Validation of the hen's egg test for micronucleus induction (HET-MN): detailed protocol including scoring atlas, historical control data and statistical analysis.

A validation exercise of the hen's egg test for micronucleus induction was finalised with a very good predictivity based on the analysis of micronuclei in peripheral erythrocytes of fertilised chicken eggs (Reisinger et al. The hen's egg test for micronucleus-induction (HET-MN): validation data set. Mutagenesis, this issue). For transparency reasons this complementary publication provides further details on the assay especially as it was the first validation study in the field of genotoxicity testing involving the use of chicken eggs. Thus, the experimental protocol is described in detail and is complemented by a scoring atlas for microscopic analysis in blood cells. In addition, general characteristics of the test system, which is able to mirror the systemic availability of test compounds, are delineated: the test compound passes the egg membrane and is taken up by the blood vessels of the underlying chorioallantoic membrane. Subsequently, it is distributed by the circulating blood, metabolised by the developing liver and the yolk sac membrane and finally excreted into the allantois, a bladder equivalent. In specific, the suitability of the test system for genotoxicity testing is shown by, inter alia, a low background DNA damage in a comprehensive historical control database. In addition, the state-of-the-art statistical method used to evaluate obtained data is delineated. It combines laboratory-specific effect threshold with the Umbrella-Williams test, a statistical model also of interest for other genotoxicity test methods.

The hen's egg test for micronucleus induction (HET-MN): validation data set.

The classical in vitro genotoxicity test battery is known to be sensitive for indicating genotoxicity. However, a high rate of 'misleading positives' was reported when three assays were combined as required by several legislations. Despite the recent optimisations of the standard in vitro tests, two gaps could hardly be addressed with assays based on 2D monolayer cell cultures: the route of exposure and a relevant intrinsic metabolic capacity to transform pro-mutagens into reactive metabolites. Following these considerations, fertilised chicken eggs have been introduced into genotoxicity testing and were combined with a classical read-out parameter, the micronucleus frequency in circulating erythrocytes, to develop the hen's egg test for micronucleus induction (HET-MN). As a major advantage, the test mirrors the systemic availability of compounds after oral exposure by reflecting certain steps of Absorption, Distribution, Metabolism, Excretion (ADME) without being considered as an animal experiment. The assay is supposed to add to a toolbox of assays to follow up on positive findings from initial testing with classical in vitro assays. We here report on a validation exercise, in which >30 chemicals were tested double-blinded in three laboratories. The specificity and sensitivity of the HET-MN were calculated to be 98 and 84%, respectively, corresponding to an overall accuracy of 91%. A detailed protocol, which includes a picture atlas detailing the cell and micronuclei analysis, is published in parallel (Maul et al. Validation of the hen's egg test for micronucleus induction (HET-MN): detailed protocol including scoring atlas, historical control data and statistical analysis).

Hen's Egg Test for Micronucleus Induction (HET-MN).

The classical in vitro genotoxicity test battery is known to be sensitive for indicating genotoxicity. However, a high rate of "misleading" positives was reported when three assays were combined as required by several legislations. Despite the recent optimizations of the standard in vitro tests, two gaps could merely be addressed with assays based on monolayer cell cultures, that is, the route of exposure and a relevant intrinsic metabolic capacity to transform chemicals into reactive metabolites. Following these considerations, fertilized chicken eggs have been introduced into genotoxicity testing and were combined with a classical readout parameter, i.e., the analysis of micronucleus frequency in erythrocytes, to develop the hen's egg test for micronucleus induction, the HET-MN. As a major advantage the test mirrors the systemic availability of compounds after oral exposure reflecting certain steps of ADME without being considered as an animal experiment. After a successful validation exercise the detailed protocol is given here.

Lung inflammation and lack of genotoxicity in the comet and micronucleus assays of industrial multiwalled carbon nanotubes Graphistrength(©) C100 after a 90-day nose-only inhalation exposure of rats.

BACKGROUND: Graphistrength (©) C100 multiwalled carbon nanotubes (MWCNT) provide superior electrical and mechanical properties for various applications. The evaluation of the intrinsic hazard properties of Graphistrength(©) C100 is an essential step for safe use. A general feature of multiwalled carbon nanotubes after inhalation or intratracheal exposures is the induction of an inflammatory reaction in the lungs sometimes associated with local genotoxic effects. METHODS: After investigating different parameters for the aerosol generation and performing a 5-day inhalation range finding study, male and female Wistar rats were exposed nose-only for 90 days to target concentrations of 0.05, 0.25 and 5.0 mg/m(3) air of Graphistrength (©) C100 and sacrificed 24 h and 90 days after the last exposure. Broncho-alveolar lavage fluid (BALF) was also collected and analyzed for inflammatory parameters. Twenty-four hours post-exposure, chromosomal aberrations in the bone marrow cells were evaluated by the micronucleus test and DNA damages in the lung, kidney and liver cells by both the standard and the human 8-oxoguanine DNA N-glycosylase 1 (hOGG1)-modified comet assay. All studies were performed according to the OECD test guidelines. RESULTS: An inflammatory lung reaction and the release of inflammatory factors in the BALF were observed in all rats exposed to 5.0 mg/m(3), associated with changes in the differential white blood cells counts. The slight changes in BALF parameters at 0.25 mg/m(3) recovered and signs of lung clearance of the MWCNT were observed. No pathological changes were observed on the pleura. Neither increase in the number of micronucleated polychromatic erythrocytes nor increase in percent DNA damage were observed at any concentration. CONCLUSIONS: Lung inflammation characteristic of an overload with insoluble particles was observed after a 90-day exposure to 5.0 mg/m(3) of Graphistrength (©) C100. Clear signs of clearance and recovery were observed at 0.25 mg/m(3). No genotoxicity was detected locally in lung and distally in bone marrow, liver and kidney. Therefore, Graphistrength (©) C100 appears of low concern in term of local and systemic genotoxicity and a No-Observed Adverse Effect Concentration (NOAEC) of 0.25 mg/m(3) (0.28 mg/m(3) as actual concentration) was established for the repeated-dose toxicity.

Lysyl oxidase activity is dysregulated during impaired alveolarization of mouse and human lungs.

RATIONALE: Disordered extracellular matrix production is a feature of bronchopulmonary dysplasia (BPD). The basis of this phenomenon is not understood. OBJECTIVES: To assess lysyl oxidase expression and activity in the injured developing lungs of newborn mice and of prematurely born infants with BPD or at risk for BPD. METHODS: Pulmonary lysyl oxidase and elastin gene and protein expression were assessed in newborn mice breathing 21 or 85% oxygen, in patients who died with BPD or were at risk for BPD, and in control patients. Signaling by transforming growth factor (TGF-beta) was preemptively blocked in mice exposed to hyperoxia using TGF-beta-neutralizing antibodies. Lysyl oxidase promoter activity was assessed using plasmids containing the lox or loxl1 promoters fused upstream of the firefly luciferase gene. MEASUREMENTS AND MAIN RESULTS: mRNA and protein levels and activity of lysyl oxidases (Lox, LoxL1, LoxL2) were elevated in the oxygen-injured lungs of newborn mice and infants with BPD or at risk for BPD. In oxygen-injured mouse lungs, increased TGF-beta signaling drove aberrant lox, but not loxl1 or loxl2, expression. Lox expression was also increased in oxygen-injured fibroblasts and pulmonary artery smooth muscle cells. CONCLUSIONS: Lysyl oxidase expression and activity are dysregulated in BPD in injured developing mouse lungs and in prematurely born infants. In developing mouse lungs, aberrant TGF-beta signaling dysregulated lysyl oxidase expression. These data support the postulate that excessive stabilization of the extracellular matrix by excessive lysyl oxidase activity might impede the normal matrix remodeling that is required for pulmonary alveolarization and thereby contribute to the pathological pulmonary features of BPD.

Heme oxygenase-2 and large-conductance Ca2+-activated K+ channels: lung vascular effects of hypoxia.

RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. A complex of heme oxygenase-2 (HO-2) and the BK channel has been suggested as a universal oxygen sensor system. OBJECTIVES: We investigated whether this complex serves as an oxygen sensor for the vascular effects of alveolar hypoxia in the lung. METHODS: The investigations were performed in chronically hypoxic mice, in isolated perfused and ventilated lungs, and on the cellular level, including HO-2- and BK-channel deficient mice. MEASUREMENTS AND MAIN RESULTS: Immunohistochemical analysis of mouse lungs identified HO-2 mainly in pulmonary arteries, the bronchial epithelium, and alveolar epithelial cells. BK channel alpha-subunit (BKalpha) immunoreactivity was found primarily in the bronchial and vascular smooth muscle layer. Immunofluorescence staining and coimmunoprecipitation suggested only a weak complexation of HO-2 and BKalpha in pulmonary arterial smooth muscle cells. The strength of acute and sustained HPV, determined in isolated perfused and ventilated lungs, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. Exposure of mice to 3 weeks of chronic hypoxia resulted in a slight down-regulation of HO-2 and no alteration in BKalpha expression. The degree of pulmonary hypertension that developed, quantified on the basis of right ventricular pressure, right-heart hypertrophy, and the degree of muscularization of precapillary pulmonary arteries, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. CONCLUSIONS: It is demonstrated that neither deletion of HO-2 nor BK channels affect acute, sustained, and chronic vascular responses to alveolar hypoxia in the lung.

Inhibition of the soluble epoxide hydrolase attenuates monocrotaline-induced pulmonary hypertension in rats.

OBJECTIVES: The soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to their less active dihydroxy derivatives. Because EETs have antiinflammatory properties, we determined whether or not inhibition of sEH attenuates disease development in the monocrotaline model of pulmonary hypertension in rats. METHODS: sEH inhibition was achieved using 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (25 mg/l) and cis- 4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (5 mg/l) administered via drinking water starting 3 days prior to monocrotaline injection (60 mg/kg). RESULTS: Monocrotaline induced the development of progressive pulmonary hypertension. sEH inhibition increased the plasma ratio of EETs to DHETs and attenuated the monocrotaline-induced increase in pulmonary artery medial wall thickness as well as the degree of vascular muscularization. Moreover, right ventricular pressure was significantly lower in the group treated with sEH inhibitors. Pulmonary sEH protein expression and sEH activity, as well as pulmonary cytochrome P450 epoxygenase activity were all impaired in monocrotaline-treated rats as compared with control animals. sEH inhibitors, however, increased the plasma ratio of EETs to dihydroxy epoxyeicosatrienoic acids. Monocrotaline induced the proliferation of pulmonary endothelial and vascular smooth muscle cells in vivo as determined by 5-Bromo-2'-deoxy-Uridine incorporation, and this effect was significantly blunted in animals treated with sEH inhibitors. Proliferation of cultured pulmonary smooth muscle cell, however, was not affected by EETs or sEH inhibitors suggesting that the in-vivo effects are a consequence of a direct EET-mediated protection against the inflammation induced by monocrotaline. CONCLUSION: sEH inhibition reduces pulmonary vascular remodeling and the development of pulmonary hypertension in the monocrotaline model of primary pulmonary hypertension in rats.

Characterization of a murine model of monocrotaline pyrrole-induced acute lung injury.

BACKGROUND: New animal models of chronic pulmonary hypertension in mice are needed. The injection of monocrotaline is an established model of pulmonary hypertension in rats. The aim of this study was to establish a murine model of pulmonary hypertension by injection of the active metabolite, monocrotaline pyrrole. METHODS: Survival studies, computed tomographic scanning, histology, bronchoalveolar lavage were performed, and arterial blood gases and hemodynamics were measured in animals which received an intravenous injection of different doses of monocrotaline pyrrole. RESULTS: Monocrotaline pyrrole induced pulmonary hypertension in Sprague Dawley rats. When injected into mice, monocrotaline pyrrole induced dose-dependant mortality in C57Bl6/N and BALB/c mice (dose range 6-15 mg/kg bodyweight). At a dose of 10 mg/kg bodyweight, mice developed a typical early-phase acute lung injury, characterized by lung edema, neutrophil influx, hypoxemia and reduced lung compliance. In the late phase, monocrotaline pyrrole injection resulted in limited lung fibrosis and no obvious pulmonary hypertension. CONCLUSION: Monocrotaline and monocrotaline pyrrole pneumotoxicity substantially differs between the animal species.

Combined tyrosine and serine/threonine kinase inhibition by sorafenib prevents progression of experimental pulmonary hypertension and myocardial remodeling.

BACKGROUND: Inhibition of tyrosine kinases, including platelet-derived growth factor receptor, can reduce pulmonary arterial pressure in experimental and clinical pulmonary hypertension. We hypothesized that inhibition of the serine/threonine kinases Raf-1 (also termed c-Raf) and b-Raf in addition to inhibition of tyrosine kinases effectively controls pulmonary vascular and right heart remodeling in pulmonary hypertension. METHODS AND RESULTS: We investigated the effects of the novel multikinase inhibitor sorafenib, which inhibits tyrosine kinases as well as serine/threonine kinases, in comparison to imatinib, a tyrosine kinase inhibitor, on hemodynamics, pulmonary and right ventricular (RV) remodeling, and downstream signaling in experimental pulmonary hypertension. Fourteen days after monocrotaline injection, male rats were treated orally for another 14 days with sorafenib (10 mg/kg per day), imatinib (50 mg/kg per day), or vehicle (n=12 to 16 per group). RV systolic pressure was decreased to 35.0+/-1.5 mm Hg by sorafenib and to 54.0+/-4.4 mm Hg by imatinib compared with placebo (82.9+/-6.0 mm Hg). In parallel, both sorafenib and imatinib reduced RV hypertrophy and pulmonary arterial muscularization. The effects of sorafenib on RV systolic pressure and RV mass were significantly greater than those of imatinib. Sorafenib prevented phosphorylation of Raf-1 and suppressed activation of the downstream ERK1/2 signaling pathway in RV myocardium and the lungs. In addition, sorafenib but not imatinib antagonized vasopressin-induced hypertrophy of the cardiomyoblast cell line H9c2. CONCLUSIONS: The multikinase inhibitor sorafenib prevents pulmonary remodeling and improves cardiac and pulmonary function in experimental pulmonary hypertension. Sorafenib exerts direct myocardial antihypertrophic effects, which appear to be mediated via inhibition of the Raf kinase pathway. The combined inhibition of tyrosine and serine/threonine kinases may provide an option to treat pulmonary arterial hypertension and associated right heart remodeling.

Role of the prostanoid EP4 receptor in iloprost-mediated vasodilatation in pulmonary hypertension.

RATIONALE: Iloprost is effective for the treatment of pulmonary hypertension. It acts through elevation of cAMP by binding to the prostacyclin receptor (IP receptor). However, there is evidence that patients with severe pulmonary hypertension have decreased expression of the IP receptor in the remodeled pulmonary arterial smooth muscle. OBJECTIVES: We hypothesized that prostanoid receptors other than the IP receptor are involved in signal transduction by iloprost. METHODS: Immunoblotting was used to detect the IP and prostanoid EP4 receptor in lung tissue from patients with idiopathic pulmonary arterial hypertension, and immunohistochemistry was used to detect these receptors in lung sections from rats treated with monocrotaline (MCT28d). Protein and mRNA were isolated from pulmonary arterial smooth muscle cells (PASMCs) from control and MCT28d rats treated with AH6809 (an EP2 receptor antagonist) and AH23848 (an EP4 receptor antagonist) in combination with iloprost. Intracellular cAMP was also assessed in these tissues. MEASUREMENTS AND MAIN RESULTS: IP receptor expression was reduced in idiopathic pulmonary arterial hypertension patient lung samples and MCT28d rat lungs compared with the controls. Reverse transcriptase-polymerase chain reaction and immunoblotting of MCT28d rat PASMC extracts revealed scant expression of the IP receptor but stable expression of EP4 receptor, compared with controls. Iloprost-induced elevation in intracellular cAMP in PASMCs was dose-dependently reduced by AH23848, but not by AH6809. CONCLUSIONS: Iloprost mediates vasodilatory functions via the EP4 receptor in the case of low IP receptor expression associated with pulmonary arterial hypertension. This is a previously unrecognized mechanism for iloprost, and illustrates that the EP4 receptor may be a novel therapeutic approach for the treatment of pulmonary arterial hypertension.

Hypoxia-dependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the pulmonary vasculature.

Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox, p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under hypoxic (10% O(2)) or normoxic (21% O(2)) conditions, an upregulation exclusively of NOX4 mRNA was observed under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression levels of NOX4, which was localized in the vessel media, were 2.5-fold upregulated. These results support an important role for NOX4 in the vascular remodeling associated with development of pulmonary hypertension.

Activation of soluble guanylate cyclase reverses experimental pulmonary hypertension and vascular remodeling.

BACKGROUND: Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. Using wild-type and homozygous endothelial nitric oxide synthase (NOS3(-/-)) knockout mice with pulmonary hypertension induced by chronic hypoxia and rats with monocrotaline-induced pulmonary hypertension, we examined whether the soluble guanylate cyclase (sGC) stimulator Bay41-2272 or the sGC activator Bay58-2667 could reverse pulmonary vascular remodeling. METHODS AND RESULTS: Both Bay41-2272 and Bay58-2667 dose-dependently inhibited the pressor response of acute hypoxia in the isolated perfused lung system. When wild-type (NOS3(+/+)) or NOS3(-/-) mice were housed under 10% oxygen conditions for 21 or 35 days, both strains developed pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling, demonstrated by an increase in fully muscularized peripheral pulmonary arteries. Treatment of wild-type mice with the activator of sGC, Bay58-2667 (10 mg/kg per day), or the stimulator of sGC, Bay41-2272 (10 mg/kg per day), after full establishment of pulmonary hypertension from day 21 to day 35 significantly reduced pulmonary hypertension, right ventricular hypertrophy, and structural remodeling of the lung vasculature. In contrast, only minor efficacy of chronic sGC activator therapies was noted in NOS3(-/-) mice. In monocrotaline-injected rats with established severe pulmonary hypertension, both compounds significantly reversed hemodynamic and structural changes. CONCLUSIONS: Activation of sGC reverses hemodynamic and structural changes associated with monocrotaline- and chronic hypoxia-induced experimental pulmonary hypertension. This effect is partially dependent on endogenous nitric oxide generated by NOS3.

Reversal of experimental pulmonary hypertension by PDGF inhibition.

Progression of pulmonary hypertension is associated with increased proliferation and migration of pulmonary vascular smooth muscle cells. PDGF is a potent mitogen and involved in this process. We now report that the PDGF receptor antagonist STI571 (imatinib) reversed advanced pulmonary vascular disease in 2 animal models of pulmonary hypertension. In rats with monocrotaline-induced pulmonary hypertension, therapy with daily administration of STI571 was started 28 days after induction of the disease. A 2-week treatment resulted in 100% survival, compared with only 50% in sham-treated rats. The changes in RV pressure, measured continuously by telemetry, and right heart hypertrophy were reversed to near-normal levels. STI571 prevented phosphorylation of the PDGF receptor and suppressed activation of downstream signaling pathways. Similar results were obtained in chronically hypoxic mice, which were treated with STI571 after full establishment of pulmonary hypertension. Moreover, expression of the PDGF receptor was found to be significantly increased in lung tissue from pulmonary arterial hypertension patients compared with healthy donor lung tissue. We conclude that STI571 reverses vascular remodeling and cor pulmonale in severe experimental pulmonary hypertension regardless of the initiating stimulus. This regimen offers a unique novel approach for antire-modeling therapy in progressed pulmonary hypertension.