Removal of xenobiotic dichlorostearic acid from phospholipids and neutral lipids in cultured human cell lines by B-oxidation and secretion of dichloromyristic acid.

Chlorinated fatty acids represent a recently discovered group of potentially hazardous organochlorine pollutants in the environment. The ability of human cells to incorporate and metabolise this type of fatty acids has never been investigated. The aim of the present study was, therefore, to investigate if two human cell lines, INT 407 and SH-SY5Y, incorporate and metabolise extracellular dichlorostearic acid. Cells were incubated with 9,10-dichlorostearic acid for 24 hr, and the amounts of chlorinated fatty acids in cells and culture medium analysed every two days for up to 6 or 10 days. Lipids were separated by solid phase extraction, transesterified to fatty acid methyl esters, and analysed by gas chromatography in combination with a halogen specific detector (GC/XSD). Dichlorostearic acid, dichloropalmitic acid and dichloromyristic acid were found in phospholipids and in neutral lipids of the INT 407 cells. Both cell lines secreted considerable amounts of dichloromyristic acid into the culture medium. Cellular or secreted metabolites shorter than dichloromyristic acid were not found. Taken together, the results suggest that human cells may (1) incorporate chlorinated fatty acids into membrane lipids and storage lipids, (2) metabolise cellular dichlorostearic acid to dichloropalmitic acid and dichloromyristic acid by B-oxidation; but that further metabolism is hindered, possibly because of the chlorine atoms, and (3) remove formed dichloromyristic acid by secretion. The removal of cellular dichloromyristic acid might represent an important cellular defence mechanism and deserves further investigations.

A comparison of sole carbon source utilization patterns and phospholipid fatty acid profiles to detect changes in the root microflora of hydroponically grown crops.

Sole carbon source utilization (SCSU) patterns and phospholipid fatty acid (PLFA) profiles were compared with respect to their potential to characterize root-inhabiting microbial communities of hydroponically grown crops. Sweet pepper (Capsicum annum cv. Evident), lettuce (Lactuca sativa cv. Grand Rapids), and four different cultivars of tomato (Lycopersicon esculentum cvs. Gitana, Armada, Aromata, and Elin) were grown in 1-L black plastic beakers placed in a cultivation chamber with artificial light. In addition to the harvest of the plants after 6 weeks, plants of one tomato cultivar, cv. Gitana, were also harvested after 4 and 8 weeks. The cultivation in this study was performed twice. Principal component analysis was used to analyze the data. Both characterization methods had the ability to discriminate between the root microflora of different plant species, cultivars, and one tomato cultivar at different ages. Differences in both SCSU patterns and PLFA profiles were larger between plant species than between cultivars, but for both methods the largest differences were between the two cultivations. Still, the differences between treatments were always due to differences in the same PLFAs in both cultivations. This was not the case for the SCSU patterns when different plant ages were studied. Furthermore, PLFA profiles showed less variation between replicates than did SCSU patterns. This larger variation observed among the SCSU data indicates that PLFA may be more useful to detect changes in the root microflora of hydroponically grown crops than the SCSU technique.

Experiences from long-term research cooperation between Costa Rican, Nicaraguan, and Swedish institutions.

This paper summarizes experiences from long-term ongoing cooperation between Swedish research institutions and institutions at the National Universities in Nicaragua and Costa Rica. 24 researchers and teachers from the Central American institutions and ten Swedish research students have been trained. In addition to three full doctoral and three licentiate (two-year PhD program) theses, the two programs have so far published 15 articles in English-language, international, refereed journals and about three times as many abstracts for conferences in more than ten countries. A "sandwich" model for training is recommended, where the southern researchers come to the wealthier partner for collaborative analyses and write-ups of the publications, while spending 50-75% of their time in their home countries for data collection. Such collaboration should be planned for a time span of at least eight years and include substantial numbers of researchers and students. Means to minimize the risk of brain drain are suggested. The collaboration has been important for the globalization of the research cultures at the participating institutions and has trained international experts.

Differentiated effects on splanchnic homeostasis by selective and non-selective endothelin receptor antagonism in porcine endotoxaemia.

1. The non-selective endothelin (ET) receptor antagonist bosentan has been shown to restore systemic and gut oxygen delivery and reverse intestinal mucosal acidosis in porcine endotoxin shock. 2. To further elucidate the specific role of the ETA as opposed to the ETB receptor and their effects in the splanchnic region a non-selective (ET(MIX)ra) A-182086 and selective ETA (ET(A)ra) PD155080 and ETB (ET(B)ra) A-192621 receptor antagonists were administered, separately or simultaneously (ET(A+B)ra) 2 h after onset of endotoxin shock. These four groups were compared to a control group receiving only endotoxin and vehicle. 3. Thirty-nine pigs were anaesthetized and catheterized for measurement of central and regional haemodynamics. A tonometer in the distal ileum was used for measurement of mucosal PCO2. Blood gases and plasma ET-1-LI levels as well as histological samples from the gut were assessed. Intervention was started 2 h after onset of endotoxemia and the experiments were terminated after 5 h. 4. Endotoxin-induced changes in systemic, gut oxygen delivery and portal hepatic vascular resistance and systemic acidosis were effectively counteracted by both ET(A+B)ra an ET(MIX)ra. ET(A)ra administration was not effective while ET(B)ra proved to be fatal as all animals in this group died prior to full time of the experiment. While both ET(A+B)ra and ET(MIX)ra improved gut oxygen delivery only the latter attenuated the profound endotoxin-induced ileal mucosal acidosis. 5. The lethal effect seen from selective ETB receptor antagonism in the current study may be due to increased ETA receptor activity as plasma levels of ET-1 is increased several fold by blocking the ETB receptor and thereby the plasma-ET-1-clearing function. Furthermore, a loss of endothelial ETB receptor vasodilating properties may also have contributed to the lethal course in the ET(B)ra group. 6. The findings in this study suggest that ET is involved in the profound endotoxin-induced disturbances in splanchnic homeostasis in porcine endotoxaemia. Furthermore, antagonism of both ETA and ETB receptors is necessary to effectively counteract these changes.

Effects on haemodynamics by selective endothelin ET(B) receptor and combined endothelin ET(A)/ET(B) receptor antagonism during endotoxin shock.

The endothelin system is highly activated during endotoxin and septic shock. To investigate this matter the selective non-peptide endothelin ET(B) receptor antagonist A-192621 ([2R-(2alpha,3beta, 4alpha)]-4-(1,3-benzodioxol-5-yl)-1-[2-[2, 6-diethylphenyl)amino]-2-oxoethyl]-2-(4-propoxy-phenyl)-3-py rrolidine carboxylic acid) was administered alone and in combination with the selective non-peptide endothelin ET(A) receptor antagonist PD 155080 (sodium 2-benzo[1, 3]dioxol-5-yl-3-benzyl-4-(4-methoxy-phenyl)-4-oxobut-2-enoat e) during established porcine endotoxin shock. Cardiopulmonary vascular function, metabolic parameters and plasma endothelin-1-like immunoreactivity levels were compared to a control group only receiving endotoxin. Administration of A-192621 alone resulted in cardiovascular collapse and death whereas combining A-192621 with PD 155080 abolished endotoxin induced pulmonary hypertension, enhanced cardiac performance and improved systemic oxygen delivery and acid-base balance. The beneficial effects of mixed endothelin ET(A)/ET(B) receptor antagonisms on the pulmonary and cardiovascular systems may result from blockage of constrictive endothelin receptors in and pulmonary circulation, reduced afterload and a direct inotropic effect. Possible mechanisms for the devastating effects by selective endothelin ET(B) receptor antagonism include increased endothelin ET(A) receptor-mediated vasoconstriction due to lack of endothelin ET(B) receptormediated vasodilation and decreased endothelin clearance from endothelin ET(B) receptor blockade. In conclusion, selective endothelin ET(B) receptor antagonism is deleterious whereas combined endothelin ET(A) and ET(B) receptor antagonism has favourable effects on haemodynamics, suggesting participation of the endothelin system in cardiopulmonary dysfunction during endotoxin shock.

Limiting life support. Experiences with a special protocol.

BACKGROUND: Different aspects of withholding or withdrawal of life-sustaining treatment have been described in the literature. However, there are few descriptions of local policies or protocols regarding the limiting of life support. The present study describes the pattern of limiting life support in a Scandinavian intensive care unit (ICU) and the experience with a special protocol for such decisions. METHOD: Prospective study of decisions to limit life support in the ICU of a tertiary referral hospital during 1 year using a special protocol and medical records. RESULTS: A decision to limit life support was documented in the special protocol in 61/1008 (6%) of the patients and in 39/79 (49%) of the patients dying in the ICU. Poor prognosis and failure to respond to treatment were the most common reasons for foregoing life-sustaining treatment. Of the 61 patients with a special protocol, 7 survived more than 3 months. The decision was altered for 5 patients towards additional limitations and for 4 patients towards fewer or no limitations. CONCLUSION: Decisions to limit life support in the ICU are common. There is a substantial survival rate. A structured clinical protocol can be used for continuous follow-up of decisions and the decision-making process.

Study of the microbial dynamics in the root environment of closed, hydroponic cultivation systems for tomato using phospholipid fatty acid profiles.

A more basic understanding of the microbial dynamics of closed, hydroponic cultivation systems is needed. We therefore initiated a study of the microbial community inhabiting the root environment, using phospholipid fatty acid (PLFA) profiles, and started to examine whether changes in the microbial population structure would result from the introduction of selected isolates of plant growth-promoting rhizobacteria (PGPR). Tomato were cultured in deep-flow systems with circulating nutrient solution. Bacteria were sampled from tomato roots at three locations, longitudinally, in the gutters of a control system and in two systems inoculated with PGPR. In the beginning of the gutters the PLFA profiles were similar in all systems, whereas the profiles differed in the gutter ends (following the direction of flow). In the control system, and in a treatment inoculated with two Gram-negative and one Gram-positive PGPR strain, the relative proportion of PLFAs characteristic to Gram-positive bacteria was highest at the end of the gutter. In a treatment inoculated only with a Gram-negative PGPR strain, the relative proportion of PLFAs characteristic of Gram-negative bacteria was highest at the end of the gutter. The results indicate a complex situation with different micro-environments distributed along the root mat. It can also be concluded that PLFA profiles may be useful tools in the study of the microbiology of closed hydroponic plant cultivation systems.

ATP leakage from ELD cells after exposure to stearic, monochlorostearic, dichlorostearic, and oleic acids.

The capacity of stearic, monochlorostearic, dichlorostearic and oleic acids to cause membrane damage was measured as their ability to induce leakage of adenosine triphosphate (ATP) from mammalian tumour cells in vitro. Chlorinated stearic acids, and oleic acid, caused ATP leakage at lower concentrations than normal stearic acid. The membrane disturbing properties are suggested to be a result of the different molecular geometries of the chlorinated stearic acids, and oleic acid, compared to non-chlorinated stearic acid.

In vivo and in vitro toxicity of fractionated fish lipids, with particular regard to their content of chlorinated organic compounds.

Six different lipid matrices (the intact lipid (IL), four lipid fractions with different polarity, and the free fatty acids (FFAs) obtained by hydrolysis of the triacylglycerol (TAG) containing fraction) were obtained from salmon (Salmo salar) and eel (Anguilla anguilla), each collected at a contaminated and a comparatively uncontaminated catch site along the coast of Scandinavia. The lipid matrices were studied in toxicological test systems representing various biological functions of different organ systems from several species and trophic levels. The results were evaluated with particular respect to the concentrations of extractable organically bound chlorine (EOC1) in the matrices tested. In some test systems, the specimens with a higher EOC1 concentration appeared to be more toxic. For example, the TAG containing fraction (F2) from Idefjord eel, having a higher EOC1 content than F2 from Oslofjord eel, reduced the number and hatchability of eggs laid by zebrafish. Both IL and F2 of Idefjord eel increased mortality and reduced the oxygen/nitrogen-ratio in blue mussels. Non-polar compounds (F1) from Bothnian Sea salmon induced 7-ethoxyresurofin O-deethylase (EROD) activity in rainbow trout hepatocytes, whereas F1 from Senja salmon did not. F1 from Bothnian Sea salmon also reduced the number of T-cells in foetal mouse thymus analagen in vitro compared with the cell number in anlagen exposed to F1 from Senja salmon. A positive correlation between EOC1 concentration and test response was found for EROD activity in rainbow trout hepatocytes and for ATP-leakage in Erlich ascites tumour cells when testing the phospolipid containing fraction (F4). However, in most test systems the fish oils, irrespective of EOC1 content, were of low toxicity, and the observed effects need to be verified in future studies.

Model system for studies of microbial dynamics at exuding surfaces such as the rhizosphere.

An autoclavable all-glass system for studying microbial dynamics at permeable surfaces is described. Standard hydrophobic or hydrophilic membranes (46-mm diameter) of various pore sizes were supported on a glass frit through which nutrient solutions were pumped by a peristaltic pump. The pump provided a precisely controlled flow at speeds of 0.5 to 500 ml of defined or natural cell exudates per h, which passed through the membrane into a receiving vessel. The construction allowed a choice of membranes, which could be modified. The system was tested with a bacterium, isolated from rape plant roots (Brassica napus L.), that was inoculated on a hydrophilic membrane filter and allowed to develop into a biofilm. A defined medium with a composition resembling that of natural rape root exudate was pumped through the membrane at 0.5 ml/h. Scanning electron microscopic examinations indicated that the inoculum formed microcolonies embedded in exopolymers evenly distributed over the membrane surface. The lipid composition and content of poly-beta-hydroxybutyrate in free-living and adhered cells were determined by gas chromatography. The bacterial consumption of amino acids in the exudate was also studied.