Effects of multi-walled carbon nanotubes on message and Micro-RNA in human lung BEAS-2B cells.

Multi-walled Carbon nanotubes (MWCNTs) lack sufficient quality cytotoxicity, toxicity, genotoxicity and genomic data on which to make environmental and regulatory decisions. Therefore, we did a multidisciplinary in vitro study of 3 MWCNTs in human lung cells (BEAS-2B) with the following endpoints: cytotoxicity, DNA damage, reactive oxygen and nitrogen species, lipid peroxidation and mRNA and microRNA expression analyses. The MWCNTs were either unfunctionalized or functionalized with either -OH or -COOH. Doses studied ranged from 0.3 to 100 ug/ml and were exposed to a human lung cell line in vitro for 72 h., with genomic studies being done from 30 ug/ml downward. Some of the genomic pathways that were altered by MWCNT exposure were NRF2 mediated oxidative stress response, DNA damage repair, nuclear excision repair, base excision repair, mitochondrial dysfunction, oxidative phosphorylation, HIF1α signaling, unfolded protein response, protein ubiquitination, ferroptosis and sirtuin signaling pathways. The data suggested that OH functionalized MWCNT caused more and larger gene/microRNA changes, followed by COOH functionalized MWCNT and unfunctionalized MWCNT being the least biologically active. From microRNA target filter analysis, there were altered signaling hubs. MYC is the only hub that altered by all 3 MWCNTs. Signaling hubs that are common to OH and COOH functionalized MWCNTs are GRB2, AR, TP63 and AGO2. The signaling hubs that were only present in OH functionalized MWCNTs are TP53, STAT3 and BRCA1. These signaling pathways and hubs we found in vitro correlated well with the published in vivo pathological effects like oxidative stress DNA damage, inflammation and cancer in MWCNTs treated mice.

Effects of multi-walled carbon nanotubes on gene and microRNA expression in human hepatocarcinoma HepG2 cells.

The usage of multi-walled carbon nanotubes (MWCNT) has increased exponentially in the past years, but, potential toxicity mechanisms are not clear. We studied the transcriptomic alterations induced by one multi-walled carbon nanotube (MWCNT) and its -OH and -COOH functionalized derivatives in human HepG2 cells. We showed that all three MWCNT treatments induced alterations in stress-related signaling pathways, inflammation-related signaling pathways, cholesterol synthesis pathways, proliferation-related pathways, senescence-related pathways and cancer-related pathways. In stress-related pathways, the acute phase response was induced in all three MWCNTs and all doses treated and ranked high. Other stress-related pathways were also related to the oxidative-induced signaling pathways, such as NRF-2 mediated oxidative stress response, hepatic fibrosis/Stella cell activation, iNOS signaling, and Hif1α signaling. Many inflammation-related pathways were altered, such as IL-8, IL-6, TNFR1, TNFR2, and NF-κB signaling pathways. These results were consistent with our previous results with exposures to the same three multi-walled carbon nanotubes in human lung BEAS-2B and also with results in mice and rats. From the microRNA target filter analysis, TXNIP & miR-128-3p interaction was present in all three MWCNT treatments, and maybe important for the induction of oxidative stress. CXCL-8 & miR-146-5p and Wee1 & miR-128-3p were only present in the cells treated with the parent and the OH-functionalized MWCNTs. These mRNA-miRNA interactions were involved in oxidative stress, inflammation, cell cycle, cholesterol biosynthesis and cancer related pathways. Target filter analysis also showed altered liver hyperplasia/hyperproliferation and hepatic cancer pathways. In short, target filter analysis complemented the transcriptomic analysis and pointed to specific gene/microRNA interactions that can help inform mechanism of action. Moreover, our study showed that the signaling pathways altered in HepG2 cells correlated well with the toxicity and carcinogenicity observed in vivo, indicating that HepG2 may be a good in vitro predictive model for MWCNT toxicity studies.

Differential genomic effects of four nano-sized and one micro-sized CeO2 particles on HepG2 cells.

The objective of this research was to perform a genomics study of five cerium oxide particles, 4 nano and one micrometer-sized particles which have been studied previously by our group with respect to cytotoxicity, biochemistry and metabolomics. Human liver carcinoma HepG2 cells were exposed to between 0.3 to 300 ug/ml of CeO2 particles for 72 hours and then total RNA was harvested. Fatty acid accumulation was observed with W4, X5, Z7 and less with Q but not Y6. The gene expression changes in the fatty acid metabolism genes correlated the fatty acid accumulation we detected in the prior metabolomics study for the CeO2 particles named W4, Y6, Z7 and Q, but not for X5. In particular, the observed genomics effects on fatty acid uptake and fatty acid oxidation offer a possible explanation of why many CeO2 particles increase cellular free fatty acid concentrations in HepG2 cells. The major genomic changes observed in this study were sirtuin, ubiquitination signaling pathways, NRF2-mediated stress response and mitochondrial dysfunction. The sirtuin pathway was affected by many CeO2 particle treatments. Sirtuin signaling itself is sensitive to oxidative stress state of the cells and may be an important contributor in CeO2 particle induced fatty acid accumulation. Ubiquitination pathway regulates many protein functions in the cells, including sirtuin signaling, NRF2 mediated stress, and mitochondrial dysfunction pathways. NRF2-mediated stress response and mitochondrial were reported to be altered in many nanoparticles treated cells. All these pathways may contribute to the fatty acid accumulation in the CeO2 particle treated cells.

Determinants of gene expression in the human liver: Impact of aging and sex on xenobiotic metabolism.

There is a need to characterize the potential susceptibility of older adults to toxicity from environmental chemical exposures. Liver xenobiotic metabolizing enzymes (XMEs) play important roles in detoxifying and eliminating xenobiotics. We examined global gene expression in the livers of young (21-45 years) and old (69+ years) men and women. Differentially expressed genes (DEG) were identified using two-way ANOVA (p ≤ 0.05). We identified 1437 and 1670 DEGs between young and old groups in men and women, respectively. Only a minor number of the total number of genes overlapped (146 genes). Aging increased or decreased pathways involved in inflammation and intermediary metabolism, respectively. Aging led to numerous changes in the expression of XME genes or genes known to control their expression (~90 genes). Out of 10 cytochrome P450s activities examined, there were increased activities of CYP1A2 and CYP2C9 enzymes in the old groups. We also identified sex-dependent genes that were more numerous in the young group (1065) than in the old group (202) and included changes in XMEs. These studies indicate that the livers from aging humans when compared to younger adults exhibit changes in XMEs that may lead to differences in the metabolism of xenobiotics.

Effects of Silver Nanoparticles and Silver Nitrate on mRNA and microRNA Expression in Human Hepatocellular Carcinoma Cells (HepG2).

In order to understand toxicity of nano silver, human hepatocellular carcinoma (HepG2) cells were treated either with silver nitrate (AgNO3) or with nano silver capped with glutathione (Ag-S) at various concentration. Differentially expressed genelists for mRNA and microRNA were obtained through Illumina RNA sequencing and DEseq data analyses. Both treatments showed non-linear dose response relationships for mRNA and microRNA. Gene expression analysis showed signaling pathways common to both nano Ag-S and AgNO3, such as cell cycle regulation, DNA damage response and cancer related pathways. But, nano Ag-S caused signaling pathway changes that were not altered by AgNO3 such as NRF2-mediated oxidative stress response inflammation, cell membrane signaling, and cell proliferation. Nano Ag-S also affected p53 signaling, survival, apoptosis, tissue repair, lipid synthesis, angiogenesis, liver fibrosis and tumor development. Several of the pathways affected by nano Ag-S are hypothesized as major contributors to nanotoxicity. MicroRNA target filter analysis revealed additional affected pathways that were not reflected in the mRNA expression response alone, including DNA damage signaling, genomic stability, ROS, cell cycle, ubiquitination, DNA methylation, cell proliferation and fibrosis for AgNO3; and cell cycle regulation, P53 signaling, cell proliferation, survival, apoptosis, tissue repair and so on for nano Ag-S. These pathways may be mediated by microRNA repression of protein translation.Our study clearly showed that the addition of microRNA profiling increased the numbers of signaling pathways discovered that affected by the treatments on HepG2 cells and gave US a better picture of the effects of these reagents in the cells.

Effects of Copper Nanoparticles on mRNA and Small RNA Expression in Human Hepatocellular Carcinoma (HepG2) Cells.

With the advancement of nanotechnology, nanoparticles are widely used in many different industrial processes and consumer products. Copper nanoparticles (Cu NPs) are among the most toxic nanomaterials. We investigated Cu NPs toxicity in Human Hepatocellular carcinoma (HepG2) cells by examining signaling pathways, and microRNA/mRNA interactions. We compared the effects of exposures to Cu NPs at various concentrations and CuCl2 was used as a control. The number of differentially expressed mRNA did not follow a linear dose-response relationship for either Cu NPs or CuCl2 treatments. The most significantly altered genes and pathways by Cu NPs exposure were NRF2 (nuclear factor erythroid 2 related factor 2)-mediated oxidative stress response, protein ubiquitination, Tumor protein p53 (p53), phase I and II metabolizing enzymes, antioxidant proteins and phase III detoxifying gene pathways.Messenger RNA-microRNA interaction from MicroRNA Target Filter Analyses revealed more signaling pathways altered in Cu NPs treated samples than transcriptomics alone, including cell proliferation, DNA methylation, endoplasmic reticulum (ER) stress, apoptosis, autophagy, reactive oxygen species, inflammation, tumorigenesis, extracellular matrix/angiogenesis and protein synthesis. In contrast, in the control (CuCl2) treated samples showed mostly changes in inflammation mainly through regulation of the Nuclear Factor Kappa-light-chain-enhancer of Activated B-cells (NFκB). Further, some RNA based parameters that showed promise as biomarkers of Cu NPs exposure including both well and lesser known genes: heme oxygenase 1 (HMOX1), heat shock protein, c-Fos proto-oncogene, DNA methyltransferases, and glutamate-cysteine ligase modifier subunit (GCLM, part of the glutathione synthesis pathway). The differences in signaling pathways altered by the Cu NPs and CuCl2 treatments suggest that the effects of the Cu NPs were not the results of nanomaterial dissolution to soluble copper ions.

The Role of Hepatic Vagal Tone in Ozone-Induced Metabolic Dysfunction in the Liver.

Air pollution has been associated with metabolic diseases and hepatic steatosis-like changes. We have shown that ozone alters liver gene expression for metabolic processes through neuroendocrine activation. This study aimed to further characterize ozone-induced changes and to determine the impact of hepatic vagotomy (HV) which reduces parasympathetic influence. Twelve-week-old male Wistar-Kyoto rats underwent HV or sham surgery 5-6 days before air or ozone exposure (0 or 1 ppm; 4 h/day for 1 or 2 days). Ozone-induced lung injury, hyperglycemia, glucose intolerance, and increases in circulating cholesterol, triglycerides, and leptin were similar in rats with HV and sham surgery. However, decreases in circulating insulin and increased HDL and LDL were observed only in ozone-exposed HV rats. Ozone exposure resulted in changed liver gene expression in both sham and HV rats (sham > HV), however, HV did not change expression in air-exposed rats. Upstream target analysis revealed that ozone-induced transcriptomic changes were similar to responses induced by glucocorticoid-mediated processes in both sham and HV rats. The directionality of ozone-induced changes reflecting cellular response to stress, metabolic pathways, and immune surveillance was similar in sham and HV rats. However, pathways regulating cell-cycle, regeneration, proliferation, cell growth, and survival were enriched by ozone in a directionally opposing manner between sham and HV rats. In conclusion, parasympathetic innervation modulated ozone-induced liver transcriptional responses for cell growth and regeneration without affecting stress-mediated metabolic changes. Thus, impaired neuroendocrine axes and parasympathetic innervation could collectively contribute to adverse effects of air pollutants on the liver.

Ozone-induced fetal growth restriction in rats is associated with sexually dimorphic placental and fetal metabolic adaptation.

OBJECTIVE: The importance of the placenta in mediating the pre- and post-natal consequences of fetal growth restriction has been increasingly recognized. However, the influence of placental sexual dimorphism on driving these outcomes has received little attention. The purpose of this study was to characterize how sex contributes to the relationship between placental metabolism and fetal programming utilizing a novel rodent model of growth restriction. METHODS: Fetal growth restriction was induced by maternal inhalation of 0.8 ppm ozone (4 h/day) during implantation receptivity (gestation days [GDs] 5 and 6) in Long-Evans rats. Control rats were exposed to filtered air. At GD 21, placental and fetal tissues were obtained for metabolic and genomic assessments. RESULTS: Growth-restricted male placentae exhibited increased mitochondrial biogenesis, increased oxygen consumption, and reduced nutrient storage. Male growth-restricted fetuses also had evidence of reduced adiposity and downregulation of hepatic metabolic signaling. In contrast, placentae from growth-restricted females had elevated markers of autophagy accompanied by an observed protection against hepatic metabolic perturbations. Despite this, growth restriction in females induced a greater number of hypothalamic gene and pathway alterations compared to growth-restricted males. CONCLUSIONS: Increases in mitochondrial metabolism in growth-restricted male placentae likely initiates a sequela of adaptations that promote poor nutrient availability and adiposity. Divergently, the female placenta expresses protective mechanisms that may serve to increase nutrient availability to support fetal metabolic development. Collectively, this work emphasizes the importance of sex in mediating alterations in placental metabolism and fetal programming.

Ozone-Induced Dysregulation of Neuroendocrine Axes Requires Adrenal-Derived Stress Hormones.

Acute ozone inhalation increases circulating stress hormones through activation of the sympathetic-adrenal-medullary and hypothalamic-pituitary-adrenal axes. Rats with adrenalectomy (AD) have attenuated ozone-induced lung responses. We hypothesized that ozone exposure will induce changes in circulating pituitary-derived hormones and global gene expression in the brainstem and hypothalamus, and that AD will ameliorate these effects. Male Wistar-Kyoto rats (13 weeks) that underwent sham surgery (SHAM) or AD were exposed to ozone (0.8 ppm) or filtered-air for 4 h. In SHAM rats, ozone exposure decreased circulating thyroid-stimulating hormone (TSH), prolactin (PRL), and luteinizing hormone (LH). AD prevented reductions in TSH and PRL, but not LH. AD increased adrenocorticotropic hormone approximately 5-fold in both air- and ozone-exposed rats. AD in air-exposed rats resulted in few significant transcriptional differences in the brainstem and hypothalamus (approximately 20 genes per tissue). In contrast, ozone-exposure in SHAM rats resulted in either increases or decreases in expression of hundreds of genes in the brainstem and hypothalamus relative to air-exposed SHAM rats (303 and 568 genes, respectively). Differentially expressed genes from ozone exposure were enriched for pathways involving hedgehog signaling, responses to alpha-interferon, hypoxia, and mTORC1, among others. Gene changes in both brain areas were analogous to those altered by corticosteroids and L-3,4-dihydroxyphenylalanine, suggesting a role for endogenous glucocorticoids and catecholamines. AD completely prevented this ozone-induced transcriptional response. These findings show that short-term ozone inhalation promotes a shift in brainstem and hypothalamic gene expression that is dependent upon the presence of circulating adrenal-derived stress hormones. This is likely to have profound downstream influence on systemic effects of ozone.

Engineering human cell spheroids to model embryonic tissue fusion in vitro.

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton's Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.

Adrenal-derived stress hormones modulate ozone-induced lung injury and inflammation.

Ozone-induced systemic effects are modulated through activation of the neuro-hormonal stress response pathway. Adrenal demedullation (DEMED) or bilateral total adrenalectomy (ADREX) inhibits systemic and pulmonary effects of acute ozone exposure. To understand the influence of adrenal-derived stress hormones in mediating ozone-induced lung injury/inflammation, we assessed global gene expression (mRNA sequencing) and selected proteins in lung tissues from male Wistar-Kyoto rats that underwent DEMED, ADREX, or sham surgery (SHAM) prior to their exposure to air or ozone (1ppm), 4h/day for 1 or 2days. Ozone exposure significantly changed the expression of over 2300 genes in lungs of SHAM rats, and these changes were markedly reduced in DEMED and ADREX rats. SHAM surgery but not DEMED or ADREX resulted in activation of multiple ozone-responsive pathways, including glucocorticoid, acute phase response, NRF2, and PI3K-AKT. Predicted targets from sequencing data showed a similarity between transcriptional changes induced by ozone and adrenergic and steroidal modulation of effects in SHAM but not ADREX rats. Ozone-induced increases in lung Il6 in SHAM rats coincided with neutrophilic inflammation, but were diminished in DEMED and ADREX rats. Although ozone exposure in SHAM rats did not significantly alter mRNA expression of Ifnγ and Il-4, the IL-4 protein and ratio of IL-4 to IFNγ (IL-4/IFNγ) proteins increased suggesting a tendency for a Th2 response. This did not occur in ADREX and DEMED rats. We demonstrate that ozone-induced lung injury and neutrophilic inflammation require the presence of circulating epinephrine and corticosterone, which transcriptionally regulates signaling mechanisms involved in this response.

Differential Genomic Effects of Six Different TiO2 Nanomaterials on Human Liver HepG2 Cells.

Human HepG2 cells were exposed to six TiO2 nanomaterials (with dry primary particle sizes ranging from 22 to 214 nm, either 0.3, 3, or 30 µg/mL) for 3 days. Some of these canonical pathways changed by nano-TiO2 in vitro treatments have been already reported in the literature, such as NRF2-mediated stress response, fatty acid metabolism, cell cycle and apoptosis, immune response, cholesterol biosynthesis, and glycolysis. But this genomic study also revealed some novel effects such as protein synthesis, protein ubiquitination, hepatic fibrosis, and cancer-related signaling pathways. More importantly, this genomic analysis of nano-TiO2 treated HepG2 cells linked some of the in vitro canonical pathways to in vivo adverse outcomes: NRF2-mediated response pathways to oxidative stress, acute phase response to inflammation, cholesterol biosynthesis to steroid hormones alteration, fatty acid metabolism changes to lipid homeostasis alteration, G2/M cell checkpoint regulation to apoptosis, and hepatic fibrosis/stellate cell activation to liver fibrosis.

Pharmacokinetic and Genomic Effects of Arsenite in Drinking Water on Mouse Lung in a 30-Day Exposure.

The 2 objectives of this subchronic study were to determine the arsenite drinking water exposure dependent increases in female C3H mouse liver and lung tissue arsenicals and to characterize the dose response (to 0, 0.05, 0.25, 1, 10, and 85 ppm arsenite in drinking water for 30 days and a purified AIN-93M diet) for genomic mouse lung expression patterns. Mouse lungs were analyzed for inorganic arsenic, monomethylated, and dimethylated arsenicals by hydride generation atomic absorption spectroscopy. The total lung mean arsenical levels were 1.4, 22.5, 30.1, 50.9, 105.3, and 316.4 ng/g lung tissue after 0, 0.05, 0.25, 1, 10, and 85 ppm, respectively. At 85 ppm, the total mean lung arsenical levels increased 14-fold and 131-fold when compared to either the lowest noncontrol dose (0.05 ppm) or the control dose, respectively. We found that arsenic exposure elicited minimal numbers of differentially expressed genes (DEGs; 77, 38, 90, 87, and 87 DEGs) after 0.05, 0.25, 1, 10, and 85 ppm, respectively, which were associated with cardiovascular disease, development, differentiation, apoptosis, proliferation, and stress response. After 30 days of arsenite exposure, this study showed monotonic increases in mouse lung arsenical (total arsenic and dimethylarsinic acid) concentrations but no clear dose-related increases in DEG numbers.

Differential Genomic Effects on Signaling Pathways by Two Different CeO2 Nanoparticles in HepG2 Cells.

To investigate genomic effects, human liver hepatocellular carcinoma (HepG2) cells were exposed for three days to two different forms of nanoparticles both composed of CeO2 (0.3, 3 and 30 µg/mL). The two CeO2 nanoparticles had dry primary particle sizes of 8 nanometers {(M) made by NanoAmor} and 58 nanometers {(L) made by Alfa Aesar} and differ in various other physical-chemical properties as well. The smaller particle has stronger antioxidant properties, probably because it has higher Ce3+ levels on the particle surface, as well as more surface area per unit weight. Nanoparticle M showed a normal dose-response pattern with 363, 633 and 1273 differentially expressed genes (DEGs) at 0.3, 3 and 30 µg/mL, respectively. In contrast, nanoparticle L showed a puzzling dose-response pattern with the most DEGs found in the lowest exposure group with 1049, 303 and 323 DEGs at 0.3, 3 and 30 µg/mL, respectively. This systems biological genomic study showed that the major altered pathways by these two nano cerium oxides were protein synthesis, stress response, proliferation/cell cycle, cytoskeleton remodeling/actin polymerization and cellular metabolism. Some of the canonical pathways affected were mTOR signaling, EIF2 signaling, fatty acid activation, G2/M DNA damage checkpoint regulation, glycolysis and protein ubiquitination. These two CeO2 nanoparticles differed considerably in their genomic effects. M is more active than L in respect to altering the pathways of mitochondrial dysfunction, acute phase response, apoptosis, 14-3-3 mediated signaling, remodeling of epithelial adherens junction signaling, actin nucleation by ARP-WASP complex, altered TCA cycle and elevated fatty acid concentrations by metabolomics. However, L is more active than M in respect to the pathways of NRF2-mediated stress response and hepatic fibrosis/hepatic stellate cell activation. One major difference in the cell response to nano M and L is that nano M caused the Warburg effect while nano L did not.

Signaling Pathways and MicroRNA Changes in Nano-TiO2 Treated Human Lung Epithelial (BEAS-2B) Cells.

The effect of titanium dioxide nanoparticles (nano-TiO2 Degussa p25) treatment of human lung epithelial cells (BEAS-2B) was examined by analyzing changes in messenger [mRNA] and microRNA [miRNA]. BEAS-2B cells were treated with 0, 3, 10, 30 or 100 µg/ml nano-TiO2 for 1 day (for mRNA analysis) or 3 days (for miRNA analysis). Differentially expressed mRNA and miRNA were analyzed using Affymetrix microarrays and Affymetrix miRNA microarrays, respectively. Although, the tested doses were not cytotoxic, there were alterations in both mRNA and miRNA expression. The expression of mRNA/miRNA changes were examined in MetaCore (GeneGo) and IPA (Ingenuity Pathway Analysis) to delineate associated canonical/signaling pathways. Canonical/signaling pathways altered by nano-TiO2 treatments included: cell cycle regulation, apoptosis, calcium signaling, translation, NRF2-mediated oxidative response, IGF1 signaling, RAS signaling, PI3K/AKT signaling, cytoskeleton remodeling, cell adhesion, BMP signaling, and inflammatory response. Many of the genes in these pathways are known to be regulated by the miRNAs whose expressions were altered by the nano-TiO2 treatment. The miRNA 17-92 cluster and let-7 miRNA family that are involved in lung cancer formation were altered by nano-TiO2 treatment. The miR-17-92 cluster, an oncogenic microRNA cluster, is induced while the tumor suppressor microRNA, let-7 family, is suppressed. The changes of let-7/KRAS signaling pathway was observed in all the doses treated. The observed changes in miRNA expression introduces an additional mechanistic dimension that supports the significance of the observed mRNA expression changes, and demonstrated that the nano-TiO2 in vitro treatment in human lung cells can cause diverse but coordinated pathway alterations associated with changes in in vivo response to tumorigenes.

Latent carcinogenicity of early-life exposure to dichloroacetic acid in mice.

Environmental exposures occurring early in life may have an important influence on cancer risk later in life. Here, we investigated carryover effects of dichloroacetic acid (DCA), a small molecule analog of pyruvate with metabolic programming properties, on age-related incidence of liver cancer. The study followed a stop-exposure/promotion design in which 4-week-old male and female B6C3F1 mice received the following treatments: deionized water alone (dH2O, control); dH2O with 0.06% phenobarbital (PB), a mouse liver tumor promoter; or DCA (1.0, 2.0 or 3.5g/l) for 10 weeks followed by dH2O or PB (n = 20-30/group/sex). Pathology and molecular assessments were performed at 98 weeks of age. In the absence of PB, early-life exposure to DCA increased the incidence and number of hepatocellular tumors in male and female mice compared with controls. Significant dose trends were observed in both sexes. At the high dose level, 10 weeks of prior DCA treatment induced comparable effects (≥85% tumor incidence and number) to those seen after continuous lifetime exposure. Prior DCA treatment did not enhance or inhibit the carcinogenic effects of PB, induce persistent liver cytotoxicity or preneoplastic changes on histopathology or alter DNA sequence variant profiles within liver tumors compared with controls. Distinct changes in liver messenger RNA and micro RNA profiles associated with prior DCA treatment were not apparent at 98 weeks. Our findings demonstrate that early-life exposure to DCA may be as carcinogenic as life-long exposures, potentially via epigenetic-mediated effects related to cellular metabolism.

Dose and temporal effects on gene expression profiles of urothelial cells from rats exposed to diuron.

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted urea herbicide that at high dietary levels (2500 ppm) induces rat urinary bladder hyperplasia after 20 weeks of exposure and neoplasia after 2 years. The effects on the urothelium after short-term exposure have not been described. The present 7-day study evaluated the dose-dependency of urothelial alterations in the urinary bladder using light microscopy, scanning electron microscopy, and genome-wide transcriptional profiling. Male Wistar rats were fed 0, 125, 500, 2500 ppm diuron for 7 days. The urinary bladder and isolated urothelial cells of these animals were processed for microscopic examination and gene expression profiling, respectively. No significant treatment-related morphologic effects were observed. The number of differentially expressed genes (DEGs) in the exposed groups increased with diuron levels. Diuron-altered genes involved in cell-to-cell interactions and tissue organization were identified in all treatment groups. After 7 days of diuron exposure, transcriptional responses were observed in the urothelium in the absence of clear morphologic changes. These morphological findings are different from those observed in a previous study in which 20 weeks of diuron exposure was associated with simple hyperplasia secondary to the persistent cytotoxicity and necrosis associated with continuous cellular regeneration. Comparison of the gene expression profiles of rats exposed to the 2500 ppm carcinogenic diuron dose for 7 days versus 20 weeks revealed few similarities between these two time points at the gene or pathway level. Taken together, these data provide insight into the dose- and temporal-dependent morphological and transcriptional changes associated with diuron exposure that may lead to the development of tumors in the rat urinary bladder.

Meta-analysis of gene expression in the mouse liver reveals biomarkers associated with inflammation increased early during aging.

Aging is associated with a loss of cellular homeostasis, a decline in physiological function and an increase in various pathologies. Employing a meta-analysis, hepatic gene expression profiles from four independent mouse aging studies were interrogated. There was little overlap in the number of genes or canonical pathways perturbed, suggesting that independent study-specific factors may play a significant role in determining age-dependent gene expression. However, 43 genes were consistently altered during aging in three or four of these studies, including those that (1) exhibited progressively increased expression starting from 12 months of age, (2) exhibited similar expression changes in models of progeria at young ages and dampened or no changes in old longevity mouse models, (3) were associated with inflammatory tertiary lymphoid neogenesis (TLN) associated with formation of ectopic lymphoid structures observed in chronically inflamed tissues, and (4) overlapped with genes perturbed by aging in brain, muscle, and lung. Surprisingly, around half of the genes altered by aging in wild-type mice exhibited similar expression changes in adult long-lived mice compared to wild-type controls, including those associated with intermediary metabolism and feminization of the male-dependent gene expression pattern. Genes unique to aging in wild-type mice included those linked to TLN.

Transcriptional ontogeny of the developing liver.

BACKGROUND: During embryogenesis the liver is derived from endodermal cells lining the digestive tract. These endodermal progenitor cells contribute to forming the parenchyma of a number of organs including the liver and pancreas. Early in organogenesis the fetal liver is populated by hematopoietic stem cells, the source for a number of blood cells including nucleated erythrocytes. A comprehensive analysis of the transcriptional changes that occur during the early stages of development to adulthood in the liver was carried out. RESULTS: We characterized gene expression changes in the developing mouse liver at gestational days (GD) 11.5, 12.5, 13.5, 14.5, 16.5, and 19 and in the neonate (postnatal day (PND) 7 and 32) compared to that in the adult liver (PND67) using full-genome microarrays. The fetal liver, and to a lesser extent the neonatal liver, exhibited dramatic differences in gene expression compared to adults. Canonical pathway analysis of the fetal liver signature demonstrated increases in functions important in cell replication and DNA fidelity whereas most metabolic pathways of intermediary metabolism were under expressed. Comparison of the dataset to a number of previously published microarray datasets revealed 1) a striking similarity between the fetal liver and that of the pancreas in both mice and humans, 2) a nucleated erythrocyte signature in the fetus and 3) under expression of most xenobiotic metabolism genes throughout development, with the exception of a number of transporters associated with either hematopoietic cells or cell proliferation in hepatocytes. CONCLUSIONS: Overall, these findings reveal the complexity of gene expression changes during liver development and maturation, and provide a foundation to predict responses to chemical and drug exposure as a function of early life-stages.

Hepatic xenobiotic metabolizing enzyme and transporter gene expression through the life stages of the mouse.

BACKGROUND: Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). No comprehensive analysis of the mRNA expression of XMETs has been carried out through life stages in any species. RESULTS: Using full-genome arrays, the mRNA expression of all XMETs and their regulatory proteins was examined during fetal (gestation day (GD) 19), neonatal (postnatal day (PND) 7), prepubescent (PND32), middle age (12 months), and old age (18 and 24 months) in the C57BL/6J (C57) mouse liver and compared to adults. Fetal and neonatal life stages exhibited dramatic differences in XMET mRNA expression compared to the relatively minor effects of old age. The total number of XMET probe sets that differed from adults was 636, 500, 84, 5, 43, and 102 for GD19, PND7, PND32, 12 months, 18 months and 24 months, respectively. At all life stages except PND32, under-expressed genes outnumbered over-expressed genes. The altered XMETs included those in all of the major metabolic and transport phases including introduction of reactive or polar groups (Phase I), conjugation (Phase II) and excretion (Phase III). In the fetus and neonate, parallel increases in expression were noted in the dioxin receptor, Nrf2 components and their regulated genes while nuclear receptors and regulated genes were generally down-regulated. Suppression of male-specific XMETs was observed at early (GD19, PND7) and to a lesser extent, later life stages (18 and 24 months). A number of female-specific XMETs exhibited a spike in expression centered at PND7. CONCLUSIONS: The analysis revealed dramatic differences in the expression of the XMETs, especially in the fetus and neonate that are partially dependent on gender-dependent factors. XMET expression can be used to predict life stage-specific responses to environmental chemicals and drugs.