A functionally distinct member of the DP family of E2F subunits.

E2F transcription factors regulate genes involved in cell-cycle progression. In mammalian cells, physiological E2F exists as an E2F/DP heterodimer. Currently, eight E2F and two DP subunits have been characterized. We report here the characterization of a new member of the DP family, DP-4. While DP-4 exhibits certain similarities with members of the DP family, it also possesses a number of significant differences. Thus, DP-4 forms a heterodimer with E2F subunits, binds to the E2F site and associates with pocket proteins including pRb. In contrast to DP-1, however, DP-4/E2F-1 complexes exhibit reduced DNA binding activity. Furthermore, DP-4 interferes with E2F-1-dependent transcription and delays cell-cycle progression. These results highlight an emerging complexity in the DP family of E2F subunits, and suggest that DP-4 may endow E2F heterodimers with distinct transcription properties.

Technical knockout, a Drosophila model of mitochondrial deafness.

Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko(25t) exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko(25t) mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity.

Production of reactive oxygen species by man-made vitreous fibres in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) or erythrocytes, isolated from human blood, were exposed to graded doses of asbestos (chrysotile), quartz, or man-made vitreous fibres (MMVF), i.e. refractory ceramic fibres (RCF), glasswool, or rockwool fibres. None of the MMVF affected either the viability of PMNL, as measured by trypan blue exclusion test, or induced haemolysis, whereas the positive controls, quartz and chrysotile, dose-dependently induced haemolysis in PMNL. MMVF did not increase the release of lactate dehydrogenase (LDH) from the PMNL, whereas the positive controls, chrysotile and quartz, induced a marked and dose-dependent release of LDH. When PMNL were exposed to MMVF, some of the fibre types slightly increased the levels of free intracellular calcium ([Ca2+]i) within the cells in a manner similar to that induced by chrysotile or quartz. All MMVF induced a dose-dependent production of reactive oxygen species (ROS) in PMNL, with RCF-induced production of ROS being the most marked. Production of ROS by MMVF seemed to depend on the availability of extracellular calcium because it could be attenuated with a Ca2+ channel blocker, verapamil, or a Ca2+ chelating agent, EGTA. Production of ROS may be a common pathway through which PMNL respond to MMVF-induced cell activation, but alterations of levels of free intracellular Ca2+ do not seem to be an absolute prerequisite for this effect. Fibre length seemed not to be an important factor in affecting the ability of MMVF to induce ROS production in PMNL. However, the balance between different elements in the fibre seemed importantly to affect the biological activity of a fibre.

Dissolution of short and long rockwool and glasswool fibers by macrophages in flowthrough cell culture.

Dissolution of MMVF (man-made vitreous fibers) by macrophages has previously been studied utilizing cell cultures in wells. A new, more dynamic method has been developed to explore the effects of macrophages on MMVF dissolution. In this method, the culture medium flows through a membrane on which the macrophages and fibers are placed. The dissolution of short and long rockwool and glasswool fibers was investigated in the present study by macrophages by assessing the dissolution of Si (silicon), Fe (iron), and Al (aluminium) from the fibers. Dissolution of these elements usually increased as a function of time. Generally, the dissolution of elements from the fibers in the flowthrough culture exceeded that observed with the culture in wells system. The dissolution of glasswool fibers was greater in medium than in cell culture, whereas the opposite was true for rockwool fibers. Dissolution of Si was greater from glasswool than from rockwool fibers, while the opposite was true for Fe and Al. Macrophages that had phagocytized fibers in flowthrough culture contained Si, and there were also precipitations with Si in the samples. The fibers in the flowthrough culture also exhibited surface changes such as breakings, pittings, etching, and peeling. The short rockwool fibers tended to fracture more than short glasswool fibers, while long glasswool fibers were more extensively broken than short glasswool fibers. The results with this new, dynamic, flowthrough culture method with macrophages demonstrate that this method provides valuable information on the abilities of macrophages to dissolve MMVF leading to subsequent morphological changes of fibers.

Comparison of cytotoxicity of man-made vitreous fibres.

The purpose of the study was to compare the cytotoxicity of man-made vitreous fibres (MMVFs): four refractory ceramic fibres (RCFs 1-4), two glasswool fibres (MMVF 10 and 11), a rockwool fibre (MMVF 21) and a slagwool fibre (MMVF 22). The ability of the fibres to induce haemolysis in sheep erythrocytes, to release lactate dehydrogenase (LDH) from rat alveolar macrophages (AM) and to increase the production of reactive oxygen metabolites (ROMs) in human polymorphonuclear leukocytes (PML) was studied. To assess the relative cytotoxicity of MMVFs, their toxicity was compared with that induced by quartz, chrysotile or titanium dioxide. MMVFs induced a modest, but dose-dependent, increase of haemolysis at doses of 0.5, 2.5 and 5.0 mg ml-1. The amount of haemolysis and LDH release induced by MMVFs was generally similar to that induced by titanium dioxide. Glasswool fibre MMVF 10 induced less LDH release from rat AM than rockwool MMVF 21 or slagwool MMVF 22 fibres, whereas glasswool fibre MMVF 11 induced less LDH release than slagwool fibre MMVF 22 (P < 0.05). All fibres also dose-dependently increased the production of ROMs at doses between 25 and 500 micrograms ml-1. The shapes of the time-courses of MMVF-induced production of ROMs suggest that the mechanisms whereby the different fibres induce ROM production may exhibit similar features. There are clear-cut differences in the potency of various MMVFs to induce cytotoxicity and oxidative burst. The present results also emphasize the importance of using several measures of toxicity when assessing the biological activity of various fibres in vitro.

Cellular effects of particles--impact of dissolution on toxicity of man-made mineral fibers.

The use of man-made vitreous fibers (MMVF) has grown rapidly because exposure to natural fibers, mainly asbestos, has proved harmful to humans. Biological activity of MMVF made of glass, rock, slag, or other minerals does not depend only on their respirability, but also on their chemical durability and persistency. In the use of MMVF, the goal is to decrease harmful effects of fibers by increasing their dissolution and removal from the lungs. The dissolution of Fe and Al from MMVF is more marked by rat alveolar macrophages (AMs) in culture than by mere medium, whereas medium is more effective than AMs in dissoluting silicon (Si) from MMVF, Fe and Al content of the fibers correlate negatively with the fiber Si dissolution by the AMs. Scanning electron micrographs show that MMVF are readily phagocytized by rat AMs in culture. The phagocytosis begins within 30 min after the onset of the exposure and continues for a 96-h observation period. Short fibers, less than 20 microns in length, are readily phagocytized by the AMs whereas longer fibers are attacked with a large number of AMs. MMVF induce also non-lethal changes in the rat AM surface morphology. Before exposure the cells have continuous membranes. The exposed AMs produce extensions which fasten them to the fibers or to other cells to form clumps or clusters of cells and fibers, each cell engulfing a part of a fiber. Over 70% of the exposed cells are viable after 96 h of exposure suggesting that MMVF are not acutely toxic rat AMs. MMVF also slightly damage cell membrane and increase the production of reactive oxygen species.

The effect of fiber length on the dissolution by macrophages of rockwool and glasswool fibers.

The effect of fiber length on the dissolution of experimental rockwool and commercial glasswood fibers in rat alveolar macrophage (AM) culture and in mere culture medium was studied. The ultrastructure of macrophages after their exposure to fibers and the suitability of macrophage-type cell line P388D1 culture in dissolution studies were also explored. The fiber samples included short (ground) and long (untreated) rockwool and glasswool fibers. The fibers were incubated in rat AM cultures, in P388D1 culture, or in mere culture medium for 4 or 8 days. The dissolution of the fibers was determined by measuring the amounts of silicon (Si), iron (Fe), and aluminum (Al) in the medium. There were no differences in the diameter of the fibers, but a clear difference existed in the length of the short and long fibers. The dissolution of Si, Fe, and Al was more pronounced from experimental rockwool than from commercial glasswool fibers. The dissolution of Si was always greater in mere culture medium than in rat AM culture. Moreover, the dissolution of Si was greater from the long fibers than from the short ones. On the contrary, the dissolution of Fe and Al in AM culture exceeded that in mere culture medium. The dissolution of Si, Fe, and Al from both fibers in P388D1 culture was similar to their dissolution in rat AM culture. The fibers were also effectively phagocytized by the macrophages. The present results together suggest that the intracellular and the extracellular dissolutions of man-made vitreous fibers differ from each other.

Dissolution of man-made vitreous fibers in rat alveolar macrophage culture and Gamble's saline solution: influence of different media and chemical composition of the fibers.

The effect of different chemical compositions of man-made vitreous fibers (MMVF) on their dissolution by alveolar macrophages (AM) in culture and in Gamble's solution was studied. The fibers were exposed to cultured rat AMs, culture medium alone; or Gamble's saline solution for 2, 4, or 8 days. The dissolution of the fibers was studied by measuring the amount of silicon (Si), iron (Fe), and aluminum (Al) in each medium. The AMs in culture dissolved Fe and Al from the fibers but the dissolution of Si was more marked in the cell culture medium without cells and in the Gamble's solution. The dissolution of Si, Fe, and Al was different for different fibers, and increased as a function of time. The Fe and Al content of the fibers correlated negatively with the dissolution of Si by AMs from the MMVF, i.e., when the content of Fe and Al of the fibers increased the dissolution of Si decreased. These results suggest that the chemical composition of MMVFs has a marked effect on their dissolution. AMs seem to affect the dissolution of Fe and Al from the fibers. This suggests that in vitro models with cells in the media rather than only culture media or saline solutions would be preferable in dissolution studies of MMVFs.

Scanning electron microscopic study on the changes in the cell surface morphology of rat alveolar macrophages after their exposure to man-made vitreous fibers.

The purpose of the present study was to investigate the morphological changes in the surface of rat alveolar macrophages (AM) after their exposure to man-made vitreous fibers (MMVF). Also the ability of rat AM to phagocytize respirable-sized MMVF was studied. The study was carried out by exposing rat AMs in a temporal fashion, for from 30 min to 96 hr, to the fibers in vitro. Scanning electron microscopy (SEM) was used to demonstrate the morphological changes and the phagocytosis of MMVF by rat AMs. Before exposure, the cells had continuous membranes with a variety of small surface features. The phagocytosis of MMVF by AMs started within 30 min after the exposure and increased as a function of time. Also the formation of prominent rufflings and blebs increased by the time. Short fibers, less than 20 microns in length, were usually phagocytized by a single AM, whereas two or more AMs usually phagocytized longer fibers. The cells produced extensions which fasten them to the fibers or to other cells to form clumps or clusters of cells and fibers, each AM engulfing a part of a fiber. Over 70% of the exposed cells were viable and still active after 96 hr of exposure. This finding suggests that the MMVF were not acutely toxic to the rat AMs in vitro. The results also reveal that the surface morphology of the AMs changed slowly during exposure of MMVF and that the cells actively phagocytized MMVF even 96 hr after the beginning of the exposure.