Biochemical effects of copper nanomaterials in human hepatocellular carcinoma (HepG2) cells.

In dose-response and structure-activity studies, human hepatic HepG2 cells were exposed for 3 days to nano Cu, nano CuO or CuCl2 (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 CuCl2 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 CuCl2 and Cu-containing nanomaterials.

Prenatal ozone exposure programs a sexually dimorphic susceptibility to high-fat diet in adolescent Long Evans rats.

Altered fetal growth, which can occur due to environmental stressors during pregnancy, may program a susceptibility to metabolic disease. Gestational exposure to the air pollutant ozone is associated with fetal growth restriction in humans and rodents. However, the impact of this early life ozone exposure on offspring metabolic risk has not yet been investigated. In this study, fetal growth restriction was induced by maternal inhalation of 0.8 ppm ozone on gestation days 5 and 6 (4 hr/day) in Long Evans rats. To uncover any metabolic inflexibility, or an impaired ability to respond to a high-fat diet (HFD), a subset of peri-adolescent male and female offspring from filtered air or ozone exposed dams were fed HFD (45% kcal from fat) for 3 days. By 6 weeks of age, male and female offspring from ozone-exposed dams were heavier than offspring from air controls. Furthermore, offspring from ozone-exposed dams had greater daily caloric consumption and reduced metabolic rate when fed HFD. In addition to energy imbalance, HFD-fed male offspring from ozone-exposed dams had dyslipidemia and increased adiposity, which was not evident in females. HFD consumption in males resulted in the activation of the protective 5'AMP-activated protein kinase (AMPKα) and sirtuin 1 (SIRT1) pathways in the liver, regardless of maternal exposure. Unlike males, ozone-exposed female offspring failed to activate these pathways, retaining hepatic triglycerides following HFD consumption that resulted in increased inflammatory gene expression and reduced insulin signaling genes. Taken together, maternal ozone exposure in early pregnancy programs impaired metabolic flexibility in offspring, which may increase susceptibility to obesity in males and hepatic dysfunction in females.

Iron and zinc homeostases in female rats with physically active and sedentary lifestyles.

To determine the effects of repeated physical activity on iron and zinc homeostases in a living system, we quantified blood and tissue levels of these two metals in sedentary and physically active Long-Evans rats. At post-natal day (PND) 22, female rats were assigned to either a sedentary or an active treatment group (n = 10/group). The physically active rats increased their use of a commercially-constructed stainless steel wire wheel so that, by the end of the study (PND 101), they were running an average of 512.8 ± 31.9 (mean ± standard error) min/night. After euthanization, plasma and aliquots of liver, lung, heart, and gastrocnemius muscle were obtained. Following digestion, non-heme iron and zinc concentrations in plasma and tissues were measured using inductively coupled plasma optical emission spectroscopy. Concentrations of both non-heme iron and zinc in plasma and liver were significantly decreased among the physically active rats relative to the sedentary animals. In the lung, both metals were increased in concentration among the physically active animals but the change in zinc did not reach significance. Similarly, tissue non-heme iron and zinc levels were both increased in heart and muscle from the physically active group. It is concluded that repeated physical activity in an animal model can be associated with a translocation of both iron and zinc from sites of storage (e.g. liver) to tissues with increased metabolism (e.g. the lung, heart, and skeletal muscle).

Ozone-induced changes in oxidative stress parameters in brain regions of adult, middle-age, and senescent Brown Norway rats.

A critical part of community based human health risk assessment following chemical exposure is identifying sources of susceptibility. Life stage is one such susceptibility. A prototypic air pollutant, ozone (O3) induces dysfunction of the pulmonary, cardiac, and nervous systems. Long-term exposure may cause oxidative stress (OS). The current study explored age-related and subchronic O3-induced changes in OS in brain regions of rats. To build a comprehensive assessment of OS-related effects of O3, a tripartite approach was implemented focusing on 1) the production of reactive oxygen species (ROS) [NADPH Quinone oxidoreductase 1, NADH Ubiquinone reductase] 2) antioxidant homeostasis [total antioxidant substances, superoxide dismutase, γ-glutamylcysteine synthetase] and 3) an assessment of oxidative damage [total aconitase and protein carbonyls]. Additionally, a neurobehavioral evaluation of motor activity was compared to these OS measures. Male Brown Norway rats (4, 12, and 24 months of age) were exposed to air or O3 (0.25 or 1 ppm) via inhalation for 6 h/day, 2 days per week for 13 weeks. A significant decrease in horizontal motor activity was noted only in 4-month old rats. Results on OS measures in frontal cortex (FC), cerebellum (CB), striatum (STR), and hippocampus (HIP) indicated life stage-related increases in ROS production, small decreases in antioxidant homeostatic mechanisms, a decrease in aconitase activity, and an increase in protein carbonyls. The effects of O3 exposure were brain area-specific, with the STR being more sensitive. Regarding life stage, the effects of O3 were greater in 4-month-old rats, which correlated with horizontal motor activity. These results indicate that OS may be increased in specific brain regions after subchronic O3 exposure, but the interactions between age and exposure along with their consequences on the brain require further investigation.

Diets enriched with coconut, fish, or olive oil modify peripheral metabolic effects of ozone in rats.

Dietary factors may modulate metabolic effects of air pollutant exposures. We hypothesized that diets enriched with coconut oil (CO), fish oil (FO), or olive oil (OO) would alter ozone-induced metabolic responses. Male Wistar-Kyoto rats (1-month-old) were fed normal diet (ND), or CO-, FO-, or OO-enriched diets. After eight weeks, animals were exposed to air or 0.8 ppm ozone, 4 h/day for 2 days. Relative to ND, CO- and OO-enriched diet increased body fat, serum triglycerides, cholesterols, and leptin, while all supplements increased liver lipid staining (OO > FO > CO). FO increased n-3, OO increased n-6/n-9, and all supplements increased saturated fatty-acids. Ozone increased total cholesterol, low-density lipoprotein, branched-chain amino acids (BCAA), induced hyperglycemia, glucose intolerance, and changed gene expression involved in energy metabolism in adipose and muscle tissue in rats fed ND. Ozone-induced glucose intolerance was exacerbated by OO-enriched diet. Ozone increased leptin in CO- and FO-enriched groups; however, BCAA increases were blunted by FO and OO. Ozone-induced inhibition of liver cholesterol biosynthesis genes in ND-fed rats was not evident in enriched dietary groups; however, genes involved in energy metabolism and glucose transport were increased in rats fed FO and OO-enriched diet. FO- and OO-enriched diets blunted ozone-induced inhibition of genes involved in adipose tissue glucose uptake and cholesterol synthesis, but exacerbated genes involved in adipose lipolysis. Ozone-induced decreases in muscle energy metabolism genes were similar in all dietary groups. In conclusion, CO-, FO-, and OO-enriched diets modified ozone-induced metabolic changes in a diet-specific manner, which could contribute to altered peripheral energy homeostasis.

Parental experiences of being approached to join multiple neonatal clinical trials: qualitative study (PARENT).

OBJECTIVE: To explore parents' perceptions and experience of being approached for enrolment of their preterm infant in more than one trial or study. DESIGN: A qualitative study involving 17 in-depth semistructured interviews, with parents who had been approached for multiple studies and who subsequently consented for their infant(s) to join at least one. Parents who declined all studies were not approached. SETTING AND PARTICIPANTS: Parents of preterm infants receiving care at one of three neonatal intensive care units in the north of England. FINDINGS: Most parents did not view concurrent participation in multiple trials or studies as a significant issue within the wider context of their infant's care. Most parents did not feel pressured into enrolling their infant into more than one study, but some suggested that participation in several provided justification for the subsequent refusal to join others, articulating feeling of guilt at saying 'no', and others appeared fatigued by multiple approaches. Parents focused on the perceived risks and benefits of each individual study and, while acknowledging that making a fully informed decision was not possible, largely agreed due to their belief in the benefits of research, trust in the health professionals caring for their baby and a range of complex personal motivations. CONCLUSIONS: Parents valued the autonomy to make decisions about participation and felt, with hindsight, that their decisions were right. Research teams could be more aware of parental feelings of guilt or gratitude that may motivate them to give consent. Similarly, the capacity of parents to fully remember details of multiple studies when they are stressed, and their infant is sick, should be taken into consideration, and continued efforts should be made to ensure ongoing consent to participation.

Ozone-induced fetal growth restriction in rats is associated with sexually dimorphic placental and fetal metabolic adaptation.

OBJECTIVE: The importance of the placenta in mediating the pre- and post-natal consequences of fetal growth restriction has been increasingly recognized. However, the influence of placental sexual dimorphism on driving these outcomes has received little attention. The purpose of this study was to characterize how sex contributes to the relationship between placental metabolism and fetal programming utilizing a novel rodent model of growth restriction. METHODS: Fetal growth restriction was induced by maternal inhalation of 0.8 ppm ozone (4 h/day) during implantation receptivity (gestation days [GDs] 5 and 6) in Long-Evans rats. Control rats were exposed to filtered air. At GD 21, placental and fetal tissues were obtained for metabolic and genomic assessments. RESULTS: Growth-restricted male placentae exhibited increased mitochondrial biogenesis, increased oxygen consumption, and reduced nutrient storage. Male growth-restricted fetuses also had evidence of reduced adiposity and downregulation of hepatic metabolic signaling. In contrast, placentae from growth-restricted females had elevated markers of autophagy accompanied by an observed protection against hepatic metabolic perturbations. Despite this, growth restriction in females induced a greater number of hypothalamic gene and pathway alterations compared to growth-restricted males. CONCLUSIONS: Increases in mitochondrial metabolism in growth-restricted male placentae likely initiates a sequela of adaptations that promote poor nutrient availability and adiposity. Divergently, the female placenta expresses protective mechanisms that may serve to increase nutrient availability to support fetal metabolic development. Collectively, this work emphasizes the importance of sex in mediating alterations in placental metabolism and fetal programming.

A single exposure to eucalyptus smoke sensitizes rats to the postprandial cardiovascular effects of a high carbohydrate oral load.

OBJECTIVE: Previous studies have shown that air pollution exposure primes the body to heightened responses to everyday stressors of the cardiovascular system. The purpose of this study was to examine the utility of postprandial responses to a high carbohydrate oral load, a cardiometabolic stressor long used to predict cardiovascular risk, in assessing the impacts of exposure to eucalyptus smoke (ES), a contributor to wildland fire air pollution in the Western coast of the United States. MATERIALS AND METHODS: Three-month-old male Sprague Dawley rats were exposed once (1 h) to filtered air (FA) or ES (700 µg/m3 fine particulate matter), generated by burning eucalyptus in a tube furnace. Rats were then fasted for six hours the following morning, and subsequently administered an oral gavage of either water or a HC suspension (70 kcal% from carbohydrate), mimicking a HC meal. Two hours post gavage, cardiovascular ultrasound, cardiac pressure-volume (PV), and baroreceptor sensitivity assessments were made, and pulmonary and systemic markers assessed. RESULTS: ES inhalation alone increased serum interleukin (IL)-4 and nasal airway levels of gamma glutamyl transferase. HC gavage alone increased blood glucose, blood pressure, and serum IL-6 and IL-13 compared to water vehicle. By contrast, only ES-exposed and HC-challenged animals had increased PV loop measures of cardiac output, ejection fraction %, dP/dtmax, dP/dtmin, and stroke work compared to ES exposure alone and/or HC challenge alone. DISCUSSION AND CONCLUSIONS: Exposure to a model wildfire air pollution source modifies cardiovascular responses to HC challenge, suggesting air pollution sensitizes the body to systemic triggers.

Two speeds of increasing milk feeds for very preterm or very low-birthweight infants: the SIFT RCT.

BACKGROUND: Observational data suggest that slowly advancing enteral feeds in preterm infants may reduce necrotising enterocolitis but increase late-onset sepsis. The Speed of Increasing milk Feeds Trial (SIFT) compared two rates of feed advancement. OBJECTIVE: To determine if faster (30 ml/kg/day) or slower (18 ml/kg/day) daily feed increments improve survival without moderate or severe disability and other morbidities in very preterm or very low-birthweight infants. DESIGN: This was a multicentre, two-arm, parallel-group, randomised controlled trial. Randomisation was via a web-hosted minimisation algorithm. It was not possible to safely and completely blind caregivers and parents. SETTING: The setting was 55 UK neonatal units, from May 2013 to June 2015. PARTICIPANTS: The participants were infants born at < 32 weeks' gestation or a weight of < 1500 g, who were receiving < 30 ml/kg/day of milk at trial enrolment. INTERVENTIONS: When clinicians were ready to start advancing feed volumes, the infant was randomised to receive daily feed increments of either 30 ml/kg/day or 18 ml/kg/day. In total, 1400 infants were allocated to fast feeds and 1404 infants were allocated to slow feeds. MAIN OUTCOME MEASURES: The primary outcome was survival without moderate or severe neurodevelopmental disability at 24 months of age, corrected for gestational age. The secondary outcomes were mortality; moderate or severe neurodevelopmental disability at 24 months corrected for gestational age; death before discharge home; microbiologically confirmed or clinically suspected late-onset sepsis; necrotising enterocolitis (Bell's stage 2 or 3); time taken to reach full milk feeds (tolerating 150 ml/kg/day for 3 consecutive days); growth from birth to discharge; duration of parenteral feeding; time in intensive care; duration of hospital stay; diagnosis of cerebral palsy by a doctor or other health professional; and individual components of the definition of moderate or severe neurodevelopmental disability. RESULTS: The results showed that survival without moderate or severe neurodevelopmental disability at 24 months occurred in 802 out of 1224 (65.5%) infants allocated to faster increments and 848 out of 1246 (68.1%) infants allocated to slower increments (adjusted risk ratio 0.96, 95% confidence interval 0.92 to 1.01). There was no significant difference between groups in the risk of the individual components of the primary outcome or in the important hospital outcomes: late-onset sepsis (adjusted risk ratio 0.96, 95% confidence interval 0.86 to 1.07) or necrotising enterocolitis (adjusted risk ratio 0.88, 95% confidence interval 0.68 to 1.16). Cost-consequence analysis showed that the faster feed increment rate was less costly but also less effective than the slower rate in terms of achieving the primary outcome, so was therefore found to not be cost-effective. Four unexpected serious adverse events were reported, two in each group. None was assessed as being causally related to the intervention. LIMITATIONS: The study could not be blinded, so care may have been affected by knowledge of allocation. Although well powered for comparisons of all infants, subgroup comparisons were underpowered. CONCLUSIONS: No clear advantage was identified for the important outcomes in very preterm or very low-birthweight infants when milk feeds were advanced in daily volume increments of 30 ml/kg/day or 18 ml/kg/day. In terms of future work, the interaction of different milk types with increments merits further examination, as may different increments in infants at the extremes of gestation or birthweight. TRIAL REGISTRATION: Current Controlled Trials ISRCTN76463425. FUNDING: This project was funded by the National Institute for Health Research Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 18. See the NIHR Journals Library website for further project information.

Some infants who are born early need to be fed through a tube into their stomach. A small volume of milk is given to begin with, which is gradually increased. To determine whether infants do better if they are fed faster or slower, this study compared increasing the milk feeds by 30 ml/kg/day with increasing the milk feeds by 18 ml/kg/day, aiming to get to full feeds (when other fluids are not needed) in 5 or 9 days. We compared results from the two groups at discharge from hospital and at 24 months of age, after correcting for prematurity. We also assessed the economic impact of the two daily feed increments, interviewed parents about taking part in multiple studies and tested methods for improving questionnaire returns. The faster-fed group reached full milk feeds sooner and needed less intravenous nutrition, and the proportion of infants developing bowel inflammation or bloodstream infection were similar. At 24 months of age, we found an unexpected increase in the risk of moderate or severe motor impairment in the faster-fed group, which is difficult to explain. We also saw that other types of disability were more frequent in the faster group, although this was not significantly different mathematically. This means that no clear advantage of increasing feeds at faster or slower rates was identified and health professionals will need to carefully consider how to increase feeds. After accepting the increased risk of disability, an economic evaluation showed that increasing milk feed volumes at a faster rate was not a cost-effective strategy. Interviews with parents showed that they valued opportunities for their infant to take part in studies, but this interaction is complex and difficult to remember at a stressful and confusing time and made worse by considering multiple studies. More questionnaires were returned when vouchers were given before rather than after receiving them.

Biochemical Effects of Silver Nanomaterials in Human Hepatocellular Carcinoma (HepG2) Cells.

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.

Fetal growth outcomes following peri-implantation exposure of Long-Evans rats to noise and ozone differ by sex.

BACKGROUND: Exposure to air pollution and high levels of noise have both been independently associated with the development of adverse pregnancy outcomes including low birth weight. However, exposure to such environmental stressors rarely occurs in isolation and is often co-localized, especially in large urban areas. METHODS: The purpose of this study was to compare the effects of combined exposure to noise (N) or ozone (O3), compared to either exposure alone. Long-Evans dams were exposed to air or 0.4 ppm ozone for 4 h on gestation day (GD) 5 and 6, coinciding with implantation receptivity. A subset of dams from each exposure group was further exposed to intermittent white noise (~ 85 dB) throughout the dark cycle following each inhalation exposure (n = 14 - 16/group). Uterine artery ultrasound was performed on GD 15 and 21. Fetal growth characteristics and indicators of placental nutrient status were measured at GD 21. RESULTS: Exposure to ozone + quiet (O3 + Q) conditions reduced uterine arterial resistance at GD 15 compared to air + quiet (A + Q) exposure, with no further reduction by GD 21. By contrast, exposure to air + noise (A + N) significantly increased uterine arterial resistance at both GD 15 and 21. Notably, while peri-implantation exposure to O3 + Q conditions reduced male fetal weight at GD 21, this effect was not observed in the air + noise (A + N) or the ozone + noise (O3 + N) exposure groups. Fetal weight in female offspring was not reduced by ozone exposure alone (O3 + Q), nor was it affected by air + noise (A + N) or by combined ozone + noise (O3 + N) exposure. CONCLUSIONS: These data indicate that exposure to ozone and noise differentially impact uterine blood flow, particularly at mid-gestation, with only ozone exposure being associated with sex-dependent fetal growth retardation in male offspring.

Exacerbation of ozone-induced pulmonary and systemic effects by ß2-adrenergic and/or glucocorticoid receptor agonist/s.

Agonists of ß2 adrenergic receptors (ß2AR) and glucocorticoid receptors (GR) are prescribed to treat pulmonary diseases. Since ozone effects are mediated through the activation of AR and GR, we hypothesized that the treatment of rats with relevant therapeutic doses of long acting ß2AR agonist (LABA; clenbuterol; CLEN) and/or GR agonist (dexamethasone; DEX) would exacerbate ozone-induced pulmonary and systemic changes. In the first study, male 12-week-old Wistar-Kyoto rats were injected intraperitoneally with vehicle (saline), CLEN (0.004 or 0.02 mg/kg), or DEX (0.02 or 0.1 mg/kg). Since dual therapy is commonly used, in the second study, rats received either saline or combined CLEN + DEX (each at 0.005 or 0.02 mg/kg) one day prior to and on both days of exposure (air or 0.8ppm ozone, 4 hr/day x 2-days). In air-exposed rats CLEN, DEX or CLEN + DEX did not induce lung injury or inflammation, however DEX and CLEN + DEX decreased circulating lymphocytes, spleen and thymus weights, increased free fatty acids (FFA) and produced hyperglycemia and glucose intolerance. Ozone exposure of vehicle-treated rats increased bronchoalveolar lavage fluid protein, albumin, neutrophils, IL-6 and TNF-α. Ozone decreased circulating lymphocytes, increased FFA, and induced hypeerglycemia  and glucose intolerance. Drug treatment did not reverse ozone-induced ventillatory changes, however, lung effects (protein and albumin leakage, inflammation, and IL-6 increase) were exacerbated by CLEN and CLEN + DEX pre-treatment in a dose-dependent manner (CLEN > CLEN + DEX). Systemic effects induced by DEX and CLEN + DEX but not CLEN in air-exposed rats were analogous to and more pronounced than those induced by ozone. These data suggest that adverse air pollution effects might be exacerbated in people receiving LABA or LABA plus glucocorticoids.

Quartz Disrupts Iron Homeostasis in Alveolar Macrophages To Impact a Pro-Inflammatory Effect.

The biological response of bronchial epithelial cells to particles is associated with a sequestration of cell metal by the particle surface and a subsequent disruption in host iron homeostasis. The macrophage is the cell type resident in the respiratory tract that is most likely to make initial contact with inhaled particles. We tested the postulates that (1) silica, a prototypical particle, disrupts iron homeostasis in alveolar macrophages (AMs); and (2) the altered iron homeostasis results in both an oxidative stress and pro-inflammatory effects. Human AMs (1.0 × 106/mL) demonstrated an increased import of iron following particle exposure with nonheme iron concentrations of 0.57 ± 0.03, 1.72 ± 0.09, 0.88 ± 0.09, and 3.21 ± 0.11 ppm in cells exposed for 4 h to media, 500 µM ferric ammonium citrate (FAC), 100 µg/mL silica, and both silica and FAC, respectively. Intracellular ferritin concentrations and iron release were similarly increased after AM exposure to FAC and silica. Silica increased oxidant generation by AMs measured using both dichlorofluorescein diacetate fluorescence and reduction of nitroblue tetrazolium salt. Concentrations of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-α in macrophage supernatant increased following 100 µg/mL silica exposure for 24 h. Treatment of AMs with 500 µM FAC decreased both oxidant generation and cytokine release associated with silica exposure, supporting a dependence of these effects on sequestration of cell metal by the particle surface. We conclude that (1) silica exposure disrupts iron homeostasis resulting in increased import, accumulation, and release of the metal; and (2) the altered iron homeostasis following silica exposure impacts oxidant generation and pro-inflammatory effects.

Ozone Exposure During Implantation Increases Serum Bioactivity in HTR-8/SVneo Trophoblasts.

Implantation is a sensitive window in reproductive development during which disruptions may increase the risk of adverse pregnancy outcomes including intrauterine growth restriction. Ozone exposure during implantation in rats reduces fetal weight near the end of gestation, potentially though impaired trophoblast migration and invasion and altered implantation. The current study characterized changes in ventilation, pulmonary injury, and circulating factors including hormonal, inflammatory, and metabolic markers related to exposure to ozone (0.4-1.2 ppm) for 4-h on gestation days 5 and 6 (window of implantation) in Long-Evans dams. To determine the effects of this exposure on trophoblast function, placental-derived, first trimester, HTR-8/SVneo cells were exposed to serum from air- or ozone (0.8 ppm×4 h)-exposed dams and examined for impacts on metabolic capacity, wound-closure, and invasion. Peri-implantation exposure to ozone induced ventilatory dysfunction and lung vascular leakage in pregnant rats, with little effect on most of the circulating markers measured. However, ozone inhalation induced a significant reduction in several serum cytokines (interferon-γ, interleukin-6, and interleukin-13). Treatment of HTR-8/SVneo trophoblasts with serum from ozone-exposed dams for 16-h downregulated metabolic capacity, wound-closure, and invasion through a Matrigel membrane compared with both air-serum and fetal bovine serum-treated cells. Ozone-serum treated cells increased the release of a critical inhibitor of invasion and angiogenesis (soluble fms-like receptor 1; sFlt1) compared with air-serum treatment. Together, our data suggest that circulating factors in the serum of pregnant rats exposed to ozone during implantation receptivity can hinder critical processes of implantation (eg, invasion and migration) and impair trophoblast metabolic capacity.

Aspirin pre-treatment modulates ozone-induced fetal growth restriction and alterations in uterine blood flow in rats.

Prenatal exposure to ozone has been linked to low birth weight in people and fetal growth restriction in rats. Clinical recommendations suggest use of low dose aspirin to lower risk of preeclampsia and intrauterine growth restriction in high-risk pregnancies, yet its utility in mitigating the postnatal effects of gestational ozone exposure is unknown. The present study investigated the possibility of low dose aspirin to mitigate the effects of ozone exposure during pregnancy. Exposure to ozone impaired uterine arterial flow and induced growth restriction in fetuses of both sexes. Aspirin treatment induced marginal improvements in ozone-induced uterine blood flow impairment. However, this resulted in a protection of fetal weight in dams given aspirin only in early pregnancy. Aspirin administration for the entirety of gestation increased placental weight and reduced antioxidant status, suggesting that prolonged exposure to low dose aspirin may induce placental inefficiency in our model of growth restriction.

Biochemical effects of some CeO2, SiO2, and TiO2 nanomaterials in HepG2 cells.

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 CeO2, three SiO2, and one TiO2-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 TiO2 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 SiO2) 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 SiO2) and glutathione peroxidase were decreased by some nanomaterials. Decreased glutathione concentration was also found after exposure to four nanomaterials (all three nano SiO2 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 SiO2 were more effective than CeO2 in reducing glutathione content, glucose 6-phosphate dehydrogenase, glutathione reductase, and superoxide dismutase activities.

Interaction of Diet and Ozone Exposure on Oxidative Stress Parameters within Specific Brain Regions of Male Brown Norway Rats.

Oxidative stress (OS) contributes to the neurological and cardio/pulmonary effects caused by adverse metabolic states and air pollutants such as ozone (O3). This study explores the interactive effects of O3 and diet (high-fructose (FRUC) or high⁻fat (FAT)) on OS in different rat brain regions. In acute exposure, there was a decrease in markers of reactive oxygen species (ROS) production in some brain regions by diet and not by O3. Total antioxidant substances (TAS) were increased in the cerebellum (CER) and frontal cortex (FC) and decreased in the striatum (STR) by both diets irrespective of O3 exposure. Protein carbonyls (PC) and total aconitase decreased in some brain regions irrespective of exposure. Following subacute exposure, an increase in markers of ROS was observed in both diet groups. TAS was increased in the FC (FAT only) and there was a clear O3 effect where TAS was increased in the FC and STR. Diet increased PC formation within the CER in the FAT group, while the hippocampus showed a decrease in PC after O3 exposure in controls. In general, these results indicate that diet/O3 did not have a global effect on brain OS parameters, but showed some brain region- and OS parameter-specific effects by diets.

Beta-2 Adrenergic and Glucocorticoid Receptor Agonists Modulate Ozone-Induced Pulmonary Protein Leakage and Inflammation in Healthy and Adrenalectomized Rats.

We have shown that acute ozone inhalation activates sympathetic-adrenal-medullary and hypothalamus-pituitary-adrenal stress axes, and adrenalectomy (AD) inhibits ozone-induced lung injury and inflammation. Therefore, we hypothesized that stress hormone receptor agonists (ß2 adrenergic-ß2AR and glucocorticoid-GR) will restore the ozone injury phenotype in AD, while exacerbating effects in sham-surgery (SH) rats. Male Wistar Kyoto rats that underwent SH or AD were treated with vehicles (saline + corn oil) or ß2AR agonist clenbuterol (CLEN, 0.2 mg/kg, i.p.) + GR agonist dexamethasone (DEX, 2 mg/kg, s.c.) for 1 day and immediately prior to each day of exposure to filtered air or ozone (0.8 ppm, 4 h/day for 1 or 2 days). Ozone-induced increases in PenH and peak-expiratory flow were exacerbated in CLEN+DEX-treated SH and AD rats. CLEN+DEX affected breath waveform in all rats. Ozone exposure in vehicle-treated SH rats increased bronchoalveolar lavage fluid (BALF) protein, N-acetyl glucosaminidase activity (macrophage activation), neutrophils, and lung cytokine expression while reducing circulating lymphocyte subpopulations. AD reduced these ozone effects in vehicle-treated rats. At the doses used herein, CLEN+DEX treatment reversed the protection offered by AD and exacerbated most ozone-induced lung effects while diminishing circulating lymphocytes. CLEN+DEX in air-exposed SH rats also induced marked protein leakage and reduced circulating lymphocytes but did not increase BALF neutrophils. In conclusion, circulating stress hormones and their receptors mediate ozone-induced vascular leakage and inflammatory cell trafficking to the lung. Those receiving ß2AR and GR agonists for chronic pulmonary diseases, or with increased circulating stress hormones due to psychosocial stresses, might have altered sensitivity to air pollution.

Acute inhalation of ozone induces DNA methylation of apelin in lungs of Long-Evans rats.

Apelin has cardiopulmonary protective properties that promote vasodilation and maintenance of the endothelial barrier. While reductions in apelin have been identified as a contributor to various lung diseases, including pulmonary edema, its role in the effect of air pollutants has not been examined. Thus, in the current study, we sought to investigate if apelin is a downstream target of inhaled ozone and if such change in expression is related to altered DNA methylation in the lung. Male, Long-Evans rats were exposed to filtered air or 1.0 ppm ozone for 4 h. Ventilation changes were assessed using whole-body plethysmography immediately following exposure, and markers of pulmonary edema and inflammation were assessed in the bronchoaveolar lavage (BAL) fluid. The enzymatic regulators of DNA methylation were measured in the lung, along with methylation and hydroxymethylation of the apelin promoter. Data showed that ozone exposure was associated with increased enhanced pause and protein leakage in the BAL fluid. Ozone exposure reduced DNA cytosine-5-methyltransferase (DNMT) activity and Dnmt3a/b gene expression. Exposure-induced upregulation of proliferating cell nuclear antigen, indicative of DNA damage, repair, and maintenance methylation. Increased methylation and reduced hydroxymethylation were measured on the apelin promoter. These epigenetic modifications accompanied ozone-induced reduction of apelin expression and development of pulmonary edema. In conclusion, epigenetic regulation, specifically increased methylation of the apelin promoter downstream of DNA damage, may lead to reductions in protective signaling of the apelinergic system, contributing to the pulmonary edema observed following the exposure to oxidant air pollution.

Acute peat smoke inhalation sensitizes rats to the postprandial cardiometabolic effects of a high fat oral load.

Wildland fire emissions cause adverse cardiopulmonary outcomes, yet controlled exposure studies to characterize health impacts of specific biomass sources have been complicated by the often latent effects of air pollution. The aim of this study was to determine if postprandial responses after a high fat challenge, long used clinically to predict cardiovascular risk, would unmask latent cardiometabolic responses in rats exposed to peat smoke, a key wildland fire air pollution source. Male Wistar Kyoto rats were exposed once (1 h) to filtered air (FA), or low (0.36 mg/m3 particulate matter) or high concentrations (3.30 mg/m3) of peat smoke, generated by burning peat from an Irish bog. Rats were then fasted overnight, and then administered an oral gavage of a HF suspension (60 kcal% from fat), mimicking a HF meal, 24 h post-exposure. In one cohort, cardiac and superior mesenteric artery function were assessed using high frequency ultrasound 2 h post gavage. In a second cohort, circulating lipids and hormones, pulmonary and systemic inflammatory markers, and circulating monocyte phenotype using flow cytometry were assessed before or 2 or 6 h after gavage. HF gavage alone elicited increases in circulating lipids characteristic of postprandial responses to a HF meal. Few effects were evident after peat exposure in un-gavaged rats. By contrast, exposure to low or high peat caused several changes relative to FA-exposed rats 2 and 6 h post HF gavage including increased heart isovolumic relaxation time, decreased serum glucose and insulin, increased CD11 b/c-expressing blood monocytes, increased serum total cholesterol, alpha-1 acid glycoprotein, and alpha-2 macroglobulin (p = 0.063), decreased serum corticosterone, and increased lung gamma-glutamyl transferase. In summary, these findings demonstrate that a HF challenge reveals effects of air pollution that may otherwise be imperceptible, particularly at low exposure levels, and suggest exposure may sensitize the body to mild inflammatory triggers.