Supernatant cytotoxicity and proteolytic activity of selected oral bacteria against human gingival fibroblasts in vitro.

The purpose of this study was to determine if endodontic bacterial act in vitro on human gingival fibroblast functions via extracellular products. The bacteria used were Prevotella nigrescens, Capnocytophaga ochracea, Peptostreptoccocus micros and Actinobacillus actinomycetemcomitans. Supernatants were collected from bacterial cultures at the beginning of the stationary phase when their density was similar. Toxins that inhibited fibroblast proliferation were found in all culture supernatants of Gram-positive or Gram-negative bacterial strains, except for Prev. nigrescens. The cytotoxicity of A. actinomycetemcomitans supernatant was about 1000 fold higher than the others. This supernatant diluted to 1/1000 led to total fibroblast growth inhibition whereas only 25% growth inhibition was obtained with Capn. ochracea and Pept. micros diluted to 1/10. Bacterial supernatant proteolytic activity was investigated in confluent fibroblast cultures that were incubated for 48 hr with each of the supernatants diluted to 1/2 except for A. actinomycetemcomitans supernatant diluted to 1/20. Indirect immunofluorescence studies of extracellular-matrix molecules, followed by immunoelectrophoretic analysis of extracts of whole-cell layers, demonstrated that only conditioned medium of Prev. nigrescens had a proteolytic activity capable of degrading the greater part of type I collagen and fibronectin fibres in the extracellular matrix.

Modulation by hypergravity of extracellular matrix macromolecules in in vitro human dermal fibroblasts.

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity (20 g) over a period of 8 days. Changes in organization of extracellular matrix molecules were seen by indirect immunofluorescence. In the fibronectin layer, bundles of fibrils were gathered together leading to a disorganisation of the normal parallel pattern of fibers seen in control cultures. Type I collagen fibrils appeared with wooly outlines in controls whereas thick fibers were closely packed in 20-g cultures. A moderate increase of type III collagen fibril density was observed. No elastic fibers were seen in control or in 20-g cultures. In the culture medium, the release of soluble elastin (ELISA) and type I and III collagens (RIA) was undisturbed. Assays of enzymes involved in the remodeling of extracellular matrix showed an increase of cellular elastase activity (10%) and a decrease of the spontaneously active collagenase. Nevertheless, the total collagenase activity, (activated by trypsin), was increased by up to 30%. These data show a significant rise of the latent collagenase activity and suggest that release of the tissue inhibitor of metalloproteinase (TIMP1) was enhanced by hypergravity.

Effects of hypergravity on the cell shape and on the organization of cytoskeleton and extracelluar matrix molecules of in vitro human dermal fibroblasts.

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity ranging from 2 to 20 g for 8 days. Changes only appeared above 15 g. The majority of 20 g-subjected cells showed fine filipods in the shape of a star whereas most control cells had rounded shapes and spread by forming lamellipodia. Indirect immunofluorescence staining of vinculin, alpha-actinin and actin stress fibers showed changes of the arrangement anchoring points of stress fibers under hypergravity. Tubulin staining showed that the centrosomal material generally located above the nucleus in control cells had migrated to the nucleus side in 20 g-exposed cells. After 8 d of culture under 20 g hypergravity the thickness of fibronectin network seemed to be increased and bundles of fibrils appeared linking ordered arrays of fibers. The fibrils of collagen I formed better delimited and thicker bundles of fibers. We may assume that 20 g hypergravity can induce changes in fibroblast cell shape, migration way, and anchorage leading to a reorganization of extracellular matrix without concomitant change of cell proliferation.

Influence of a long duration exposure, 69 months, to the space flight factors in Artemia cysts, tobacco and rice seeds.

Three french laboratories have participated in the Free Flyer Biostack experiment. Artemia cysts, tobacco seeds and rice caryopsis and embryos were used. Biological objects in monolayers were dead. In opposite, a large fraction of samples used in bulk survived. A stimulatory effect occurred in the first steps of development in Artemia cysts. In fact, the larval survival was unchanged or slightly reduced. In tobacco a drastic decrease in germination and survival rate was observed. Space flight did not induce genetic changes. In rice, results depend on the variety which was investigated; the growth rate stimulation in flight samples is discussed with respect to controls.

Effects of hypergravity on adherent human cells.

In recent years, accumulating evidence has shown that microgravity or hypergravity may affect cell growth and differentiation. Since it is not easy to carry out researches in space or to simulate weightlessness on earth, we conducted experiments on simulated hypergravity (2 to 15 g) by using a centrifuge (radius: 80 cm; speed motor: 180 rpm). We looked for the effects of chronic hypergravity (7 to 10 days) on cultures of three human cell lines: lung or dermic fibroblasts and lung adenocarcinoma A 549 cells. The results showed a significant decrease (10-20%, P<0.05) in cell proliferation connected to a significant decrease (20-50%, P<0.01) in culture DNA content under hypergravity, but only for lung fibroblasts. The protein content was never disturbed. Dermic fibroblast elastase activity was enhanced (8-13%, P<0.02) under 15 g. Total phospholipid content as well as relative amounts of phospholipid components, analysed by thin layer chromatography, were unchanged in A 549 cells.

Effects of hypergravity on lung carcinoma cells maintained in continuous organotypic culture.

The effects of hypergravity levels ranging from 1 to 15 g were studied on A549 lung adenocarcinoma cell line, cultivated as nodules. This organotypic culture model preserves as closely as possible the cellular structures and differentiation functions of the in vivo situation. Nodules submitted to hypergravity conditions for 27 d did not show any change of cell growth, protein and DNA contents, compared with controls. Also, cellular differentiation, as regards intracellular phospholipid composition and more particularly phosphatidylcholine content, appeared undisturbed. The only obvious effect of hypergravity was a modification of the structural organization, with a disappearance of the large alveoli present at the surrounding of control nodules and the development of a dense cellular mass instead.

Investigations of the effects of cosmic rays on Artemia cysts and tobacco seeds; results of Exobloc II experiment, flown aboard Biocosmos 1887.

Artemia (Brine shrimp) cysts and tobacco seeds, dormant biological material devoid of metabolic activity, were flown aboard the Soviet Biocosmos 1887 in order to investigate the effects of cosmic rays. Artemia cysts and tobacco seeds were used in bulk or in monolayers sandwiched with track detectors. Biological and physical units were located outside and inside the spacecraft. Stacks included lead shielding in order to expose the objects to different doses of radiation. Total dosimetry was performed using thermoluminescent detectors. In spite of low levels of doses, the space flight resulted in a decrease in developmental capacity of Artemia cysts, and in a higher mutation rate in tobacco seeds. The more obvious responses occurred, in both cases, in biological objects exposed to the highest doses. These results are compared to those of previous space experiments.

Space environmental factors affecting responses to radiation at the cellular level.

Previous space experiments suggest a high value for the RBE of cosmic radiation. A possible explanation could be a change in cell radiosensitivity due to a combined effect of radiation and other factors related to the space environment and to the space flight. Results of the EXOBLOC II experiment support this assumption. On earth, vibrations or accelerations applied before or after irradiation can change the responses to radiation. Microgravity could be the main factor affecting the radiosensitivity and DNA repair but this hypothesis must be confirmed by additional experiments.

Stimulating effect of space flight factors on Artemia cysts: comparison with irradiation by gamma rays.

The Artemia cyst, a gastrula in dormant state, is a very suitable material to investigate the individual effects of HZE cosmic particles. Monolayers of Artemia cysts, sandwiched with nuclear emulsions, flew aboard the Soviet biosatellite Cosmos 1129. The space flight stimulated the developmental capacity expressed by higher percentages of emergence, hatching, and alive nauplii at day 4-5. A greater mean life span was reported in Artemias developed from Artemia cysts hit by the cosmic heavy ions. On Earth, Artemia cysts were exposed to 1, 10, 100, 200 and 400 Gy of gamma (gamma) rays. A stimulating effect on developmental capacity was observed for 10 Gy; the mean life span was significantly increased for this dose. These results are discussed in comparison with previous investigations performed on Earth and in space.

Radiation-induced changes in late effects and in developmental capacities of exposed artemia cysts.

Artemia dry cysts from a Californian bisexual strain used in several space experiments were irradiated with 60Co gamma rays. The three cyst populations experimented could be differentiated according to their development and survival rates. The variations observed for both of these criteria were related to the age of the cysts and the selection technique. The study of radiosensitivity based on LD50 value showed that the highest radiosensitivity differences were related to the cyst selection technique and not to the age. Furthermore, the three cyst populations showed that radio-induced lethal effects were enhanced, or appeared with time, namely following the delay between irradiation and the cyst development study. The observation of late effects after irradiations or after space flights show the difficulties encountered in assessing radiative risks during long duration space flights.

Changes in developmental capacity of artemia cyst and chromosomal aberrations in lettuce seeds flown aboard Salyut-7 (Biobloc III experiment).

This paper gives the results of investigations performed on the first container (A) of the Biobloc III experiment, flown aboard the orbital station Salyut 7 for 40 days. The space flight resulted in a decreased developmental capacity of Arterlia cysts, hit or not hit by the HZE particles. No effect was observed in cysts in bulk. A synergetic effect of microgravity and gamma pre irradiation is described. The germination of in-flight lettuce seeds was decreased. The space flight resulted also in a higher percentage of cells with chromosomal aberrations. Relations between biological response, TEL and location of HZE particles are discussed.

Results on Artemia cysts, lettuce and tobacco seeds in the Biobloc 4 experiment flown aboard the Soviet Biosatellite Cosmos 1129.

Artemia cysts, lettuce and tobacco seeds were flown aboard the Cosmos 1129 for 19 days. A correlative method was used in order to determine the passage of cosmic heavy ions (HZE particles) through the biological test objects. This space flight resulted in a decrease on hatchability, nucleic acid and protein synthesis in hydrated Artemia cysts. HZE particle effects on plant cellular chromosomes are confirmed. In tobacco seeds, a stimulating effect on germination rate and a higher frequency of abnormalities were observed. Dormant biological objects are a very suitable material to study cosmic ray effects: these objects can be arranged in monolayers and sandwiched between visual track detectors in order to determine the passage of the cosmic heavy ions (HZE particles). On the other hand this method allows us to study effects of microgravity and those of the protonic component of cosmic rays in the objects not hit by the HZE articles.

Effects of 645 MeV and 9.2 GeV protons on Artemia eggs.

Developmental capacities of Artemia eggs have been studied after exposure to 645 MeV or 9.2 GeV protons. Effects of proton irradiation were studied in comparison with 60Co gamma ray irradiation, endpoints being emergence, hatching and 4-5 day old live nauplii percentages. Effectiveness of 645 MeV protons is greater than that of 9.2 GeV protons. R.b.e. values calculated for nauplius survival is 2.3 for 645 MeV protons and 1.5 for 9.2 GeV protons. These results can be taken into account in radiation hazard estimation during space flights.

Development capacity of artemia cysts and lettuce seeds flown in Cosmos 936 and directly exposed to cosmic rays.

Developmental capacity of Artemia cysts and chromosomal aberration frequency in lettuce seeds, flown aboard Cosmos 936 have been investigated. Biological objects were located inside or outside the spacecraft. Lettuce seeds were stuck on plastic plates and sandwiched in cellulose sheets in order to discriminate the objects hit by the cosmic heavy ions from the ones not hit. The absorbed dose inside the spacecraft was about 650 mrad. Biological objects were located at different levels in the "outside" container; absorbed doses were 1.5 rad for lettuce seeds and 30 rad for Artemia cysts. There was no change in hatch-ability of Artemia cysts located inside or outside the spacecraft when eggs were tested 2-4 months after recovery, but a marked decrease was observed in eggs 9 months after the space flight. Survival of larvae and adults was normal in "inside" eggs, but decreased in "outside" eggs. Space flight also resulted in a higher frequency of single chromosomal aberrations in cells developed from "inside and outside", hit and non-hit seeds. This highest percentage of multiple chromosomal aberrations occurred in seeds hit by the heavy ions.

Radiobiological investigations in Cosmos 782 space flight (Biobloc SF1 experiment).

Several biological objects were flown in Cosmos 782 in order to investigate the effects of HZE cosmic particles and other environmental factors of space. Space flight results in chromosomic aberrations in lettuce seeds, decreased germination rate and increased frequency of abnormalities in tobacco seeds and decreased developmental capacity in Artemia eggs. In lettuce and tobacco seedlings, changes were observed not only in seeds hit by heavy ions but also in nonhit seeds. The results indicate that exposure to the space environment can induce important changes in biological objects and emphasize the usefulness of investigations carried out on organisms less complex than mammals.

Preliminary results of the Biobloc experiment on the Cosmos 782 flight: effects of cosmic rays on brine shrimp eggs and tobacco seeds.

The aim of the Biobloc experiment is to determine the biological effects of cosmic rays. Biobloc is a stack made up of biological layers and physical detectors (nuclear emulsions and plastic). Artemia eggs and tobacco seeds are used by French workers. Irradiation by cosmic heavy ions induces a late developmental inhibition of Artemia eggs. A light increase of emergence rate is observed for the non-hit eggs. The results are compared with those of previous space experiments. Investigations on tobacco seeds are in progress.

[Influence of various physical factors on the developmental capacities of the ovum of Artemia salina]. / Influence de différents facteurs physiques sur les capacités de développement de l'oeuf d'Artemia salina

Artemia salina dry eggs exhibit a great resistance to different physical factors: acceleration and vibration, low temperature, high vacuum and magnetic fields. Vibration test, alone or combined with acceleration test decreases the Artemia salina egg developmental capacity. Nevertheless, the differences are small and much lower than those observed in previous space experiments. The viability of Artemia eggs is not impaired by low temperature, high vacuum exposure and by high magnetic fields (15, 25 and 150 Koersted) or by earth magnetic field shielding.