Deletion of IL-1ß exacerbates acrylamide-induced neurotoxicity in mice.

Acrylamide is a neurotoxicant in human and experimental animals. Interleukin-1ß (IL-1ß) is a proinflammatory cytokine known as a critical component of brain reaction to any insult or neurodegenerative pathologies, though its role in electrophile-induced neurotoxicity remains elusive. The aim of this study was to investigate the role of IL-1ß in acrylamide-induced neurotoxicity in mice. Ten-week-old male wild-type and IL-1ß knock-out mice were allocated into 3 groups each and exposed to acrylamide at 0, 12.5, 25 mg/kg body weight by oral gavage for 28 days. Compared with wild-type mice, the results showed a significant increase in landing foot spread test and a significant decrease in density of cortical noradrenergic axons in IL-1ß KO mice exposed to acrylamide at 25 mg/kg body weight. Exposure to acrylamide at 25 mg/kg significantly increased cortical gene expression of Gclc, Gpx1, and Gpx4 in wild-type mice but decreased them in IL-1ß KO mice. The same exposure level significantly increased total glutathione and oxidized glutathione (GSSG) in the cerebellum of wild-type mice but neither changed total glutathione nor decreased GSSG in the cerebellum of IL-1ß KO mice. The basal level of malondialdehyde in the cerebellum was higher in IL-1ß KO mice than in wild-type mice. The results suggest that IL-1ß protects the mouse brain against acrylamide-induced neurotoxicity, probably through suppression of oxidative stress by glutathione synthesis and peroxidation. This unexpected result provides new insight on the protective role of IL-1ß in acrylamide-induced neurotoxicity.

CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis.

BACKGROUND: TSP1 (thrombospondin-1)-a well-known angiogenesis inhibitor-mediates differential effects via interacting with cell surface receptors including CD36 (cluster of differentiation) and CD47. However, the role of TSP1 in regulating lymphangiogenesis is not clear. Our previous study suggested the importance of cell-specific CD47 blockade in limiting atherosclerosis. Further, our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we aimed to investigate the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerosis. METHODS: Murine atherosclerotic and nonatherosclerotic arteries were utilized to investigate TSP1 expression using Western blotting and immunostaining. LEC-specific knockout mice were used to determine the in vivo role of LEC Cd47 in lymphangiogenesis and atherosclerosis. Various in vitro cell-based assays, in vivo Matrigel plug implantation, molecular biological techniques, and immunohistological approaches were used to evaluate the underlying signaling mechanisms. RESULTS: Elevated TSP1 expression was observed in mouse atherosclerotic aortic tissue compared with nonatherosclerotic control tissue. TSP1 at pathological concentrations suppressed both in vitro and in vivo lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF (vascular endothelial growth factor)-C-induced AKT and eNOS activation in LEC and attenuated NO (nitric oxide) production. Further, CD47 silencing in LEC prevented the effects of TSP1 on lymphangiogenic AKT-eNOS signaling and lymphangiogenesis. Atheroprone AAV (adeno-associated virus) 8-PCSK9-injected LEC-specific Cd47 knockout mice (Cd47ΔLEC) had reduced atherosclerosis in both aorta and aortic root compared with control mice (Cd47ΔWT). However, no differences in metabolic parameters including body weight, plasma total cholesterol levels, and fasting blood glucose were observed. Additional immunostaining experiments performed on aortic root cross-sections indicated higher lymphatic vessel density in Cd47ΔLEC mice in comparison to controls. CONCLUSIONS: These findings demonstrate that TSP1 inhibits lymphangiogenesis via activation of CD47 in LEC, and loss of LEC Cd47 attenuates atherosclerotic lesion formation. Collectively, these results identify LEC CD47 as a potential therapeutic target in atherosclerosis.

Role of Nrf2 in 1,2-dichloropropane-induced cell proliferation and DNA damage in the mouse liver.

1,2-Dichloropropane (1,2-DCP) is recognized as the causative chemical of occupational cholangiocarcinoma in printing workers in Japan. However, the cellular and molecular mechanisms of 1,2-DCP-induced carcinogenesis remains elusive. The present study investigated cellular proliferation, DNA damage, apoptosis, and expression of antioxidant and proinflammatory genes in the liver of mice exposed daily to 1,2-DCP for 5 weeks, and the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in these responses. Wild-type and Nrf2-knockout (Nrf2-/-) mice were administered 1,2-DCP by gastric gavage, and then the livers were collected for analysis. Immunohistochemistry for BrdU or Ki67 and TUNEL assay revealed that exposure to 1,2-DCP dose-dependently increased proliferative cholangiocytes, whereas decreased apoptotic cholangiocytes in wild-type mice but not in Nrf2-/- mice. Western blot and quantitative real-time PCR showed that exposure to 1,2-DCP increased the levels of DNA double-strand break marker γ-H2AX and mRNA expression levels of NQO1, xCT, GSTM1, and G6PD in the livers of wild-type mice in a dose-dependent manner, but no such changes were noted in Nrf2-/- mice. 1,2-DCP increased glutathione levels in the liver of both the wild-type and Nrf2-/- mice, suggesting that an Nrf2-independent mechanism contributes to 1,2-DCP-induced increase in glutathione level. In conclusion, the study demonstrated that exposure to 1,2-DCP induced proliferation but reduced apoptosis in cholangiocytes, and induced double-strand DNA breaks and upregulation of antioxidant genes in the liver in an Nrf2-dependent manner. The study suggests a role of Nrf2 in 1,2-DCP-induced cell proliferation, antiapoptotic effect, and DNA damage, which are recognized as key characteristics of carcinogens.

Transcriptome analysis of human cholangiocytes exposed to carcinogenic 1,2-dichloropropane in the presence of macrophages in vitro.

1,2-Dichloropropane (1,2-DCP), a synthetic organic solvent, has been implicated in causality of cholangiocarcinoma (bile duct cancer). 1,2-DCP-induced occupational cholangiocarcinoma show a different carcinogenic process compared to common cholangiocarcinoma, but its mechanism remains elusive. We reported previously that exposure of MMNK-1 cholangiocytes co-cultured with THP-1 macrophages, but not monocultured MMNK-1 cholangiocytes, to 1,2-DCP induced activation-induced cytidine deaminase (AID) expression, DNA damage and ROS production. The aim of this study was to identify relevant biological processes or target genes expressed in response to 1,2-DCP, using an in vitro system where cholangiocytes are co-cultured with macrophages. The co-cultured cells were exposed to 1,2-DCP at 0, 0.1 or 0.4 mM for 24 h, and then the cell lysates were assessed by transcriptome analysis. 1,2-DCP upregulated the expression of base excision repair genes in MMNK-1 cholangiocytes in the co-cultures, whereas it upregulated the expression of cell cycle-related genes in THP-1 macrophages. Activation of the base excision repair pathway might result from the previously observed DNA damage in MMNK-1 cholangiocytes co-cultured with THP-1 macrophages, although involvement of other mechanisms such as DNA replication, cell death or other types of DNA repair was not disproved. Cross talk interactions between cholangiocytes and macrophages leading to DNA damage in the cholangiocytes should be explored.

Role of Macrophages in Cytotoxicity, Reactive Oxygen Species Production and DNA Damage in 1,2-Dichloropropane-Exposed Human Cholangiocytes In Vitro.

1,2-Dichloropropane (1,2-DCP), a synthetic chlorinated organic compound, was extensively used in the past in offset color proof-printing. In 2014, the International Agency for Research on Cancer (IARC) reclassified 1,2-DCP from its initial Group 3 to Group 1. Prior to the reclassification, cholangiocarcinoma was diagnosed in a group of workers exposed to 1,2 -DCP in an offset color proof-printing company in Japan. In comparison with other forms of cholangiocarcinoma, 1,2-DCP-induced cholangiocarcinoma was of early onset and accompanied by extensive pre-cancerous lesions in large bile ducts. However, the mechanism of 1,2-DCP-induced cholangiocarcinoma is poorly understood. Inflammatory cell proliferation was observed in various sites of the bile duct in the noncancerous hepatic tissues of the 1,2-DCP-induced cholangiocarcinoma. The aim of this study was to enhance our understanding of the mechanism of 1,2-DCP-related cholangiocarcinogenesis. We applied an in vitro system to investigate the effects of 1,2-DCP, using MMNK-1 cholangiocytes cultured alone or with THP-1 macrophages. The cultured cells were exposed to 1,2-DCP at 0, 0.1, 0.2, 0.4, and 0.8 mM for 24 h, and then assessed for cell proliferation, cell cytotoxicity, DNA damage, and ROS production. Exposure to 1,2-DCP increased proliferation of MMNK-1 cholangiocytes cultured alone, but not those cultured with macrophages. 1,2-DCP also increased LDH cytotoxicity, DNA damage, and ROS production in MMNK-1 cholangiocytes co-cultured with macrophages but not those cultured alone. 1,2-DCP increased TNFα and IL-1ß protein expression in macrophages. The results highlight the role of macrophages in enhancing the effects of 1,2-DCP on cytotoxicity, ROS production, and DNA damage in cholangiocytes.

Nrf2 Activation Attenuates Acrylamide-Induced Neuropathy in Mice.

Acrylamide is a well characterized neurotoxicant known to cause neuropathy and encephalopathy in humans and experimental animals. To investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in acrylamide-induced neuropathy, male C57Bl/6JJcl adult mice were exposed to acrylamide at 0, 200 or 300 ppm in drinking water and co-administered with subcutaneous injections of sulforaphane, a known activator of the Nrf2 signaling pathway at 0 or 25 mg/kg body weight daily for 4 weeks. Assessments for neurotoxicity, hepatotoxicity, oxidative stress as well as messenger RNA-expression analysis for Nrf2-antioxidant and pro-inflammatory cytokine genes were conducted. Relative to mice exposed only to acrylamide, co-administration of sulforaphane protected against acrylamide-induced neurotoxic effects such as increase in landing foot spread or decrease in density of noradrenergic axons as well as hepatic necrosis and hemorrhage. Moreover, co-administration of sulforaphane enhanced acrylamide-induced mRNA upregulation of Nrf2 and its downstream antioxidant proteins and suppressed acrylamide-induced mRNA upregulation of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) in the cerebral cortex. The results demonstrate that activation of the Nrf2 signaling pathway by co-treatment of sulforaphane provides protection against acrylamide-induced neurotoxicity through suppression of oxidative stress and inflammation. Nrf2 remains an important target for the strategic prevention of acrylamide-induced neurotoxicity.

Genetic ablation of Nrf2 exacerbates neurotoxic effects of acrylamide in mice.

Acrylamide (ACR), a recognized neurotoxicant in humans and experimental animals, is widely used in industry and in food generated through Maillard reaction. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of the cellular defense system and activates antioxidants and cytoprotective genes. The exact roles of Nrf2 in environmental electrophile-induced neurotoxicity is poorly understood. The aim of this study was to determine the roles of Nrf2 in ACR-induced neurotoxicity including degeneration of monoaminergic axons and sensorimotor dysfunction. Male 10-week-old C57BL/6JJcl Nrf2-knockout mice and wild type (WT) counterparts were each divided into four groups of 12 and provided with drinking water containing acrylamide at 0, 67, 110 or 200 ppm for four weeks. The effects of acrylamide were examined by landing foot spread test, immunohistochemistry for noradrenaline (NA) and serotonin (5-HT)-containing axons and Iba1-positive microglia in the prefrontal cortex as well as quantitative real-time polymerase chain reaction (qRT-PCR) on antioxidant, proinflammatory and anti-inflammatory genes in the prefrontal cortex. Relative to the wild type, exposure of Nrf2-knockout mice to acrylamide increased hindlimb splay length, microglial area and process length as well as decreasing the density of NA and 5-HT-immunoreactive axons to a greater extent. Moreover, deletion of Nrf2 gene suppressed acrylamide-induced mRNA upregulation of Nrf2-antioxidants, NAD(P): quinone oxidoreductase 1 (NQO1), superoxide dismutase-1 (SOD-1) and heme oxygenase-1 (HO-1) as well as anti-inflammatory markers such as, arginase-1 (Arg1), found in the inflammatory zone-1 (Fizz1), chitinase-like 3 (Chi3l3), interleukin-4 receptor alpha (IL-4Rα), cluster of differentiation  206 (CD206) and transforming growth factor beta-1 (TGFß1) while enhancing acrylamide-induced upregulation of pro-inflammatory cytokines, interleukin-1 beta (IL-1ß), tumor necrosis-alpha (TNF-α) and inducible nitric oxide synthase (iNOS) in the prefrontal cortex. The results demonstrate susceptibility of mice lacking the Nrf2 gene to acrylamide-induced neurotoxicity and neuroinflammation with the activation of microglia. Moreover, the results suggest the role of Nrf2 not only in induction of antioxidant gene expression, but also in suppression of proinflammatory cytokine gene expression.