Dexfenfluramine discontinuous treatment does not worsen hypoxia-induced pulmonary vascular remodeling but activates RhoA/ROCK pathway: consequences on pulmonary hypertension.

The anorectic drug, dexfenfluramine has been associated with an increase in the relative risk of developing pulmonary hypertension. 5-hydroxytryptamine (5-HT) is a mitogen for smooth muscle cell, an effect that relies on 5-HT transporter expression and which has been proposed to explain pulmonary side effect of dexfenfluramine, and more particularly its effect on vascular remodeling. However recent data supported a major role of pulmonary artery vasoconstriction through the RhoA/Rho-kinase pathway. We questioned whether or not anorectic treatment aggravates pulmonary hypertension through vascular remodeling and if RhoA/Rho-kinase (ROCK) was potentially involved. In rats exposed to hypoxia, concomitant dexfenfluramine treatment (5 mg/kg/day, i.v.) for 4 weeks had no effect on pulmonary hypertension development. When exposure to 2 weeks of chronic hypoxia followed discontinuation of dexfenfluramine treatment (dexfenfluramine-hypoxic rats), echocardiographic parameters of pulmonary artery flow and right ventricle were further altered (P<0.05) as well as right ventricle systolic pressure was further increased (P<0.001) when compared to hypoxic rats treated with vehicle (hypoxic rats). However, the total number of muscularized distal pulmonary arteries artery was similar in dexfenfluramine-hypoxic vs. hypoxic rats (P>0.05). Western blot, RT-PCR and immunofluorescence analysis revealed a greater expression of 5-HT transporter and ROCK, as well as a greater activation of RhoA in dexfenfluramine-hypoxic rats compared to hypoxic rats. These data show that increased 5-HT transporter expression that follows dexfenfluramine discontinuation is not associated to a greater vascular remodeling despite worsening the development of pulmonary hypertension. Furthermore dexfenfluramine discontinuation promotes a greater RhoA/ROCK pathway activation. This pathway, involved in many cardiovascular diseases, might explain the cardiac and pulmonary toxicity of serotoninergic agonists.

Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug toxicology and safety pharmacology.

BACKGROUND: Echocardiography (EC) is a method used for the investigation of cardiac morphology and function. Two-dimensional EC gives a visualisation of the morphology of the heart. M-mode EC allows heart function to be monitored. Pulsed Doppler EC is the method of choice to measure blood flows. OBJECTIVE: To describe the information EC can provide for cardiovascular investigation in laboratory animals, with a special focus on the potential helpfulness of EC in preclinical toxicology and safety pharmacology. METHODS: This review includes publications describing the methodology of EC and its application to several animal species used in biological experimentation. RESULTS/CONCLUSION: EC has been established in dogs, monkeys, rodents, rabbits and pigs. As demonstrated by experiments in different species, EC can be particularly helpful in toxicology and safety pharmacology, based on the amount of information it can give on the causes and consequences of drug adverse effects on the cardiovascular system. Furthermore, EC does not require any surgery and is therefore a key refinement compared to invasive methods generally used for investigating the cardiovascular function in laboratory animals. Despite some limitations of the method (the need for trained people, time required for an accurate EC recording, lack of current validation), EC should be further developed in preclinical toxicology and safety pharmacology.

Cardiovascular toxicity of minoxidil in the marmoset.

The aim of the experiments was to assess the toxicity of minoxidil, a potent vasodilator, in marmosets. The animals were treated either at escalating doses from 2 to 40 mg/kg, escalating doses from 40 to 200 mg/kg or single doses of 150 mg/kg or 200 mg/kg. ECG recording and echocardiographic examination were conducted before and 1h after treatment. Necropsy and histopathology were performed 24h after the last dose. The treatment with minoxidil induced myocardial necrosis, coronary arteriopathy and degeneration of renal tubules in animals treated with 150 mg/kg or 200 mg/kg. Myocardial necrosis associated with fibrosis in some animals was located mainly in the left and right ventricles (including papillary muscles), but also in the right atrium, left atrium and/or interventricular septum. Arteriopathy was observed in small coronary arteries of the right or left atrium. ECG and echocardiographic examinations showed that in animals treated with 150 mg/kg or 200 mg/kg, there were positive chronotropic and inotropic effects that compensated for the hypotensive effect of the drug and were considered to have played a key role in the pathogenesis of the cardiovascular lesions. The cardiotoxicity of minoxidil in marmosets was similar to that described in dogs, but occurred at much higher doses. In conclusion minoxidil produced cardiovascular toxicity in the marmoset, which was probably due to the marked changes in the cardiac function associated with exaggerated pharmacological effects of the compound. The marmosets were found to be less sensitive than dogs to the cardiotoxicity of minoxidil.

Characterisation of the vascular and inflammatory lesions induced by the PDE4 inhibitor CI-1044 in the dog.

Phosphodiesterase 4 (PDE4) inhibitors are potential therapeutic agents but vascular injury and perivascular inflammation occurs frequently during preclinical toxicology testing of these drugs. The lesions induced by PDE4 inhibitors have been described mainly in rats but there is limited data available for monkeys and no data for dogs. Here we present the toxicological profile of CI-1044, a PDE4 inhibitor, administered orally to dogs. Dogs were treated for 4 days with 5, 10, 20 or 50 mg/kg of CI-1044, and a group of dogs was submitted to a 4-week recovery period after treatment with 20 mg/kg. CI-1044 induced disseminated vascular necrosis and inflammation in various organs/tissues from 20 mg/(kg day). The nasal turbinates and the scrotal skin were the most sensitive tissues but lesions were also observed in the stomach, heart, kidneys and, to a lower extent, in the liver, mesenteric lymph nodes, adrenals and lung. The inflammation was mainly characterized by an infiltration of polynuclear neutrophils, oedema and necrosis. The inflammation observed microscopically correlated with marked increases in serum amyloid A and C-reactive protein. Variations in these acute phase response proteins were detected 24 h after the first dose and were further increased over the course of the treatment. The vascular and inflammatory lesions were reversible over 4 weeks. In conclusion, the lesions induced by the PDE4 inhibitor CI-1044 in dogs differed from the haemodynamically mediated coronary arteritis reported with PDE3 inhibitors.

Impact of study design on proarrhythmia prediction in the SCREENIT rabbit isolated heart model.

INTRODUCTION: Prediction of the propensity of a compound to induce Torsades de Pointes continues to be a formidable challenge to the pharmaceutical industry. Development of an in vitro model for assessment of proarrhythmic potential offers the advantage of higher throughput and reduced compound quantity requirements when compared to in vivo studies. A rabbit isolated heart model (SCREENIT) has been reported to identify compounds with proarrhythmic potential based on the observance of compound-induced triangulation and instability of the monophasic action potential (MAP), ectopic beats, and reverse-use dependence of prolongation of the MAP duration. Previous reports have indicated that this model qualitatively identifies proarrhythmic compounds and suggest the use of this model to assign safety margins for human clinical use. The intent of this series of studies was to evaluate the impact of study design on the proarrhythmic concentration predicted by this model. METHODS: Nine compounds of varying proarrhythmic potential and a negative control were tested in a blinded fashion using a series of different experimental protocols: Compounds were tested at multiple concentration ranges and extended perfusion times were also evaluated. RESULTS: In general when the dataset is viewed as a whole, the model did identify proarrhythmic compounds, however the concentration at which action potential prolongation, triangulation, instability, reverse-use dependence and ectopic beats occurred often varied based on the concentration range selected. Further analysis using extended compound perfusion times demonstrated that variability may be due in part to lack of adequate equilibration of compound with the cardiac tissue. DISCUSSION: We report that the model correctly identified proarrhythmic agents in a qualitative manner, but that study design impacts the proarrhythmic concentration derived from the model.

Preclinical cardiac safety assessment of drugs.

The heart is a frequent site of toxicity of pharmaceutical compounds in humans, and when developing a new drug it is critical to conduct a thorough preclinical evaluation of its possible adverse effects on cardiac structure and function. Changes in cardiac morphology such as myocardial necrosis, hypertrophy or valvulopathy are assessed in regulatory toxicity studies in laboratory animals, although specific models may be needed for a more accurate detection of the risk. The potential proarrhythmic risk of new drugs is a major subject of concern and needs to be fully addressed before treatment of volunteers or patients takes place. In vitro assays are conducted to determine the effects on cardiac ion channels, in particular I(Kr) potassium channel antagonism. Prolongation of the QT interval is assessed in vivo, generally in telemetered dogs. Together, these two tests are considered to detect most arrhythmic drugs. The results of this core battery can be refined by additional studies, in particular assays on isolated cardiac tissues determining changes in cardiac action potential duration, shape and variability over time. Triggering of arrhythmia is assessed in hypokalaemic dogs with artificially created bradycardia, or in vitro in isolated whole hearts. The proarrhythmic risk of the new compound is then evaluated by integrating the results of these different tests. Drug adverse effects on cardiac electrophysiological function, in particular impulse formation and conduction, are evaluated through changes in ECG, generally recorded in dogs, pigs or monkeys. Changes in cardiac contractility occurring either as a primary effect of the drug on cardiac function or as a consequence of cardiac lesions should also be carefully assessed. In telemetered or anaesthetised animals, cardiac contractility is evaluated by measurement of left ventricular pressure and its first derivative over time. Echocardiography allows non-invasive measurement of drug-induced changes in ventricular wall movements and cardiac haemodynamics indicative of effects on contractility. In conclusion, a reliable and accurate evaluation of the cardiac safety of a new pharmaceutical agent is based on the results of in vitro tests, with overall moderate to high throughput, and in vivo experiments assessing the effects of the drug on the heart in its physiological environment. The specific sensitivities of the animals used in these assays to cardiac adverse effects should also be considered. The final evaluation of the cardiac risk is therefore based on an integrated analysis of the results from a battery of tests.

Investigation of the molecular mechanisms preceding PDE4 inhibitor-induced vasculopathy in rats: tissue inhibitor of metalloproteinase 1, a potential predictive biomarker.

Phosphodiesterase (PDE) 4 inhibitors are a class of drugs that can provide novel therapies for asthma and chronic obstructive pulmonary disease. Their development is frequently hampered by the induction of vascular toxicity in rat mesenteric tissue during preclinical studies. Whereas these vascular lesions in rats have been well characterized histologically, little is known about their pathogenesis and in turn, sensitive and specific biomarkers for preclinical and clinical monitoring do not exist. In order to investigate the early molecular mechanisms underlying vascular injury, time-course studies were performed by treating rats for 2-24 h with high doses of the PDE4 inhibitor CI-1044. Transcriptomics analyses in mesenteric tissue were performed using oligonucleotide microarray and real-time RT-PCR technologies and compared to histopathological observations. In addition, protein measurements were performed in serum samples to identify soluble biomarkers of vascular injury. Our results indicate that molecular alterations preceded the histological observations of inflammatory and necrotic lesions in mesenteric arteries. Some gene expression changes suggest that the development of the lesions could follow a primary modulation of the vascular tone in response to the pharmacological effect of the compound. Activation of genes coding for pro- and antioxidant enzymes, cytokines, adhesion molecules, and tissue inhibitor of metalloproteinase 1 (TIMP-1) indicates that biomechanical stimuli may contribute to vascular oxidant stress, inflammation, and tissue remodeling. TIMP-1 appeared to be an early and sensitive predictive biomarker of the inflammatory and the tissue remodeling components of PDE4 inhibitor-induced vascular injury.

Continuous inhalation of carbon monoxide induces right ventricle ischemia and dysfunction in rats with hypoxic pulmonary hypertension.

We aimed to investigate the toxicity of carbon monoxide (CO) in rats with right ventricle (RV) remodeling induced by hypoxic pulmonary hypertension (PHT). A group of Wistar rats was exposed to 3-wk hypobaric hypoxia (H). A second group was exposed to 50 ppm CO for 1 wk (CO). A third group was exposed to chronic hypoxia including 50 ppm CO during the third week (H+CO). These groups were compared with controls. RV and left ventricle (LV) functions were assessed by echocardiography and transparietal catheterization. Ventricular perfusion was estimated with the fluorescent microsphere method. Results were confirmed by histology. PHT induced RV hypertrophy and function enhancement. In the H group, RV shortening fraction (RVSF; 71 +/- 12% vs. 41 +/- 2%) and RV end-systolic pressure (RVESP; 54 +/- 6 vs. 19 +/- 2 mmHg) were increased compared with controls. Moreover, myocardial perfusion was increased in the RV (36 +/- 2% vs. 22 +/- 2%) and decreased in the LV (64 +/- 3% vs. 78 +/- 2%). In the H+CO group, RVSF (45 +/- 3% vs. 71 +/- 12%) and RVESP (38 +/- 3 vs. 54 +/- 6 mmHg) were decreased compared with the H group. RV perfusion was decreased in the H+CO group compared with the H group (21 +/- 5% vs. 36 +/- 2%), and LV perfusion was increased (79 +/- 5% vs. 64 +/- 3%). PHT and RV hypertrophy were still present in the H+CO group, and fibroses localized in the RV were detected. Similar lesions were observed in an additional group exposed simultaneously to hypoxia and 50 ppm CO over 3 wk. We demonstrated that rats with established PHT were more sensitive to CO, which dramatically alters the RV adaptive response to PHT, leading to ischemic lesions.

Hepatotoxin-induced hypertyrosinemia and its toxicological significance.

A (1)H Nuclear Magnetic Resonance (NMR) spectroscopic investigation of the effects of single doses of four model hepatotoxins on male Sprague-Dawley rats showed that hypertyrosinemia was induced by three of the treatments (ethionine 300 mg/kg, galactosamine hydrochloride 800 mg/kg and isoniazid 400 mg/kg) but not by the fourth (thioacetamide 200 mg/kg). Concomitant histopathological and clinical chemistry analyses showed that hypertyrosinemia could occur with or without substantial hepatic damage and that substantial hepatic damage could occur without hypertyrosinemia. However, in the rats dosed with galactosamine hydrochloride, which showed highly variable amounts of liver damage at ca. 24 h after dosing, a clear relationship was found between the degree of hypertyrosinemia and the extent of the hepatic necrosis induced. In line with the cause of clinically observed Type II Tyrosinemia, we consider that the critical event in the onset of hepatotoxin-induced hypertyrosinemia is likely to be a reduction in hepatic tyrosine aminotransferase (TAT) activity. We discuss mechanisms by which TAT activity could be lost with special consideration given to pyridoxal 5'-phosphate (P5P) depletion and to the inhibition of protein synthesis. This analysis may have implications for the interpretation of clinical measures of liver status such as Fischer's ratio and the branched-chain tyrosine ratio (BTR).

Altered gene expression in rat mesenteric tissue following in vivo exposure to a phosphodiesterase 4 inhibitor.

Vascular injury is a relatively common finding during the pre-clinical toxicity testing of drugs. The mechanisms of the injury are poorly understood and in turn, sensitive and specific biomarkers for pre-clinical and clinical monitoring do not exist. The present study was undertaken to investigate the molecular mechanisms of drug-induced vascular injury in mesenteric tissue of rats treated with the selective phosphodiesterase 4 (PDE4) inhibitor CI-1044. In a time-course study, male Sprague Dawley rats were given daily doses of 40 or 80 mg/kg for 1, 2 or 3 successive days and were euthanized the following day. Gene expression profiles in mesenteric tissue were determined using Affymetrix RG_U34A microarrays and fibrinogen and cytokine measurements were performed in blood samples. Hierarchical clustering analysis produced a clear pattern separation of the animals with inflammation, animal with inflammation and necrosis and animals without any lesion. Genes associated with inflammation, procoagulation, extracellular matrix remodeling were up-regulated. An altered expression of genes involved in vascular tone regulation, lipid and glucose metabolism was also observed. Selected genes expression changes were confirmed by TaqMan real-time RT-PCR. The inflammatory process was also detected in the bloodstream at the protein level since fibrinogen, IL6 and IL1beta concentrations were increased in treated animals. Overall, the present study reveals several molecular changes supporting the hypothesis by which PDE4 inhibitor-induced vascular lesions in rats are triggered by an inflammatory mechanism and/or a vascular tone dysregulation.

Dexfenfluramine does not worsen but moderates progression of chronic hypoxia-induced pulmonary hypertension.

This study shows for the first time, that dexfenfluramine, a 5-HT(2) receptor agonist, attenuates the development of chronic hypoxia-induced pulmonary hypertension. Chronic exposure to hypoxia, 4 weeks, induced hypoxic pulmonary hypertension in adult rat as haemodynamic and cardiac measurements showed significant modifications in right ventricle parameters (free wall right ventricle thickness; pulmonary acceleration time and velocity time integral) in chronic hypoxic control when compared to normoxic control animals. We observed that free wall right ventricle thickness and pulmonary velocity time integral were significantly less in chronic hypoxic rats treated with dexfenfluramine when compared to chronic hypoxic control rats. Similarly, rats exposed to chronic hypoxia exhibited an increase in both right ventricle pressure and weight by comparison to normoxic control animals but those variations were significantly diminished in dexfenfluramine-treated rats, indicating the moderating influence exerted by dexfenfluramine on chronic hypoxia-induced pulmonary hypertension and cardiac alterations. Thus, we report here the ability of dexfenfluramine to limit chronic hypoxia-induced pulmonary hypertension, emphasizing the importance of the time after the dexfenfluramine treatment discontinuation to assess the influence of this 5-HT receptor agonist on the development of chronic hypoxia-induced pulmonary hypertension.

An integrated metabonomic approach to describe temporal metabolic disregulation induced in the rat by the model hepatotoxin allyl formate.

The time-related metabolic events in rat liver, plasma, and urine following hepatotoxic insult with allyl formate (75 mg/kg) were studied using a combination of high-resolution liquid state and magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopic methods together with pattern recognition analysis. The metabonomics results were compared with the results of conventional plasma chemistry and histopathological assessments of liver damage. Various degrees of liver damage were observed in different animals, and this variation was reflected in all of the analyses. Furthermore, each analysis revealed a high degree of functional and structural recovery by the end of the study. The allyl formate-induced changes included hepatocellular necrosis, hepatic lipidosis, decreased liver glycogen and glucose, decreased plasma lipids, increased plasma creatine and tyrosine, increased urinary taurine and creatine, and decreased urinary TCA cycle intermediates. The observed reductions in hepatic glycogen and glucose suggest increased glucose utilization and are consistent with the expected depletion of hepatic ATP following mitochondrial impairment, assuming that there is a consequent increase in energy production from glycolysis. The increase in plasma tyrosine is consistent with impaired protein synthesis, a known consequence of ATP depletion. Partial least squares-based cross-correlation of the variation in the liver and plasma NMR profiles indicated that the allyl formate-induced increase in liver lipids correlated with the decrease in plasma lipids. This suggests disruption in lipid transport from the liver to plasma, which could arise through impaired apolipoprotein synthesis, as with ethionine.

Pharmaco-metabonomic phenotyping and personalized drug treatment.

There is a clear case for drug treatments to be selected according to the characteristics of an individual patient, in order to improve efficacy and reduce the number and severity of adverse drug reactions. However, such personalization of drug treatments requires the ability to predict how different individuals will respond to a particular drug/dose combination. After initial optimism, there is increasing recognition of the limitations of the pharmacogenomic approach, which does not take account of important environmental influences on drug absorption, distribution, metabolism and excretion. For instance, a major factor underlying inter-individual variation in drug effects is variation in metabolic phenotype, which is influenced not only by genotype but also by environmental factors such as nutritional status, the gut microbiota, age, disease and the co- or pre-administration of other drugs. Thus, although genetic variation is clearly important, it seems unlikely that personalized drug therapy will be enabled for a wide range of major diseases using genomic knowledge alone. Here we describe an alternative and conceptually new 'pharmaco-metabonomic' approach to personalizing drug treatment, which uses a combination of pre-dose metabolite profiling and chemometrics to model and predict the responses of individual subjects. We provide proof-of-principle for this new approach, which is sensitive to both genetic and environmental influences, with a study of paracetamol (acetaminophen) administered to rats. We show pre-dose prediction of an aspect of the urinary drug metabolite profile and an association between pre-dose urinary composition and the extent of liver damage sustained after paracetamol administration.

Cardiac safety strategies. 25-26 October 2005, the Radisson SAS Hotel, Nice, France.

This meeting was organised by IIR Life Sciences. It was chaired by Brian Guth, (head of General Pharmacology at Boehringer Ingelheim Pharma) and brought together scientists and clinicians from the pharmaceutical industry, university and regulatory agencies. The meeting presented emerging trends in cardiac safety, including its regulatory context pertaining to ICH S7A, S7B and E14. ICH S7A and S7B highlight the importance of the hERG test and telemetric studies in non-rodents. ICH E14 describes the clinical 'thorough QT study' that is required by the FDA for any new drug. Marked physiological variability in QT interval over time can be observed, partly as a result of fluctuation in autonomic tone. Beat-to-beat QT variability and T-wave morphology should be considered as a part of an integrated estimate of proarrhythmic risk. A case study illustrated the predictivity of preclinical data for proarrhythmic risk in humans, showing the importance of evaluating QT effects in patients to establish a safety margin.

Doppler tissue imaging in assessment of pulmonary hypertension-induced right ventricle dysfunction.

We aimed to assess the accuracy of Doppler tissue imaging (DTI) in detecting right ventricle (RV) dysfunction and electromechanical coupling alteration following pulmonary hypertension (PHT) in rat. PHT was induced by chronic hypoxia exposure (hypoxic PHT) or monocrotaline treatment (monocrotaline PHT). In both PHT models, we observed transparietal RV pressure increase and remodeling, including hypertrophy and dilation. Conventional echocardiography provided evidence for pulmonary outflow impairment with midsystolic notch and acceleration time decrease in PHT groups (21.7 +/- 1.6 and 13.2 +/- 2.9 ms in hypoxic and monocrotaline PHT groups vs. 28.1 +/- 1.0 ms in control). RV shortening fraction was decreased in the monocrotaline PHT group compared with the hypoxic PHT and control groups. Combining conventional Doppler and DTI was more helpful to detect RV diastolic dysfunction in the monocrotaline PHT group (E/Ea ratio = 17.0 +/- 1.4) compared with the hypoxic PHT and control groups (11.5 +/- 0.7 and 10.2 +/- 0.4, respectively). Tei index measured using DTI highlighted global RV dysfunction in the monocrotaline PHT group (1.36 +/- 0.24 vs. 0.92 +/- 0.05 and 0.86 +/- 0.05 in the hypoxic PHT and control groups, respectively). Q-Sm time measured from the onset of Q wave to the onset of DTI Sm wave was increased in both PHT groups. PHT-induced electromechanical coupling alteration was confirmed by in vitro activation-contraction delay measurements on isolated RV papillary muscle, and both Q-Sm time and activation-contraction delay were correlated with PHT severity. We demonstrated that Q-Sm time measured in DTI was an easily and convenient index to detect early RV electromechanical coupling alteration in both moderate and severe PHT.

Effect of milrinone on echocardiographic parameters after single dose in Beagle dogs and relationship with drug-induced cardiotoxicity.

The aim of this study was to further investigate the mechanism of development of cardiac lesions occurring under treatment with milrinone in dogs, by using echocardiography for assessing the effects of this drug on cardiac function. Milrinone is a cAMP phosphodiesterase 3 inhibitor having positive inotropic and vasodilatory effects. We treated groups of three dogs with milrinone at a single dose of 0.5 or 1 mg/kg and recorded M-mode and Doppler parameters at different time points before and after treatment. The hearts of the high-dose animals were histopathologically examined. The treatment with milrinone at 1 mg/kg produced mild cardiac lesions at two different locations. In the left ventricle, haemorrhages in the subendocardium and myocardium occurred in all three dogs. In the right atrium, subepicardial haemorrhages occurred in one dog and inflammation of the epicardium was observed in two dogs. These lesions were considered to be related to changes in the cardiac function, which were investigated by echocardiography. Milrinone treatment produced a moderate tachycardia and changes in M-mode parameters indicating an increase in contractility, in particular, a decrease in end-systolic volume, an increase in ejection fraction and an increase in the rate of circumferential fiber shortening. In addition, there was an increase in the maximal aortic flow velocity evaluated by Doppler measurements, which is thought to represent a haemodynamic correlate of an increase in left ventricular contractility. This increase in myocardial work is considered to play a key role in the development of the lesions observed in the left ventricle. Doppler measurements also revealed changes in the right atrioventricular flow, probably resulting from cardiac stimulation produced by milrinone. In particular, there was an increase in the Vmax of the A-wave of the tricuspid flow, suggesting an increase in contractility of the right atrium. This change, by increasing blood flow in atrial wall, may be involved in the induction of the lesions observed in the right atrium. In conclusion, Doppler and M-mode echocardiography are useful tools to assess haemodynamic changes occurring upon treatment with vasodilators or cardiac stimulants in order to further understand the mechanism of development of cardiac lesions produced by such compounds.

Hepatotoxin-induced hypercreatinaemia and hypercreatinuria: their relationship to one another, to liver damage and to weakened nutritional status.

Hypercreatinuria is a well-known feature of liver and testicular toxicity and we have recently proposed that hepatotoxin-induced hypercreatinuria would arise as a consequence of increased cysteine synthesis associated with the provision of protective substances (glutathione and/or taurine). Here a direct relationship between hepatotoxin-induced hypercreatinaemia and hypercreatinuria is shown and the possible relationships of hepatotoxin-induced hypercreatinaemia and hypercreatinuria to hepatic damage and to weakened nutritional status are examined. Male Sprague-Dawley rats were dosed with a variety of model hepatotoxins at two dose levels per toxin. Blood plasma samples taken at 24 h post-dosing and urine samples collected from 24-31 h post-dosing were analysed by (1)H NMR spectroscopy. Both hypercreatinaemia and hypercreatinuria were found in rats dosed with allyl formate (75 mg/kg), chlorpromazine (30 and 60 mg/kg), alpha-naphthylisothiocyanate (ANIT, 100 mg/kg) and thioacetamide (200 mg/kg), whilst significant hypercreatinuria, but not hypercreatinaemia, was found after dosing with thioacetamide (50 mg/kg). Neither hypercreatinaemia nor hypercreatinuria were found after dosing with allyl formate (25 mg/kg), ethionine (300 and 1000 mg/kg) or ANIT (30 mg/kg). Reduced feeding is known to cause hypercreatinuria in rats and, of the four hepatotoxins that induced hypercreatinaemia and hypercreatinuria at the given time-points, two, chlorpromazine and ANIT, also affected nutritional status with ketosis being clearly identifiable from the plasma (1)H NMR spectra. Thus, the creatine changes induced by ANIT and chlorpromazine are potentially attributable, in whole or in part, to reduced feeding rather than to liver effects alone and, consequently, the results were examined with and without inclusion of the ANIT and chlorpromazine data. With all of the data included, there were eight out of ten points of correspondence between the incidence of hypercreatinaemia and/or hypercreatinuria and the incidence of increases in plasma alanine aminotransferase (ALT) activity. At the same time there were nine out of ten points of correspondence between the incidence of hypercreatinaemia and/or hypercreatinuria and the incidence of increases in plasma aspartate aminotransferase (AST) activity. However, with the ANIT and chlorpromazine data excluded there was complete (six out of six points) correspondence between the incidence of hypercreatinaemia and/or hypercreatinuria and the incidence of increases in plasma AST and ALT in the remaining data. Likewise, with all of the data included, there was some apparent correlation (correlation coefficient, r=0.80) between the group mean levels of plasma AST and plasma creatine when expressed relative to the mean values for controls sampled at the same time-point. However, with the ANIT and chlorpromazine data excluded, that correlation coefficient was increased to 0.95. The findings of these studies suggest that the ANIT- and chlorpromazine-induced creatine changes may have been caused by reduced feeding rather than by liver toxicity. The allyl formate and thioacetamide data indicate that hepatocellular necrosis is accompanied by increases in plasma and urinary creatine, and suggest the possibility of a quantitative relationship between the increases in plasma AST and the increases in plasma creatine that are associated with hepatocellular necrosis. The ethionine and ANIT data suggest that fatty liver (steatosis) and cholestatic damage may not be associated with hypercreatinaemia and hypercreatinuria.

Use of M-mode and Doppler echocardiography to investigate the cardiotoxicity of minoxidil in beagle dogs.

Doppler and M-mode echocardiography (EC) were used to investigate the effects of minoxidil on the cardiac function of the dog and potentially to clarify the pathogenesis of cardiac lesions, in particular the necrotic lesion in the left ventricle and the haemorrhagic lesion in the right atrium. Groups of three dogs were treated with a single oral dose of 0.5 or 2 mg/kg minoxidil or control vehicle, and M-mode and Doppler parameters were recorded at different time points before as well as 1, 3 and 24 h after treatment. The treatment produced a number of changes in M-mode parameters that indicate an increase in left ventricle contractility, in particular, increases in the percentage of thickening of the left ventricle wall during systole and in ejection fraction, and decrease of systolic volume. There was also a decrease in diastolic volume, which indicates a decrease in filling of the left ventricle probably due to the tachycardia and subsequent decrease in inter-systolic time. Doppler EC showed an increase in the velocity of the aortic flow, which indicates an increase in cardiac contractility. There was also a mild increase in stroke volume, which together with the tachycardia resulted in a marked increase in cardiac output. Together, Doppler and M-mode recordings gave evidence of an increase in the contractility of the left ventricle. This change is consistent with the generally accepted mechanism for the development of the left ventricle lesion induced by minoxidil. Minoxidil also produced changes in atrio-ventricular flows. The velocity and/or acceleration of E- and A-waves of the mitral and tricuspid flows increased, and the E/A ratio decreased. The changes in the E-wave indicate a faster diastole of the ventricle probably to compensate for the decrease in inter-systolic time. The changes in A wave are characteristic of an increased amplitude and velocity of the atrial contraction. This latter change is much more marked for tricuspid than for mitral flow. For both flows the E/A ratio decreased, which indicates that the contraction of the atria plays an increased role in ventricle filling after minoxidil treatment. This stimulation of atrial contraction that we evaluate with Doppler EC may play a key role in the development of the atrial lesion produced by minoxidil. The fact that the change is more marked in the right than in the left atrium may explain why the lesion occurs only in the right atrium in dogs. This study showed, therefore, that Doppler EC associated with M-mode EC is a useful method for obtaining pertinent information on the pathogenesis of the left ventricle lesion induced by haemodynamic mechanisms. Moreover, Doppler EC allowed the assessment of changes in the function of the right atrium that may be involved in the development of the right atrial lesion.

An hypothesis for a mechanism underlying hepatotoxin-induced hypercreatinuria.

As part of a wider metabonomic investigation into the early detection and discrimination of site-specific hepatotoxicity, male Sprague-Dawley rats were dosed with the model hepatotoxins allyl formate, ethionine and alpha-naphthylisothiocyanate (ANIT). Urine samples collected pre- and post-dose were examined by (1)H nuclear magnetic resonance (NMR) spectroscopy and the toxin-induced changes in urinary taurine and creatine excretion were quantified. Hypertaurinuria and hypercreatinuria were observed following allyl formate dosing, hypertaurinuria with no change in creatine excretion was observed after ethionine dosing, and hypotaurinuria and hypercreatinuria were observed after ANIT dosing. These changes are indicative of different effects on liver and it has been previously suggested that some hepatotoxin-induced changes in urinary taurine excretion may be due to altered hepatic cysteine utilisation. A related hypothesis is now presented that would explain the selective hypercreatinuria in terms of increased cysteine synthesis.

Type B Ventricular Preexcitation With Abnormal Contraction of the Ventricular Septum in a Dog.

A 14monthold female beagle had ventricular preexcitation (VP).The finding was characterized by a wide positive QRS complex with a tall notched R wave in leads I, II, III, and aVF, an inverted QRS complex in leads aVR and aVL, a Q wave in lead I, and a short PR interval. Compression of the carotid sinus caused an anticipated marked decrease in heart rate, but did not reveal latent electrocardiographic abnormalities. We did not detect evidence of ventricular hypertrophy during echocardiography. Examination of the M-mode image indicated abnormal movements of the septum. There were 2 sharp waves at each systole instead of a single wider wave that is seen for clinically normal dogs. To further characterize this ECG finding, the affected dog and 2 clinically normal female beagles (positive control dogs) were given atropine (0.025 mg/kg of body weight, i. v.) Increases in heart rate, relative to values obtained before atropine administration, were evident in all 3 dogs. Increase in heart rate in the dog with VP appeared sooner after injection than in the clinically normal dogs; it was evident at the conclusion of the atropine injection. When the increase in heart rate was maximal in the affected dog (3 min after atropine administration), notching of the R wave disappeared, and the QRS duration decreased to about 60 ms. Echocardiographically, atropine produced a decrease in end diastolic, end systolic and stroke volumes in all treated dogs, which was similar between clinically normal dogs and the dog with VP. Atropine administration also was associated with a decrease in the percentage of thickening of the septum in the dog with VP, but not in the clinically normal dogs. We did not detect histopathologic abnormalities in the heart of the dog with VP.