Gap-junctional coupling measured by flow cytometry.

A method is described which reliably quantifies the degree of intercellular communication via gap junctions by combining a dye-loading technique with fluorescence-activated flow cytometry. Our experiments expand former measurements of other groups by analyzing the time- and density-dependent onset of coupling with a fixed ratio of donor to recipient cells. The high sensitivity of this technique provides a better resolution than the microelectrode technique and allows the detection of small changes in gap-junctional coupling by examining a large number of cells in a single experiment. Suspended cells were loaded with the membrane-permeable dye calcein AM, which is intracellularly hydrolyzed by nonspecific esterases, and the resulting polyanionic calcein is thus trapped inside these donor cells. Gap junctions, however, are permeable for this fluorescent dye, as can be observed when suspended donor cells are added to recipient cells (i.e., monolayer cultures) in which case cell-cell contact is established within less than 60 min. In addition, one of these two cell populations can also be stained with a membrane-resident dye (e.g., DiI), which facilitates the identification of different cell populations (donors, recipients, and noncoupled cells) not only by epifluorescence microscopy but also by flow cytometry. Our analyses reveal that junctional coupling depends not only on the connexin type (homo- or heterotypic junction) but also on the origin (species) of the contacting cells (homo- or heterospecific contact). We confirm earlier reports in which homotypic-homospecific coupling was demonstrated with different techniques in connexin-transfected HeLa and RIN cells as well as in BICR/M1R(k) and 3T3/SV40 cells. In contrast to other publications, we show that a significant heterotypic-homospecific coupling between Cx40- and Cx43-HeLa transfectants can be resolved, whereas no coupling was detected for heterotypic-heterospecific contacts between Cx40-HeLa transfectants and the Cx43-expressing cell lines BICR/M1R(k), 3T3/SV40, and RIN.

Bioeffects of diagnostic ultrasound in vitro.

Biological effects induced by ultrasound were frequently reported for continuous wave (cw) mode. Thresholds for the onset of bioeffects of pulsed ultrasound, starting from diagnostic conditions, have not yet been defined by standardized in vitro models. We therefore investigated the effects of pulsed ultrasound on cultured cells using diagnostic ultrasound devices, a selfmade transducer and a sonochemical laboratory reactor tunable from pulsed diagnostic conditions to cw ultrasound. Additionally, we determined physical parameters of the ultrasonic field by different types of hydrophones. Sonochemical reactions and the effects induced by the ultrasonic fields in cultured cells indicated a threshold for bioeffects.

Disturbance of cellular calcium homeostasis by in vitro application of shock waves.

Extracorporeally generated shock waves used in lithotripsy of urinary and biliary stones exhibit tissue lesions in vivo and destroy or damage cells in vitro. The involvement of cavitation-generated free radicals in these harmful effects is discussed controversially. We investigated changes in cytoplasmic calcium concentration and intracellular calcium localization after shock-wave treatment of suspended cell cultures using flow cytometry and electron microscopy and present evidence for the disturbance of mitochondrial Ca2+ a sequestration and, therefore, for a chemically induced cell injury.

Quality targets for concentrations of hazardous substances in surface waters in Germany.

During the past several years, in Germany, a federal government/federal states working group prepared a "guide document for the derivation of quality targets designed to protect inland surface waters against hazardous substances." In May 1993 the 40th Conference of Environment Ministers (Umweltministerkonferenz) adopted the guide document subject to further trial. Based on the guide document it will be possible to define water quality requirements in the field of water resources management, e.g., protection of aquatic communities and safeguarding of drinking water supply, as a complementing approach to existing statutory emission control regulations. The quality targets derived on the basis of the guide document define concentrations of hazardous substances in water, suspended particulate matter or sediments which should not be exceeded (guide values). According to current scientific knowledge, compliance with the quality targets ensures that a hazard to the protection-meriting uses considered need not be feared. Where quality targets are found to be exceeded, the causes should be explored. On that basis water pollution control and sanitation priorities can be defined more appropriately. In addition, the outlined approach to deriving quality targets provides a basis for characterizing water quality and is helpful in priority-setting with respect to analytical methods to be optimized.

Reduced cavitation-induced cellular damage by the antioxidative effect of vitamin E.

Fragmentation of human urinary and biliary stones by shock waves in extracorporeal lithotripsy is accompanied by tissue damage. Both the fragmentation as well as the side effects are often attributed to cavitation. The hazardous potential of cavitation is not only of a physical nature but also of a chemical nature, because of the generation of free radicals, e.g. .OH, .H and .O2. After the application of shock waves, we have demonstrated cavitation-generated free radicals in cell-free solutions and also in the surviving and intact suspended MGH-U1 cells by hydroethidine measurements. Under electron microscopical inspection, the same cells exhibited perinuclear cisternae, damaged mitochondria and numerous intracellular vacuoles. The contribution of free radicals to cell damage was investigated by reducing the vitamin E level in rats by a tocopherol free diet and by incubating L1210 cells in a tocopherol enriched medium. After 250 shock waves, ex vivo erythrocytes revealed a 75% increase in total cell disruption over cells from non-depleted rats. The in vitro experiments with L1210 cells exhibited a moderate protection by the addition of this scavenger of free radicals.

[Employment of continuous biotest systems for monitoring the Rhine river]. / Einsatz kontinuierlicher Biotestsyteme zur Uberwachung des Rheins.

As a part of the work of the German Commission for the Prevention of Pollution of the River Rhine biological test automats are developed and proved in a research project. The joint project "Development, Testing and Implementation of Biotests for the Monitoring of the River Rhine" sponsored by the Federal Ministry for Research and Technology (BMFT) and the Federal Environmental Agency (UBA), aims to provide a scientific and technical basis for an improved Rhine monitoring system. To realize a biological effect monitoring with functional subjects at the trophic levels of producers, primary and secondary consumers and destruents, the test spectrum includes algae, mussels, daphnia, fishes, and bacteria. Moreover, at the suborganismic level a test system with plant protoplasts is tested. Assessment schemes for the evaluation of measuring data and alarm situations, and limit values for releasing alarms are defined by the Working Group "Biotests for the Monitoring of the River Rhine" (WIR). By means of applicability criteria the most suitable biotests are chosen and recommended for the use on the River Rhine. The first results are presented in this paper.

Effects of various organic chemicals on the regreening of red colored Chlorella zofingiensis.

Under nitrogen-deficient culture conditions Chlorella zofingiensis decomposes the chlorophylls, synthesizes secondary cartenoids, and thus develops red colored resting stages (akinetes). On transfer of the akinetes into nitrogen-containing fresh culture medium the algae regreen completely within a short period. The results of the experiments presented in this study show that the regreening process reacts with high sensitivity to the addition of environmentally hazardous chemicals. This system proved to be much more sensitive than other methods of testing for ecotoxicity. This screening test is universally applicable and offers, in addition, the possibility of testing volatile compounds since it is carried out in air-tight culture vessels. A phytotoxicity of 50% calculated as inhibition of the chlorophyll biosynthesis during exposure for 48 hr was found for paraquat at concentrations of 0.15 microM (added as methysulfate) and 0.28 microM (added as chloride) and for pentachlorophenol (PCP) at 0.16 microM. The chemicals 2- and 4-nitrophenol, however, proved to be much less toxic. Here, a 50% inhibition of chlorophyll biosynthesis was observed at 49 and 75 microM. When exposed to 4-chloroaniline even concentrations of 200 microM were not sufficient to cause a 50% inhibition of the regreening process. In additional experiments the influence of increasing salt concentrations (NaCl stress) and simultaneous exposure to the toxicants was examined. A step-by-step increase in the salinity of the experimental medium resulted in a drastic reduction of the toxicity caused by the addition of paraquat. This effect was much less pronounced when PCP was employed as toxicant. A different result was obtained with 2-nitrophenol: Here the toxic action was slightly enhanced. Differences in the time-dependent toxic response were also observed using the diverse toxicants. Probable causes are discussed.