In vitro effect of r-verapamil on acute myelogenous leukemia blast cells: studies of cytokine secretion and cytokine-dependent blast proliferation

The in vitro effect of the dextroisomer r-verapamil on blast cells derived from patients with acute myelogenous leukemia (AML) was studied. R-verapamil caused a dose-dependent inhibition of AML blast proliferation in the presence of stem-cell factor, leukemia inhibitory factor, interleukin 4, interleukin 6, and interleukin 10 when these cytokines were tested both alone and in different combinations. R-verapamil also inhibited the growth of clonogenic AML blast cells. The antiproliferative effect was not specific for AML blast cells, because r-verapamil also inhibited cytokine-dependent proliferation of blast cells derived from patients with acute lymphoblastic leukemia. The inhibitory effects of r-verapamil and anti-IL1 serum were additive, suggesting that the antiproliferative effect of r-verapamil does not depend solely on inhibition of IL1-mediated effects. Although r-verapamil inhibited spontaneous AML blast proliferation, for a majority of patients it caused only minimal, if any, inhibition of spontaneous cytokine secretion (IL1, IL1, TNF, IL6) by AML blast cells. Thus, although inhibition of IL1 effects may contribute in certain patients to the antiproliferative effect of r-verapamil, mechanisms other than IL1 inhibition seem to be more important in mediating the effects of r-verapamil.Abbreviations ALL Acute lymphocytic leukemia - AML acute myelogenous leukemia - cpm counts per minute - ELISA enzyme-linked immunosorbent assay - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor - IL interleukin - IF leukemia inhibitory factor - PBMC peripheral blood mononuclear cells - RR relative response - SCF stem cell factor - TNF tumor necrosis factor      

Plasma concentrations and renal clearance of orotic acid in argininosuccinic acid synthetase deficiency

Argininosuccinic acid synthetase deficiency (ASD) is a rare disorder of urea cycle metabolism, with pronounced citrullinemia and orotic aciduria being characteristic biochemical features. To further investigate the role of plasma orotic acid and its possible use for monitoring the metabolic status in ASD, we determined plasma orotic acid, amino acid, and ammonium levels in plasma samples collected over a period of 3 years from a patient who is now 8 years of age. Orotic acid plasma concentrations varied widely from less than 1 μmol/l to more than 60 μmol/l. The renal clearance of orotic acid was eightfold the glomerular filtration rate, thus supporting an active mechanism underlying the excretion of this pyrimidine. Data obtained during a metabolic crisis yielded a statistically significant linear correlation of orotic acid plasma levels with those of glutamine and ammonium, which are generally accepted for assessment of the successful treatment of this disorder. Our data revealed no advantage of plasma orotic acid concentrations over the established amino acids (glutamine and arginine) and ammonium for determining acute treatment responses. Since several effects of high levels of orotic acid have been described in mammals, further research is necessary to assess a possible contribution of orotic acid to the pathogenesis of ASD and the use of plasma orotic acid levels in the long-term monitoring of these patients. Received: 3 November 1998 / Revised: 3 May 1999 / Accepted: 3 May 1999     

Detection of polycyclic aromatic hydrocarbon metabolites by high-pressure liquid chromatography after purification on immunoaffinity columns in urine from occupationally exposed workers

Objective: The objective in our study was to quantitate benzo[a]pyrene (B[a]P) metabolites by a combination of immunoaffinity chromatography and high-pressure liquid chromatography (HPLC) with fluorescence detection in urine from workers exposed to high levels of polycyclic aromatic hydrocarbons (PAH). Furthermore, by the simultaneous quantitation of 1-hydroxypyrene, the correlation between the B[a]P-tetrol and 1-hydroxypyrene would provide a means of evaluating the validity of 1-hydroxypyrene as a surrogate biomarker for occupational exposure to the potent carcinogen B[a]P in an electrode paste plant. Methods: The study was carried out at an electrode paste plant that produces electrode paste for Söderberg electrodes. A total of 34 pre- and post-shift urine samples and 17 personal air samples were collected from 17 workers during a normal work week. The concentration of 1-hydroxypyrene was measured in all urine samples. A recent method of quantitating B[a]P-r-7, t-8, t-9, c-10-tetrol in urine of humans exposed to low levels of PAH has been described. A modified version of this method involving purification of urine samples on immunoaffinity columns and HPLC analysis with fluorescence detection was used on urine samples from workers exposed to high levels of PAH. A monoclonal antibody (8E11) with binding affinity to B[a]P-tetrols was used. This antibody also binds several PAH-DNA adducts and metabolites, including 1-hydroxypyrene. Gas chromatography/mass spectroscopy (GC/MS) was also used for identification of metabolites isolated by HPLC fractionation. Results: From personal air sampling the mean exposure to particulate PAHs was 38?μg/m³. The mean concentration of urinary 1-hydroxypyrene was 3.9?μmol/mol creatinine in preshift samples and 10.2?μmol/mol creatinine in postshift samples. We could not identify detectable amounts of urinary B[a]P-tetrol by HPLC or fluorescence spectroscopy after purification on immunoaffinity columns. However, in the HPLC analysis we identified several hydroxyphenantrene metabolites that were detected at relatively high concentrations in all of the workers' urine samples. We could not separate 2- and 3-hydroxyphenanthrene (2?+?3-OH-Phe) in peak 1, and peak 2 contained both 1- and 9-hydroxyphenanthrene (1?+?9-OH-Phe). The phenanthrene metabolites were mainly conjugated to glucuronic acid and sulfate. There was a significant correlation between the 1-hydroxypyrene concentration and 2?+?3-OH-Phe (r?=?0.73) and 1?+?9-OH-Phe (r?=?0.64) in the urine samples. 1-Hydroxypyrene was measured in all post-shift urine samples but was not significantly correlated with workplace pyrene exposure, indicating that skin exposure is an important route of pyrene exposure in this factory. As with 1-hydroxypyrene, dermal PAH uptake may also account for the poor correlation between 2?+?3- and 1?+?9-OH-Phe and ambient phenanthrene. Discussion: Since dermal uptake is likely to be important in occupational PAH exposure in addition to inhalation, estimation of total PAH exposure is best achieved by quantitation of PAHs excreted into body fluids. However, it remains unclear whether there might be a difference in uptake and urinary excretion of 3-ring, 4-ring, or 5-ring PAHs and in the correlation between these metabolites and ambient-air PAH measurements. In summary, using immunaffinity chromatography, we did not find detectable amounts of B[a]P-tetrol in urine from workers occupationally exposed to PAH. However, by an HPLC/immunoaffinity method, relatively high amounts of 1-hydroxypyrene as well as 2?+?3- and 1?+?9-OH-Phe were quantitated in the urine samples, both of which are relevant as biomarkers of PAH exposure.     

Chronic treatment with epidermal growth factor stimulates growth of the urinary tract in the rat

Twenty-four male Wistar rats, 8 weeks old, were allocated into three groups and treated with human recombinant epidermal growth factor (EGF) administered subcutancously in doses of 0, 30, and 150 g/kg per day for 4 weeks. Blood sampling was done every 2nd week and urine sampling was done for 2 consecutive days every week. The most striking finding was that the ureters were dose dependently enlarged, due to growth of all layers of the ureteric wall. The urothelium of the bladder showed considerable hyperplasticity with a widening of the basal proliferative compartment and a normal differentiation pattern as observed by the expression of carbohydrate epitopes, characterized with lectinohistochemistry. Blood examination revealed a decrease in blood haemoglobin concentration and a slight increase in serum creatinine concentration in the high-dose group. There were no effects of EGF on the urinary excretion of electrolytes, proteins, and endogenous EGF.     

Biological monitoring of isocyanates and related amines

Summary Five men were exposed to toluene diisocyanate (TDI) atmospheres for 7.5 h. The TDI atmospheres were generated by a gas-phase permeation method, and the exposures were performed in an 8-m3 stainless-steel test chamber. The mean air concentration of TDI was ca. 40 g/m³, which corresponds to the threshold limit value (TLV) of Sweden. The inhaled doses of 2,4- and 2,6-TDI were ca. 120 g. TDI in the test chamber air was determined by an HPLC method using the 9-(N-methyl-aminomethyl)-anthracene reagent and by a continuous-monitoring filter-tape instrument. After hydrolysis of plasma and urine, the related amines, 2,4- and 2,6-toluenediamine 2,4-, and 2,6-TDA), were determined as pentafluoropropionic anhydride (PFPA) derivatives by capillary gas-chromatography using selected ion monitoring (SIM) in the electron-impact mode. The urinary elimination of the TDAs showed a possible biphasic pattern, with rapid first phases for 2,4-TDA (mean t _1/2 for the concentration in urine, 1.9 h) and for 2,6-TDA (mean t _1/2 for the concentration in urine, 1.6 h). The cumulative amount of 2,4-TDA excreted in urine within 28 h ranged from 8% to 14% of the estimated dose of 2,4-TDI, and the cumulative amount of 2,6-TDA in urine ranged from 14% to 18% of the 2,6-TDI dose. The average urinary level of 2,4-TDA was 5 g/l in the 6 to 8-h sample (range 2.8–9.6 g/l), and the corresponding value for 2,6-TDA was 8.6 g/l (range, 5.6–16.6 g/l). Biological monitoring of exposure to 2,4- and 2,6-TDI by analysis of 2,4- and 2,6-TDA in urine is feasible.     

Characteristics of pentobarbital discrimination in the gerbil: Transfer and antagonism

Experiment 1. Gerbils were trained in a T-shaped maze to discriminate the effects produced by pentobarbital (P-barb. 15 mg/kg, i.p.) and the effects of saline. The response, a left or right turn in the maze, was thus contingent upon the prevailing training condition (P-barb. or saline). The criterion of performing 8 correct first trial choices in 10 consecutive sessions was reached within 20 training sessions. Tests with descending doses of P-barb. yielded an ED₅₀ of 9 mg/kg. Tests with phenobarbital (40 mg/kg) or diazepam (2 and 4 mg/kg) solely maintained the drug response. P-barb. discrimination was reversed by megimide (ED₅₀: 8.5–9.6 mg/kg) and metrazol (ED₅₀: 24.9–27.9 mg/kg). Thus megimide was approximately 3 times more effective than metrazol. Metrazol (40 and 80 mg/kg) also counteracted the phenobarbital and diazepam response. Picrotoxin (2.5 and 5 mg/kg) was less effective whereas caffeine (100 mg/kg) and piracetam (100–1000 mg/kg) did not upset P-barb. discrimination. Experiment 2. Naive gerbils had to discriminate mixtures of P-barb. (15 mg/kg) plus either 40 or 80 mg/kg of metrazol from saline already at the start of the discriminative training. The drug combinations produced discriminable effects since most gerbils reached the acquisition criterion (8/10), although more slowly than gerbils trained with P-barb. solely. Gerbils trained without a drug stimulus (saline vs. saline) never attained the criterion during 60 consecutive sessions. In conclusion, reversal of established discrimination (Expt. 1) does not necessarily mean that the same drug combination lacks discriminable effects as demonstrated in Experiment 2.     

Schwangerschaft bei Konnektivitiden

Zusammenfassung Schwangerschaften bei Frauen mit Konnektivitiden sind häufig mit Risiken für Mutter und Kind verbunden. Ausschlaggebend sind der Umfang der Organbeteiligung, das Vorhandensein von Autoantikörpern und die Art der medikamentösen Behandlung. In dieser Übersicht werden die Besonderheiten einer Schwangerschaft bei rheumatoider Arthritis, systemischem Lupus erythematodes und ankylosierender Spondylitis vorgestellt. Risiken für die Mutter sind aktive Krankheit oder ein Schub während der Schwangerschaft. Risiken für den Schwangerschaftsverlauf und das Kind sind spontaner Abort, Präeklampsie, intrauterine Wachstumsstörung, Prämaturität und Krankheit des Neugeborenen. Das Vorliegen von Antiphospholipidantikörpern und Antikörpern gegen SS-A und SS-B erfordert eine sorgfältige Überwachung der Schwangerschaft. Eine medikamentöse Therapie erfolgt bei aktiver Krankheit während der Schwangerschaft oder prophylaktisch, um einen Schub zu vermeiden. Bei Konnektivitiden sollten die Schwangerschaften geplant sein und in einem Zustand der Remission und bei stabiler medikamentöser Therapie erfolgen. Regelmäßige Kontrolle und Überwachung des Fetus sind Bedingung für einen unkomplizierten Schwangerschaftsverlauf.     

Effects of the Environmental Oestrogens Bisphenol A,Tetrachlorobisphenol A,Tetrabromobisphenol A, 4‐Hydroxybiphenyl and 4,4′‐Dihydroxybiphenyl on Oestrogen Receptor Binding,Cell Proliferation and Regulation of Oestrogen Sensitive Proteins in the Human Breast Cancer Cell Line MCF‐7

Abstract: Bisphenol A is extensively used in the manufacturing of epoxy resins and polycarbonate plastics, whereas several brominated and chlorinated analogues are used as flame retardants and intermediates in the plastic industry. Due to the structural relationship between these chemicals and the high production volumes, we wanted to characterize and compare their potential oestrogen‐like potency using several end‐points in MCF‐7 cells: induction of pS2 protein and progesterone receptor, reduction of oestrogen receptor level, and stimulation of cell growth. Bisphenol A, tetrachloro‐ and tetrabromo‐bisphenol A, 4‐hydroxybiphenyl and 4,4′‐dihydroxybiphenyl all showed oestrogen‐like properties in MCF‐7 cells. The chemicals tested had affinity to the oestrogen receptor isolated from MCF‐7 cells, although their EC₅₀s were 1,000 to 80,000 times higher than the EC₅₀ of 17β‐oestradiol. Bisphenol A and 4‐hydroxybiphenyl induced cell growth in MCF‐7 cells, and the highest test concentrations induced responses, apparently exceeding the cell growth induced by 17β‐oestradiol. The other chemicals tested induced less than 50% of the maximum 17β‐oestradiol‐stimulated cell growth. Bisphenol A, 4‐hydroxybiphenyl, tetrabromobisphenol A and tetrachlorobisphenol A all increased the level of the oestrogen‐regulated proteins, progesterone receptor and pS2, whereas 4,4′‐dihydroxybiphenyl showed no such effect. Bisphenol A was the only chemical tested that clearly mimicked 17β‐oestradiol in its ability to reduce the level of cytosolic oestrogen receptors in MCF‐7 cells. By measuring several oestrogen‐dependent endpoints it seems that some xeno‐oestrogens cause an imbalanced oestrogen‐response. Their ability and potency in mimicking 17β‐oestrogen in one parameter is not necessarily accompanied by a similar effect in another oestrogen‐linked parameter.     

Intensity modulated radiation therapy with electrons using algorithm based energy/range selection methods.

BACKGROUND AND PURPOSE: In recent years photon intensity modulated radiation therapy (IMRT) has gained attention due to its ability to improve conformity of dose distributions. A potential advantage of electron-IMRT is that the dose fall off in the depth dose curve makes it possible to modulate the dose distribution in the direction of the beam by selecting different electron energies. This paper examines the use of a computer based energy selection in combination with the IMRT technique to optimise the electron dose distribution. MATERIALS AND METHODS: One centimetre square electron beamlets ranging from 2.5 to 50 MeV were pre-calculated in water using Monte Carlo methods. A modified IMRT optimisation tool was then used to find an optimum mix of electron energies and intensities. The main principles used are illustrated in some simple geometries and tested on two clinical cases of post-operated ca. mam. RESULTS: It is clearly illustrated that the energy optimisation procedure lowers the dose to lung and heart and makes the dose in the target more homogeneous. Increasing the energy at steep gradients compensates for lack of target coverage at beam edges and steep gradients. Comparison with a clinically acceptable four segment plan indicates the advantage of the used electron IMRT technique. CONCLUSIONS: Using an intensity optimised mix of computer selected electron energies has the potential to improve electron treatments for mastectomy patients with good target coverage and reduced dose to normal tissue such as lung and heart.     

Impact of target reproducibility on tumor dose in stereotactic radiotherapy of targets in the lung and liver.

BACKGROUND AND PURPOSE: Previous analyses of target reproducibility in extracranial stereotactic radiotherapy have revealed standard security margins for planning target volume (PTV) definition of 5mm in axial and 5-10mm in longitudinal direction. In this study the reproducibility of the clinical target volume (CTV) of lung and liver tumors within the PTV over the complete course of hypofractionated treatment is evaluated. The impact of target mobility on dose to the CTV is assessed by dose-volume histograms (DVH). MATERIALS AND METHODS: Twenty-two pulmonary and 21 hepatic targets were treated with three stereotactic fractions of 10 Gy to the PTV-enclosing 100%-isodose with normalization to 150% at the isocenter. A conformal dose distribution was related to the PTV, which was defined by margins of 5-10mm added to the CTV. Prior to each fraction a computed tomography (CT)-simulation over the complete target volume was performed resulting in a total of 60 CT-simulations for lung and 58 CT-simulations for hepatic targets. The CTV from each CT-simulation was segmented and matched with the CT-study used for treatment planning. A DVH of the simulated CTV was calculated for each fraction. The target coverage (TC) of dose to the simulated CTV was defined as the proportion of the CTV receiving at least the reference dose (100%). RESULTS: A decrease of TC to <95% was found in 3/60 simulations (5%) of pulmonary and 7/58 simulations (12%) of hepatic targets. In two of 22 pulmonary targets (9%) and in four of 21 hepatic targets (19%) a TC of <95% occurred in at least one fraction. At risk for a decreased TC <95% were pulmonary targets with increased breathing mobility and hepatic targets with a CTV exceeding 100 cm(3). CONCLUSIONS: Target reproducibility was precise within the reference isodose in 91% of lung and 81% of liver tumors with a TC of the complete CTV >or=95% at each fraction of treatment. Pulmonary targets with increased breathing mobility and liver tumors >100 cm(3) are at risk for target deviation exceeding the standard security margins for PTV-definition at least for one fraction and require individual evaluation of sufficient margins.