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Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET. [ABSTRACT FROM AUTHOR]

Fluoroquinolones and tetracyclines are known as β-diketone antibiotics (DKAs) because of bearing a diketone group in their molecular structure. DKAs are the most widely used antibiotics to prevent generation of disease in humans and animals and to suppress bacterial growth in aquaculture. In recent years, overuse of DKAs has caused serious environmental risk due to their pseudo-persistence in the environment, even though their half-lives are not long. So far, no reports were concerned with the joint immunotoxicity of DKAs. Herein, we reported on the immunotoxicity of DKAs on zebrafish after a 3-month DKAs exposure using transcriptomic techniques. According to transcriptome sequencing, 10 differentially expressed genes were screened out among the genes related to KEGG pathways with high enrichment. The identified 7 genes showed to be consistent between RNA-seq and qRT-PCR. Due to DKAs exposure, the content or activity for a series of immune-related biomarkers (Complement 3, lysozyme, IgM and AKP) showed the inconsistent changing trends as compared with the control group. Histopathological observations showed that the number of goblet cells increased sharply, the columnar epithelial cells swelled, the nucleus became slender in intestinal villi, and numerous brown metachromatic granules occurred in spleens of DKAs-exposed groups. Overall, both detection of biomarkers and histopathological observation corroborated that chronic DKAs exposure could result in abnormal expression of immune genes and enzymes, and variable levels of damage to immune-related organs. These complex effects of DKAs may lead to zebrafish dysfunction and occurrence of diseases related to the immune system. [ABSTRACT FROM AUTHOR]

Carrageenan has been widely used as food additive for decades and therefore, an extended oral data set is available in the public domain. Less data are available for other routes of administration, especially intranasal administration. The current publication describes the non-clinical safety and toxicity of native (non-degraded) iota-carrageenan when applied intranasally or via inhalation. Intranasally applied iota-carrageenan is a topically applied, locally acting compound with no need of systemic bioavailability for the drug’s action. Animal experiments included repeated dose local tolerance and toxicity studies with intranasally applied 0.12% iota-carrageenan for 7 or 28 days in New Zealand White rabbits and nebulized 0.12% iota-carrageenan administered to F344 rats for 7 days. Permeation studies revealed no penetration of iota-carrageenan across nasal mucosa, demonstrating that iota-carrageenan does not reach the blood stream. Consistent with this, no relevant toxic or secondary pharmacological effects due to systemic exposure were observed in the rabbit or rat repeated dose toxicity studies. Data do not provide any evidence for local intolerance or toxicity, when carrageenan is applied intranasally or by inhalation. No signs for immunogenicity or immunotoxicity have been observed in the in vivo studies. This is substantiated by in vitro assays showing no stimulation of a panel of pro-inflammatory cytokines by iota-carrageenan. In conclusion, 0.12% iota-carrageenan is safe for clinical use via intranasal application. [ABSTRACT FROM AUTHOR]

Perinatal exposure to the food contaminant bisphenol A (BPA) in rats induces long lasting adverse effects on intestinal immune homeostasis. This study was aimed at examining the immune response to dietary antigens and the clearance of parasites in young rats at the end of perinatal exposure to a low dose of BPA. Female rats were fed with BPA [5 µg/kg of body weight/day] or vehicle from gestational day 15 to pup weaning. Juvenile female offspring (day (D)25) were used to analyze immune cell populations, humoral and cellular responses after oral tolerance or immunization protocol to ovalbumin (OVA), and susceptibility to infection by the intestinal nematode Nippostrongylus brasiliensis (N. brasiliensis). Anti-OVA IgG titers following either oral tolerance or immunization were not affected after BPA perinatal exposure, while a sharp decrease in OVA-induced IFNγ secretion occurred in spleen and mesenteric lymph nodes (MLN) of OVA-immunized rats. These results are consistent with a decreased number of helper T cells, regulatory T cells and dendritic cells in spleen and MLN of BPA-exposed rats. The lack of cellular response to antigens questioned the ability of BPA-exposed rats to clear intestinal infections. A 1.5-fold increase in N. brasiliensis living larvae was observed in the intestine of BPA-exposed rats compared to controls due to an inappropriate Th1/Th2 cytokine production in infected jejunal tissues. These results show that perinatal BPA exposure impairs cellular response to food antigens, and increases susceptibility to intestinal parasitic infection in the juveniles. This emphasized the maturing immune system during perinatal period highly sensitive to low dose exposure to BPA, altering innate and adaptative immune response capacities in early life. [ABSTRACT FROM AUTHOR]

Silver materials have been widely used in diverse fields. However, their toxicity and their mechanism, especially in different forms, have not been studied sufficiently. Thus, cytotoxicity, apoptosis, and interleukin-1beta (IL-1β) production were investigated using macrophage-like THP-1 cells in the presence of Ag microparticles (AgMPs, 2.7 µm), Ag submicroparticles (AgSMPs, 150 nm), and Ag wires (AgWs, 274 nm×5.3 µm). The levels of cytotoxicity, apoptosis, and IL-1β production by AgWs were higher than those by the other two AgSMPs and AgMPs. This trend was also observed with each step of the signaling mechanism for IL-1β production, which is a single pathway affiliated with ROS generation or lysosomal rupture or both, cathepsin B, caspase-1 (NALP3 inflammasome), and finally IL-1β production in THP-1 cells. All these results suggest that, for development of safe and effective silver materials, the shape or form of silver materials should be considered, especially for macrophage cell lines because epithelial cell lines are not overly sensitive to silver materials. [ABSTRACT FROM AUTHOR]

Background: Microcystins LR (MC-LR) are hepatotoxic cyanotoxins that have been shown to induce reproductive toxicity, and Hypothalamic–Pituitary–Gonadal Axis (HPG) is responsible for the control of reproductive functions. However, few studies have been performed to evaluate the effects of MC-LR on HPG axis. This study aimed to investigate the MC-LR-induced toxicity in the reproductive system of mouse and focus on the HPG axis. Methods: Adult male C57BL/6 mice were exposed to various concentrations of MC-LR (0, 3.75, 7.50, 15.00 and 30.00 µg/kg body weight per day) for 1 to 14 days, and it was found that exposure to different concentrations of MC-LR significantly disturbed sperm production in the mice testes in a dose- and time-dependent manner. To elucidate the associated possible mechanisms, the serum levels of testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assessed. Meanwhile, PCR assays were employed to detect alterations in a series of genes involved in HPG axis, such as FSH, LH, gonadotropin-releasing hormone (GnRH) and their complement receptors. Furthermore, the effect of MC-LR on the viability and testosterone production of Leydig cells were tested in vitro. Results: MC-LR significantly impaired the spermatogenesis of mice possibly through the direct or indirect inhibition of GnRH synthesis at the hypothalamic level, which resulted in reduction of serum levels of LH that lead to suppression of testosterone production in the testis of mice. Conclusions: MC-LR may be a GnRH toxin that would disrupt the reproductive system of mice. [ABSTRACT FROM AUTHOR]

Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×105 twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8+ IL17+ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants. [ABSTRACT FROM AUTHOR]

Human exposure to arsenic in drinking water is known to contribute to many different health outcomes such as cancer, diabetes, and cardiopulmonary disease. Several epidemiological studies suggest that T cell function is also altered by drinking water arsenic exposure. However, it is unclear how individual responses differ to various levels of exposure to arsenic. Our laboratory has recently identified differential responses of human peripheral blood mononuclear cell (HPMBC) T cells as measured by polyclonal T cell activation by mitogens during sodium arsenite exposure. T cells from certain healthy individuals exposed to various concentrations (1–100 nM) of arsenite in vitro showed a dose-dependent suppression at these extremely low concentrations (∼0.1–10 ppb) of arsenite, whereas other individuals were not suppressed at low concentrations. In a series of more than 30 normal donors, two individuals were found to be sensitive to low concentration (10 nM equivalent ∼1 ppb drinking water exposure) to sodium arsenite-induced inhibition of T cell proliferation produced by phytohemagglutinin (PHA) and anti-CD3/anti-CD28. In an arsenite-susceptible individual, arsenite suppressed the activation of Th1 (Tbet) cells, and decreased the percentage of cells in the double positive Th17 (RORγt) and Treg (FoxP3) population. While the majority of normal blood donors tested were not susceptible to inhibition of proliferation at the 1–100 nM concentrations of As+3, it was found that all donors were sensitive to suppression by 100 nM monomethylarsonous acid (MMA+3), a key metabolite of arsenite. Thus, our studies demonstrate for the first time that low ppb-equivalent concentrations of As+3 are immunosuppressive to HPBMC T cells in some individuals, but that most donor HPBMC are sensitive to suppression by MMA+3 at environmentally relevant exposure levels. [ABSTRACT FROM AUTHOR]

Exposure to the herbicide Atrazine (ATR) can cause immunotoxicity, apart from other adverse consequences for animal and human health. We aimed at elucidating the apoptotic mechanisms involved in immunotoxicity of ATR and their attenuation by Melatonin (MEL). Young Swiss mice were divided into control, ATR and MEL+ATR groups based on daily (x14) intraperitoneal administration of the vehicle (normal saline), ATR (100 mg/kg body weight) and MEL (20 mg/kg body weight) with ATR. Isolated splenocytes were processed for detection of apoptosis by Annexin V-FITC and TUNEL assays, and endoplasmic reticulum (ER) stress by immunostaining. Key proteins involved in apoptosis, ER stress and autophagy were quantified by immunoblotting. ATR treatment resulted in Fas-mediated activation of caspases 8 and 3 and inactivation of PARP1 which were inhibited significantly by co-treatment with MEL. MEL also attenuated the ATR-induced, p53 independent mitochondrial apoptosis through upregulation of E2F-1 and PUMA and suppression of their downstream target Bax. An excessive ER stress triggered by ATR through overexpression of ATF-6α, spliced XBP-1, CREB-2 and GADD153 signals was reversed by MEL. MEL also reversed the ATR-induced impairment of autophagy which was indicated by a decline in BECN-1, along with significant enhancement in LC3B-II and p62 expressions. Induction of mitochondrial apoptosis, ER stress and autophagy dysregulation provide a new insight into the mechanism of ATR immunotoxicity. The cytoprotective role of MEL, on the other hand, was defined by attenuation of ER stress, Fas-mediated and p53 independent mitochondria-mediated apoptosis as well as autophagy signals. [ABSTRACT FROM AUTHOR]

Polyaniline nanomaterial (nPANI) is getting popular in many industrial fields due to its conductivity and stability. The fate and effect of nPANI in the environment is of paramount importance towards its technological applications. In this work, the cytotoxicity of nPANI, which was prepared by rapid surface polymerization, was studied on rat celiac macrophages. Cell viability of macrophages treated with various concentrations of nPANI and different periods ranging from 24 to 72 hours was tested by a MTT assay. Damages of nPANI to structures of macrophages were evaluated according to the exposure level of cellular reactive oxygen species (ROS) and change of mitochondrial membrane potential (MMP). We observed no significant effects of nPANI on the survival, ROS level and MMP loss of macrophages at concentrations up to 1 µg/ml. However, higher dose of nPANI (10 µg/ml or above) induced cell death, changes of ROS level and MMP. In addition, an increase in the expression level of caspase-3 protein and its activated form was detected in a Western blot assay under the high dose exposure of nPANI. All together, our experimental results suggest that the hazardous potential of nPANI on macrophages is time- and dose-dependent and high dose of nPANI can induce cell apoptosis through caspase-3 mediated pathway. [ABSTRACT FROM AUTHOR]