The diagnosis and management of drug-induced liver injury (DILI) is hindered by the limited utility of traditional clinical chemistries. It has recently been shown that hepatotoxicants can produce compound-specific changes in the peripheral blood (PB) transcriptome in rodents, suggesting that the blood transcriptome might provide new biomarkers of DILI. To investigate in humans, we used DNA microarrays as well as serum metabolomic methods to characterize changes in the transcriptome and metabolome in serial PB samples obtained from six healthy adults treated with a 4-g bolus dose of acetaminophen (APAP) and from three receiving placebo. Treatment did not cause liver injury as assessed by traditional liver chemistries. However, 48 hours after exposure, treated subjects showed marked down-regulation of genes involved in oxidative phosphorylation/mitochondrial function that was not observed in the placebos ( P < 1.66E-19). The magnitude of down-regulation was positively correlated with the percent of APAP converted to the reactive metabolite N -acetyl- p -benzoquinone-imide (NAPQI) ( r = 0.739; P = 0.058). In addition, unbiased analysis of the serum metabolome revealed an increase in serum lactate from 24 to 72 hours postdosing in the treated subjects alone ( P < 0.005). Similar PB transcriptome changes were observed in human overdose patients and rats receiving toxic doses. Conclusion: The single 4-g APAP dose produced a transcriptome signature in PB cells characterized by down-regulation of oxidative phosphorylation genes accompanied by increased serum lactate. Similar gene expression changes were observed in rats and several patients after consuming hepatotoxic doses of APAP. The timing of the changes and the correlation with NAPQI production are consistent with mechanisms known to underlie APAP hepatoxicity. These studies support the further exploration of the blood transcriptome for biomarkers of DILI. (Hepatology 2010.)

Deficiency in Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in humans is associated with the development of congenital hypothyroidism. However, the functions of GLIS3 in the thyroid gland and the mechanism by which GLIS3 dysfunction causes hypothyroidism are unknown. In the current study, we demonstrate that GLIS3 acts downstream of thyroid-stimulating hormone (TSH) and TSH receptor (TSHR) and is indispensable for TSH/TSHR-mediated proliferation of thyroid follicular cells and biosynthesis of thyroid hormone. Using ChIP-Seq and promoter analysis, we demonstrate that GLIS3 is critical for the transcriptional activation of several genes required for thyroid hormone biosynthesis, including the iodide transporters Nis and Pds, both of which showed enhanced GLIS3 binding at their promoters. The repression of cell proliferation of GLIS3-deficient thyroid follicular cells was due to the inhibition of TSH-mediated activation of the mTOR complex 1/ribosomal protein S6 (mTORC1/RPS6) pathway as well as the reduced expression of several cell division–related genes regulated directly by GLIS3. Consequently, GLIS3 deficiency in a murine model prevented the development of goiter as well as the induction of inflammatory and fibrotic genes during chronic elevation of circulating TSH. Our study identifies GLIS3 as a key regulator of TSH/TSHR-mediated thyroid hormone biosynthesis and proliferation of thyroid follicular cells and uncovers a mechanism by which GLIS3 deficiency causes neonatal hypothyroidism and prevents goiter development.

This report details the standardized experimental design and the different data streams that were collected (histopathology, clinical chemistry, hematology and gene expression from the target tissue (liver) and a bio-available tissue (blood)) after treatment with eight known hepatotoxicants (at multiple time points and doses with multiple biological replicates). The results of the study demonstrate the classification of histopathological differences, likely reflecting differences in mechanisms of cell-specific toxicity, using either liver tissue or blood transcriptomic data.

Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25–2.5 μM cisplatin (CisPt) or 12.5–100 μM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 μM CisPt from 3 to 96 h expressing 2000–5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity.

Approximately 1 in 10 newborns are born preterm and require supplemental oxygen (O₂) in an extrauterine environment following birth. Supplemental O₂ can induce oxidative stress that can impair mitochondrial function, resulting in lung injury and increased risk in early life pulmonary diseases. The nuclear factor-erythroid 2 related factor 2 (NRF2) protects the cells from oxidative stress by regulating the expression of genes containing antioxidant response elements and many mitochondrial-associated genes. In this study, we compared Nrf2-deficient (Nrf2^−/−) and wild-type (Nrf2^+/+) mice to define the role of NRF2 in lung mitochondrial genomic features in late embryonic development in mice (embryonic days, E13.5 and E18.5) versus birth (postnatal day 0, PND0). We also determined whether NRF2 protects lung mitochondrial genome parameters in postnatal mice exposed to a 72 h hyperoxia environment. We found Nrf2^−/− embryonic lungs were characterized by decreases in mtDNA copies from E13.5 to E18.5. Interestingly, Nrf2^−/− heteroplasmy frequency was significantly higher than Nrf2^+/+ at E18.5, though this effect reversed at PND0. In postnatal mice exposed to hyperoxia, we identified three- to four-fold increases in mitochondria-encoded mitochondrial genes, which regulate oxidative phosphorylation. Overall, our findings demonstrate a potentially critical role of NRF2 in mediating long-term effects of hyperoxia on mitochondrial function.