Many efficacious cancer treatments cause significant cardiac morbidity, yet biomarkers or functional indices of early damage, which would allow monitoring and intervention, are lacking. In this study, we have utilized a rat model of progressive doxorubicin (DOX)-induced cardiomyopathy, applying multiple approaches, including cardiac magnetic resonance imaging (MRI), to provide the most comprehensive characterization to date of the timecourse of serological, pathological, and functional events underlying this toxicity. Hannover Wistar rats were dosed with 1.25 mg/kg DOX weekly for 8 weeks followed by a 4 week off-dosing "recovery" period. Electron microscopy of the myocardium revealed subcellular degeneration and marked mitochondrial changes after a single dose. Histopathological analysis revealed progressive cardiomyocyte degeneration, hypertrophy/cytomegaly, and extensive vacuolation after two doses. Extensive replacement fibrosis (quantified by Sirius red staining) developed during the off-dosing period. Functional indices assessed by cardiac MRI (including left ventricular ejection fraction (LVEF), cardiac output, and E/A ratio) declined progressively, reaching statistical significance after two doses and culminating in "clinical" LV dysfunction by 12 weeks. Significant increases in peak myocardial contrast enhancement and serological cardiac troponin I (cTnI) emerged after eight doses, importantly preceding the LVEF decline to <50%. Troponin I levels positively correlated with delayed and peak gadolinium contrast enhancement, histopathological grading, and diastolic dysfunction. In summary, subcellular cardiomyocyte degeneration was the earliest marker, followed by progressive functional decline and histopathological manifestations. Myocardial contrast enhancement and elevations in cTnI occurred later. However, all indices predated "clinical" LV dysfunction and thus warrant further evaluation as predictive biomarkers.
Until now, there has been limited information on the effects of smoking on atherogenesis and senescence in the context of lipoprotein parameters, particularly in young smokers who have smoked fewer than 10 cigarettes per day for 3 years. In this study, lipoprotein profiles and functions were compared between smoker (n = 21) and control groups (n = 20). In the smoking group, ferric ion reduction abilities of serum and high-density lipoprotein (HDL) fractions were significantly reduced, and low-density lipoprotein (LDL) was severely oxidized. All lipoprotein particles from the smoker group showed higher advanced glycated end products with more triglyceride (TG) content compared with the control group. Lipoproteins from smokers showed faster agarose gel electromobility as well as greater smear band intensity in SDS-PAGE due to oxidation and glycation. LDL from smokers was more sensitive to oxidation and promoted foam cell formation in macrophages. Gel filtration column chromatography revealed that the protein and cholesterol peaks of VLDL and LDL were elevated in the smoker group, whereas those of HDL were reduced. Human dermal fibroblast cells from the smoker group showed severe senescence following treatment with HDL2 and HDL3. Although HDL from young smokers showed impaired antioxidant ability, smaller particle size, and increased TG content, cholesteryl ester transfer protein activities were greatly enhanced in the serum and HDL fractions of the smoker group. In conclusion, smoking can cause production of dysfunctional lipoproteins having a smaller particle size that exacerbate senescence and atherogenic progress due to oxidation and glycation.
There is a growing body of evidence that exposure to endocrine disrupting chemicals and to estrogenic compounds in particular can affect the testis and male fertility. In the present study, the constitutive expression of steroidogenic and non-steroidogenic cytochrome P450 (CYP) and related enzymes in adult rat testis, and their regulation by estradiol and bisphenol A, were investigated. CYP1B1, CYP2A1, NADPH-cytochrome P450 oxidoreductase (POR) and microsomal epoxide hydrolase (mEH) proteins, together with CYP17A1 and 3β-hydroxysteroid dehydrogenase (HSD3B), were detected by immunoblot analysis in testicular microsomes prepared from untreated adult Sprague Dawley rats. In contrast, CYP1A, CYP2B, CYP2E, CYP2D, CYP2C, CYP3A, and CYP4A enzymes were not detected. Immunofluorescence staining of cryosections of perfusion-fixed testes showed that CYP1B1, CYP2A1, CYP17A1, and HSD3B were expressed exclusively or mainly in interstitial cells, whereas mEH and POR protein staining was detected both in interstitial cells and in seminiferous tubules. Testicular CYP1B1 and CYP2A1 protein levels were decreased following treatment of adult rats with estradiol benzoate at 0.004, 0.04, 0.4, or 4 μmol/kg/day or bisphenol A at 400 or 800 μmol/kg/day, for 14 days, whereas expression of HSD3B was unaffected. Testicular CYP17A1, POR, and mEH protein expression was also downregulated at the three highest dosages of estradiol benzoate and at both dosages of bisphenol A. The present study is the first to establish the cellular localization of CYP1B1, mEH, and POR in rat testis and to demonstrate the suppressive effect of bisphenol A on testicular CYP1B1, CYP2A1, mEH, and POR protein levels.
Organophosphorus pesticides are the most widely used pesticides in modern agricultural systems to ensure good harvests. Isocarbophos (ICP), with a potent acetylcholinesterase inhibitory effect is widely utilized to control a variety of leaf-eating and soil insects. However, the characteristics of the bioactivation and detoxification of ICP in humans remain unclear. In this study, the oxidative metabolism, esterase hydrolysis, and chiral inversion of ICP in human liver microsomes (HLMs) were investigated with the aid of a stereoselective LC/MS/MS method. The depletion of ICP in HLMs was faster in the absence of carboxylesterase inhibitor (BNPP) than in the presence of NADPH and BNPP, with t1/2 of 5.2 and 90 min, respectively. Carboxylesterase was found to be responsible for the hydrolysis of ICP, the major metabolic pathway. CYP3A4, CYP1A2, CYP2D6, CYP2C9, and CYP2C19 were all involved in the secondary metabolism pathway of desulfuration of ICP. Flavin-containing monooxygenase (FMO) did not contribute to the clearance of ICP. The hydrolysis and desulfuration of (±)ICP, (+)ICP, and (–)ICP in HLMs follow Michaelis-Menten kinetics. Individual enantiomers of ICP and its oxidative desulfuration metabolite isocarbophos oxon (ICPO) were found to be inhibitors of acetylcholinesterases at different extents. For example, (±)ICPO is more potent than ICP (IC50 0.031μM vs. 192μM), whereas (+)ICPO is more potent than (–)ICPO (IC50 0.017μM vs. 1.55μM). Given the finding of rapid hydrolysis of ICP and low abundance of oxidative metabolites presence in human liver, the current study highlights that human liver has a greater capacity for detoxification of ICP.
Chlorpyrifos is a pesticide that is metabolically activated to chlorpyrifos oxon (acetylcholinesterase inhibitor) primarily by the cytochrome P450 2B (CYP2B) enzyme subfamily in the liver and brain. We have previously shown that intracerebroventricular pretreatment with a CYP2B inhibitor, C8-Xanthate, can block chlorpyrifos toxicity. Here, we assessed whether delayed introduction of C8-Xanthate would still reduce toxicity and whether peripheral administration of C8-Xanthate could also inhibit chlorpyrifos activation in the brain and block toxicity. Male rats (N = 4–5/group) were either pretreated with C8-Xanthate (40 μg intracerebroventricular or 5 mg/kg intraperitoneal), or vehicle (ACSF or saline, respectively), 24 h before chlorpyrifos treatment (125 mg/kg subcutaneous) and then treated daily with inhibitor or vehicle until 7 days post-chlorpyrifos treatment. Additional groups received vehicle pretreatment, switching to C8-Xanthate 1, 2, 3, or 4 days after chlorpyrifos and then continuing with daily C8-Xanthate treatment until 7 days post-chlorpyrifos treatment. Neurotoxicity was assessed at baseline (before chlorpyrifos) and then daily after chlorpyrifos, using behavioral assessments (e.g., gait score). Neurochemical assays (e.g., serum and brain chlorpyrifos) were performed at the end of study. Pretreatment with C8-Xanthate completely prevented chlorpyrifos toxicity, and delayed introduction of C8-Xanthate reduced toxicity, even when started up to 4 days after chlorpyrifos treatment. Discontinuation of C8-Xanthate treatment 7 days post-chlorpyrifos treatment did not result in the reappearance of toxicity, tested through 10 days after chlorpyrifos treatment. These findings suggest that CYP2B inhibitor treatment, even days after chlorpyrifos exposure, and using a peripheral delivery route, may be useful as a therapeutic approach to reduce chlorpyrifos toxicity.
Many studies have reported associations between air pollution particles with an aerodynamic diameter <2.5 μm (fine particulate matter (PM)) and adverse cardiovascular effects. However, there is an increased concern that so-called ultrafine PM which comprises the smallest fraction of fine PM (aerodynamic diameter <0.1 μm) may be disproportionately toxic relative to the 0.1–2.5 μm fraction. Ultrafine PM is not routinely measured in state monitoring networks and is not homogenously dispersed throughout an airshed but rather located in hot spots such as near combustion sources (e.g., roads), making it difficult for epidemiology studies to associate exposure to ultrafine PM with adverse health effects. Thirty four middle-aged individuals with metabolic syndrome were exposed for 2 h while at rest in a randomized crossover design to clean air and concentrated ambient ultrafine particles (UCAPS) for 2 h. To further define potential risk, study individuals carrying the null allele for GSTM1 (a prominent antioxidant gene) were identified by genotyping. Blood was obtained immediately prior to exposure, and at 1 and 20 h afterward. Continuous Holter monitoring began immediately prior to exposure and continued for 24 h. Based on changes we observed in previous CAPS studies, we hypothesized that ultrafine CAPS would cause changes in markers of blood inflammation and fibrinolysis as well as changes in heart rate variability and cardiac repolarization. GSTM1 null individuals had altered cardiac repolarization as seen by a change in QRS complexity following exposure to UCAPS and both the entire study population as well as GSTM1 null individuals had increased QT duration. Blood plasminogen and thrombomodulin were decreased in the whole population following UCAPS exposure, whereas C-reactive protein (CRP) and SAA were increased. This controlled human exposure study is the first to show that ambient ultrafine particles can cause cardiovascular changes in people with metabolic syndrome, which affects nearly a quarter of the U.S. adult population.
Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that has been implicated in modulating aromatase enzyme function with the potential to interrupt normal reproductive function. The aim of this study was to use a fish model, Fundulus heteroclitus, to assess whether BaP exposure adversely impacts reproduction. Adult fish were exposed to waterborne BaP nominal concentrations of (0, 1, or 10 μg/l) for 28 days. Males and females were combined for the second half of the exposure (days 14–28) in order to quantitate egg production and fertilization success. Egg fertilization and subsequent hatching success of F1 embryos was significantly decreased by the high dose of BaP. In males, both gonad weight and plasma testosterone concentrations were significantly reduced compared to controls by 10 μg/l BaP. Histopathological examination of testes including spermatogonia, spermatocyte and spermatid cyst areas, percentage of cysts per phase, and area of spermatozoa per seminiferous tubule were not significantly affected. Other biomarkers, including male liver weight, liver vitellogenin (vtg) mRNA expression and sperm concentrations, were also not affected. In females, estradiol concentrations were significantly reduced after BaP exposure, but egg production, gonad weight, liver weight, vtg expression and oocyte maturation were not altered. Steroid concentrations in Fundulus larvae from exposed parents at 1 and 3 weeks posthatch were not significantly changed. BaP exposure at these environmentally relevant concentrations caused negative alterations particularly in male fish to both biochemical and phenotypic biomarkers associated with reproduction and multigenerational embryo survival.
Air pollution exposure is associated with cardiovascular events triggered by clot formation. Endothelial activation and initiation of coagulation are pathophysiological mechanisms that could link inhaled air pollutants to vascular events. Here we investigated the underlying mechanisms of increased endothelial cell procoagulant activity following exposure to soluble components of ultrafine particles (soluble UF). Human coronary artery endothelial cells (HCAEC) were exposed to soluble UF and assessed for their ability to trigger procoagulant activity in platelet-free plasma. Exposed HCAEC triggered earlier thrombin generation and faster fibrin clot formation, which was abolished by an anti-tissue factor (TF) antibody, indicating TF-dependent effects. Soluble UF exposure increased TF mRNA expression without compensatory increases in key anticoagulant proteins. To identify early events that regulate TF expression, we measured endothelial H2O2 production following soluble UF exposure and identified the enzymatic source. Soluble UF exposure increased endothelial H2O2 production, and antioxidants attenuated UF-induced upregulation of TF, linking the procoagulant responses to reactive oxygen species (ROS) formation. Chemical inhibitors and RNA silencing showed that NOX-4, an important endothelial source of H2O2, was involved in UF-induced upregulation of TF mRNA. These data indicate that soluble UF exposure induces endothelial cell procoagulant activity, which involves de novo TF synthesis, ROS production, and the NOX-4 enzyme. These findings provide mechanistic insight into the adverse cardiovascular effects associated with air pollution exposure.
The p53 tumor suppressor protein plays an essential role in cellular integrity and inactivation of the TP53 gene by mutation is the most frequent alteration in human cancer. As loss of p53 function is associated with increased genetic instability, it is important in genotoxicity testing to explore the role of p53 competency. In vitro model systems for genotoxicity testing are sometimes prone to misleading positive results; some of this loss of predictivity may be caused by p53 inactivation in some cell models. To explore whether impaired p53 function plays a role in mutation sensitivity, TK6 cells (p53 competent) and NH32 cells (p53 deficient) were treated with two known genotoxicants, mitomycin C (MMC) and cytosine arabinoside (araC). Chromosomal damage was assessed in the low dose region by an automated micronucleus system and p53 activity was investigated by gene and protein expression analysis. Cell cycle progression studies were also assessed. Low levels of micronucleus and p53 induction were observed in TK6 cells treated with MMC. On the other hand, higher levels of micronucleus and p53 induction were shown in TK6 cells treated with araC and a G1/S arrest was observed after araC treatment. p53 deficient NH32 cells showed an increased sensitivity of micronucleus (MN) induction after araC treatment compared with TK6 cells and less of an active G1/S phase checkpoint. Thus, impaired p53 function sensitizes cells to genotoxicants and plays a central role in the DNA damage response. This data has clear importance for safety assessment of genotoxicity and shows how crucial p53 competence is.
DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, H2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objective of this study was to assess if H2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified H2AX foci manually and automatically. In addition, total H2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. We show that (1) all genotoxic agents induced H2AX dose-dependently whereas nongenotoxic agents do not; (2) H2AX was observed for genotoxicants in the cytotoxic dose range, revealing a correlation between cytotoxicity and H2AX for genotoxic agents; for nongenotoxic agents cytotoxicity was not related to H2AX; (3) manual scoring of H2AX and automated scoring provided comparable results, the automated scoring was faster and investigator independent; (4) data obtained by foci counting and flow cytometry showed a high correlation, suggesting that H2AX scoring by flow cytometry has the potential for high-throughput analysis. However, the microscopic evaluation can provide additional information as to foci size, distribution, colocalization and background staining; (5) H2AX foci were colocalized with 53BP1 and Rad51, supporting the notion that they represent true DSBs. Collectively, the automated analysis of H2AX foci allows for rapid determination of genetic damage in mammalian cells. The data revealed that the induction of H2AX by genotoxicants is related to loss of viability and support H2AX as a reliable bio-indicator for pretoxic DNA damage.
The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia/reperfusion injury, and many other pathological conditions. DNA strand breaks caused by ROS lead to the activation of poly(ADP-ribose)polymerase-1 (PARP-1), the excessive activation of which can result in cell death. We have utilized a model in which 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes ROS-dependent cell death in human renal proximal tubule epithelial cells (HK-2), to further elucidate the role of PARP-1 in ROS-dependent cell death. TGHQ-induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca2+ concentrations, and subsequent nonapoptotic cell death in both a PARP- and Ca2+-dependent manner. Thus, inhibition of PARP-1 with PJ34 completely blocked TGHQ-mediated accumulation of poly(ADP-ribose) polymers and NAD consumption, and delayed HK-2 cell death. In contrast, chelation of intracellular Ca2+ with BAPTA completely abrogated TGHQ-induced cell death. Ca2+ chelation also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca2+ homeostasis. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP-dependent cell death. Thus, pathways coupling PARP-1 hyperactivation to cell death are likely to be context-dependent, and therapeutic strategies designed to target PARP-1 need to recognize such variability. Our studies provide new insights into PARP-1-mediated nonapoptotic cell death, during which PARP-1 hyperactivation and elevations in intracellular Ca2+ are reciprocally coupled to amplify ROS-induced nonapoptotic cell death.
We set out to better understand the signal transduction pathways that mediate liver tumor promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxn ("dioxin"). To this end, we first employed congenic mice homozygous for either the Ahrb1 or Ahrd alleles (encoding an aryl hydrocarbon receptor (AHR) with high or low binding affinity for dioxin, respectively) and demonstrated that hepatocellular tumor promotion in response to dioxin segregated with the Ahr locus. Once we had genetic evidence for the importance of AHR signaling, we then asked if tumor promotion by dioxin was influenced by "interleukin-1 (IL-1)-like" inflammatory cytokines. The importance of this question arose from our earlier observation that aspects of the acute hepatocellular toxicity of dioxin are dependent upon IL1-like cytokine signaling. To address this issue, we employed a triple knock-out (TKO) mouse model with null alleles at the loci encoding the three relevant receptors for tumor necrosis factors α and β and IL-1α and IL-1β (i.e., null alleles at the Tnfrsf1a, Tnfrsf1b, and Il-1r1 loci). The observation that TKO mice were resistant to the tumor promoting effects of dioxin in liver suggests that inflammatory cytokines play an important step in dioxin mediated liver tumor promotion in the mouse. Collectively, these data support the idea that the mechanism of dioxin acute hepatotoxicity and its activity as a promoter in a mouse two stage liver cancer model may be similar, i.e., tumor promotion by dioxin, like acute hepatotoxicity, are mediated by the linked action of two receptor systems, the AHR and the receptors for the "IL-1-like" cytokines.
Drug-induced liver injury (DILI) is an important clinical problem. Here, we used a genomics approach to in detail investigate the hypothesis that critical drug-induced toxicity pathways act in synergy with the pro-inflammatory cytokine tumor necrosis factor α (TNFα) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ~80 DILI compounds in primary human hepatocytes. Compounds displaying weak or no TNFα synergism, namely ketoconazole, nefazodone, and methotrexate, failed to synchronously induce both pathways. The ER stress induced was primarily related to protein kinase R-like ER kinase (PERK) and activating transcription factor 4 (ATF4) activation and subsequent expression of C/EBP homologous protein (CHOP), which was all independent of TNFα signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision-cut human liver slices. Targeted RNA interference studies revealed that whereas ER stress signaling through inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6) acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNFα-induced apoptosis. Whereas inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNFα cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNFα-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing toward caspase-8-dependent TNFα-induced apoptosis.
The brain appears to be a target of air pollution. This study aimed to further ascertain behavioral and neurobiological mechanisms of our previously observed preference for immediate reward (Allen, J. L., Conrad, K., Oberdorster, G., Johnston, C. J., Sleezer, B., and Cory-Slechta, D. A. (2013). Developmental exposure to concentrated ambient particles and preference for immediate reward in mice. Environ. Health Perspect. 121, 32–38), a phenotype consistent with impulsivity, in mice developmentally exposed to inhaled ultrafine particles. It examined the impact of postnatal and/or adult concentrated ambient ultrafine particles (CAPS) or filtered air on another behavior thought to reflect impulsivity, Fixed interval (FI) schedule-controlled performance, and extended the assessment to learning/memory (novel object recognition (NOR)), and locomotor activity to assist in understanding behavioral mechanisms of action. In addition, levels of brain monoamines and amino acids, and markers of glial presence and activation (GFAP, IBA-1) were assessed in mesocorticolimbic brain regions mediating these cognitive functions. This design produced four treatment groups/sex of postnatal/adult exposure: Air/Air, Air/CAPS, CAPS/Air, and CAPS/CAPS. FI performance was adversely influenced by CAPS/Air in males, but by Air/CAPS in females, effects that appeared to reflect corresponding changes in brain mesocorticolimbic dopamine/glutamate systems that mediate FI performance. Both sexes showed impaired short-term memory on the NOR. Mechanistically, cortical and hippocampal changes in amino acids raised the potential for excitotoxicity, and persistent glial activation was seen in frontal cortex and corpus callosum of both sexes. Collectively, neurodevelopment and/or adulthood CAPS can produce enduring and sex-dependent neurotoxicity. Although mechanisms of these effects remain to be fully elucidated, findings suggest that neurodevelopment and/or adulthood air pollution exposure may represent a significant underexplored risk factor for central nervous system diseases/disorders and thus a significant public health threat even beyond current appreciation.
Parkinson's disease (PD) is the second most common neurodegenerative disease. Much data has linked the etiology of PD to a variety of environmental factors. The majority of cases are thought to arise from a combination of genetic susceptibility and environmental factors. Chronic exposures to dietary factors, including meat, have been identified as potential risk factors. Although heterocyclic amines that are produced during high-temperature meat cooking are known to be carcinogenic, their effect on the nervous system has yet to be studied in depth. In this study, we investigated neurotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a highly abundant heterocyclic amine in cooked meat, in vitro. We tested toxicity of PhIP and the two major phase I metabolites, N-OH-PhIP and 4'-OH-PhIP, using primary mesencephalic cultures from rat embryos. This culture system contains both dopaminergic and nondopaminergic neurons, which allows specificity of neurotoxicity to be readily examined. We find that exposure to PhIP or N-OH-PhIP is selectively toxic to dopaminergic neurons in primary cultures, resulting in a decreased percentage of dopaminergic neurons. Neurite length is decreased in surviving dopaminergic neurons. Exposure to 4'-OH-PhIP did not produce significant neurotoxicity. PhIP treatment also increased formation of oxidative damage markers, 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine in dopaminergic neurons. Pretreatment with N-acetylcysteine was protective. Finally, treatment with blueberry extract, a dietary factor with known antioxidant and other protective mechanisms, prevented PhIP-induced toxicity. Collectively, our study suggests, for the first time, that PhIP is selectively toxic to dopaminergic neurons likely through inducing oxidative stress.
Concerns have been raised regarding the long-term impacts of early life exposure to the ubiquitous environmental contaminant bisphenol A (BPA) on brain organization. Because BPA has been reported to affect estrogen signaling, and steroid hormones play a critical role in brain sexual differentiation, there is also concern that BPA exposure could alter neural sex differences. Here, we examine the impact of subchronic exposure from gestation to adulthood to oral doses of BPA below the current no-observed-adverse-effect level (NOAEL) of 5 mg/kg body weight (bw)/day on estrogen receptor (ESR) expression in sexually dimorphic brain regions of prepubertal and adult female rats. The dams were gavaged daily with vehicle (0.3% carboxymethylcellulose), 2.5, 25, 260, or 2700 μg BPA/kg bw/day, or 0.5 or 5.0 μg ethinyl estradiol (EE)/kg bw/day from gestational day 6 until labor began. Offspring were then gavaged directly from the day after birth until the day before scheduled sacrifice on postnatal days 21 or 90. Using in situ hybridization, one or more BPA doses produced significant decreases in Esr1 expression in the juvenile female rat anteroventral periventricular nucleus (AVPV) of the hypothalamus and significant decreases in Esr2 expression in the adult female rat AVPV and medial preoptic area (MPOA), relative to vehicle controls. BPA did not simply reproduce EE effects, indicating that BPA is not acting solely as an estrogen mimic. The possible consequences of long-term changes in hypothalamic ESR expression resulting from subchronic low dose BPA exposure on neuroendocrine effects are discussed and being addressed in ongoing, related work.
Cigarette smoke is associated with chronic and enhanced pulmonary inflammation characterized by increased cytokine production and leukocyte recruitment to the lung. Although the aryl hydrocarbon receptor (AhR) is well-known to mediate toxic effects of manmade environmental contaminants, the AhR has emerged as a suppressor of acute cigarette smoke-induced neutrophilia by a mechanism involving the NF-B protein RelB. Yet individuals who smoke often smoke for many years and vary in their cigarette consumption. As there is currently no information on the AhR prevention of lung inflammation, including neutrophilia, due to varied and prolonged exposure regimes, we exposed control and AhR–/– mice to cigarette smoke for 2 weeks (subchronic exposure) utilizing low and high exposure protocols and evaluated pulmonary inflammation. Subchronic cigarette smoke exposure significantly increased pulmonary neutrophilia dose-dependently in AhR–/– mice. Surprisingly, there was no difference between smoke-exposed AhR+/– and AhR–/– mice in the expression of cytokines associated with neutrophil recruitment. Expression of pulmonary intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule involved in neutrophil migration and retention, was higher in pulmonary endothelial cells from AhR–/– mice. Although total lung RelB expression was increased by cigarette smoke, nuclear RelB was significantly lower in subchronically exposed AhR–/– mice. Inhibition of AhR activity by CH-223191 in endothelial cells potentiated ICAM-1 expression and prevented RelB nuclear translocation but had no effect on neutrophil adhesion. These data support that genetic absence of the AhR contributes to heightened pulmonary neutrophilia in response to ongoing cigarette smoke exposure. Interindividual variations in AhR expression may enhance the susceptibility to cigarette smoke-induced diseases.
Cross-linking of proteins has been exploited by the food industry to change food texture and functionality but the effects of these manipulations on food allergenicity still remain unclear. To model the safety assessment of these food biopolymers, we created cross-linked bovine β-lactoglobulin (CL-BLG) by laccase treatment. The purpose of the present study was to compare the immunogenicity and allergenicity of CL-BLG with native BLG in a mouse model of food allergy. First, BALB/c mice were intragastrically sensitized and orally challenged with BLG or CL-BLG and BLG-specific serum antibodies and splenic leukocyte cytokine production and cell proliferation were measured. Hereafter, epithelial protein uptake was monitored in vitro and in vivo and the effects of BLG cross-linking on interactions with dendritic cells were analyzed in vitro. Sensitization of mice with CL-BLG resulted in higher levels of IgE, IgG1, and IgG2a. In contrast, a subsequent oral challenge with CL-BLG resulted in lower mast cell degranulation. Cross-linking of BLG reduced its epithelial uptake but promoted sampling through Peyer's patches. Differences in endocytosis by dendritic cells (DCs) and in vitro endolysosomal processing were observed between BLG and CL-BLG. CL-BLG primed DCs induced higher Th2 response in vitro. Cross-linking of BLG increased its sensitizing capacity, implying that the assessment of highly polymerized food proteins is of clinical importance in food allergy. Moreover, manufacturers of foods or therapeutic proteins should pay considerate attention to the health risk of protein aggregation.
Early phase drug development relies on primary human hepatocytes for studies of drug metabolism, cytotoxicity, and drug-drug interactions. However, primary human hepatocytes rapidly lose metabolic functions ex vivo and are refractory to expansion in culture and thus are limited in quantity. Hepatocytes derived from human pluripotent stem cells (either embryonic stem (ES) or induced pluripotent stem (iPS) cells), have the potential to overcome many of the limitations of primary human hepatocytes, but to date the use of human pluripotent stem cell-derived hepatocytes has been limited by poor enzyme inducibility and immature metabolic function. Here, we present a simple suspension culture of aggregates of ES cell-derived hepatocytes that compared to conventional monolayer adherent culture significantly increases induction of CYP 1A2 by omeprazole and 3A4 by rifampicin. Using liquid chromatography-tandem mass spectrometry, we further show that ES cell-derived hepatocytes in aggregate culture convert omeprazole and rifampicin to their human-specific metabolites. We also show that these cells convert acetaminophen (APAP) to its cytotoxic metabolite (N-acetyl-p-benzoquinone imine (NAPQI)), although they fail to perform APAP glucuronidation. In summary, we show that human pluripotent stem cell-derived hepatocytes in aggregate culture display improved enzymatic inducibility and metabolic function and is a promising step toward a simple, scalable system, but nonetheless will require further improvements to completely replace primary human hepatocytes in drug development.