Early microRNA responses in rodent liver mediated by furan exposure establish dose thresholds for later adverse outcomes.

To reduce reliance on long-term in vivo studies, short-term data linking early molecular-based measurements to later adverse health effects is needed. Although transcriptional-based benchmark dose (BMDT) modeling has been used to estimate potencies and stratify chemicals based on potential to induce later-life effects, dose-responsive epigenetic alterations have not been routinely considered. Here, we evaluated the utility of microRNA (miRNA) profiling in mouse liver and blood, as well as in mouse primary hepatocytes in vitro, to indicate mechanisms of liver perturbation due to short-term exposure of the known rodent liver hepatotoxicant and carcinogen, furan. Benchmark dose modeling of miRNA measurements (BMDmiR) were compared to the referent transcriptional (BMDT) and apical (BMDA) estimates. These analyses indicate a robust dose response for 34 miRNAs to furan and involvement of p53-linked pathways in furan-mediated hepatotoxicity, supporting mRNA and apical measurements. Liver-sourced miRNAs were also altered in the blood and primary hepatocytes. Overall, these results indicate mechanistic involvement of miRNA in furan carcinogenicity and provide evidence of their potential utility as accessible biomarkers of exposure and disease.

Circulating MicroRNAs, Polychlorinated Biphenyls, and Environmental Liver Disease in the Anniston Community Health Survey.

BACKGROUND: Polychlorinated biphenyl (PCB) exposures have been associated with liver injury in human cohorts, and steatohepatitis with liver necrosis in model systems. MicroRNAs (miRs) maintain cellular homeostasis and may regulate the response to environmental stress. OBJECTIVES: We tested the hypothesis that specific miRs are associated with liver disease and PCB exposures in a residential cohort. METHODS: Sixty-eight targeted hepatotoxicity miRs were measured in archived serum from 734 PCB-exposed participants in the cross-sectional Anniston Community Health Survey. Necrotic and other liver disease categories were defined by serum keratin 18 (K18) biomarkers. Associations were determined between exposure biomarkers (35 ortho-substituted PCB congeners) and disease biomarkers (highly expressed miRs or previously measured cytokines), and Ingenuity Pathway Analysis was performed. RESULTS: The necrotic liver disease category was associated with four up-regulated miRs (miR-99a-5p, miR-122-5p, miR-192-5p, and miR-320a) and five down-regulated miRs (let-7d-5p, miR-17-5p, miR-24-3p, miR-197-3p, and miR-221-3p). Twenty-two miRs were associated with the other liver disease category or with K18 measurements. Eleven miRs were associated with 24 PCBs, most commonly congeners with anti-estrogenic activities. Most of the exposure-associated miRs were associated with at least one serum hepatocyte death, pro-inflammatory cytokine or insulin resistance bioarker, or with both. Within each biomarker category, associations were strongest for the liver-specific miR-122-5p. Pathways of liver toxicity that were identified included inflammation/hepatitis, hyperplasia/hyperproliferation, cirrhosis, and hepatocellular carcinoma. Tumor protein p53 and tumor necrosis factor α were well integrated within the top identified networks. DISCUSSION: These results support the human hepatotoxicity of environmental PCB exposures while elucidating potential modes of PCB action. The MiR-derived liquid liver biopsy represents a promising new technique for environmental hepatology cohort studies. https://doi.org/10.1289/EHP9467.

Early microRNA indicators of PPARα pathway activation in the liver.

MicroRNAs (miRNAs) are short non-coding RNA species that play key roles in post-transcriptional regulation of gene expression. MiRNAs also serve as a promising source of early biomarkers for different environmental exposures and health effects, although there is limited information linking miRNA changes to specific target pathways. In this study, we measured liver miRNAs in male B6C3F1 mice exposed to a known chemical activator of the peroxisome proliferator-activated receptor alpha (PPARα) pathway, di(2-ethylhexyl) phthalate (DEHP), for 7 and 28 days at concentrations of 0, 750, 1500, 3000, or 6000 ppm in feed. At the highest dose tested, DEHP altered 61 miRNAs after 7 days and 171 miRNAs after 28 days of exposure, with 48 overlapping miRNAs between timepoints. Analysis of these 48 common miRNAs indicated enrichment in PPARα-related targets and other pathways related to liver injury and cancer. Four of the 10 miRNAs exhibiting a clear dose trend were linked to the PPARα pathway: mmu-miRs-125a-5p, -182-5p, -20a-5p, and -378a-3p. mmu-miRs-182-5p and -378a-3p were subsequently measured using digital drop PCR across a dose range for DEHP and two related phthalates with weaker PPARα activity, di-n-octyl phthalate and n-butyl benzyl phthalate, following 7-day exposures. Analysis of mmu-miRs-182-5p and -378a-3p by transcriptional benchmark dose analysis correctly identified DEHP as having the greatest potency. However, benchmark dose estimates for DEHP based on these miRNAs (average 163; range 126-202 mg/kg-day) were higher on average than values for PPARα target genes (average 74; range 29-183 mg/kg-day). These findings identify putative miRNA biomarkers of PPARα pathway activity and suggest that early miRNA changes may be used to stratify chemical potency.

Mitochondrial oxidative capacity and NAD+ biosynthesis are reduced in human sarcopenia across ethnicities.

The causes of impaired skeletal muscle mass and strength during aging are well-studied in healthy populations. Less is known on pathological age-related muscle wasting and weakness termed sarcopenia, which directly impacts physical autonomy and survival. Here, we compare genome-wide transcriptional changes of sarcopenia versus age-matched controls in muscle biopsies from 119 older men from Singapore, Hertfordshire UK and Jamaica. Individuals with sarcopenia reproducibly demonstrate a prominent transcriptional signature of mitochondrial bioenergetic dysfunction in skeletal muscle, with low PGC-1α/ERRα signalling, and downregulation of oxidative phosphorylation and mitochondrial proteostasis genes. These changes translate functionally into fewer mitochondria, reduced mitochondrial respiratory complex expression and activity, and low NAD+ levels through perturbed NAD+ biosynthesis and salvage in sarcopenic muscle. We provide an integrated molecular profile of human sarcopenia across ethnicities, demonstrating a fundamental role of altered mitochondrial metabolism in the pathological loss of skeletal muscle mass and function in older people.

Ozone-Induced Vascular Contractility and Pulmonary Injury Are Differentially Impacted by Diets Enriched With Coconut Oil, Fish Oil, and Olive Oil.

Fish, olive, and coconut oil dietary supplementation have several cardioprotective benefits, but it is not established if they protect against air pollution-induced adverse effects. We hypothesized that these dietary supplements would attenuate ozone-induced systemic and pulmonary effects. Male Wistar Kyoto rats were fed either a normal diet, or a diet supplemented with fish, olive, or coconut oil for 8 weeks. Animals were then exposed to air or ozone (0.8 ppm), 4 h/day for 2 days. Ozone exposure increased phenylephrine-induced aortic vasocontraction, which was completely abolished in rats fed the fish oil diet. Despite this cardioprotective effect, the fish oil diet increased baseline levels of bronchoalveolar lavage fluid (BALF) markers of lung injury and inflammation. Ozone-induced pulmonary injury/inflammation were comparable in rats on normal, coconut oil, and olive oil diets with altered expression of markers in animals fed the fish oil diet. Fish oil, regardless of exposure, led to enlarged, foamy macrophages in the BALF that coincided with decreased pulmonary mRNA expression of cholesterol transporters, cholesterol receptors, and nuclear receptors. Serum microRNA profile was assessed and demonstrated marked depletion of a variety of microRNAs in animals fed the fish oil diet, several of which were of splenic origin. No ozone-specific changes were noted. Collectively, these data indicate that although fish oil offered vascular protection from ozone exposure, it increased pulmonary injury/inflammation and impaired lipid transport mechanisms resulting in foamy macrophage accumulation, demonstrating the need to be cognizant of potential off-target pulmonary effects that might offset the overall benefit of this vasoprotective supplement.

Factors influencing platelet clumping during peripheral blood hematopoietic stem cell collection.

BACKGROUND: Platelet clumping is a common occurrence during peripheral blood hematopoietic stem cell (HSC) collection using the Spectra Optia mononuclear cell (MNC) protocol. If clumping persists, it may prevent continuation of the collection and interfere with proper MNC separation. This study is the first to report the incidence of clumping, identify precollection factors associated with platelet clumping, and describe the degree to which platelet clumping interferes with HSC product yield. STUDY DESIGN AND METHODS: In total, 258 HSC collections performed on 116 patients using the Optia MNC protocol were reviewed. Collections utilized heparin in anticoagulant citrate dextrose to facilitate large-volume leukapheresis. Linear and logistic regression models were utilized to determine which precollection factors were predictive of platelet clumping and whether clumping was associated with product yield or collection efficiency. RESULTS: Platelet clumping was observed in 63% of collections. Multivariable analysis revealed that a lower white blood cell count was an independent predictor of clumping occurrence. Chemotherapy mobilization and a lower peripheral blood CD34+ cell count were predictors of the degree of clumping. Procedures with clumping had higher collection efficiency but lower blood volume processed on average, resulting in no difference in collection yields. Citrate toxicity did not correlate with clumping. CONCLUSION: Although platelet clumping is a common technical problem seen during HSC collection, the total CD34+ cell-collection yields were not affected by clumping. WBC count, mobilization approach, and peripheral blood CD34+ cell count can help predict clumping and potentially drive interventions to proactively manage clumping.

Oncogene expression profiles in K6/ODC mouse skin and papillomas following a chronic exposure to monomethylarsonous acid.

We have previously observed that a chronic drinking water exposure to monomethylarsonous acid [MMA(III)], a cellular metabolite of inorganic arsenic, increases tumor frequency in the skin of keratin VI/ornithine decarboxylase (K6/ODC) transgenic mice. To characterize gene expression profiles predictive of MMA(III) exposure and mode of action of carcinogenesis, skin and papilloma RNA was isolated from K6/ODC mice administered 0, 10, 50, and 100 ppm MMA(III) in their drinking water for 26 weeks. Following RNA processing, the resulting cRNA samples were hybridized to Affymetrix Mouse Genome 430A 2.0 GeneChips(R). Micoarray data were normalized using MAS 5.0 software, and statistically significant genes were determined using a regularized t-test. Significant changes in bZIP transcription factors, MAP kinase signaling, chromatin remodeling, and lipid metabolism gene transcripts were observed following MMA(III) exposure as determined using the Database for Annotation, Visualization and Integrated Discovery 2.1 (DAVID) (Dennis et al., Genome Biol 2003;4(5):P3). MMA(III) also caused dose-dependent changes in multiple Rho guanine nucleotide triphosphatase (GTPase) and cell cycle related genes as determined by linear regression analyses. Observed increases in transcript abundance of Fosl1, Myc, and Rac1 oncogenes in mouse skin support previous reports on the inducibility of these oncogenes in response to arsenic and support the relevance of these genomic changes in skin tumor induction in the K6/ODC mouse model.

Transcriptional changes associated with reduced spontaneous liver tumor incidence in mice chronically exposed to high dose arsenic.

Exposure of male C3H mice in utero (from gestational days 8-18) to 85ppm sodium arsenite via the dams' drinking water has previously been shown to increase liver tumor incidence by 2 years of age. However, in our companion study (Ahlborn et al., 2009), continuous exposure to 85ppm sodium arsenic (from gestational day 8 to postnatal day 365) did not result in increased tumor incidence, but rather in a significant reduction (0% tumor incidence). The purpose of the present study was to examine the gene expression responses that may lead to the apparent protective effect of continuous arsenic exposure. Genes in many functional categories including cellular growth and proliferation, gene expression, cell death, oxidative stress, protein ubiquitination, and mitochondrial dysfunction were altered by continuous arsenic treatment. Many of these genes are known to be involved in liver cancer. One such gene associated with rodent hepatocarcinogenesis, Scd1, encodes stearoyl-CoA desaturase and was down-regulated by continuous arsenic treatment. An overlap between the genes in our study affected by continuous arsenic exposure and those from the literature affected by long-term caloric restriction suggests that reduction in the spontaneous tumor incidence under both conditions may involve similar gene pathways such as fatty acid metabolism, apoptosis, and stress response.

Impact of life stage and duration of exposure on arsenic-induced proliferative lesions and neoplasia in C3H mice.

Epidemiological studies suggest that chronic exposure to inorganic arsenic is associated with cancer of the skin, urinary bladder and lung as well as the kidney and liver. Previous experimental studies have demonstrated increased incidence of liver, lung, ovary, and uterine tumors in mice exposed to 85 ppm (approximately 8 mg/kg) inorganic arsenic during gestation. To further characterize age susceptibility to arsenic carcinogenesis we administered 85 ppm inorganic arsenic in drinking water to C3H mice during gestation, prior to pubescence and post-pubescence to compare proliferative lesion and tumor outcomes over a one-year exposure period. Inorganic arsenic significantly increased the incidence of hyperplasia in urinary bladder (48%) and oviduct (36%) in female mice exposed prior to pubescence (beginning on postnatal day 21 and extending through one year) compared to control mice (19 and 5%, respectively). Arsenic also increased the incidence of hyperplasia in urinary bladder (28%) of female mice continuously exposed to arsenic (beginning on gestation day 8 and extending though one year) compared to gestation only exposed mice (0%). In contrast, inorganic arsenic significantly decreased the incidence of tumors in liver (0%) and adrenal glands (0%) of male mice continuously exposed from gestation through one year, as compared to levels in control (30 and 65%, respectively) and gestation only (33 and 55%, respectively) exposed mice. Together, these results suggest that continuous inorganic arsenic exposure at 85 ppm from gestation through one year increases the incidence and severity of urogenital proliferative lesions in female mice and decreases the incidence of liver and adrenal tumors in male mice. The paradoxical nature of these effects may be related to altered lipid metabolism, the effective dose in each target organ, and/or the shorter one-year observational period.

Dose response evaluation of gene expression profiles in the skin of K6/ODC mice exposed to sodium arsenite.

Chronic drinking water exposure to inorganic arsenic and its metabolites increases tumor frequency in the skin of K6/ODC transgenic mice. To identify potential biomarkers and modes of action for this skin tumorigenicity, we characterized gene expression profiles from analysis of K6/ODC mice administered 0, 0.05, 0.25, 1.0 and 10 ppm sodium arsenite in their drinking water for 4 weeks. Following exposure, total RNA was isolated from mouse skin and processed to biotin-labeled cRNA for microarray analyses. Skin gene expression was analyzed with Affymetrix Mouse Genome 430A 2.0 GeneChips, and pathway analysis was conducted with DAVID (NIH), Ingenuity Systems and MetaCore's GeneGo. Differential expression of several key genes was verified through qPCR. Only the highest dose (10 ppm) resulted in significantly altered KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, including MAPK, regulation of actin cytoskeleton, Wnt, Jak-Stat, Tight junction, Toll-like, phosphatidylinositol and insulin signaling pathways. Approximately 20 genes exhibited a dose response, including several genes known to be associated with carcinogenesis or tumor progression including cyclin D1, CLIC4, Ephrin A1, STAT3 and DNA methyltransferase 3a. Although transcription changes in all identified genes have not previously been linked to arsenic carcinogenesis, their association with carcinogenesis in other systems suggests that these genes may play a role in the early stages of arsenic-induced skin carcinogenesis and can be considered potential biomarkers.

Folate deficiency enhances arsenic effects on expression of genes involved in epidermal differentiation in transgenic K6/ODC mouse skin.

Chronic arsenic exposure in humans is associated with cancers of the skin, lung, bladder and other tissues. There is evidence that folate deficiency may increase susceptibility to arsenic effects, including skin lesions. K6/ODC mice develop skin tumors when exposed to 10ppm sodium arsenite for 5 months. In the current study, K6/ODC mice maintained on either a folate deficient or folate sufficient diet were exposed to 0, 1, or 10ppm sodium arsenite in the drinking water for 30 days. Total RNA was isolated from skin samples and gene expression analyzed using Affymetrix Mouse 430 2.0 GeneChips. Data from 24 samples, with 4 mice in each of the 6 treatment groups, were RMA normalized and analyzed by two-way ANOVA using GeneSpring. Top gene ontology (GO) categories for genes responding significantly to both arsenic treatment and folate deficiency include nucleotide metabolism and cell organization and biogenesis. For many of these genes, folate deficiency magnifies the response to arsenic treatment. In particular, expression of markers of epidermal differentiation, e.g., loricrin, small proline rich proteins and involucrin, was significantly reduced by arsenic in the folate sufficient animals, and reduced further or at a lower arsenic dose in the folate deficient animals. In addition, expression of a number of epidermal cell growth/proliferation genes and cellular movement genes was altered. These results indicate that arsenic disrupts the normal balance of cell proliferation and differentiation, and that folate deficiency exacerbates these effects, consistent with the view that folate deficiency is a nutritional susceptibility factor for arsenic-induced skin tumorigenesis.

Toxicity profiles in mice treated with hepatotumorigenic and non-hepatotumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil.

Conazoles comprise a class of fungicides used in agriculture and as pharmaceutical products. The fungicidal properties of conazoles are due to their inhibition of ergosterol biosynthesis. Certain conazoles are tumorigenic in rodents; both propiconazole and triadimefon are hepatotoxic and hepatotumorigenic in mice, while myclobutanil is not a mouse liver tumorigen. As a component of a large-scale study aimed at determining the mode(s) of action for tumorigenic conazoles, we report the results from comparative evaluations of liver and body weights, liver histopathology, cell proliferation, cytochrome P450 (CYP) activity, and serum cholesterol, high-density lipoprotein and triglyceride levels after exposure to propiconazole, triadimefon, and myclobutanil. Male CD-1 mice were treated in the feed for 4, 30, or 90 days with triadimefon (0, 100, 500, or 1800 ppm), propiconazole (0, 100, 500, or 2500 ppm) or myclobutanil (0, 100, 500, or 2000 ppm). Alkoxyresorufin O-dealkylation (AROD) assays indicated that all 3 chemicals induced similar patterns of dose-related increases in metabolizing enzyme activity. PROD activities exceeded those of MROD, and EROD with propiconazole inducing the highest activities of PROD. Mice had similar patterns of dose-dependent increases in hepatocyte hypertrophy after exposure to the 3 conazoles. High-dose exposures to propiconazole and myclobutanil, but not triadimefon, were associated with early (4 days) increases in cell proliferation. All the chemicals at high doses reduced serum cholesterol and high-density lipoprotein (HDL) levels at 30 days of treatment, while only triadimefon had this effect at 4 days of treatment and only myclobutanil and propiconazole at 90 days of treatment. Overall, the tumorigenic and nontumorigenic conazoles induced similar effects on mouse liver CYP enzyme activities and pathology. There was no specific pattern of tissue responses that could consistently be used to differentiate the tumorigenic conazoles, propiconazole, and triadimefon, from the nontumorigenic myclobutanil. These findings serve to anchor other transcriptional profiling studies aimed at probing differences in key events and modes of action for tumorigenic and nontumorigenic conazoles.

Toxicity profiles in rats treated with tumorigenic and nontumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil.

Conazoles are a class of azole based fungicides used in agriculture and as pharmaceutical products. They have a common mode of antifungal action through inhibition of ergosterol biosynthesis. Some members of this class have been shown to be hepatotoxic and will induce mouse hepatocellular tumors and/or rat thyroid follicular cell tumors. The particular mode of toxic and tumorigenic action for these compounds is not known, however it has been proposed that triadimefon-induced rat thyroid tumors arise through the specific mechanism of increased TSH. The present study was designed to identify commonalities of effects across the different conazoles and to determine unique features of the tissue responses that suggest a toxicity pathway and a mode of action for the observed thyroid response for triadimefon. Male Wistar/Han rats were treated with triadimefon (100, 500, 1800 ppm), propiconazole (100, 500, 2500 ppm), or myclobutanil (100, 500, 2000 ppm) in feed for 4, 30, or 90 days. The rats were evaluated for clinical signs, body and liver weight, histopathology of thyroid and liver, hepatic metabolizing enzyme activity, and serum T3, T4, TSH, and cholesterol levels. There was a dose-dependent increase in liver weight but not body weight for all treatments. The indication of cytochrome induction, pentoxyresorufin O-dealkylation (PROD) activity, had a dose-related increase at all time points for all conazoles. Uridine diphopho-glucuronosyl transferase (UDPGT), the T4 metabolizing enzyme measured as glucuronidation of 1-naphthol, was induced to the same extent after 30 and 90 days for all three conazoles. Livers from all high dose treated rats had centrilobular hepatocyte hypertrophy after 4 days, while only triadimefon and propiconazole treated rats had hepatocyte hypertrophy after 30 days, and only triadimefon treated rats had hepatocyte hypertrophy after 90 days. Thyroid follicular cell hypertrophy, increased follicular cell proliferation, and colloid depletion were present only after 30 days in rats treated with the high dose of triadimefon. A dose-dependent decrease in T4 was present after 4 days with all 3 compounds but only the high doses of propiconazole and triadimefon produced decreased T4 after 30 days. T3 was decreased after high-dose triadimefon after 4 days and in a dose-dependent manner for all compounds after 30 days. Thyroid hormone levels did not differ from control values after 90 days and TSH was not increased in any exposure group. A unique pattern of toxic responses was not identified for each conazole and the hypothesized mode of action for triadimefon-induced thyroid gland tumors was not supported by the data.

Molecular biomarkers of oxidative stress associated with bromate carcinogenicity.

Potassium bromate (KBrO3) is a chemical oxidizing agent found in drinking water as a disinfection byproduct of surface water ozonation. Chronic exposures to KBrO3 cause renal cell tumors in rats, hamsters and mice and thyroid and testicular mesothelial tumors in rats. Experimental evidence indicates that bromate mediates toxicological effects via the induction of oxidative stress. To investigate the contribution of oxidative stress in KBrO3-induced cancer, male F344 rats were administered KBrO3 in their drinking water at multiple concentrations for 2-100 weeks. Gene expression analyses were performed on kidney, thyroid and mesothelial cell RNA. Families of mRNA transcripts differentially expressed with respect to bromate treatment included multiple cancer, cell death, ion transport and oxidative stress genes. Multiple glutathione metabolism genes were up-regulated in kidney following carcinogenic (400 mg/L) but not non-carcinogenic (20 mg/L) bromate exposures. 8-Oxodeoxyguanosine glycosylase (Ogg1) mRNA was up-regulated in response to bromate treatment in kidney but not thyroid. A dramatic decrease in global gene expression changes was observed following 1mg/L compared to 20 mg/L bromate exposures. In a separate study oxygen-18 (18O) labeled KBrO3 was administered to male rats by oral gavage and tissues were analyzed for 18O deposition. Tissue enrichment of 18O was observed at 5 and 24 h post-KBr18O3 exposure with the highest enrichment occurring in the liver followed by the kidney, thyroid and testes. The kidney dose response observed was biphasic showing similar statistical increases in 18O deposition between 0.25 and 50 mg/L (equivalent dose) KBr18O3 followed by a much greater increase above 50 mg/L. These results suggest that carcinogenic doses of potassium bromate require attainment of a threshold at which oxidation of tissues occurs and that gene expression profiles may be predictive of these physiological changes in renal homeostasis.

Tribromomethane exposure and dietary folate deficiency in the formation of aberrant crypt foci in the colons of F344/N rats.

Folate and folic acid are forms of the B vitamin that are involved in the synthesis, repair, and functioning of DNA and are required for the production and maintenance of cells. Low levels of folate have been associated with several forms of cancer, including colon cancer. Aberrant crypt foci (ACF), identified as putative precursor lesions in the development of colon cancer, have been induced by the drinking water disinfection by-product, tribromomethane (TBM). To investigate whether ACF induced by TBM could be promoted by a diet devoid of dietary folate, male F344/N rats were exposed to 500 mg/l of TBM in drinking water and fed either a normal or no folate diet (NFD) for 26 weeks. At the conclusion of the study, colons were excised and examined for ACF. Rats exposed to TBM and fed a NFD, evident by significantly reduced serum folate concentrations and elevated serum homocysteine levels, had significant increases of ACF when compared to rats exposed to TBM and fed a normal diet. This study highlights the important role that diet, especially folate intake, represents in protecting the colon against TBM-induced ACF.