Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30–70 % for pre-adipocyte proliferation; this was 40–80 % for triglyceride accumulation induced by pyraclostrobin, 80–100 % for tributyltin, and 80–100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30–70 % active determinations for pyraclostrobin, 30–50 % for tributyltin, and 20–40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.Over-arching project supported by grants [R01 ES016099 to HMS; R00 ES030405 to CDK] from the National Institute of Environmental Health Sciences (NIEHS); University of Turin; European Union's Horizon 2020 research and innovation program under grant agreement GOLIATH No. 825489; Brunel University London; NIEHS (1K22ES026208 and R01ES027863); NIEHS (Z0ES102785); Spanish Institute of Health Carlos III (grant FIS-PI16/01812)

PCB11 Metabolite, 3,3’-Dichlorobiphenyl-4-ol, Exposure Alters the Expression of Genes Governing Fatty Acid Metabolism in the Absence of Functional Sirtuin 3: Examining the Contribution of MnSOD

Although the production of polychlorinated biphenyls (PCBs) is prohibited, the inadvertent production of certain lower-chlorinated PCB congeners still threatens human health. We and others have identified 3,3’-dichlorobiphenyl (PCB11) and its metabolite, 3,3’-dichlorobiphenyl-4-ol (4OH-PCB11), in human blood, and there is a correlation between exposure to this metabolite and mitochondrial oxidative stress in mammalian cells. Here, we evaluated the downstream effects of 4OH-PCB11 on mitochondrial metabolism and function in the presence and absence of functional Sirtuin 3 (SIRT3), a mitochondrial fidelity protein that protects redox homeostasis. A 24 h exposure to 3 μM 4OH-PCB11 significantly decreased the cellular growth and mitochondrial membrane potential of SIRT3-knockout mouse embryonic fibroblasts (MEFs). Only wild-type cells demonstrated an increase in Manganese superoxide dismutase (MnSOD) activity in response to 4OH-PCB11–induced oxidative injury. This suggests the presence of a SIRT3-mediated post-translational modification to MnSOD, which was impaired in SIRT3-knockout MEFs, which counters the PCB insult. We found that 4OH-PCB11 increased mitochondrial respiration and endogenous fatty-acid oxidation-associated oxygen consumption in SIRT3-knockout MEFs; this appeared to occur because the cells exhausted their reserve respiratory capacity. To determine whether these changes in mitochondrial respiration were accompanied by similar changes in the regulation of fatty acid metabolism, we performed quantitative real-time polymerase chain reaction (qRT-PCR) after a 24 h treatment with 4OH-PCB11. In SIRT3-knockout MEFs, 4OH-PCB11 significantly increased the expression of ten genes controlling fatty acid biosynthesis, metabolism, and transport. When we overexpressed MnSOD in these cells, the expression of six of these genes returned to the baseline level, suggesting that the protective role of SIRT3 against 4OH-PCB11 is partially governed by MnSOD activity

Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes

The hepatotoxicity of acetaminophen (APAP) occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO) and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1) inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP), a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein), reactive oxygen formation (superoxide), loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction. Keywords: Acetaminophen, Neuronal nitric oxide, Oxidative stress, Mitochondri

The Role of Sirtuin 3 in Radiation-Induced Long-Term Persistent Liver Injury

In patients with abdominal region cancers, ionizing radiation (IR)-induced long-term liver injury is a major limiting factor in the use of radiotherapy. Previously, the major mitochondrial deacetylase, sirtuin 3 (SIRT3), has been implicated to play an important role in the development of acute liver injury after total body irradiation but no studies to date have examined the role of SIRT3 in liver’s chronic response to radiation. In the current study, ten-month-old Sirt3−/− and Sirt3+/+ male mice received 24 Gy radiation targeted to liver. Six months after exposure, irradiated Sirt3−/− mice livers demonstrated histopathological elevations in inflammatory infiltration, the loss of mature bile ducts and higher DNA damage (TUNEL) as well as protein oxidation (3-nitrotyrosine). In addition, increased expression of inflammatory chemokines (IL-6, IL-1β, TGF-β) and fibrotic factors (Procollagen 1, α-SMA) were also measured in Sirt3−/− mice following 24 Gy IR. The alterations measured in enzymatic activities of catalase, glutathione peroxidase, and glutathione reductase in the livers of irradiated Sirt3−/− mice also implied that hydrogen peroxide and hydroperoxide sensitive signaling cascades in the absence of SIRT3 might contribute to the IR-induced long-term liver injury

Intratracheal injection of adenovirus containing the human MNSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis

Purpose: A dose and volume limiting factor in radiation treatment of thoracic cancer is the development of fibrosis in normal lung. The goal of the present study was to determine whether expression prior to irradiation of a transgene for human manganese superoxide dismutase (MnSOD) or human copper/zinc superoxide dismutase (Cu/ZnSOD) protects against irradiation- induced lung damage in mice. Methods and Materials: Athymic Nude (Nu/J) mice were intratracheally injected with 109 plaque-forming units (PFU) of a replication-incompetent mutant adenovirus construct containing the gene for either human MnSOD, human copper/zinc superoxide dismutase (Cu/ZnSOD) or LacZ. Four days later the mice were irradiated to the pulmonary cavity to doses of 850, 900, or 950 cGy. To demonstrate adenoviral infection, nested reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out with primers specific for either human MnSOD or Cu/ZnSOD transgene on freshly explanted lung, trachea, or alveolar type II cells, and immunohistochemistry was used to measure LacZ expression. RNA was extracted on day 0, 1, 4, or 7 after 850 cGy of irradiation from lungs of mice that had previously received adenovirus or had no treatment. Slot blot analysis was performed to quantitate RNA expression for IL-1, tumor necrosis factor (TNF)-α, TGF-β, MnSOD, or Cu/ZnSOD. Lung tissue was explanted and tested for biochemical activity of MnSOD or Cu/ZnSOD after adenovirus injection. Other mice were sacrificed 132 days after irradiation, lungs excised, frozen in OCT, (polyvinyl alcohol, polyethylene glycol mixture) sectioned, H and E stained, and evaluated for percent of the lung demonstrating organizing alveolitis. Results: Mice injected intratracheally with adenovirus containing the gene for human MnSOD had significantly reduced chronic lung irradiation damage following 950 cGy, compared to control mice or mice injected with adenovirus containing the gene for human Cu/ZnSOD or LacZ. Immunohistochemistry for LacZ protein in adenovirus LacZ (Ad-LacZ)-injected mice demonstrated expression of LacZ in both the upper and lower airway. Nested RT-PCR showed lung expression of MnSOD and Cu/ZnSOD for at least 11 days following infection with each respective adenovirus construct. Nested RT-PCR using primers specific for human MnSOD demonstrated increased expression of the human MnSOD transgene in the trachea and alveolar type II cells 4 days after virus injection on the day of irradiation. At this time point, increased biochemical activity of MnSOD and Cu/ZnSOD respectively, was detected in lungs from these two adenovirus groups, compared to each other or to control or adenovirus LacZ mice. Slot blot analysis of RNA from lungs of mice in each group following 850 cGy irradiation demonstrated decreased expression of mRNA for interleukin-I (IL-1), TNF-α, and transforming growth factor-beta (TGF-β) in the MnSOD adenovirus-injected mice, compared to irradiated control, LacZ, or Cu/ZnSOD adenovirus-injected, irradiated mice. Mice receiving adenovirus MnSOD showed decreased organizing alveolitis at 132 days in all three dose groups, compared to irradiated control or Ad-LacZ, or Ad-Cu/ZnSOD mice. Conclusions: Overexpression of MnSOD in the lungs of mice prior to irradiation prevents irradiation-induced acute and chronic damage quantitated as decreased levels of mRNA for IL-1, TNF-α, and TGF-β in the days immediately following irradiation, and decrease in the percent of lung demonstrating fibrosis or organizing alveolitis at 132 days. These data provide a rational basis for development of gene therapy as a method of protection of the normal lung from acute and chronic sequellae of ionizing irradiation

Effective diagnosis of genetic disease by computational phenotype analysis of the disease-associated genome

Less than half of patients with suspected genetic disease receive a molecular diagnosis. We have therefore integrated next-generation sequencing (NGS), bioinformatics, and clinical data into an effective diagnostic workflow. We used variants in the 2741 established Mendelian disease genes [the disease-associated genome (DAG)] to develop a targeted enrichment DAG panel (7.1 Mb), which achieves a coverage of 20-fold or better for 98% of bases. Furthermore, we established a computational method [Phenotypic Interpretation of eXomes (PhenIX)] that evaluated and ranked variants based on pathogenicity and semantic similarity of patients' phenotype described by Human Phenotype Ontology (HPO) terms to those of 3991 Mendelian diseases. In computer simulations, ranking genes based on the variant score put the true gene in first place less than 5% of the time; PhenIX placed the correct gene in first place more than 86% of the time. In a retrospective test of PhenIX on 52 patients with previously identified mutations and known diagnoses, the correct gene achieved a mean rank of 2.1. In a prospective study on 40 individuals without a diagnosis, PhenIX analysis enabled a diagnosis in 11 cases (28%, at a mean rank of 2.4). Thus, the NGS of the DAG followed by phenotype-driven bioinformatic analysis allows quick and effective differential diagnostics in medical genetics.status: publishe

Spanning Trees with Bounded Number of Branch Vertices

We introduce the following combinatorial optimization problem: Given a connected graph G, find a spanning tree T of G with the smallest number of branchv ertices (vertices of degree 3 or more in T). The problem is motivated by new technologies in the realm of optical networks. We investigate algorithmic and combinatorial aspects of the problem