New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

SENP2 knockdown in human adipocytes reduces glucose metabolism and lipid accumulation, while increases lipid oxidation.

Adipose tissue is one of the main regulative sites for energy metabolism. Excess lipid storage and expansion of white adipose tissue (WAT) is the primary contributor to obesity, a strong predisposing factor for development of insulin resistance. Sentrin-specific protease (SENP) 2 has been shown to play a role in metabolism in murine fat and skeletal muscle cells, and we have previously demonstrated its role in energy metabolism of human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism in primary human fat cells by using cultured primary human adipocytes to knock down the SENP2 gene. Glucose uptake and oxidation, as well as accumulation and distribution of oleic acid into complex lipids were decreased, while oleic acid oxidation was increased in SENP2-knockdown cells compared to control adipocytes. Furthermore, lipogenesis was reduced by SENP2-knockdown in adipocytes. Although TAG accumulation relative to total uptake was unchanged, there was increased mRNA expression of metabolically relevant genes such as UCP1 and PPARGC1A and mRNA and proteomic data revealed increased levels of mRNA and proteins related to mitochondrial function by SENP2-knockdown. In conclusion, SENP2 is an important regulator of energy metabolism in primary human adipocytes and its knockdown reduce glucose metabolism and lipid accumulation, while increasing lipid oxidation in human adipocytes.

Pancreatic Cancer Cell-Conditioned, Human-Derived Primary Myotubes Display Increased Leucine Turnover, Increased Lipid Accumulation, and Reduced Glucose Uptake.

Metabolic alterations occurring in cancer cells have been seen to also occur in other tissues than cancerous tissue. For instance, cachexia, peripheral insulin resistance, or both are commonly seen in patients with cancer. We explored differences in substrate use in myotubes conditioned with the medium from a pancreatic cancer cell line, PANC-1, or primary human pancreatic cells, hPECs. Protein turnover was assessed using scintillation proximity assay, glucose and oleic acid handling were analyzed by substrate oxidation assay. We performed qPCR to study gene expression and immunoblotting and proteomic analyses to study protein expression. PANC-1-conditioned myotubes had an imbalance in protein turnover with decreased accumulation, increased decay, and decreased MYH2 gene expression. Glucose uptake decreased despite increased insulin-stimulated Akt phosphorylation. Fatty acid uptake increased, whereas fatty acid oxidation was unchanged, leading to accumulation of intracellular lipids (TAG) in PANC-1-conditioned myotubes. Secretome analyses revealed increased release of growth factors and growth factor receptor from PANC-1 cells, potentially affecting muscle cell metabolism. Myotubes exposed to pancreatic cancer cell medium displayed altered energy metabolism with increased protein/leucine turnover and lipid accumulation, while glucose uptake and oxidation reduced. This indicates production and release of substances from pancreatic cancer cells affecting skeletal muscle.

Experimental Models for Cold Exposure of Muscle in vitroand in vivo.

Work in cold environments may have a significant impact on occupational health. In these and similar situations, cold exposure localized to the extremities may reduce the temperature of underlying tissues. To investigate the molecular effects of cold exposure in muscle, and since adequate methods were missing, we established two experimental cold exposure models: 1) In vitroexposure to cold (18°C) or control temperature (37°C) of cultured human skeletal muscle cells (myotubes); and 2) unilateral cold exposure of hind limb skeletal muscle in anesthetized rats (intramuscular temperature 18°C), with contralateral control (37°C). This methodology enables studies of muscle responses to local cold exposures at the level of gene expression, but also other molecular outcomes. Graphical abstract.

SENP2 is vital for optimal insulin signaling and insulin-stimulated glycogen synthesis in human skeletal muscle cells.

Sentrin-specific protease (SENP) 2 has been suggested as a possible novel drug target for the treatment of obesity and type 2 diabetes mellitus after observations of a palmitate-induced increase in SENP2 that lead to increased fatty acid oxidation and improved insulin sensitivity in skeletal muscle cells from mice. However, no precedent research has examined the role of SENP2 in human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism as well as insulin sensitivity in human skeletal muscle using cultured primary human myotubes. Acute (4 â€‹h) oleic acid oxidation was reduced in SENP2-knockdown (SENP2-KD) cells compared to control cells, with no difference in uptake. After prelabeling (24 â€‹h) with oleic acid, total lipid content and incorporation into triacylglycerol was decreased, while incorporation into other lipids, as well as complete oxidation and ß-oxidation was increased in SENP2-KD cells. Basal glucose uptake (i.e., not under insulin-stimulated conditions) was higher in SENP2-KD cells, whereas oxidation was similar to control myotubes. Further, basal glycogen synthesis was not different in SENP2-KD myotubes, but both insulin-stimulated glycogen synthesis and AktSer473 phosphorylation was completely blunted in SENP2-KD cells. In conclusion, SENP2 plays an important role in fatty acid and glucose metabolism in human myotubes. Interestingly, it also appears to have a pivotal role in regulating myotube insulin sensitivity. Future studies should examine the role of SENP2 in regulation of insulin sensitivity in other tissues and in vivo, defining the potential for SENP2 as a drug target.

Novel methods for cold exposure of skeletal muscle in vivo and in vitro show temperature-dependent myokine production.

Proteins secreted from skeletal muscle serving a signalling role have been termed myokines. Many of the myokines are exercise factors, produced and released in response to muscle activity. Cold exposures affecting muscle may occur in recreational, occupational and therapeutic settings. Whether muscle temperature independently affects myokine profile, is still to be elucidated. We hypothesized that manipulating muscle temperature by means of external cooling would change myokine production and release. In the present study we have established new models for cold exposure of muscle in vivo and in vitro where rat hind limb or cultured human myotubes were cooled to 18 °C. After a recovery period, muscle tissue, cells and culture media were harvested for further analysis by qPCR and immunoassays. Expression of several myokine genes were significantly increased after cold exposure in both models: in rat muscle, mRNA levels of CCL2 (p = 0.04), VEGFA (p = 0.02), CXCL1 (p = 0.02) and RBM3 (p = 0.02) increased while mRNA levels of IL-6 (p = 0.03) were decreased; in human myotubes, mRNA levels of IL6 (p = 0.01), CXCL8 (p = 0.04), VEGFA (p = 0.03) and CXCL1 (p < 0.01) were significantly increased, as well as intracellular protein levels of IL-8 (CXCL8 gene product; p < 0.01). The corresponding effect on myokine secretion was not observed, on the contrary, IL-8 (p = 0.02) and VEGF (VEGFA gene product) p < 0.01) concentrations in culture media were reduced after cold exposure in vitro. In conclusion, cold exposure of muscle in vivo and in vitro had an effect on the production and release of several known exercise-related myokines. Myokine expression at the level of mRNA and protein was increased by cold exposure, whereas secretion tended to be decreased.

Pancreatic cancer cells show lower oleic acid oxidation and their conditioned medium inhibits oleic acid oxidation in human myotubes.

BACKGROUND: /Objectives: We aimed to metabolically compare healthy primary human pancreatic epithelial cells (hPEC) to a pancreatic cancer cell line (PANC-1) and explore the effect on energy metabolism of exposing primary human myotubes to conditioned medium from hPEC and PANC-1 cells. METHODS: Differences in metabolism were examined with radiolabeled glucose, oleic acid and lactic acid, and by qPCR. Mass spectrometry-based proteomics was used to study global protein secretion from the two cell types. Pathway analyses were performed. RESULTS: PANC-1 cells tended to have higher glucose uptake, production of lactic acid, and glucose oxidation compared to hPEC cells. PANC-1 cells had higher uptake but lower oxidation of oleic acid, and mitochondrial reserve capacity from oleic acid was lower in PANC-1 cells. These differences in energy metabolism were reflected by differences in gene expressions and pathway analyses of the secretome. Conditioned medium from PANC-1 cells attenuated oleic acid oxidation in primary human myotubes. CONCLUSIONS: Metabolic characterization of the PANC-1 cells revealed a glycolytic phenotype since they had an active glucose oxidation. Furthermore, PANC-1 cells showed a lower oleic acid oxidation and secreted a high amount of proteins into conditioned medium that also induced a reduced oleic acid oxidation in myotubes.