Cigarette smoking significantly alters sperm DNA methylation patterns.

Numerous health consequences of tobacco smoke exposure have been characterized, and the effects of smoking on traditional measures of male fertility are well described. However, a growing body of data indicates that pre-conception paternal smoking also confers increased risk for a number of morbidities on offspring. The mechanism for this increased risk has not been elucidated, but it is likely mediated, at least in part, through epigenetic modifications transmitted through spermatozoa. In this study, we investigated the impact of cigarette smoke exposure on sperm DNA methylation patterns in 78 men who smoke and 78 never-smokers using the Infinium Human Methylation 450 beadchip. We investigated two models of DNA methylation alterations: (i) consistently altered methylation at specific CpGs or within specific genomic regions and (ii) stochastic DNA methylation alterations manifest as increased variability in genome-wide methylation patterns in men who smoke. We identified 141 significantly differentially methylated CpGs associated with smoking. In addition, we identified a trend toward increased variance in methylation patterns genome-wide in sperm DNA from men who smoke compared with never-smokers. These findings of widespread DNA methylation alterations are consistent with the broad range of offspring heath disparities associated with pre-conception paternal smoke exposure and warrant further investigation to identify the specific mechanism by which sperm DNA methylation perturbation confers risk to offspring health and whether these changes can be transmitted to offspring and transgenerationally.

The role of NADPH oxidase in carotid body arterial chemoreceptors.

O(2)-sensing in the carotid body occurs in neuroectoderm-derived type I glomus cells where hypoxia elicits a complex chemotransduction cascade involving membrane depolarization, Ca(2+) entry and the release of excitatory neurotransmitters. Efforts to understand the exquisite O(2)-sensitivity of these cells currently focus on the coupling between local P(O2) and the open-closed state of K(+)-channels. Amongst multiple competing hypotheses is the notion that K(+)-channel activity is mediated by a phagocytic-like multisubunit enzyme, NADPH oxidase, which produces reactive oxygen species (ROS) in proportion to the prevailing P(O2). In O(2)-sensitive cells of lung neuroepithelial bodies (NEB), multiple studies confirm that ROS levels decrease in hypoxia, and that E(M) and K(+)-channel activity are indeed controlled by ROS produced by NADPH oxidase. However, recent studies in our laboratories suggest that ROS generated by a non-phagocyte isoform of the oxidase are important contributors to chemotransduction, but that their role in type I cells differs fundamentally from the mechanism utilized by NEB chemoreceptors. Data indicate that in response to hypoxia, NADPH oxidase activity is increased in type I cells, and further, that increased ROS levels generated in response to low-O(2) facilitate cell repolarization via specific subsets of K(+)-channels.

Effect of p47phox gene deletion on ROS production and oxygen sensing in mouse carotid body chemoreceptor cells.

Membrane potential in oxygen-sensitive type I cells in carotid body is controlled by diverse sets of voltage-dependent and -independent K(+) channels. Coupling of Po(2) to the open-closed state of channels may involve production of reactive oxygen species (ROS) by NADPH oxidase. One hypothesis suggests that ROS are produced in proportion to the prevailing Po(2) and a subset of K(+) channels closes as ROS levels decrease. We evaluated ROS levels in normal and p47(phox) gene-deleted [NADPH oxidase knockout (KO)] type I cells using the ROS-sensitive dye dihydroethidium (DHE). In normal cells, hypoxia elicited an increase in ROS, which was blocked by the specific NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, 3 mM). KO type I cells did not respond to hypoxia, but the mitochondrial uncoupler azide (5 microM) elicited increased fluorescence in both normal and KO cells. Hypoxia had no effect on ROS production in sensory and sympathetic neurons. Methodological control experiments showed that stimulation of neutrophils with a cocktail containing the chemotactic peptide N-formyl-Met-Leu-Phe (1 microM), arachidonic acid (10 microM), and cytochalasin B (5 microg/ml) elicited a rapid increase in DHE fluorescence. This response was blocked by the NADPH oxidase inhibitor diphenyleneiodonium (10 microM). KO neutrophils did not respond; however, azide (5 microM) elicited a rapid increase in fluorescence. Physiological studies in type I cells demonstrated that hypoxia evoked an enhanced depression of K+ current and increased intracellular Ca2+ levels in KO vs. normal cells. Moreover, AEBSF potentiated hypoxia-induced increases in intracellular Ca2+ and enhanced the depression of K+ current in low O(2). Our findings suggest that local compartmental increases in oxidase activity and ROS production inhibit the activity of type I cells by facilitating K+ channel activity in hypoxia.

Polymorphisms in the 5'-UTR of the tissue factor gene are associated with altered expression in human endothelial cells.

BACKGROUND: Enhanced tissue factor (TF) expression mediates many disease processes. Recently, four completely concordant polymorphisms were detected in the 5'-UTR of the TF gene. Three were single base changes and one was an 18-bp insertion/deletion at -1208. OBJECTIVES: This study was undertaken to determine if the I-allele or the D-allele would associate with elevated TF expression in human umbilical vein endothelial cells (HUVEC). METHODS: HUVEC were genotyped by polymerase chain reaction for 18-bp insert status. TF expression was induced by interleukin (IL)-1 or phorbol 12-myristate 13-acetate (PMA). Total TF activity was determined by a one-stage clotting assay and surface TF activity by a chromogenic assay. Protein binding differences between the I- and D-alleles were examined by gel shift assays. RESULTS: IL-1- or PMA-induced total TF activity in D-allele HUVEC was increased 2.0-2.5-fold above that seen in II HUVEC. Surface clotting activity in D-allele cells was 1.3-1.7-fold greater than in II-allele cultures. Experiments with consensus site mutation oligos suggested that the 18-bp insert creates GATA and CCAAT-enhancer binding protein (C/EBP) transcription factor recognition sites. CONCLUSIONS: The D-allele is associated with enhanced TF activity in HUVEC. The differences in TF expression between the alleles may be due to variant transcription factor binding in the -1208 region. Further studies are warranted to investigate whether the D-allele is associated with increased incidence of pathological processes that involve TF.

Redox signaling of NF-kappaB by membrane NAD(P)H oxidases in normal and malignant cells.

Evidence is rapidly accumulating that low-activity NAD(P)H oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates. In this review we discuss evidence that signaling NAD(P)H oxidases in part influence normal and malignant cell division by activating the redox-regulated transcription factor nuclear factor kappaB. The roles of growth-regulatory NAD(P)H oxidases in human airway smooth muscle and malignant melanoma are used as examples.

Improved method for measurement of human plasma xanthine oxidoreductase activity.

The XOR activity in human plasma was measured by quantifying the XOR-derived uric acid (UA) in plasma using the high-performance liquid chromatography (HPLC) equipped with a UV detector. Chromatographic separation consisted of the mobile phase (a mixture of 0.1% trifluoroacetic acid in Milli-Q water and 0.085% trifluoroacetic acid in acetonitrile in a mix ratio of 99:1) running through a Zorbax StableBond SB-C(18) column at a flow-rate of 1 ml/min. Deproteinization with heat-treatment of plasma samples after the reaction was used in the assay to avoid splitting of the UA and xanthine peaks caused by acid deproteinization that could interfere the accurate determination of human plasma XOR activity in our case. Based on the examination of the dependence of XOR activity on added amounts of xanthine and reaction times, the amount of xanthine and reaction time for XOR activity assay were determined to prevent the errors caused by the limiting effect of substrates and plateau phase of the reaction. Using this method, human plasma XOR activities of 25 healthy people were measured. The average human plasma XOR activity was 2.1+/-0.8 (x10(-3) U/ml).

Role of components of the phagocytic NADPH oxidase in oxygen sensing.

It has been hypothesized that O(2) sensing in type I cells of the carotid body and erythropoietin (EPO)-producing cells of the kidney involves protein components identical to the NADPH oxidase system responsible for the respiratory burst of phagocytes. In the present study, we evaluated O(2) sensing in mice with null mutant genotypes for two components of the phagocytic oxidase. Whole body plethysmography was used to study unanesthetized, unrestrained mice. When exposed to an acute hypoxic stimulus, gp91(phox)-null mutant and wild-type mice increased their minute ventilation by similar amounts. In contrast, p47(phox)-null mutant mice demonstrated increases in minute ventilation in response to hypoxia that exceeded that of their wild-type counterparts: 98.0 +/- 18.0 vs. 20.0 +/- 13.0% (n = 11, P = 0.003). In vitro recordings of carotid sinus nerve (CSN) activity demonstrated that resting (basal) neural activity was marginally elevated in p47(phox)-null mutant mice. With hypoxic challenge, mean CSN discharge was 1.5-fold greater in p47(phox)-null mutant than in wild-type mice: 109.61 +/- 13.29 vs. 72.54 +/- 7.65 impulses/s (n = 8 and 7, respectively, P = 0.026). Consequently, the hypoxia-evoked CSN discharge (stimulus-basal) was approximately 58% larger in p47(phox)-null mutant mice. Quantities of EPO mRNA in kidney were similar in gp91(phox)- and p47(phox)-null mutant mice and their respective wild-type controls exposed to hypobaric hypoxia for 72 h. These findings confirm the previous observation that absence of the gp91(phox) component of the phagocytic NADPH oxidase does not alter the O(2)-sensing mechanism of the carotid body. However, absence of the p47(phox) component significantly potentiates ventilatory and chemoreceptor responses to hypoxia. O(2) sensing in EPO-producing cells of the kidney appears to be independent of the gp91(phox) and p47(phox) components of the phagocytic NADPH oxidase.

Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice.

Various heme-containing proteins have been proposed as primary molecular O(2) sensors for hypoxia-sensitive type I cells in the mammalian carotid body. One set of data in particular supports the involvement of a cytochrome b NADPH oxidase that is commonly found in neutrophils. Subunits of this enzyme have been immunocytochemically localized in type I cells, and diphenyleneiodonium, an inhibitor of the oxidase, increases carotid body chemoreceptor activity. The present study evaluated immunocytochemical and functional properties of carotid bodies from normal mice and from mice with a disrupted gp91 phagocytic oxidase (gp91(phox)) DNA sequence gene knockout (KO), a gene that codes for a subunit of the neutrophilic form of NADPH oxidase. Immunostaining for tyrosine hydroxylase, a signature marker antigen for type I cells, was found in groups or lobules of cells displaying morphological features typical of the O(2)-sensitive cells in other species, and the incidence of tyrosine hydroxylase-immunopositive cells was similar in carotid bodies from both strains of mice. Studies of whole cell K(+) currents also revealed identical current-voltage relationships and current depression by hypoxia in type I cells dissociated from normal vs. KO animals. Likewise, hypoxia-evoked increases in intracellular Ca(2+) concentration were not significantly different for normal and KO type I cells. The whole organ response to hypoxia was evaluated in recordings of carotid sinus nerve activity in vitro. In these experiments, responses elicited by hypoxia and by the classic chemoreceptor stimulant nicotine were also indistinguishable in normal vs. KO preparations. Our data demonstrate that carotid body function remains intact after sequence disruption of the gp91(phox) gene. These findings are not in accord with the hypothesis that the phagocytic form of NADPH oxidase acts as a primary O(2) sensor in arterial chemoreception.

Granulomatous pneumonitis following bone marrow transplantation.

We describe the rare occurrence of a granulomatous pneumonitis seen in a patient following allogeneic bone marrow transplantation. Interestingly sarcoidosis was diagnosed in the marrow donor less than a year after donating his bone marrow.

Gadolinium chloride inhibits pulmonary macrophage influx and prevents O(2)-induced pulmonary hypertension in the neonatal rat.

Newborn rats exposed to 60% O(2) for 14 d demonstrated a bronchopulmonary dysplasia-like lung morphology and pulmonary hypertension. A 21-aminosteroid antioxidant, U74389G, attenuated both pulmonary hypertension and macrophage accumulation in the O(2)-exposed lungs. To determine whether macrophage accumulation played an essential role in the development of pulmonary hypertension in this model, pups were treated with gadolinium chloride (GdCl(3)) to reduce lung macrophage content. Treatment of 60% O(2)-exposed animals with GdCl(3) prevented right ventricular hypertrophy (p < 0.05) and smooth muscle hyperplasia around pulmonary vessels, but had no effect on morphologic changes in the lung parenchyma. In addition, GdCl(3) inhibited 60% O(2)-mediated increases in endothelin-1, 8-isoprostane, and nitrotyrosine residues. Organotypic cultures of fetal rat distal lung cells were subjected to cyclical mechanical strain to assess the potential role of GdCl(3)-induced blockade of stretch-mediated cation channels in these effects. Mechanical strain caused a moderate increase of endothelin-1 (p < 0.05), which was unaffected by GdCl(3), but had no effect on 8-isoprostane or nitric oxide synthesis. A critical role for endothelin-1 in O(2)-mediated pulmonary hypertension was confirmed using the combined endothelin receptor antagonist SB217242. We concluded that pulmonary macrophage accumulation, in response to 60% O(2), mediated pulmonary hypertension through up-regulation of endothelin-1.

Cloning and characterization of the cDNA and gene for human epitheliasin.

Previously, we reported cloning and characterization of the mouse gene, epitheliasin. In the present work we cloned the cDNA of the full-length human orthologue and characterized its gene including 2 kb of 5' flanking sequence. Analysis of epitheliasin gene expression in adult tissues shows that it is expressed as 3.4 kb and 2 kb transcripts. The major 3.4 kb transcript is observed in the following order: prostate > colon > small intestine > pancreas > kidney > lung > liver. Epitheliasin transcripts in fetal tissues are observed only in kidney and lung. In situ hybridization analysis of tissues revealed that epitheliasin was preferentially expressed in epithelial cells. The gene consists of 14 exons and 13 introns based on comparison with its cDNA sequence. In the 5' flanking region, we identified two transcription start sites and three CpG islands encompassing a number of potential regulatory elements including SP1, SREBP, GRE/PRE and ERE. The region upstream of the transcription sites lacks a TATA box but contains an initiator-like element as well as a downstream promoter-like element. In vitro experiments with lymph node carcinoma of prostate (LNCaP) cells revealed that the epitheliasin gene was induced by androgens and the induction was not blocked by cycloheximide indicating that the induction required no intermediate protein factors. Immunoprecipitation analysis showed that androgens strongly increased epitheliasin protein levels.

Reactive oxygen species from NAD(P)H:quinone oxidoreductase constitutively activate NF-kappaB in malignant melanoma cells.

The transcription factor nuclear factor-kappaB (NF-kappaB) is constitutively activated in malignancies from enhanced activity of inhibitor of NF-kappaB (IkappaB) kinase, with accelerated IkappaBalpha degradation. We studied whether redox signaling might stimulate these events. Cultured melanoma cells generated superoxide anions (O(2)(-)) without serum stimulation. O(2)(-) generation was reduced by the NAD(P)H:quinone oxidoreductase (NQO) inhibitor dicumarol and the quinone analog capsaicin, suggesting that electron transfer from NQO through a quinone-mediated pathway may be an important source of endogenous reactive oxygen species (ROS) in tumor cells. Treatment of malignant melanoma cells with the H(2)O(2) scavenger catalase, the sulfhydryl donor N-acetylcysteine, the glutathione peroxidase mimetic ebselen, or dicumarol decreased NF-kappaB activation. Catalase, N-acetylcysteine, ebselen, dicumarol, and capsaicin also inhibited growth of melanoma and other malignant cell lines. These results raise the possibility that ROS produced endogenously by mechanisms involving NQO can constitutively activate NF-kappaB in an autocrine fashion and suggest the potential for new antioxidant strategies for interruption of oxidant signaling of melanoma cell growth.

Nonanticoagulant heparin inhibits NF-kappaB activation and attenuates myocardial reperfusion injury.

Heparin reduces ischemia-reperfusion injury to myocardium. This effect has been attributed to complement inhibition, but heparin also has other activities that might diminish ischemia-reperfusion. To further probe these mechanisms, we compared heparin or an o-desulfated nonanticoagulant heparin with greatly reduced anticomplement activity. When given at the time of coronary artery reperfusion in a canine model of myocardial infarction, heparin or o-desulfated heparin equally reduced neutrophil adherence to ischemic-reperfused coronary artery endothelium, influx of neutrophils into ischemic-reperfused myocardium, myocardial necrosis, and release of creatine kinase into plasma. Heparin or o-desulfated heparin also prevented dysfunction of endothelial-dependent coronary relaxation following ischemic injury. In addition, heparin and o-desulfated heparin inhibited translocation of the transcription nuclear factor-kappaB (NF-kappaB) from the cytoplasm to the nucleus in human endothelial cells and decreased NF-kappaB DNA binding in human endothelium and ischemic-reperfused rat myocardium. Thus heparin and nonanticoagulant heparin decrease ischemia-reperfusion injury by disrupting multiple levels of the inflammatory cascade, including the novel observation that heparins inhibit activation of the proinflammatory transcription factor NF-kappaB.

Repressed expression of the human xanthine oxidoreductase gene. E-box and TATA-like elements restrict ground state transcriptional activity.

Studies were initiated to address the basis for the low xanthine oxidoreductase (XOR) activity in humans relative to nonprimate mammalian species. The expression of the XOR in humans is strikingly lower than in mice, and both transcription rates and core promoter activity of the gene are repressed. Analysis of human XOR promoter activity in hepatocytes and vascular endothelial cells showed that the region from -258 to -1 contains both repressor and activator binding regions regulating core promoter activity. The region between -138 and -1 is necessary and sufficient for initiating, and the region between -258 and -228 is critical for restricting core promoter activity. Within the latter region, site-directed mutations identified a consensus sequence "acacaggtgtgg" (-242 to -230) that contains an E-box that binds a repressor. In addition, the TATA-like element is also required to restrict promoter activity and TFIID binds to this site. The results demonstrate that both an E-box and TATA-like element are required to restrict gene activity. A model is proposed to account for human XOR regulation.

Cloning, genomic organization, chromosomal assignment and expression of a novel mosaic serine proteinase: epitheliasin.

We report the isolation of a cDNA encoding a novel murine serine proteinase, epitheliasin. The cDNA spans 1753 bp and encodes a mosaic protein with a calculated molecular mass of 53529 Da. Its domains include a cytoplasmic tail, a type II transmembrane domain, a low-density lipoprotein receptor class A domain, a cysteine rich scavenger receptor-like domain and a serine proteinase domain. The proteinase portion domain shows 46-53% identity with mouse neurotrypsin, acrosin, hepsin and enteropeptidase. The gene, located in the telomeric region in the long arm of mouse chromosome 16, consists of 14 exons and 13 introns and spans approximately 18 kb. Epitheliasin is expressed primarily in the apical surfaces of renal tubular and airway epithelial cells.

Requirement for reactive oxygen species in serum-induced and platelet-derived growth factor-induced growth of airway smooth muscle.

Reactive oxygen species have been recently identified as important mediators of mitogenic signaling in a number of cell types. We therefore explored their role in mediating mitogenesis of airway smooth muscle. The antioxidants catalase, N-acetylcysteine, and probucol significantly reduced proliferation in primary cultures of rat tracheal smooth muscle stimulated with fetal bovine serum or platelet-derived growth factor, without affecting cell viability or inducing apoptosis. N-Acetylcysteine also significantly reduced serum-stimulated elevation of c-Fos but did not prevent the normal mitogen-induced increase in c-fos mRNA. Fractionation of ribosomes by sucrose density centrifugation and subsequent dot-blot Northern analysis revealed that antioxidants reduced incorporation of c-fos mRNA into the heaviest polyribosomes, suggesting redox regulation of c-fos mRNA translation. Serum treatment of monolayers produced a small but reproducibly significant rise in superoxide dismutase-inhibitable reduction of ferricytochrome c by myocyte monolayers. Serum-induced ferricytochrome c reduction, cellular proliferation, and c-Fos elevation were decreased by the flavoprotein-dependent enzyme inhibitor dipheyleneiodonium. Growth responses to fetal bovine serum and superoxide dismutase-inhibitable reduction of ferricytochrome c were not different between cultured tracheal myocytes from wild-type versus gp91 phagocyte oxidase null mice. These results suggest that mitogen stimulation of airway smooth muscle induces signal transduction of cell proliferation that is in part dependent on generation of partially reduced oxygen species, generated by an NADH or NADPH oxidoreductase that is different from the oxidase in phagocytic cells.