of drug-induced liver injury.

N-acetylcysteine (NAC) is the treatment of choice for acetaminophen poisoning; standard 72-h oral or 21-h intravenous protocols are most frequently used. There is controversy regarding which protocol is optimal and whether the full treatment course is always necessary. It would be challenging to address these questions in a clinical trial. We used DILIsym, a mechanistic simulation of drug-induced liver injury, to investigate optimal NAC treatment after a single acetaminophen overdose for an average patient and a sample population (n = 957). For patients presenting within 24 h of ingestion, we found that the oral NAC protocol preserves more hepatocytes than the 21-h intravenous protocol. In various modeled scenarios, we found that the 21-h NAC infusion is often too short, whereas the full 72-h oral course is often unnecessary. We found that there is generally a good correlation between the time taken to reach peak serum alanine aminotransferase (ALT) and the time taken to clear N-acetyl-p-benzoquinone imine (NAPQI) from the liver. We also found that the most frequently used treatment nomograms underestimate the risk for patients presenting within 8 h of overdose ingestion. V(max) for acetaminophen bioactivation to NAPQI was the most important variable in the model in determining interpatient differences in susceptibility. In conclusion, DILIsym predicts that the oral NAC treatment protocol, or an intravenous protocol with identical dosing, is superior to the 21-h intravenous protocol and ALT is the optimal available biomarker for discontinuation of the therapy. The modeling also suggests that modification of the current treatment nomograms should be considered.

In vitro to in vivo extrapolation and species response comparisons for drug-induced liver injury (DILI) using DILIsym™: a mechanistic, mathematical model of DILI.

Drug-induced liver injury (DILI) is not only a major concern for all patients requiring drug therapy, but also for the pharmaceutical industry. Many new in vitro assays and pre-clinical animal models are being developed to help screen compounds for the potential to cause DILI. This study demonstrates that mechanistic, mathematical modeling offers a method for interpreting and extrapolating results. The DILIsym™ model (version 1A), a mathematical representation of DILI, was combined with in vitro data for the model hepatotoxicant methapyrilene (MP) to carry out an in vitro to in vivo extrapolation. In addition, simulations comparing DILI responses across species illustrated how modeling can aid in selecting the most appropriate pre-clinical species for safety testing results relevant to humans. The parameter inputs used to predict DILI for MP were restricted to in vitro inputs solely related to ADME (absorption, distribution, metabolism, elimination) processes. MP toxicity was correctly predicted to occur in rats, but was not apparent in the simulations for humans and mice (consistent with literature). When the hepatotoxicity of MP and acetaminophen (APAP) was compared across rats, mice, and humans at an equivalent dose, the species most susceptible to APAP was not susceptible to MP, and vice versa. Furthermore, consideration of variability in simulated population samples (SimPops™) provided confidence in the predictions and allowed examination of the biological parameters most predictive of outcome. Differences in model sensitivity to the parameters were related to species differences, but the severity of DILI for each drug/species combination was also an important factor.

A Collaborative Initiative to Establish Genomic Biomarkers for Assessing Tumorigenic Potential to Reduce Reliance on Conventional Rodent Carcinogenicity Studies.

There is growing recognition across broad sectors of the scientific community that use of genomic biomarkers has the potential to reduce the need for conventional rodent carcinogenicity studies of industrial chemicals, agrochemicals, and pharmaceuticals through a weight-of-evidence approach. These biomarkers fall into 2 major categories: (1) sets of gene transcripts that can identify distinct tumorigenic mechanisms of action; and (2) cancer driver gene mutations indicative of rapidly expanding growth-advantaged clonal cell populations. This call-to-action article describes a collaborative approach launched to develop and qualify biomarker gene expression panels that measure widely accepted molecular pathways linked to tumorigenesis and their activation levels to predict tumorigenic doses of chemicals from short-term exposures. Growing evidence suggests that application of such biomarker panels in short-term exposure rodent studies can identify both tumorigenic hazard and tumorigenic activation levels for chemical-induced carcinogenicity. In the future, this approach will be expanded to include methodologies examining mutations in key cancer driver gene mutation hotspots as biomarkers of both genotoxic and nongenotoxic chemical tumor risk. Analytical, technical, and biological validation studies of these complementary genomic tools are being undertaken by multisector and multidisciplinary collaborative teams within the Health and Environmental Sciences Institute. Success from these efforts will facilitate the transition from current heavy reliance on conventional 2-year rodent carcinogenicity studies to more rapid animal- and resource-sparing approaches for mechanism-based carcinogenicity evaluation supporting internal and regulatory decision-making.

Urinary miRNA Biomarkers of Drug-Induced Kidney Injury and Their Site Specificity Within the Nephron.

Drug-induced kidney injury (DIKI) is a major concern in both drug development and clinical practice. There is an unmet need for biomarkers of glomerular damage and more distal renal injury in the loop of Henle and the collecting duct (CD). A cross-laboratory program to identify and characterize urinary microRNA (miRNA) patterns reflecting tissue- or pathology-specific DIKI was conducted. The overall goal was to propose miRNA biomarker candidates for DIKI that could supplement information provided by protein kidney biomarkers in urine. Rats were treated with nephrotoxicants causing injury to distinct nephron segments: the glomerulus, proximal tubule, thick ascending limb (TAL) of the loop of Henle and CD. Meta-analysis identified miR-192-5p as a potential proximal tubule-specific urinary miRNA candidate. This result was supported by data obtained in laser capture microdissection nephron segments showing that miR-192-5p expression was enriched in the proximal tubule. Discriminative miRNAs including miR-221-3p and -222-3p were increased in urine from rats treated with TAL versus proximal tubule toxicants in accordance with their expression localization in the kidney. Urinary miR-210-3p increased up to 40-fold upon treatment with TAL toxicants and was also enriched in laser capture microdissection samples containing TAL and/or CD versus proximal tubule. miR-23a-3p was enriched in the glomerulus and was increased in urine from rats treated with doxorubicin, a glomerular toxicant, but not with toxicants affecting other nephron segments. Taken together these results suggest that urinary miRNA panels sourced from specific nephron regions may be useful to discriminate the pathology of toxicant-induced lesions in the kidney, thereby contributing to DIKI biomarker development needs for industry, clinical, and regulatory use.

Urinary MicroRNAs in Environmental Health: Biomarkers of Emergent Kidney Injury and Disease.

PURPOSE OF REVIEW: There is a critical need for sensitive biomarkers of renal disease and progression. Micro(mi)RNAs are attractive as next-generation biomarkers in kidney disease, particularly as urine miRNAs can inform kidney function and cellular integrity. This review summarizes recent epidemiologic and toxicologic advances using urinary miRNAs and exosomal miRNAs as novel biomarkers of chemical exposure and of kidney damage and disease. RECENT FINDINGS: Urine miRNA biomarkers offer improved stability over protein in stored samples, relative ease of collection and quantitation, and conserved sequence homology across species. Particularly in the case of emergent environmental health threats such as chronic kidney disease of unknown origin, urinary miRNAs hold promise as biomarkers of disease and/or exposure. We present evidence to address scientific knowledge gaps, comment on the relevance of urine-derived miRNAs in environmental health research, and discuss limitations and recommendations for future directions needed to advance miRNA biomarker strategies.

Nitrosative Stress and Lipid Homeostasis as a Mechanism for Zileuton Hepatotoxicity and Resistance in Genetically Sensitive Mice.

Zileuton is an orally active inhibitor of leukotriene synthesis for maintenance treatment of asthma, for which clinical usage has been associated with idiosyncratic liver injury. Mechanistic understanding of zileuton toxicity is hampered by the rarity of the cases and lack of an animal model. A promising model for mechanistic study of rare liver injury is the Diversity Outbred (J:DO) mouse population, with genetic variation similar to that found in humans. In this study, female DO mice were administered zileuton or vehicle daily for 7 days (i.g.). Serum liver enzymes were elevated in the zileuton group, with marked interindividual variability in response. Zileuton exposure-induced findings in susceptible DO mice included microvesicular fatty change, hepatocellular mitosis, and hepatocellular necrosis. Inducible nitric oxide synthase and nitrotyrosine abundance were increased in livers of animals with necrosis and those with fatty change, implicating nitrosative stress as a possible injury mechanism. Conversely, DO mice lacking adverse liver pathology following zileuton exposure experienced decreased hepatic concentrations of resistin and increased concentrations of insulin and leptin, providing potential clues into mechanisms of toxicity resistance. Transcriptome pathway analysis highlighted mitochondrial dysfunction and altered fatty acid oxidation as key molecular perturbations associated with zileuton exposure, and suggested that interindividual differences in cytochrome P450 metabolism, glutathione-mediated detoxification, and farnesoid X receptor signaling may contribute to zileuton-induced liver injury (ZILI). Taken together, DO mice provided a platform for investigating mechanisms of toxicity and resistance in context of ZILI which may lead to targeted therapeutic interventions.

Mapping the Effects of Genetic Variation on Chromatin State and Gene Expression Reveals Loci That Control Ground State Pluripotency.

Mouse embryonic stem cells (mESCs) cultured in the presence of LIF occupy a ground state with highly active pluripotency-associated transcriptional and epigenetic circuitry. However, ground state pluripotency in some inbred strain backgrounds is unstable in the absence of ERK1/2 and GSK3 inhibition. Using an unbiased genetic approach, we dissect the basis of this divergent response to extracellular cues by profiling gene expression and chromatin accessibility in 170 genetically heterogeneous mESCs. We map thousands of loci affecting chromatin accessibility and/or transcript abundance, including 10 QTL hotspots where genetic variation at a single locus coordinates the regulation of genes throughout the genome. For one hotspot, we identify a single enhancer variant ∼10 kb upstream of Lifr associated with chromatin accessibility and mediating a cascade of molecular events affecting pluripotency. We validate causation through reciprocal allele swaps, demonstrating the functional consequences of noncoding variation in gene regulatory networks that stabilize pluripotent states in vitro.

Replication and narrowing of gene expression quantitative trait loci using inbred mice.

Gene expression quantitative trait locus (eQTL) mapping has become a powerful tool in systems biology. While many authors have made important discoveries using this approach, one persistent challenge in eQTL studies is the selection of loci and genes that should receive further biological investigation. In this study we compared eQTL generated from gene expression profiling in the livers of two panels of mouse strains: 41 BXD recombinant inbred and 36 Mouse Diversity Panel (MDP) strains. Cis-eQTL, loci in which the transcript and its maximum QTL are colocated, have been shown to be more reproducible than trans-eQTL, which are not colocated with the transcript. We observed that between 9.9 and 12.1% of cis-eQTL and between 2.0 and 12.6% of trans-eQTL replicated between the two panels depending on the degree of statistical stringency. Notably, a significant eQTL hotspot on distal chromosome 12 observed in the BXD panel was reproduced in the MDP. Furthermore, the shorter linkage disequilibrium in the MDP strains allowed us to considerably narrow the locus and limit the number of candidate genes to a cluster of Serpin genes, which code for extracellular proteases. We conclude that this strategy has some utility in increasing confidence and resolution in eQTL mapping studies; however, due to the high false-positive rate in the MDP, eQTL mapping in inbred strains is best carried out in combination with an eQTL linkage study.

Mouse population-based evaluation of urinary protein and miRNA biomarker performance associated with cisplatin renal injury.

Discovery and qualification of novel biomarkers with improved specificity and sensitivity for detection of xenobiotic-induced injuries is an area of active research across multiple sectors. However, the majority of efforts in this arena have used genetically limited rodent stocks that lack variability in xenobiotic responses inherent to genetically heterogeneous human populations. In this study, genetically diverse Diversity Outbred (DO) mice were used as a surrogate for human clinical populations to investigate performance of urinary kidney biomarkers against classical preclinical kidney injury biomarkers (blood urea nitrogen [BUN] and serum creatinine). In this study, cisplatin was used as a paradigm kidney toxicant, with female adult DO mice exposed to a single injection (5 mg/kg) of cisplatin or vehicle and necropsied 72 h post-exposure and 18 h following overnight urine collection. Interindividual variability in kidney toxicity was observed, with DO mice experiencing either no tubule cell necrosis or minimal-mild necrosis. A panel of urinary protein biomarkers and profiled miRNAs were assessed by receiver-operating characteristic curves as to their ability to distinguish non-responder versus responder animals, as defined by histopathological evidence of renal tubule cell necrosis. A surprising outcome of these studies was that BUN was elevated alongside of urinary miRNA and protein biomarkers in animals with grade 2 proximal tubular cell necrosis; but not elevated with grade 1 necrosis. These studies demonstrate a novel approach for using a rodent population to better assess sensitivity of candidate biomarkers, especially for proposed clinical applications. Impact statement Recent studies have indicated that several urinary proteins and miRNA species may be suitable as biomarkers for acute kidney injury. A major focus on biomarker qualification is demonstrating improved specificity and sensitivity over gold standard tests. In this study, a mouse population model, Diversity Outbred mice, was used to demonstrate that neither the urinary protein markers nor the miRNA species assayed in urine could reliably detect low severity kidney injury better than blood urea nitrogen. This study has implications for use of these biomarkers in the clinic, where interindividual heterogeneity is present within patient populations and for which the underlying tissue pathology may not be known.

New Rodent Population Models May Inform Human Health Risk Assessment and Identification of Genetic Susceptibility to Environmental Exposures.

BACKGROUND: This paper provides an introduction for environmental health scientists to emerging population-based rodent resources. Mouse reference populations provide an opportunity to model environmental exposures and gene-environment interactions in human disease and to inform human health risk assessment. OBJECTIVES: This review will describe several mouse populations for toxicity assessment, including older models such as the Mouse Diversity Panel (MDP), and newer models that include the Collaborative Cross (CC) and Diversity Outbred (DO) models. METHODS: This review will outline the features of the MDP, CC, and DO mouse models and will discuss published case studies investigating the use of these mouse population resources in each step of the risk assessment paradigm. DISCUSSION: These unique resources have the potential to be powerful tools for generating hypotheses related to gene-environment interplay in human disease, performing controlled exposure studies to understand the differential responses in humans for susceptibility or resistance to environmental exposures, and identifying gene variants that influence sensitivity to toxicity and disease states. CONCLUSIONS: These new resources offer substantial advances to classical toxicity testing paradigms by including genetically sensitive individuals that may inform toxicity risks for sensitive subpopulations. Both in vivo and complementary in vitro resources provide platforms with which to reduce uncertainty by providing population-level data around biological variability. https://doi.org/10.1289/EHP1274.