Hepatotoxicity of silver nanoparticles: Benchmark concentration modeling of an in vitro transcriptomics study in human iPSC-derived hepatocytes.

Despite two decades of research on silver nanoparticle (AgNP) toxicity, a safe threshold for exposure has not yet been established, albeit being critically needed for risk assessment and regulatory decision-making. Traditionally, a point-of-departure (PoD) value is derived from dose response of apical endpoints in animal studies using either the no-observed-adverse-effect level (NOAEL) approach, or benchmark dose (BMD) modeling. To develop new approach methodologies (NAMs) to inform human risk assessment of AgNPs, we conducted a concentration response modeling of the transcriptomic changes in hepatocytes derived from human induced pluripotent stem cells (iPSCs) after being exposed to a wide range concentration (0.01-25 µg/ml) of AgNPs for 24 h. A plausible transcriptomic PoD of 0.21 µg/ml was derived for a pathway related to the mode-of-action (MOA) of AgNPs, and a more conservative PoD of 0.10 µg/ml for a gene ontology (GO) term not apparently associated with the MOA of AgNPs. A reference dose (RfD) could be calculated from either of the PoDs as a safe threshold for AgNP exposure. The current study illustrates the usefulness of in vitro transcriptomic concentration response study using human cells as a NAM for toxicity study of chemicals that lack adequate toxicity data to inform human risk assessment.

Oxidative DNA damage contributes to usnic acid-induced toxicity in human induced pluripotent stem cell-derived hepatocytes.

Dietary supplements containing usnic acid have been increasingly marketed for weight loss over the past decades, even though incidences of severe hepatotoxicity and acute liver failure due to their overuse have been reported. To date, the toxic mechanism of usnic acid-induced liver injury at the molecular level still remains to be fully elucidated. Here, we conducted a transcriptomic study on usnic acid using a novel in vitro hepatotoxicity model employing human induced pluripotent stem cell (iPSC)-derived hepatocytes. Treatment with 20 µM usnic acid for 24 h caused 4272 differentially expressed genes (DEGs) in the cells. Ingenuity Pathway Analysis (IPA) based on the DEGs and gene set enrichment analysis (GSEA) using the whole transcriptome expression data concordantly revealed several signaling pathways and biological processes that, when taken together, suggest that usnic acid caused oxidative stress and DNA damage in the cells, which further led to cell cycle arrest and eventually resulted in cell death through apoptosis. These transcriptomic findings were subsequently corroborated by a variety of cellular assays, including reactive oxygen species (ROS) generation and glutathione (GSH) depletion, DNA damage (pH2AX detection and 8-hydroxy-2'-deoxyguanosine [8-OH-dg] assay), cell cycle analysis, and caspase 3/7 activity. Collectively, the results of the current study accord with previous in vivo and in vitro findings, provide further evidence that oxidative stress-caused DNA damage contributes to usnic acid-induced hepatotoxicity, shed new light on molecular mechanisms of usnic acid-induced hepatotoxicity, and demonstrate the usefulness of iPSC-derived hepatocytes as an in vitro model for hepatotoxicity testing and prediction.

Axes of visual adaptation in the ecologically diverse family Cichlidae.

The family Cichlidae contains approximately 2000 species that live in diverse freshwater habitats including murky lakes, turbid rivers, and clear lakes from both the Old and New Worlds. Their visual systems are similarly diverse and have evolved specific sensitivities that differ along several axes of variation. Variation in cornea and lens transmission affect which wavelengths reach the retina. Variation in photoreceptor number and distribution affect brightness sensitivity, spectral sensitivity and resolution. Probably their most dynamic characteristic is the variation in visual pigment peak sensitivities. Visual pigments can be altered through changes in chromophore, opsin sequence and opsin expression. Opsin expression varies by altering which of the seven available cone opsins in their genomes are turned on. These opsins can even be coexpressed to produce seemingly infinitely tunable cone sensitivities. Both chromophore and opsin expression can vary on either rapid (hours or days), slower (seasonal or ontogenetic) or evolutionary timescales. Such visual system shifts have enabled cichlids to adapt to different habitats and foraging styles. Through both short term plasticity and longer evolutionary adaptations, cichlids have proven to be ecologically successful and an excellent model for studying organismal adaptation.

A mouse model of Proteus syndrome.

Proteus syndrome is a mosaic, progressive overgrowth disorder caused by a somatic activating variant c.49G > A p.(E17K) in AKT1. The presentation in affected individuals is variable, with a diversity of tissues demonstrating abnormalities. Common manifestations include skin and bony overgrowth, vascular malformations (VMs), cysts and benign tumors. We used two methods to create mouse models that had endogenously-regulated mosaic expression of the Proteus syndrome variant. Variant allele fractions (VAFs) ranged from 0% to 50% across numerous tissues in 44 Proteus syndrome mice. Mice were phenotypically heterogeneous with lesions rarely observed before 12 months of age. VMs were the most frequent finding with a total of 69 found in 29 of 44 Proteus syndrome mice. Twenty-eight cysts and ectasia, frequently biliary, were seen in 22 of 44 Proteus syndrome mice. Varying levels of mammary hyperplasia were seen in 10 of 16 female Proteus syndrome mice with other localized regions of hyperplasia and stromal expansion noted in several additional animals. Interestingly, 27 of 31 Proteus syndrome animals had non-zero blood VAF that is in contrast to the human disorder where it is rarely seen in peripheral blood. Identification of variant-positive cells by green fluorescent protein (GFP) staining in chimeric Proteus syndrome mice showed that in some lesions, hyperplastic cells were predominantly GFP/Akt1E17K-positive and showed increased pAKT signal compared to GFP-negative cells. However, hyperplastic mammary epithelium was a mixture of GFP/Akt1E17K-positive and negative cells with some GFP/Akt1E17K-negative cells also having increased pAKT signal suggesting that the variant-positive cells can induce lesion formation in a non-cell autonomous manner.

Adult plasticity in African cichlids: Rapid changes in opsin expression in response to environmental light differences.

Phenotypic plasticity allows organisms to adapt quickly to local environmental conditions and could facilitate adaptive radiations. Cichlids have recently undergone an adaptive radiation in Lake Malawi where they inhabit diverse light environments and tune their visual sensitivity through differences in cone opsin expression. While cichlid opsin expression is known to be plastic over development, whether adults remain plastic is unknown. Adult plasticity in visual tuning could play a role in cichlid radiations by enabling survival in changing environments and facilitating invasion into novel environments. Here we examine the existence of and temporal changes in adult visual plasticity of two closely related species. In complementary experiments, wild adult Metriaclima mbenji from Lake Malawi were moved to the lab under UV-deficient fluorescent lighting; while lab raised M. benetos were placed under UV-rich lighting designed to mimic light conditions in the wild. Surprisingly, adult cichlids in both experiments showed significant changes in the expression of the UV-sensitive single cone opsin, SWS1, in only 3 days. Modeling quantum catches in the light environments revealed a possible link between the light available to the SWS1 visual pigment and SWS1 expression. We conclude that adult cichlids can undergo rapid and significant changes in opsin expression in response to environmental light shifts that are relevant to their habitat and evolutionary history in Lake Malawi. This could have contributed to the rapid divergence characteristic of these fantastic fishes.

Comparison on the mechanism and potency of hepatotoxicity among hemp extract and its four major constituent cannabinoids.

Cannabidiol (CBD) has been reported to induce hepatotoxicity in clinical trials and research studies; however, little is known about the safety of other nonintoxicating cannabinoids. New approach methodologies (NAMs) based on bioinformatic analysis of high-throughput transcriptomic data are gaining increasing importance in risk assessment and regulatory decision-making of data-poor chemicals. In the current study, we conducted a concentration response transcriptomic analysis of hemp extract and its four major constituent cannabinoids [CBD, cannabichromene (CBC), cannabigerol (CBG), and cannabinol (CBN)] in hepatocytes derived from human induced pluripotent stem cells (iPSCs). Each compound impacted a distinctive combination of biological functions and pathways. However, all the cannabinoids impaired liver metabolism and caused oxidative stress in the cells. Benchmark concentration (BMC) analysis showed potencies in transcriptional activity of the cannabinoids were in the order of CBN > CBD > CBC > CBG, consistent with the order of their cytotoxicity IC50 values. Patterns of transcriptomic changes induced by hemp extract and its median overall BMC were very similar to CBD but differed significantly from other cannabinoids, suggesting that potential adverse effects of hemp extract were largely due to its major constituent CBD. Lastly, transcriptomic point-of-departure (tPoD) values were determined for each of the compounds, with the value for CBD (0.106 µM) being concordant with a previously reported one derived from apical endpoints of clinical and animal studies. Taken together, the current study demonstrates the potential utility of transcriptomic BMC analysis as a NAM for hazard assessment of data-poor chemicals, improves our understanding of the possible health effects of hemp extract and its constituent cannabinoids, and provides important tPoD data that could contribute to inform human safety assessment of these cannabinoid compounds.

Allelic heterogeneity of Proteus syndrome.

Proteus syndrome is a mosaic disorder that can cause progressive postnatal overgrowth of nearly any organ or tissue. To date, Proteus syndrome has been exclusively associated with the mosaic c.49G > A p.(Glu17Lys) pathogenic variant in AKT1, a variant that is also present in many cancers. Here we describe an individual with severe Proteus syndrome who died at 7.5 yr of age from combined parenchymal and restrictive pulmonary disease. Remarkably, this individual was found to harbor a mosaic c.49_50delinsAG p.(Glu17Arg) variant in AKT1 at a variant allele fraction that ranged from <0.01 to 0.46 in fibroblasts established from an overgrown digit. This variant was demonstrated to be constitutively activating by phosphorylation of AKT(S473). These data document allelic heterogeneity for Proteus syndrome. We recommend that individuals with a potential clinical diagnosis of Proteus syndrome who are negative for the p.(Glu17Lys) variant be tested for other variants in AKT1.

Diurnal variation in opsin expression and common housekeeping genes necessitates comprehensive normalization methods for quantitative real-time PCR analyses.

To determine the visual sensitivities of an organism of interest, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is often used to quantify expression of the light-sensitive opsins in the retina. While qRT-PCR is an affordable, high-throughput method for measuring expression, it comes with inherent normalization issues that affect the interpretation of results, especially as opsin expression can vary greatly based on developmental stage, light environment or diurnal cycles. We tested for diurnal cycles of opsin expression over a period of 24 hr at 1-hr increments and examined how normalization affects a data set with fluctuating expression levels using qRT-PCR and transcriptome data from the retinae of the cichlid Pelmatolapia mariae. We compared five methods of normalizing opsin expression relative to (a) the average of three stably expressed housekeeping genes (Ube2z, EF1-α and ß-actin), (b) total RNA concentration, (c) GNAT2, (the cone-specific subunit of transducin), (d) total opsin expression and (e) only opsins expressed in the same cone type. Normalizing by proportion of cone type produced the least variation and would be best for removing time-of-day variation. In contrast, normalizing by housekeeping genes produced the highest daily variation in expression and demonstrated that the peak of cone opsin expression was in the late afternoon. A weighted correlation network analysis showed that the expression of different cone opsins follows a very similar daily cycle. With the knowledge of how these normalization methods affect opsin expression data, we make recommendations for designing sampling approaches and quantification methods based upon the scientific question being examined.

Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome.

A somatic activating mutation in AKT1, c.49G>A, pGlu17Lys, that results in elevated AKT signaling in mutation-positive cells, is responsible for the mosaic overgrowth condition, Proteus syndrome. ARQ 092 is an allosteric pan-AKT inhibitor under development for treatment in cancer. We tested the efficacy of this drug for suppressing AKT signaling in cells and tissues from patients with Proteus syndrome. ARQ 092 reduced phosphorylation of AKT and downstream targets of AKT in a concentration-dependent manner in as little as two hours. While AKT signaling was suppressed with ARQ 092 treatment, cells retained their ability to respond to growth factor stimulation by increasing pAKT levels proportionally to untreated cells. At concentrations sufficient to decrease AKT signaling, little reduction in cell viability was seen. These results indicate that ARQ 092 can suppress AKT signaling and warrants further development as a therapeutic option for patients with Proteus syndrome.