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In dose-response and structure-activity studies, human hepatic HepG2 cells were exposed for 3 days to nano Cu, nano CuO or CuCl 2 (ions) at doses between 0.1 and 30 ug/ml (approximately the no observable adverse effect level to a high degree of cytotoxicity). Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. With nano Cu and nano CuO, few indications of cytotoxicity were observed between 0.1 and 3 ug/ml. In respect to dose, lactate dehydrogenase and aspartate transaminase were the most sensitive cytotoxicity parameters. The next most responsive parameters were alanine aminotransferase, glutathione reductase, glucose 6-phosphate dehydrogenase, and protein concentration. The medium responsive parameters were superoxide dismutase, gamma glutamyltranspeptidase, total bilirubin, and microalbumin. The parameters glutathione peroxidase, glutathione reductase, and protein were all altered by nano Cu and nano CuO but not by CuCl 2 exposures. Our chief observations were (1) significant decreases in glucose 6-phosphate dehydrogenase and glutathione reductase was observed at doses below the doses that show high cytotoxicity, (2) even high cytotoxicity did not induce large changes in some study parameters (e.g., alkaline phosphatase, catalase, microalbumin, total bilirubin, thioredoxin reductase, and triglycerides), (3) even though many significant biochemical effects happen only at doses showing varying degrees of cytotoxicity, it was not clear that cytotoxicity alone caused all of the observed significant biochemical effects, and (4) the decreased glucose 6-phosphate dehydrogenase and glutathione reductase support the view that oxidative stress is a main toxicity pathway of CuCl 2 and Cu-containing nanomaterials., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Alterations in physiological processes in pancreas have been associated with various metabolic dysfunctions and can result from environmental exposures, such as chemicals and diet. It was reported that environmental vinyl chloride (VC) exposure, a common industrial organochlorine and environmental pollutant, significantly exacerbated metabolic-related phenotypes in mice fed concurrently with high-fat diet (HFD) but not low-fat diet (LFD). However, little is known about the role of the pancreas in this interplay, especially at a proteomic level. The present study was undertaken to examine the protein responses to VC exposure in pancreas tissues of C57BL/6J mice fed LFD or HFD, with focus on the investigation of protein expression and/or phosphorylation levels of key protein biomarkers of carbohydrate, lipid, and energy metabolism, oxidative stress and detoxification, insulin secretion and regulation, cell growth, development, and communication, immunological responses and inflammation, and biomarkers of pancreatic diseases and cancers. We found that the protein alterations may indicate diet-mediated susceptibility in mouse pancreas induced by HFD to concurrent exposure of low levels of inhaled VC. These proteome biomarkers may lead to a better understanding of pancreas-mediated adaptive or adverse response and susceptibility to metabolic disease., (Published by Oxford University Press on behalf of the Society of Toxicology 2023.)

In dose-response and structure-activity studies, human hepatic HepG2 cells were exposed to between 0.01 and 300 ug/ml of different silver nanomaterials and AgNO₃ for 3 days. Treatment chemicals included a custom synthesized rod shaped nano Ag, a glutathione capped nano Ag, polyvinylpyrrolidone (PVP) capped nano Ag (75 nm) from Nanocomposix and AgNO₃. Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function and oxidative stress. Few indications of cytotoxicity were observed between 0.1 ug/ml and 6 ug/ml of any nano Ag. At 10 ug/ml and above, Ag containing nanomaterials caused a moderate to severe degree of cytotoxicity in HepG2 cells. Lactate dehydrogenase and aspartate transaminase activity alterations were the most sensitive cytotoxicity parameters. Some biochemical parameters were altered by exposures to both nano Ag and AgNO₃ (statistically significant increases in alkaline phosphatase, gamma glutamyltranspeptidase, glutathione peroxidase and triglycerides; in contrast both glutathione reductase and HepG2 protein concentration were both decreased). Three parameters were significantly altered by nano Ag but not by AgNO₃ (decreases in glucose 6-phosphate dehydrogenase and thioredoxin reductase and increases in catalase). Cytotoxicity per se did not appear to fully explain the patterns of biological responses observed. Some of the observations with the three nano Ag (increases in alkaline phosphatase, catalase, gamma glutamyltranspeptidase, as well as decreases in glucose 6-phosphate dehydrogenase and glutathione reductase) are in the same direction as HepG2 responses to other nanomaterials composed of TiO₂, CeO₂, SiO₂, CuO and Cu. Therefore, these biochemical responses may be due to micropinocytosis of nanomaterials, membrane damage, oxidative stress and/or cytotoxicity. Decreased G6PDH (by all three nano Ag forms) and GRD activity (only nano Ag R did not cause decreases) support and are consistent with the oxidative stress theory of Ag nanomaterial action.

The potential mammalian hepatotoxicity of nanomaterials was explored in dose-response and structure-activity studies in human hepatic HepG2 cells exposed to between 10 and 1000 μg/ml of five different CeO 2 , three SiO 2 , and one TiO 2 -based particles for 3 days. Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. Few indications of cytotoxicity were observed between 10 and 30 μg/ml. In the 100 to 300 μg/ml exposure range, a moderate degree of cytotoxicity was often observed. At 1000 μg/ml exposures, all but TiO 2 showed a high degree of cytotoxicity. Cytotoxicity per se did not seem to fully explain the observed patterns of biochemical parameters. Four nanomaterials (all three SiO 2 ) decreased glucose 6-phosphate dehydrogenase activity with some significant decreases observed at 30 μg/ml. In the range of 100 to 1000 μg/ml, the activities of glutathione reductase (by all three SiO 2 ) and glutathione peroxidase were decreased by some nanomaterials. Decreased glutathione concentration was also found after exposure to four nanomaterials (all three nano SiO 2 particles). In this study, the more responsive and informative assays were glucose 6-phosphate dehydrogenase, glutathione reductase, superoxide dismutase, lactate dehydrogenase, and aspartate transaminase. In this study, there were six factors that contribute to oxidative stress observed in nanomaterials exposed to hepatocytes (decreased glutathione content, reduced glucose 6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and increased catalase activities). With respect to structure-activity, nanomaterials of SiO 2 were more effective than CeO 2 in reducing glutathione content, glucose 6-phosphate dehydrogenase, glutathione reductase, and superoxide dismutase activities.

Neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3, have been implicated in the pathogenesis of many features and symptoms of asthma. The role of neurotrophins in fungal allergic asthma, however, is unknown. Repeated pulmonary challenge with Penicillium chrysogenum extract (PCE) induces dose-dependent allergic asthma-like responses in mice. The aim of this study was to investigate whether neurotrophins are involved in the PCE-induced allergic airway response in mice. Mice were exposed to 10, 20, 50, or 70 microg PCE by involuntary aspiration 4 times over 1 mo. Bronchial alveolar lavage fluid (BALF) was collected immediately before and after the final exposure. The levels of NGF, NT-3, and NT-4 were determined by enzyme-linked immunosorbent assay (ELISA). The lungs were fixed and processed for immunohistochemical examination of NGF production. PCE-exposed mice had dose-dependent increases in NGF, NT-3, and NT-4 in both BALF and sera. Exposures to PCE produced elevation in positive immunohistochemical staining for NGF in the airway epithelium and smooth muscle cells, in addition to infiltrated cells such as mononuclear cells, eosinophils, and macrophages. Taken together, this is the first study to link fungal allergic asthma in an experimental model with enhanced production of neurotrophins in the airways, and suggests that neurotrophins may play a role in the etiology of mold-induced asthma in humans.

Indoor mold has been associated with the development of allergic asthma. Penicillium chrysogenum, a common indoor mold, is known to have several allergens and can induce allergic responses in a mouse model of allergic penicilliosis. Our hypothesis is that soluble components of P. chrysogenum (PCE) can dose-dependently induce responses typical of allergic asthma in BALB/c mice. Mice were exposed to 10, 20, 50, or 70 microg of PCE by involuntary aspiration four times over a 4-week period. Serum and bronchoalveolar lavage fluid (BALF) were collected before (day 0), and at days 1 and 3 following the final exposure. PCE-exposed mice demonstrated dose-dependent increases in: BALF total cell numbers including eosinophil, serum and BALF total IgE levels, BALF IL-5 levels, and increased severity of histopathologic lesions. A single exposure to the highest dose of PCE resulted in edema and cellular damage but not immune responses. Four exposures to Metarhizium anisopliae crude antigen (10 microg, positive control) resulted in equivalent or greater allergic asthma-like responses than those demonstrated by multiple exposures to 50 or 70 microg of PCE. Multiple exposures to 70 microg of PCE showed increased allergen-triggered immediate respiratory responses as well as non-specific airway hyperresponsiveness to methacholine as assessed by barometric whole-body plethysmography. Taken together, repeated pulmonary challenge with P. chrysogenum extract induced dose-dependent allergic asthma-like responses in mice.

Environmental exposure to Stachybotrys chartarum has been associated with multiple adverse health effects in humans. The goal of this study was to assess soluble components of this fungus for their ability to cause an asthma-like response in a BALB/c mouse model. Five isolates of S. chartarum were combined and extracted to form a crude antigen preparation (S. chartarum extract 1 [SCE-1]). Female BALB/c mice were sensitized by involuntary aspiration of SCE-1 and subsequently reexposed at 2, 3, and 4 weeks. To distinguish immune from nonspecific inflammatory effects, mice were exposed to 3 doses of Hanks' balanced salt solution (HBSS) and a final dose of SCE-1; or to 4 doses of bovine serum albumin (BSA) as a negative control protein. Serum and bronchoalveolar lavage fluid (BALF) were collected before the fourth aspiration (Day 0), and at Days 1, 3, and 7 following the final exposure, and lungs were fixed for histopathological examination. SCE-1-exposed mice displayed increased BALF total protein on Days 0, 1, and 3 and increased lactate dehydrogenase (LDH) at Days 1 and 3 only, compared to HBSS controls. BALF total cell numbers were elevated on each day, and differential counts of BALF cells showed neutrophilia on Day 1, marked eosinophilia on all days, and increased numbers of lymphocytes at Days 1, 3, and 7. Serum and BALF total IgE levels were elevated at all days, and BALF IL-5 levels were greatly increased (7-fold) on Day 1. Mice exposed to a single dose of SCE-1 exhibited inflammatory responses but not allergic responses, while BSA-treated mice showed neither inflammatory nor allergic responses. Histopathology confirmed the biochemical findings. Barometric whole-body plethysmography was performed 10 min prior to (baseline) and one h following each aspiration exposure in a second group of mice, to assess immediate respiratory responses. Airway hyperresponsiveness to increasing concentrations of nebulized methacholine (MCh) was assessed on Days 1 and 3 following the fourth aspiration exposure. Exposure to HBSS or BSA did not alter baseline enhanced pause (PenH) values or PenH following the aspiration exposures, nor did it cause an increase in airway responsiveness to MCh. Exposure to SCE-1 resulted in a 4.7-fold increase in PenH over baseline after the third exposure, increasing to 5.6-fold after the final exposure, and increased responsiveness to a 32 mg/ml MCh aerosol challenge. We conclude that multiple respiratory exposures to SCE-1 cause responses typical of allergic airway disease in this mouse model. However, BSA was nonallergenic and did not generate respiratory physiological responses when administered by aspiration.