Implementation of the European health interview survey (EHIS) into the German health update (GEDA).

BACKGROUND: This methodological paper describes the integration of the 'European Health Interview Survey wave 2' (EHIS 2) into the 'German Health Update' 2014/2015 (GEDA 2014/2015-EHIS). METHODS: GEDA 2014/2015-EHIS is a cross-sectional health survey. A two-stage stratified cluster sampling approach was used to recruit persons aged 15 years and older with permanent residence in Germany. Two different modes of data collection were used, self-administered web questionnaire and self-administered paper questionnaire. The survey instrument implemented the EHIS 2 modules on health status, health care use, health determinants and social background variables and additional national questions. Data processing was conducted according to the quality and validation rules specified by Eurostat. RESULTS: In total, 24,824 questionnaires were completed. The response rate was 27.6%. The two-stage cluster sample method seems to have been successful in achieving a sample with high representativeness. The final micro data file was inspected, approved and certified by Eurostat. Access to micro data of the EHIS 2 can be provided by Eurostat via research contract and to the GEDA 2014/2015-EHIS public use file by the Research Data Centre of the Robert Koch Institute. First EHIS 2 results are available at the Eurostat website. CONCLUSIONS: Integrating a multinational health survey into an existing national health monitoring system was a challenge in Germany. The national survey methodology for conducting the survey had to be further developed in order to meet the overarching goal of harmonizing the health information from national statistical offices and public health research institutes across the European Union. The harmonized EHIS 2 data source will profoundly impact international public health research in the near future. The next EHIS wave 3 will be conducted around 2019.

Race to accumulate evidence for few and many saccade alternatives: an exception to speed-accuracy trade-off.

Hick's law states that increasing the number of response alternatives increases reaction time. Lawrence and colleagues report an exception to the law, whereby more alternatives lead to shorter saccadic reaction times (SRTs). Usher and McClelland (Psychol Rev 108(3):550-592. doi: 10.1037/0033-295X.108.3.550 , 2001) predict such an anti-Hick's effect when accuracy is not prioritized in a task, which should result in higher error rates with more response alternatives, and in turn to a shorter right tail of the SRT distribution. In the current study, we aim to replicate the original controversial findings and we compare them to these predictions by examining error rates and SRT distributions. Two experiments were conducted where participants made rapid eye movements to one of few or many alternatives. In Experiment 1, the saccade target was an onset and participants started either with few or many possible target locations and then alternated between conditions. An anti-Hick's effect emerged only when participants had started with a small set-size block. In Experiment 2, placeholders were displayed at the possible target locations and independent groups were used. A reliable anti-Hick's effect in SRTs was observed. However, results did not meet the stated predictions: anticipations and false direction errors were never more frequent when the set size was larger and SRT differences between the two set-size conditions were not more pronounced at the slower end of the distributions. In line with Lawrence and colleagues, we speculate that initial motor preparation, and the subsequent inhibition to counteract a premature response, may induce the anti-Hick's effect.

Antibiotic Bactericidal Activity Is Countered by Maintaining pH Homeostasis in Mycobacterium smegmatis.

Antibiotics target specific biosynthetic processes essential for bacterial growth. It is intriguing that several commonalities connect the bactericidal activity of seemingly disparate antibiotics, such as the numerous conditions that confer broad-spectrum antibiotic tolerance. Whether antibiotics kill in a manner unique to their specific targets or by a universal mechanism is a critical and contested subject. Herein, we demonstrate that the bactericidal activity of diverse antibiotics against Mycobacterium smegmatis and four evolutionarily divergent bacterial pathogens was blocked by conditions that worked to maintain intracellular pH homeostasis. Single-cell pH analysis demonstrated that antibiotics increased the cytosolic pH of M. smegmatis, while conditions that promoted proton entry into the cytosol prevented intracellular alkalization and antibiotic killing. These findings led to a hypothesis that posits antibiotic lethality occurs when antibiotics obstruct ATP-consuming biosynthetic processes while metabolically driven proton efflux is sustained despite the loss of proton influx via ATP synthase. Consequently, without a concomitant reduction in respiratory proton efflux, cell death occurs due to intracellular alkalization. Our findings indicate the effects of antibiotics on pH homeostasis should be considered a potential mechanism contributing to antibiotic lethality. IMPORTANCE Since the discovery of antibiotics, mortality due to bacterial infection has decreased dramatically. However, infections from difficult to treat bacteria such as Mycobacterium tuberculosis and multidrug-resistant pathogens have been on the rise. An understanding of the cascade of events that leads to cell death downstream of specific drug-target interactions is not well understood. We have discovered that killing by several classes of antibiotics was stopped by maintaining pH balance within the bacterial cell, consistent with a shared mechanism of antibiotic killing. Our findings suggest a mechanism of antibiotic killing that stems from the antibiotic's ability to increase the pH within bacterial cells by disrupting proton entry without affecting proton pumping out of cells. Knowledge of the core mechanism necessary for antibiotic killing could have a significant impact on the development of new lethal antibiotics and for the treatment of recalcitrant and drug-resistant pathogens.

[Health status of people with a migrant background and impact of socio-economic factors: First results of the German Health Interview and Examination Survey for Adults (DEGS1)]. / Die gesundheitliche Lage von Menschen mit Migrationshintergrund und die Bedeutung des sozioökonomischen Status : Erste Ergebnisse der Studie zur Gesundheit Erwachsener in Deutschland (DEGS1).

People with a migrant background (PMB) have specific health-related risk factors and resources compared to the non-migrant population (NMP). The analysis focuses on the relationship between migrant background and health and health-related behavior. Moreover, the study analyses whether socio-economic status (SES) contributes to the explanation of differences between PMB and the NMP. The research is based on the German Health Interview and Examination Survey for Adults (DEGS1) (2008-2012, n = 8151). The population for cross-sectional analyses contains 1107 PMB (weighted 19.8 %). The research question is addressed on the basis of nine exemplary health outcomes. All analyses are gender specific and make a distinction between first and second generation PMB. Logistic regression is calculated adjusting for age and SES. The results reveal clear gender-specific patterns: For women, differences are statistically significant mainly for first generation PMB. Compared to the NMP their self-assessed health status is lower, they are less physically active, consume less alcohol, feel less informed about cancer screening programs and make less use of preventive health services. However, daily smoking is more prevalent in second generation women. For men, differences are statistically significant for first and second generation PMB. Men with a migrant background show more symptoms of depression, consume less alcohol and feel less informed about cancer screening programs. After adjusting for SES the impact of migrant background on health status and health-related behavior largely remains stable. The study shows that the DEGS1 data offers valuable results and new insights into the health status of people with a migrant background. The use of this data for further research requires a differentiated approach to the concept of migrant background and a careful interpretation of results.

Mechanical strength of specific bonds acting isolated or in pairs: a case study on engineered proteins.

The dynamic strength of multiple specific bonds exposed to external mechanical force is of significant interest for the understanding of biological adhesion. Exploiting the well-established FLAG tag technology, we engineered model proteins exhibiting no, one, or two identical binding sites for a monoclonal antibody. Bonds between these engineered proteins and the antibody were studied with dynamic force spectroscopy. On single bonds between a FLAG-tag and the antibody, we observed two regimes corresponding to two different activated complexes, that is, two intermediate states along the reaction path for bond breakage. Dynamic force spectroscopy on double bonds showed the same two regimes. The actual yield forces of double bonds slightly exceeded those of single bonds. A simplified kinetic model with analytical solutions was developed and used to interpret the measured spectra.

Longitudinal single-cell cytokine responses reveal recurrent autoimmune myelin reactivity in relapsing--remitting multiple sclerosis patients.

The relationship between multiple sclerosis (MS) disease activity and myelin protein-induced cytokine responses over time is not elucidated. We addressed this relationship by examining longitudinal cytokine responses to myelin proteins every three months for one year, in the context of gadolinium (gad)-enhancing brain lesions and of clinical relapses. The ELISPOT assay was used to determine the ex vivo cytokine production in response to nine amino acid long peptides spanning the entire proteolipid protein (PLP) and myelin basic protein (MBP) molecules in relapsing-remitting (RR) MS patients and matched healthy controls. We identified three longitudinal levels of myelin-induced cytokine secretion by adding up the positive responses for all PLP or MBP peptides obtained for five timepoints, at three-month intervals: low reactivity (< 200 cumulative cytokine-secreting cells), isolated peptide reactivity (201-450 cumulative cytokine-secreting cells) and recurrent protein-wide bursts of cytokine reactivity (> 451 cumulative cytokine-secreting cells). The majority of MS patients showed recurrent bursts to PLP and MBP. In contrast, controls showed a more even distribution between all levels of cytokine reactivity. The majority of patients with gad-enhancing lesions showed PLP/IFN gamma and MBP/IFN gamma recurrent burst responses. This is the first longitudinal study on MS patients in which nine amino acid long myelin peptides are used to reveal the broad range of PLP- and MBP-peptide cytokine reactivity across the whole molecule of these two major myelin proteins. This study also reveals the extremely dynamic nature of the immune reactivity to numerous regions of myelin, which can fluctuate dramatically over time. Such fluctuation could hamper the efficacy of antigen-based therapies for MS.

[Pyoderma gangrenosum and orbital pseudotumor]. / Pyroderma gangraenosum und Pseudotumor orbitae.

Pyoderma gangrenosum belongs to the chronic, neutrophilic and necrotic dermatoses. These very painful, inflammable and purulent skin-ulcers often arise after injury or surgical intervention. About half of the patients suffer from systemic disease, for example hematological, gastrointestinal or rheumatic diseases. Our report includes a 77-year-old female with a multilocular emergence of pyoderma gangrenosum following surgery for an orbital pseudotumor. An additional lesion was detected on her anterior neck. Because the possibility of wound-infection was suspected, the lesion was treated locally and with a systemic antibiotic therapy. After this treatment the condition of the wound deteriorated. Upon dermatological examination, pyoderma gangrenosum was identified and a immunosuppressive therapy with corticosteroids and cyclosporin was initiated. As a result of this treatment the lesion healed slowly--with the formation of typical, atrophic and reticular scars. A connection between the pyoderma gangrenosum and the orbital pseudotumor can, on the basis of similar clinical and structural characteristics, not be excluded. A related disease was not diagnosed in this patient. Recurrence can be predicted in patients who have had skin injuries, therefore strict observation is required.

Comparative metabolism and kinetics of coumarin in mice and rats.

Coumarin, a well recognized rat hepatotoxicant, also causes acute, selective necrosis of terminal bronchiolar Clara cells in the mouse lung. Further, chronic oral gavage administration of coumarin at 200 mg/kg, a dose that causes Clara cell death, resulted in a statistically significant increased incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. In contrast, mouse lung tumors were not observed at the 100 and 50 mg/kg dose levels in the oral gavage study, or in CD-1 mice following chronic intake of coumarin at levels equivalent to 276 mg/kg in diet. The current studies were designed to determine the impact of oral gavage vs dietary administration on the pharmacokinetics and metabolism of coumarin in CD-1 and B6C3F1 mice and F344 rats. Following the administration of 200 mg/kg 14C-coumarin via oral gavage, lung C(max) values (total 14C-associated radioactivity) were five- and 37-fold greater than those resulting from a 50 mg/kg oral gavage dose or 1000 ppm in diet, respectively. Coumarin (200 mg/kg) pharmacokinetics and metabolism was also examined in F344 rats following oral gavage dosing. Total 14C-coumarin associated radioactivity in plasma was 3.5-fold lower than in the mouse, and the plasma half-life in rats was five-times longer than in mice. Using non-radiolabeled compound (200 mg/kg), coumarin and products of the coumarin 3,4-epoxidation pathway were quantitated in plasma and urine after oral gavage administration to mice and rats. 7-Hydroxycoumarin (7-HC) was quantitated in mouse plasma and urine. o-Hydroxyphenylacetic acid (o-HPAA) reached a concentration of 37 microg/ml in plasma, and accounted for 41% of the dose in the urine, whereas the C(max) for 7-hydroxycoumarin was 3 microg/ml, and represented 7% of the administered dose. In the rat, the plasma C(max) for o-HPAA was 6 microg/ml, and accounted for 12% of the dose. The coumarin C(max) in rat plasma was comparable to that in mouse. Coumarin 3,4-epoxide (CE) and its rearrangement product o-hydroxyphenylacetaldehyde (o-HPA) and o-hydroxyphenylethanol (o-HPE), were not detected at any time point in plasma or urine. This analysis of coumarin and CE pharmacokinetics in rodents suggests that the differential tumor response in the mouse oral gavage and dietary bioassays is a function of the route of exposure, whereas species differences in lung toxicity between mice and rats result from heightened local bioactivation in the mouse lung.

Pyoderma gangraenosum und Pseudotumor orbitae.

Pyoderma gangrenosum belongs to the chronic, neutrophilic and necrotic dermatoses. These very painful, inflammable and purulent skin-ulcers often arise after injury or surgical intervention. About half of the patients suffer from systemic disease, for example hematological, gastrointestinal or rheumatic diseases.Our report includes a 77-year-old female with a multilocular emergence of pyoderma gangrenosum following surgery for an orbital pseudotumor.An additional lesion was detected on her anterior neck. Because the possibility of wound-infection was suspected, the lesion was treated locally and with a systemic antibiotic therapy. After this treatment, the condition of the wound deteriorated. Upon dermatological examination, pyoderma gangrenosum was identified and a immunosuppressive therapy with corticosteroids and cyclosporin was initiated. As a result of this treatment, the lesion healed slowly - with the formation of typical, atrophic and reticular scars.A connection between the pyoderma gangrenosum and the orbital pseudotumor can, on the basis of similar clinical and structural characteristics, not be excluded. A related disease was not diagnosed in this patient. Recurrence can be predicted in patients who have had skin injuries, therefore strict observation is required.

[Postoperative pyoderma gangrenosum]. / Das postoperative Pyoderma gangraenosum.

Post-traumatic pyoderma gangrenosum is an ulceronecrotizing dermatosis that is rare and therefore often misinterpreted. After excision of an orbit pseudotumor by otorhinolaryngological surgeons, a 77-year-old woman developed a multifocal ulcerous wound dehiscence. Although the case history, development and clinical picture were characteristic, the correct diagnosis was established and successfully treated 6 months later by a consulting dermatologist. Postoperative pyoderma gangrenosum should be taken into consideration if a sterile, painful, chronically progressive, ulcerative and sludgy defect develops after surgery. The preoperative investigation should include pyoderma gangrenosum in the personal history and take associated diseases into account (e.g., inflammatory bowel diseases, blood diseases, rheumatological diseases and vasculitis) to provide prophylactic immunosuppressive pharmacotherapy. In minimally invasive or cosmetic surgery these aspects may have a forensic impact, as pyoderma gangrenosum heals with atrophic scars. The purpose of this paper was to describe the clinical characteristics of pyoderma gangrenosum, its prevention and its therapy.

In vitro kinetics of coumarin 3,4-epoxidation: application to species differences in toxicity and carcinogenicity.

Coumarin, a natural product and fragrance ingredient, is a well recognized rat liver toxicant, and dietary administration at toxic dosages increased the incidence of rat cholangiocarcinomas and parenchymal liver-cell tumors in a chronic bioassay. Hepatotoxicity in rats is site- and species-specific, and is thought to result from the formation of coumarin 3,4-epoxide and its rearrangement product, o-hydroxyphenylacetaldehyde (o-HPA). The goals of the current study were to describe the in vitro kinetics of the metabolic activation of coumarin, and determine whether species differences in susceptibility to liver injury correlate with coumarin bioactivation determined in vitro. Coumarin 3,4-epoxidation was quantified via the formation of o-HPA in pooled hepatic microsomes from female B6C3F1 mice, male F344 rats, and individual humans (n = 12 subjects), and the apparent kinetic constants for o-HPA production were calculated using nonlinear regression and fitting to either a one-enzyme or two-enzyme model. Eadie-Hofstee analyses indicated that o-HPA formation was biphasic in both rat and mouse liver. Although the apparent high affinity K:(m) in rat and mouse liver microsomes was 38.9 and 47.2 microM, respectively, the overall rate of o-HPA formation was far greater in mouse than in rat liver microsomes. Furthermore, the total clearance (CL(int)) of coumarin via o-HPA formation in mouse liver microsomes was 4-fold greater than in rat liver microsomes. Since mice are relatively resistant to hepatotoxicity, the data indicated that rates of o-HPA formation in rat and mouse liver microsomes were not directly predictive of liver toxicity in vivo, and further suggested that o-HPA detoxification played a role in modulating coumarin-mediated toxicity. The current studies also indicated that coumarin 3,4-epoxidation in human hepatic microsomes was minimal. In human liver microsomes (n = 12), the kinetics of o-HPA formation were best described by a single enzyme model, with the K(m) for o-HPA formation ranging from 1320-7420 microM. In the most active human sample, the intrinsic clearance of coumarin via the 3,4-epoxidation pathway was 1/9 and 1/38 that of the rat and mouse, respectively. The in vitro kinetics of o-HPA formation, and in particular, the large quantities of coumarin required for o-HPA production in human liver microsomes, strongly suggest that humans are unlikely to produce toxicologically relevant concentrations of this metabolite following low level coumarin exposures.

o-hydroxyphenylacetaldehyde is a hepatotoxic metabolite of coumarin.

o-Hydroxyphenylacetaldehyde (o-HPA), the product of coumarin 3, 4-epoxide, was synthesized and its contribution to the hepatotoxic effects of coumarin in the rat was determined. The relative toxicity of coumarin and o-HPA were initially assessed in Chinese hamster ovary K1 (CHO K1) cells, a cell line that does not contain cytochrome P450. In CHO K1 cells, o-HPA-mediated toxicity greatly exceeded that of coumarin. CHO K1 cell viability, determined via the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), was decreased by 95 and 6% in cultures containing o-HPA and coumarin (4 mM), respectively. Coumarin and o-HPA were then incubated in metabolically competent primary rat hepatocyte cultures. Cell viability was determined via the reduction of MTT, and lactic dehydrogenase (LDH) release was used as a measure of cytotoxicity. Concentration-dependent decreases in cell viability and increased LDH release were observed using 0.2 to 0.8 mM o-HPA and coumarin, with coumarin being consistently less toxic than o-HPA. Cell viability was decreased by 11 and 50% at 0.5 mM coumarin or o-HPA, respectively. Hepatocyte LDH release increased 5-fold after a 6-h exposure to 0.8 mM o-HPA, corresponding to a greater than 90% loss of cell viability in these cultures. In contrast, 0.8 mM coumarin decreased cell viability by 60%, an effect likely due to the conversion of coumarin to coumarin epoxide and o-HPA. Furthermore, 3-hydroxycoumarin (0.8 mM), which is not a product of coumarin epoxidation, had no effect on cell viability or hepatocellular LDH release. These studies demonstrate that metabolically active rat hepatocytes convert coumarin into toxic metabolites, and strongly suggest that o-HPA and coumarin 3, 4-epoxide mediate the toxicity of coumarin in rodents in vivo.

Metastasising malignant lymphoma mimicking necrotising and hyperplastic gingivostomatitis.

This paper presents the case of a 65 year-old woman suffering from recurrent oral aphthoid ulcers which rapidly evolved towards hyperplastic and ulcerated lesions over the entire floor of the mouth. The initial lesions were interpreted as non-specific aphthoid ulcers. Later, a tentative diagnosis of necrotising stomatitis with secondary reactive proliferating epithelial hyperplasia was made. The clinical symptoms and the immuno-phenotyping of lymphocytes circulating in the peripheral blood suggested the diagnosis of CD30-positive large cell anaplastic lymphoma. The biopsy showed only a pseudoepitheliomatous hyperplasia, reactive infiltrates and no lymphoma cells. The disease ran a fulminant course leading to death within 4 weeks due to acute gastro-intestinal bleeding. Autopsy revealed infiltrates of CD30+ large cell anaplastic lymphoma in a submandibular lymph node, in a thrombus stenosing the right subclavian vein, in the spleen, the anterior and posterior gastric wall as well as in the depth of the tumour on the floor of the mouth. The clinical and histopathological spectrum of CD30+ large cell anaplastic lymphoma is considerably variable. The particular feature of pseudoepitheliomatous hyperplasia has been reported especially in CD30+ anaplastic large cell lymphomas. An early correct diagnosis is rendered difficult in insufficient biopsy size, becauses this type of lymphoma often simulates other inflammatory or neoplastic skin diseases. Thus, with a necrotising and hyperplastic gingivostomatitis, the diagnosis of a CD30+ anaplastic large cell lymphoma should be considered.

Development of tolerance to Clara cell necrosis with repeat administration of coumarin.

Coumarin was identified as a mouse-lung carcinogen following oral gavage administration in a chronic bioassay, and was shown to cause the selective necrosis of terminal bronchiolar Clara (non-ciliated bronchiolar epithelial) cells in the mouse lung after acute administration. After oral gavage, a similar effect was not observed in the terminal bronchioles of rats, suggesting that coumarin-mediated Clara cell toxicity is a species-specific effect. Using coumarin dosages (50 and 200 mg/kg) and a dosing schedule modeled after the chronic bioassay, the current study examined the effects of repeated coumarin administration in mouse lung. A single dosage of coumarin (200 mg/kg) caused swelling of Clara cells and necrosis in mouse-lung terminal bronchioles. However, after 5 consecutive oral doses of coumarin (200 mg/kg), the mouse lung became tolerant to coumarin, and although areas of bronchiolar epithelial flattening and hyperplasia were noted, Clara cell necrosis was not observed. After 10 doses of coumarin, mouse lungs appeared nearly normal. Coumarin-mediated Clara cell injury is thought to result from the cytochrome P450-catalyzed formation of coumarin 3,4-epoxide and Western analysis of whole mouse lung microsomal P450 content indicated that, commensurate with Clara cell necrosis, many P450s were decreased. However, P450 levels appeared qualitatively normal in lung microsomes from tolerant mice. Similarly, coumarin epoxidation and 7-hydroxylation rates in whole lung microsomes from tolerant animals were similar to controls. To determine if animals tolerant to coumarin were tolerant to other Clara cell toxicants, a single toxic dose of naphthalene (200 mg/kg) was administered to coumarin-tolerant mice. Coumarin pretreatment reduced naphthalene-mediated Clara cell toxicity, supporting the hypothesis that tolerance may result from general biochemical and molecular changes and not exclusively from alterations in chemical metabolism.

Baroreflex sensitivity and heart rate variability in coronary disease compared to dilated cardiomyopathy.

This study was designed to compare baroreceptor sensitivity and heart rate variability as measures of cardiac autonomic tone in patients with coronary disease (CAD, n = 49) and idiopathic dilated cardiomyopathy (IDC, n = 130). Time domain heart rate variability, including SDNN, SDANN, and pNN50, was determined during 24-hour Holter ECG. Baroreflex sensitivity was analyzed nonivasively using the phenylephrine method. Baroreflex sensitivity and heart rate variability were comparable between patients with CAD versus IDC (baroreflex sensitivity: 6.1 +/- 3 vs 6.9 +/- 5 ms/mmHg; SDNN: 97 +/- 40 vs 114 +/- 41 ms; SDANN: 83 +/- 33 vs 99 +/- 41 ms; pNN50: 3.9 +/- 4 vs 9.6 +/- 13 ms, P = NS for all comparisons). Likewise, a subgroup analysis of patients with a left ventricular ejection fraction (LVEF) < or = 30% showed no significant difference in baroreceptor sensitivity and heart rate variability between IDC and CAD patients. Patients with CAD and an LVEF > 30% had a decreased heart rate variability but not a decreased baroreflex sensitivity compared to patients with IDC and LVEF > 30% (baroreflex sensitivity: 6.4 +/- 4 vs 8.3 +/- 6 ms/mmHg, P = NS; SDNN: 98 +/- 19 vs 128 +/- 42 ms, P < 0.05; SDANN: 86 +/- 21 vs 112 +/- 43 ms, P < 0.05; pNN50: 4.2 +/- 3 vs 12.3 +/- 8 ms, P < 0.05). Patients with a markedly depressed LVEF show comparable alterations in cardiac autonomic tone whether they have CAD or IDC. Patients with CAD and preserved LV function, however, have a decreased heart rate variability compared to patients with IDC and preserved LV function. The prognostic significance of these findings will be determined prospectively in a large patient cohort at our institution.

Noninvasive arrhythmia risk stratification in idiopathic dilated cardiomyopathy: design and first results of the Marburg Cardiomyopathy Study.

The Marburg Cardiomyopathy Study (MACAS) is a prospective, observational study designed to determine the value of the following potential noninvasive arrhythmia risk predictors in at least 200 patients with idiopathic dilated cardiomyopathy (IDC) over a 5-year follow-up period: NYHA-class, left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter, left bundle branch block and atrial fibrillation on ECG, QT/JT dispersion on 12-lead ECG, signal-averaged ECG, ventricular arrhythmias and heart rate variability (HRV) on 24-hour Holter ECG, baroreflex sensitivity, and microvolt T wave alternans during exercise. This article describes the findings among the first 159 patients with IDCs enrolled in MACAS until May 1998 (40 women, 119 men; age: 49 +/- 12 years; LVEF: 32 +/- 10%). Twenty-nine patients (18%) had atrial fibrillation and 130 patients (82%) were in sinus rhythm. Patients with sinus rhythm were further stratified according to LVEF < 30% (n = 54) versus LVEF > or = 30% (n = 76). Compared to patients with LVEF > or = 30%, patients with LVEF < 30% more often had left bundle branch block (43% vs 25%, P < 0.05), nonsustained VT (44% vs 22%, P < 0.05), decreased HRV (SDNN: 95 +/- 39 vs 128 +/- 42 ms, P < 0.01), decreased baroreflex sensitivity (5.6 +/- 4 vs 8.3 +/- 6 ms/mmHg, P < 0.01), and T wave alternans (59% vs 37%, P < 0.05). The prognostic significance of these findings will be determined by multivariate Cox analysis at the end of a 5-year follow-up. Primary endpoints in MACAS are overall mortality and arrhythmic events (i.e., sustained VT or VF, or sudden cardiac death).

Selective Clara cell injury in mouse lung following acute administration of coumarin.

Coumarin is a known hepatotoxicant in laboratory animals, particularly rats. However, the mouse lung was identified as a major target organ in a chronic bioassay, with an oral gavage dosage of 200 mg/kg coumarin increasing the incidence of alveolar/bronchiolar adenomas and carcinomas. The purpose of the present work was to determine whether coumarin was acutely toxic in the mouse and rat lung. Male and female B6C3F1 mice were dosed orally by gavage with coumarin at 0, 10, 20, 50, 100, 150, and 200 mg/kg and lung toxicity was determined 24 h later by histological evaluation. The results indicated that coumarin dosages >/= 150 mg/kg caused selective injury to Clara cells in the distal bronchiolar epithelium. The time course of this injury was studied from 6 h to 7 days after a single dosage of coumarin (200 mg/kg). At 12 h after dosing, Clara cell swelling was apparent along with the onset of necrosis and bronchiolar epithelial disorganization. At 24-48 h, necrotic Clara cells were observed sloughed into the lumens of the terminal bronchioles, with concomitant thinning of the epithelium and flattening of the remaining ciliated cells. By 72-96 h, there was epithelial hypertrophy and hyperplasia, and by 7 days after dosing, the Clara cells had regenerated and the bronchiolar epithelial architecture appeared nearly normal. Unlike the mouse, oral administration of coumarin (200 mg/kg) caused severe hepatotoxicity in male F344 rats, seen histologically as centrilobular necrosis and associated with increases, up to 140-fold, in serum ALT, AST, and SDH levels. Clara cell toxicity was not observed in the distal bronchioles of treated rats. However, in the upper airways, coumarin treatment produced generalized epithelial necrosis involving both ciliated and nonciliated cells. 3,4-Dihydrocoumarin (DHC), which is not a mouse lung carcinogen, did not cause Clara cell injury when dosed to mice at 800 mg/kg. This finding suggests, because DHC lacks a 3,4-double bond, that bioactivation of coumarin to a 3,4-epoxide intermediate may contribute to mouse lung Clara cell toxicity. Collectively, the results indicate that coumarin is a Clara cell toxicant and establish the mouse lung as a target organ for coumarin toxicity. These new findings lay the foundation for studies to determine the mechanisms of coumarin-induced toxicity and carcincogenicity and to define the relevance of these effects to humans.

Synthesis and reactivity of coumarin 3,4-epoxide.

Coumarin is used widely as a fragrance constituent and is administered clinically in the treatment of certain lymphedemas and malignancies. Although toxicity occurs only rarely in humans treated clinically with high-dose coumarin, it is well established that coumarin is hepatotoxic in the rat. This species difference in susceptibility to toxicity reflects the disparate metabolic processes occurring in humans and rodents. In humans, coumarin is converted extensively via cytochrome P450 2A6 to the nontoxic 7-hydroxycoumarin metabolite. In contrast, coumarin 3,4-epoxidation is thought to predominate in rodent species, resulting in the formation of several potentially toxic metabolites. Coumarin epoxide is thought to be highly unstable and has not been isolated synthetically or as a microsomal product. To address this issue, coumarin 3,4-epoxide was synthesized, and its stability and fate have been determined. Coumarin 3,4-epoxide was prepared by reacting coumarin with dimethyldioxirane. The epoxide was stable in organic solvents and survived conditions required for analysis by gas chromotography. Its structure was confirmed via 1H-NMR and gas chromatography-mass spectrometry-infrared spectroscopy (GC-MS-IR). In contrast, coumarin 3,4-epoxide was unstable in aqueous solution, converting within 20 sec to a ring-opened compound. Using GC-MS-IR analysis, the single coumarin 3,4-epoxide product was identified as o-hydroxyphenylacetaldehyde (o-HPA). Although other investigators have suggested that 3-hydroxycoumarin is an intermediate in o-HPA formation from coumarin 3,4-epoxide, we have demonstrated that 3-hydroxycoumarin, incubated in an aqueous system or with liver microsomal proteins, does not form o-HPA. Thus, the results of the present work establish that coumarin 3,4-epoxide can be synthesized and that o-HPA, which has previously been shown to be a prominent coumarin metabolite in rat liver microsomal incubations, is formed directly from coumarin 3,4-epoxide. These results suggest that both coumarin 3,4-epoxide and o-HPA may contribute to the hepatotoxicity of coumarin.

Escherichia coli expression and substrate specificities of canine cytochrome P450 3A12 and rabbit cytochrome P450 3A6.

High level Escherichia coli expression of cytochromes P450 3A12 and 3A6 has facilitated the characterization of proteins which exhibit limited activity as purified hepatic enzymes in reconstituted systems. Three 3A12 and two 3A6 constructs modified at the 5'-end to encode the bovine 17 alpha-sequence (Barnes et al., Proc. Natl. Acad. Sci. U.S.A. 88: 5597-5601, 1991), or related sequences, exhibited expression levels ranging from 2 to 89 nmol of cytochrome P450 liter-1. Recombinant canine 3A12 catalyzed steroid 6 beta-hydroxylation and erythromycin demethylation at rates comparable to those obtained in phenobarbital-induced canine liver microsomes. In contrast, 3A12 troleandomycin demethylase activity (2.5 nmol/min/nmol) was significantly lower than that of canine phenobarbital-induced liver microsomes (6.6 nmol/min/nmol). This difference in activity suggests that at least two 3A forms, which may differ functionally, are present within the canine liver. Purification of recombinant rabbit 3A6 revealed that homogeneous and E. coli-solubilized membrane preparations of 3A6 exhibit similar metabolic rates and identical substrate specificities; 3A activity was modulated by 25 microM alpha-naphthoflavone, which stimulated an unidentified progesterone metabolite 9-fold in 3A6 reconstituted systems in contrast to the 4-fold stimulation of 3A12. Furthermore, 25 microM alpha-naphthoflavone inhibited erythromycin demethylation 64 and 33% by purified recombinant 3A6- or 3A6-solubilized membrane fractions, respectively; 3A12-mediated erythromycin demethylation in solubilized membrane fractions was resistant to flavonoid inhibition. These results indicate that, although 3A substrate specificities are highly conserved between species, functional differences exist between canine 3A12 and rabbit 3A6, which may be utilized to better understand 3A structure-function relationships.

Suppression of rat hepatic microsomal cytochromes P450 by cyclophosphamide is correlated with plasma thyroid hormone levels and displays differential strain sensitivity.

Strain differences in cytochrome P450 (P450) expression were investigated in Sprague-Dawley (SDs) compared with Fischer 344s (F344s) rats after administration of cyclophosphamide (CPA). Animals received a single dose of CPA with sacrifice occurring 6 days post-treatment. At 130 mg/kg, male F344s displayed a greater sensitivity to CPA, as evidenced by a 68% loss of total hepatic microsomal P450 compared with only 35% in SDs. The most dramatic change in P450 was the loss of 2C11 (84% in F344s, 52% in SDs). In the SD, individual rat 2C11 activity was correlated (r2 = 0.76), with the level of plasma thyroxine in that animal. In male F344s administered CPA at 50 mg/kg, 43 and 44% losses in 2C11 activity (P < .05) and thyroxine (P < .01), respectively, were observed, whereas activities characteristic of P450s 2C11, 3A2, 2A2, 2C6 and 2E1/1A2 were unaffected in SDs at this dose. CPA also produced suppression of P450 in female SDs, including female-specific 2C12. Correlation was observed between the loss of P450 expression and change in body weight after treatment in both male and female animals, suggesting that CPA downregulates P450 expression secondary to decreased caloric intake. The anorectic effect of CPA is believed to result from potent central nervous system stimulation, accompanied by a state of adaptive hypothyroidism. It has been reported that CPA produces "feminization" of P450 expression in male rats. However, our findings suggest the alternative explanation that the effects of CPA on P450 expression result from decreased caloric intake.