Monocrotaline pneumotoxicity in mice

Lung injury induced in rats by the pyrrolizidine alkaloid monocrotaline is a well-documented model of pulmonary hypertension. To our knowledge, however, monocrotaline-induced cardiopulmonary injury has rarely been described and has never been quantitated in mice. In the present study, adult male mice received 2.4, 4.8, or 24.0 mg monocrotaline/kg body weight/day in the drinking water continuously for 6 weeks. These doses represent 1, 2, and 10 times the severely pneumotoxic regimen in rats. Pulmonary endothelial function was monitored by right lung angiotensin converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI₂) and thromboxane (TXA₂) production. Light and electron microscopy were performed on the left lungs. Cardiac right ventricular hypertrophy was evaluated by the right ventricle to left ventricle plus septum weight ratio (RV/LV + S). Monocrotalinetreated mice exhibited a dose-dependent decrease in lung ACE and PLA activities and an increase in PGI₂ and TXA₂ production, indicative of endothelial dysfunction. However, these responses were significant only after the highest monocrotaline dose. Light and electron microscopy revealed dosedependent pulmonary inflammatory and exudative reactions. Unlike previous studies in rats, however, monocrotaline-treated mice developed relatively little lung fibrosis, cardiomegaly, or right ventricular hypertrophy, and no occlusive medial thickening of the pulmonary arteries, even at the highest dose level. These and previous data indicate that there are quantitative biochemical and qualitative morphological differences between mice and rats with respect to monocrotaline pneumotoxicity. Furthermore, in monocrotaline-treated mice (but not in rats) there appears to be a dissociation between lung endothelial dysfunction and inflammation on the one hand, and pulmonary hypertension and fibrosis on the other.     

Capsaicin pre- and post-treatment on rat monocrotaline pneumotoxicity

Monocrotaline (MCT) produces respiratory dysfunction, pulmonary hypertension (PH), and right ventricular hypertrophy (RVH) in rats. Tachykinins, such as substance P (SP) and neurokinin A (NKA), may mediate these effects. The purpose of this study was to investigate the length of tachykinin depletion (via capsaicin treatment) is needed to prevent (or attenuate) PH and/or RVH. Six groups of rats were injected subcutaneously with saline (3 ml/kg); capsaicin followed by saline or MCT (60 mg/kg); or MCT followed 7, 11, or 14 days later by capsaicin. Capsaicin (cumulative dose, 500 mg/kg) was given over a period of 4-5 days. Respiratory function, pulmonary vascular parameters, lung tachykinin levels, and tracheal neutral endopeptidase (NEP) activity were measured 21 days after MCT or saline injection. Capsaicin significantly decreased lung levels of SP but not NKA. Both capsaicin pretreatment and posttreatment blocked the following MCT-induced alterations: increases in lung SP and airway constriction; decreases in tracheal NEP activity and dynamic respiratory compliance. Administration of capsaicin before or 7 days after MCT blocked MCT-induced PH and RVH. The above data suggest that the early tachykinin-mediated airway dysfunction requires only transient elevated tachykinins, while progression of late tachykinin-mediated effects (PH and RVH) requires elevated tachykinins for more than one week.     

Potentiation of carbon tetrachloride-induced hepatotoxicity and pneumotoxicity by pyridine

Induction of P450HE1 by pyridine was compared with that by ethanol, and the resulting potentiation of the pneumotoxicity and hepato-toxicity following carbon tetrachloride inhalation by pyridine was examined. Rats were treated with ethanol as either a 10% solution in the drinking water or as a daily bolus (3 ml/kg, ip) dose for 7 days or one bolus dose of pyridine (200 mg/kg, ip) and compared for P450IIE1 apoprotein content by immunoblot analysis. Ethanol in the drinking water and pyridine elevated both hepatic and pulmonary P450IIE1 apoprotein content, but bolus dose ethanol did not. The induction was greatest in the pyridine group. In the interaction study, rats were treated with pyridine (200 mg/kg, ip) and 12 hours later were exposed to CC1₄ (8000 ppm for 3 hours). Pulmonary injury and hepatic damage were assessed 24 hours later by bronchoalveolar lavage fluid (BALF) analysis [γ-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH), and total protein] and serum sorbitol dehydrogenase (SDH) activity, respectively. Pyridine alone had no effect on BALF or SDH but enhanced GGT and LDH release into the BALF and SDH release into the serum when compared with CC1₄ exposure alone. Evaluation of the liver at the light microscopic level revealed characteristic CCl₄-induced centrilobular necrosis which was potentiated by pyridine. No changes were observed in the lung by light microscopic evaluation. Pyridine induced pulmonary and hepatic microsomal apoprotein levels of cytochrome P450IIE1 two- and 2- to sixfold, respectively. Exposure to CC1₄ decreased hepatic but not pulmonary P450IIE1 levels. Induction of cytochrome P450IIE1 by pyridine increases the bioactivation of CC1₄ in both the liver and lung, leading to enhanced toxicity.     

Hepato- and pneumotoxicity of pyrrolizidine alkaloids and derivatives in relation to molecular structure.

62 pyrrolizidine alkaloids and derivatives have been screened for acute and chronic hepato- and pneumotoxicity by the single dose method previously described. This procedure is satisfactory for the compounds of medium to high hepatotoxicity but failed to detect toxicity in certain other compounds of known, low hepatotoxicity. New findings significant in relation to hepatotoxicity are as follows: (i) On a molar basis, diesters of heliotridine and retronecine are about 4 times as toxic as the respective mono-esters and heliotridine esters are 2-4 times as toxic as retronecine esters. (ii) Crotanecine esters are less toxic than retronecine esters, and the 6,9-diester madurensine, 2-4 times less toxic than the 7,9-diester anacrotine (the difference being ascribed to there being only one reactive alkylating centre in the toxic metabolite from madurensine). (iii) Hepatotoxicity was confirmed for 7-angelylheliotridine but not observed for 9-angelyheliotridine and 7- and 9-angelylretronecine. (iv) Other significant compounds failing to induce hepatotoxicity were 9-pivalyl- and 7,9-dipivalyheliotridine, the alpha- and beta-epoxides of monocrotaline, 7-angelyl-1-methylenepyrrolizidine and the methiodides of monocrotaline and senecionine. The following compounds are readily converted by rat liver microsomes in vitro into dehydroheliotridine (or dehydroretronecine): 7- and 9-angelyheliotridine, 7- and 9-angelylretronecine, 7,9-dipivalylheliotridine and otosenine. 7,9-Divalerylheliotridine, the alpha- and beta-epoxides of monocrotaline, and retusamine yield pyrrolic metabolites more slowly. The preparation and characterisation of several alkaloid derivatives are described. Chronic lung lesions were produced by most compounds which gave chronic liver lesions, although a higher dose was required in some instances. This requirement may sometimes mean that chronic lung lesions cannot be induced because of the intervention of acute or peracute deaths. Apart from this factor, structure activity requirements for pneumotoxicity are the same as for hepatotoxicity, consistent with their being both caused by the same toxic metabolites.     

Metabolic Changes Precede the Development of Pulmonary Hypertension in the Monocrotaline Exposed Rat Lung

There is increasing interest in the potential for metabolic profiling to evaluate the progression of pulmonary hypertension (PH). However, a detailed analysis of the metabolic changes in lungs at the early stage of PH, characterized by increased pulmonary artery pressure but prior to the development of right ventricle hypertrophy and failure, is lacking in a preclinical animal model of PH. Thus, we undertook a study using rats 14 days after exposure to monocrotaline (MCT), to determine whether we could identify early stage metabolic changes prior to the manifestation of developed PH. We observed changes in multiple pathways associated with the development of PH, including activated glycolysis, increased markers of proliferation, disruptions in carnitine homeostasis, increased inflammatory and fibrosis biomarkers, and a reduction in glutathione biosynthesis. Further, our global metabolic profile data compare favorably with prior work carried out in humans with PH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Thus, we suggest that despite its limitations it can still serve as a useful preclinical model for the study of PH.     

Therapeutic Efficacy of Valproic Acid in a Combined Monocrotaline and Chronic Hypoxia Rat Model of Severe Pulmonary Hypertension

Background

Pulmonary hypertension (PH) is a serious disease with poor prognosis. Reports show that cells in remodeled pulmonary arteries of PH patients have similar characteristics to cancer cells, such as exuberant inflammation, increased proliferation, and decreased apoptosis. An ideal strategy for developing PH therapies is to directly target pulmonary vascular remodeling. High levels of histone deacetylase (HDAC) expression and activity are found in certain cancers, and research has shown the potential of HDAC inhibitors in repressing tumor growth via anti-inflammatory and anti-proliferative effects. To date, little is known about the effectiveness of HDAC inhibitors against pulmonary vascular remodeling in severe PH.

Objective

To investigate whether class I HDAC inhibitors suppress or reverse the development of severe PH in rats.

Methods

Male Sprague-Dawley rats were injected with a single, subcutaneous dose of monocrotaline (60mg/kg), and were exposed to chronic hypoxia to induce severe PH. Valproic acid, a class I HDAC inhibitor, was administered to rats daily via gastric gavage (300mg/kg) in a PH prevention study (during the first 3 weeks) or a PH reversal study (from 3 to 5 weeks). At the end of experiment, hemodynamic indices were measured, ventricular hypertrophy indices were calculated and vascular remodeling phenotypes were analyzed. Results: After 3 weeks exposure to a combined stimulation of monocrotaline and chronic hypoxia, rats exhibited a reduced body weight, elevated right ventricular systolic pressure, an increased Fulton index, right ventricle weight ratio, medial wall thickness and muscularized peripheral pulmonary arteries. These parameters for PH evaluation were exacerbated from 3 to 5 weeks. Daily administration of valproic acid therapy prevented and partially reversed the development of severe PH in rats, and decreased inflammation and proliferation in remodeled pulmonary arteries.

Conclusion

These data show that class I HDAC inhibitors may be effective for treating severe PH.     

Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells

A single injection of monocrotaline produces a pulmonary insult in rats with a phenotype similar to human primary pulmonary hypertension. Although extensively used as a model, the mechanism(s) by which this chemical insult mimics a condition with genetic and environmental links remains an enigma, although formation of protein adducts has been implicated. Monocrotaline (MCT) is non-toxic and must undergo hepatic dehydrogenation to the soft electrophile monocrotaline pyrrole as prerequisite to damaging endothelial cells lining arterioles at remote pulmonary sites. In this report we extend our earlier investigation (J. Biol. Chem. 2000, 275, 29091-29099) by examining protein adducts to lower abundance adducts, a pI range not covered before, and subcellular localization of adduct-forming proteins associated with plasma membranes. Human pulmonary artery endothelial cells were exposed to [(14)C]MCT pyrrole (MCTP) and protein targets were identified using 2-DE with IPG 4-11. Adducted proteins were identified by pI, apparent molecular weight, and PMF using MALDI-TOF MS. Results of this study show that the majority of adducts form on proteins that contain reactive thiols in a CXXC motif, such as protein disulfide isomerase A(3) (ERp57), protein disulfide isomerase (PDI), and endothelial PDI. These same proteins were the major adduct-forming proteins associated with the plasma membrane. Other proteins found to be targets were thioredoxin, galectin-1, reticulocalbin 1 and 3, cytoskeletal tropomyosin, mitochondrial ATP synthase beta-chain, annexin A2 and cofilin-1. For the first time, MCTP adducts were observed on proteins not known to contain cysteine residues. However, known reactive proteins including nucleophosmin did not form detectable adducts, potentially indicating that MCTP did not reach the interior of nucleus to the same extent as other cellular sites. These findings suggest that molecular events underlying MCTP toxicity are initiated at the plasma membrane or readily accessible subcellular regions including the cytosol and membranes of the endoplasmic reticulum and mitochondria.     

Inhalation of the BKCa-Opener NS1619 Attenuates Right Ventricular Pressure and Improves Oxygenation in the Rat Monocrotaline Model of Pulmonary Hypertension

Background

Right heart failure is a fatal consequence of chronic pulmonary hypertension (PH). The development of PH is characterized by increased proliferation of vascular cells, in particular pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells. In the course of PH, an escalated right ventricular (RV) afterload occurs, which leads to increased perioperative morbidity and mortality. BK_Ca channels are ubiquitously expressed in vascular smooth muscle cells and their opening induces cell membrane hyperpolarization followed by vasodilation. Moreover, BK activation induces anti-proliferative effects in a multitude of cell types. On this basis, we hypothesized that treatment with the nebulized BK channel opener NS1619 might be a therapy option for pulmonary hypertension and tested this in rats.

Methods

(1) Rats received monocrotaline injection for PH induction. Twenty-four days later, rats were anesthetized and NS1619 or the solvent was administered by inhalation. Systemic hemodynamic parameters, RV hemodynamic parameters, and blood gas analyses were measured before as well as 30 and 120 minutes after inhalation. (2) Rat PASMCs were stimulated with PDGF-BB in the presence and absence of NS1619. AKT, ERK1 and ERK2 activation were investigated by western blot analyses, and relative cell number was determined 48 hours after stimulation.

Results

Inhalation of a 12 µM and 100 µM NS1619 solution significantly reduced RV pressure without affecting systemic arterial pressure. Blood gas analyses demonstrated significantly reduced carbon dioxide and improved oxygenation in NS1619-treated animals pointing towards a considerable pulmonary shunt-reducing effect. In PASMC’s, NS1619 (100 µM) significantly attenuated PASMC proliferation by a pathway independent of AKT and ERK1/2 activation.

Conclusion

NS1619 inhalation reduces RV pressure and improves oxygen supply and its application inhibits PASMC proliferation in vitro. Hence, BK opening might be a novel option for the treatment of pulmonary hypertension.     

Mutagenicity tests in mice

Summary The dominant lethal method has been described and the control data of C3H inbred mice and (101xC3H)F₁ hybrids were analysed. It could be demonstrated that infectious diseases and the number of implantations per uterus can influence the embryonic mortality. Other tested parameters such as 0.9% NaCl solution, aqua dest., and paternal and maternal age have within a certain frame no effect on the embryonic mortality. The heterogeneity does not permit a pooling of the controls and suggests on the other hand that each experiment should have a sufficient number of own controls to enable a decision over the mutagenicity of a compound.(Direktor: Prof. Dr. F. Vogel)     

Assessing hoarding in mice

Hoarding is a species-typical behavior shown by rodents, as well as other animals. By hoarding, the rodent secures a food supply for times of emergency (for example, when threatened by a predator) or for times of seasonal adversity such as winter. Scatter hoarding, as seen typically in squirrels and birds, involves placing small caches of food in hidden places, generally underground. Most rodents, however, hoard a supply of food in or near the home base--for example, in 'larders' near the sleeping quarters in a burrow. In the laboratory, measurement of hoarding involves simply weighing the food transported into the home cage from an external source, but the route to that source must be secure and animal-proof; for example, there should be no holes large enough to permit escape of a mouse, and no weak points that could be enlarged by gnawing. A suitable and easily constructed apparatus is described in the protocol. Hoarding has been shown to be sensitive to brain lesions and pharmacological agents, and is a suitable test for species-typical behavior in genetically modified mice.     

Retro-orbital injections in mice

Intravenous vascular access is technically challenging in the adult mouse and even more challenging in neonatal mice. The authors describe the technique of retro-orbital injection of the venous sinus in the adult and neonatal mouse. This technique is a useful alternative to tail vein injection for the administration of non-tumorigenic compounds. The authors report that they have routinely used this technique in the adult mouse to administer volumes up to 150?μl without incident. Administration of retro-orbital injections is more challenging in neonatal mice but can reliably deliver volumes up to 10?μl.     

Transposons reanimated in mice

Transposons have proved valuable in the genetic modification of many organisms but not mammals. New work reported by Ding et al. (2005) in this issue of Cell and by Collier et al. (2005) and Dupuy et al. (2005) in a recent issue of Nature now reveals that insertional mutagenesis in mammalian cells is possible thanks to modified derivatives of the piggyBac and Sleeping Beauty DNA transposons. However, the slow rate of proliferation of DNA transposons suggests that derivatives of the L1 retrotransposon might be more powerful for mutagenesis of germline cells. In either case, the future of transposon-driven genetic analyses of mice and other mammals looks promising.     

Oxidative stress in mice

The aim of this study was to analyze the effect of high dietary Fe on liver antioxidant status in mice fed a corn-oil-enriched diet. Male Balb/c mice were fed for 3 wk with a standard diet enriched with 5% by weight of corn oil with adequate Fe (FCO diet) or supplemented with 1% carbonyl Fe (FCOFe diet). The control group was fed a standard diet. The high-Fe diet induced a twofold increase of hepatic Fe level. However, an increase of thymic Fe level has been induced solely by dietary fat. The hepatic copper (Cu) level slightly decreased in the FCO diet. In the spleen, the high-Fe diet-induced increase of Fe level was negatively correlated with the Cu level. The antioxidant status was influenced by both dietary fat and Fe. Mice fed corn-oil-enriched diets had a higher concentration of thiobarbituric acid-reactive substances (TBARS), with a greater increase in the FCOFe diet. Fatty acid analysis showed decreased n−3 and n−6/n−3 ratio, particularly in the FCOFe diet. Hepatic Cu/Zn superoxide dismutase (CuZn-SOD) activity was decreased in FCO diet, and Fe supplementation caused a further decrease in the enzyme activity. These results suggest that feeding with corn oil-enriched diet increases oxidative damage by decreasing antioxidant enzyme defense. The high-Fe diet additionally affects the antioxidant defense system, further increasing the tissue's susceptibility to lipid peroxidation. Additionally, both corn-oil- and Fe-enriched diets have increased the Cu requirement in mice.     

Cocaine kindling in mice

Previous studies proposed the involvement of theN-methyl-D-aspartate (NMDA) type of glutamate receptors in the development of sensitization to the convulsive effect of cocaine (cocaine kindling). The present study was undertaken to determine, first, if cocaine kindling is associated with enhanced sensitivity of the NMDA receptor to the convulsive response ofN-methyl-D,L-aspartate (NMDLA), and second, whether in vivo modulation of nitric oxide synthase (NOS) function regulates the development of cocaine kindling. The following results were observed:

1. Cocaine-kindled animals were significantly more susceptible to the convulsive effect of the NMDA receptor agonist NMDLA than saline controls;
2. Pretreatment with the NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 100 mg/kg; ip) blocked the development of cocaine kindling;
3. The protective effect of L-NAME was partially reversed with the coadministration of the NOS substrate,L-arginine (300 mg/kg; ip), but notD-arginine; and
4. L-Arginine (300 mg/kg; ip), but notD-arginine, amplified the development of cocaine kindling. Taken together, these findings suggest that supersensitivity of the NMDA receptor and activation of NOS may underlie the development of cocaine kindling.