Pulmonary inflammation in rats after intratracheal instillation of quartz, amorphous SiO2, carbon black, and coal dust and the influence of poly-2-vinylpyridine-N-oxide (PVNO).

Effects of poly-2-vinylpyridine-N-oxide (PVNO) were investigated in numerous in vivo and in vitro studies published in the nineteen sixties and seventies. These studies showed that PVNO inhibited development of fibrosis from quartz dust and improved lung clearance of quartz after inhalation exposure. Ameliorating effects of PVNO were observed also for pulmonary damage from colloidal SiO2 and organic substances, and the fibrogenic inflammation caused by carrageenan. Although it is not proven that silicosis is a precondition for quartz-induced lung tumours, we investigated the hypothesis that PVNO could reduce the lung tumour risk from quartz in rats. A carcinogenicity study was therefore started in rats with the main focus on the quantitative relationships among pulmonary inflammation, fibrosis and neoplasia caused by intratracheal instillation of 3 mg quartz DQ 12 with or without additional subcutaneous PVNO treatment. Other study groups were treated with multiple dust instillations, i.e. 30 instillations of 0.5 mg amorphous SiO2 at intervals of 2 weeks, 10 instillations of 0.5 mg of ultrafine carbon black or 1 mg coal at weekly intervals. The analyses of the bronchoalveolar lavage fluid (BALF) 9 months after start of the life-time study showed that the aim of producing similar levels of increased enzyme concentrations in the four groups treated with quartz/PVNO, amorphous SiO2, carbon black and coal was achieved. A 2.5- to 7.7-fold increase for lactate dehydrogenase (LDH), total protein, alkaline phosphatase and gamma-glutamyl transferase (gamma-GT) was found in these groups as compared to the control. In contrast, quartz treatment without PVNO increased the LDH level up to 24-fold and of total protein to 13-fold. However, the cell counts in the BALF were not so much different in all five groups, i.e. quartz without PVNO (leukocytes: 480.000, PMN: 190.000), quartz with PVNO (leukocytes: 300.000, PMN: 100.000), amorphous SiO2 (leukocytes: 570.000, PMN: 315.000), carbon black (leukocytes: 390.000, PMN: 150.000) and coal (leukocytes: 200.000, PMN: 65.000). Histopathological investigations after four weeks and three months revealed that the used PVNO sample was active in the quartz and amorphous SiO2 groups and markedly reduced the incidences or severity of several pulmonary changes such as macrophage accumulation, inflammatory cell infiltration, interstitial fibrosis, bronchiolo-alveolar hyperplasia, alveolar lipoproteinosis and amorphous SiO2 -induced granulomatous alveolitis/interstitial fibrotic granulomas. Also in the lung-associated lymph nodes (LALN), PVNO treatment significantly reduced the incidence and severity of inflammation in both quartz and amorphous SiO2 groups as evidenced by the presence of well-circumscribed aggregates of intact particle-laden macrophages without signs of degeneration and accompanying granulocytic infiltration and fibrosis. Immunological investigations at the 9 months timepoint on the in vitro production of reactive nitrogen (RNI) or oxygen (ROI) intermediates and tumour necrosis factor (TNF-alpha) from BALF-derived cells indicated a diminished responsiveness to LPS in all particle treatment groups. A diminished production of ROI was also found in the quartz, carbon black, and coal dust groups, respectively, as compared to the values seen in the quartz/PVNO- and amorphous SiO2 treated groups. Treatment with quartz plus PVNO restored the capability of the cells to respond to LPS as compared to the treatment with quartz alone. TNF-alpha production was diminished in the groups treated with quartz, carbon black, and coal dust alone whereas in the quartz/PVNO- and amorphous SiO2-treated groups an elevated TNF-alpha production was seen. These results led to the conclusion that only amorphous SiO2 did not affect the "normal" ability of the cells to respond to LPS and that PVNO protected the cells from a toxic effect of the quartz particles.

DNA damage in lung epithelial cells isolated from rats exposed to quartz: role of surface reactivity and neutrophilic inflammation.

Respirable quartz has been classified as a human lung carcinogen (IARC, 1997). However, the mechanisms involved in quartz-induced carcinogenesis remain unclear. The aim of the present study was to investigate acute DNA damage in epithelial lung cells from rats exposed to quartz. Since surface reactivity is considered to play a crucial role in the toxicity of quartz, the effect of surface modifying agents polyvinylpyridine-N-oxide (PVNO) and aluminium lactate (AL) was evaluated. Therefore, rats were instilled with quartz (DQ12, 2 mg/rat) or quartz treated with PVNO or AL. After 3 days animals were killed and brochoalveolar lavage (BAL) was performed to evaluate inflammatory cell influx. BAL-fluid levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP) and total protein were used as lung damage markers. Neutrophil activation was assessed by myeloperoxidase (MPO) measurement, and total antioxidant capacity of the BAL-fluid was determined using the TEAC (trolox equivalent antioxidant capacity) assay. Lung epithelial cells were isolated and DNA strand breakage was determined by single cell gel electrophoresis (comet assay). DNA damage was significantly increased in epithelial cells from rats instilled with DQ12, whereas no enhanced DNA strand breakage was observed when quartz was treated with PVNO or AL. Total protein, LDH and TEAC were increased in rats treated with native quartz, and this was inhibited by both coatings. A significant correlation between neutrophil numbers and MPO levels was observed, indicating neutrophil activation. Inhibition of DNA damage by both coatings was paralleled by a reduction of neutrophil influx as well as MPO activity. In this study we provide evidence that modification of the particle surface prevents DNA strand breakage in epithelial lung cells from quartz-exposed rats. Furthermore, the present data show the feasibility of our in vivo model to evaluate the role of inflammation, antioxidant status, and cytotoxicity in particle-induced DNA damage.

Studies on the toxic effects of quartz and a mycobacterial glycolipid, trehalose 6,6'-dimycolate.

Quartz and trehalose 6,6'-dimycolate (TDM) both potentiate tuberculosis and have toxicities that depend on surface crystalline structures. Investigations were undertaken to determine if TDM can kill macrophages and produce hemolysis in a fashion similar to that of quartz and if quartz can induce granulomas similar to those induced by TDM. Trehalose 6,6'-dimycolate was spread as a molecular monolayer on the surface of tissue culture dishes adjacent to areas of uncoated plastic for comparison. Murine peritoneal macrophages were killed within hours by contact with the TDM monolayer, while those on adjacent areas of uncoated plastic remained viable and spread normally. The membranes of erythrocytes were also damaged by contact with the monolayer of TDM. This damage was inhibited by poly-2-vinyl-pyridine-N-oxide, an inhibitor of hydrogen bonding that blocks quartz induced hemolysis. These data suggest that TDM damages membranes via an adhesive mechanism similar to that of quartz. Furthermore, injections of quartz particles into mice induce acute granulomatous reactions similar to those induced by TDM. These data indicate that TDM and quartz have certain similarities in their mechanisms of action and that these similarities may be of importance in the pathogenesis of tuberculosis.

The effects of tetrandrine (TT) and polyvinylpyridine-N-oxide (PVNO) on gene expression of type I and type III collagens during experimental silicosis.

In the screening tests of drugs for silicosis in our laboratory, we found that TT, a type of alkaloid isolated from Stephania tetrandra, could inhibit the development of experimental silicosis of rats and the synthesis of collagen in rat lung. Chest X-rays of silicotic patients treated with TT for 1-3 years showed obvious changes. The silicotic nodules became smaller and shadows became clearer. PVNO was proved to have anti-silicotic effect on animal and clinically. This presentation reports the effect of them on collagen mRNA. Dot blot results showed that alpha 1 (I) and alpha 1 (III) mRNA levels increased significantly at 60 and 120 days after the rats were exposed to silica dust. The mRNA levels went down at 1 and 3 months after treated by TT and PVNO. In situ hybridization observation revealed that the silver grains of Type I and Type III collagen were scattered within the fibroblasts in cellular nodules and in thickened interstitium of silicosis tissue. The amounts of mRNA silver grains decreased in the lung tissue treated by TT and PVNO. It was suggested that TT and PVNO may inhibit the gene expression of collagen during silicosis.

Polyvinylpyridine-N-oxide and carboxymethyl cellulose inhibit mineral dust-induced production of reactive oxygen species by human macrophages.

Polyvinylpyridine-N-oxide (PVPNO) and carboxymethyl cellulose (CMC) are known to inhibit the cytotoxic effects of quartz particles and chrysotile asbestos fibers, respectively. The effect of PVPNO and CMC on mineral dust-induced production of reactive oxygen species (ROS) by human monocyte-derived macrophages was studied using lucigenin-dependent chemiluminescence. Ten micrograms of PVPNO inhibited the ROS production induced by 100 micrograms of quartz completely, but caused only 25% inhibition of the response to 100 micrograms of chrysotile. Ten micrograms of CMC inhibited the chrysotile-induced ROS production to 70%, but had a weak effect on the responses to quartz. Neither PVPNO nor CMC caused any inhibition of the ROS responses to serum-opsonized zymosan. PVPNO was bound to both quartz and chrysotile, but no binding of CMC to either dust could be detected. Neither PVPNO nor CMC had any superoxide scavenging effect. In conclusion, PVPNO and CMC seem to selectively modify mineral dust surface properties, which are of importance in the induction of ROS production by phagocytes. These observations may be useful in describing the pathogenesis of mineral dust-induced diseases, and may also have therapeutic implications.

Effect of two particle surface-modifying agents, polyvinylpyridine-N-oxide and carboxymethylcellulose, on the quartz and asbestos mineral fiber-induced production of reactive oxygen metabolites by human polymorphonuclear leukocytes.

We studied the capacity of quartz and asbestos fibers to induce the generation of reactive oxygen metabolites in human polymorphonuclear leukocytes (PMNs) with a chemiluminescence (CL) assay. On an equal weight basis, the particulates induced CL in the following order of magnitude: chrysotile, quartz greater than amosite, crocidolite, greater than anthophyllite, wollastonite. The intensity of CL correlated positively with the Alcian blue (a cationic dye) binding capacity of the particles. Polyvinylpyridine-N-oxide (0.5 microgram/ml) inhibited completely the CL induced by quartz but had little effect on the CL induced by asbestos fibers. Carboxymethylcellulose (1.0 microgram/ml), however, reduced the CL caused by chrysotile asbestos but had no effect on the CL induced by the other particulates. Our results suggest that in addition to length and diameter, the effect of quartz and asbestos fibers on inflammatory cells will depend on surface characteristics, including the charge of the particles.

Carcinogenicity studies on natural and man-made fibres with the intraperitoneal test in rats.

Female Wistar rats were injected intraperitoneally (i.p.) with a suspension of 11 fibrous and 3 granular dusts. A dose of 0.25 mg actinolite or UICC chrysotile induced tumours of the peritoneum in more than 50% of the animals. Even 0.05 and 0.01 mg proved to be carcinogenic, although no adhesions of the abdominal organs could be observed. The findings are in conflict with the hypothesis that a scar is always the morphological precondition for the development of an asbestos-induced tumour. Actinolite injected i.p. in a solution of polyvinylpyridine-N-oxide gave a lower tumour incidence than when suspended only in saline, possibly due to inactivation of the fibre surface. Persistent glass fibres were less effective than actinolite having a similar fibre size distribution. On the other hand, relatively thick basalt fibres and ceramic fibres gave higher tumour incidences than expected. Wollastonite fibres were not carcinogenic, probably because of their low durability. Large amounts of polyvinylchloride, alpha-ferric oxide hydrate and wood dust also led only to adhesions of the abdominal organs and fibrosis; a definite carcinogenic effect was not detected.

Quartz-dust-induced production of reactive oxygen metabolites by human granulocytes.

We studied the effect of quartz on the production of reactive oxygen species by human polymorphonuclear leukocytes (PMN) in vitro by a chemiluminescence (CL) assay. Quartz caused a rapid dose-dependent CL response in the cells. Diamond dust used as an inert control did not stimulate the production of reactive oxygen metabolite by PMN. The quartz-induced activation of oxygen metabolism was also demonstrated by measuring oxygen consumption, nitroblue tetrazolium reduction, and superoxide and hydrogen peroxide production by PMN. Poly-vinyl-pyridine N-oxide (a quartz surface modifying agent) completely abolished the quartz-induced response, but had no effect on opsonized zymosan-induced CL response of PMN. The effect of N-acetylcysteine (a known antioxidant) was inhibitory to the CL formation induced by both quartz and opsonized zymosan. Our results are in agreement with the hypothesis that quartz-induced production of reactive oxygen metabolites is a possible mechanism by which quartz dust produces chronic inflammation and tissue injury of the lung. Agents interfering with the generation of reactive oxygen metabolites may provide a rationale for treatment of mineral-dust-induced pulmonary disease.

Titanium dioxide induced chemiluminescence of human polymorphonuclear leukocytes.

The ability of different titanium dioxides (TiO2) to induce production of reactive oxygen metabolites by human polymorphonuclear leukocytes was studied. Pure rutile or anatase preparations show only a weak chemiluminescent response. Surface-modified TiO2 causes a strong chemiluminescent response with a biphasic configuration resembling that of quartz. Sonication of the dust suspensions resulted in a strong enhancement of the chemiluminescent response, with each dust preparation showing approximately equal maximal activity. However, coated TiO2 still exhibited a different mode of cell activation. The chemiluminescence-inducing activity of the different TiO2 studied did not correlate with their hemolytic activity. As polyvinyl-pyridin-N-oxide (PVPNO) inhibits the chemiluminescence induced by coated TiO2 samples, it seems that both particle size and surface structure determine the mode and intensity of activation of human PMNL by TiO2. The results point out the need for in vivo testing and comparison of different TiO2 preparations.

The prophylactic use of polyvinylpyridine-N-oxide (PVNO) in baboons exposed to quartz dust.

Twelve baboons were exposed to a quartz dust cloud. Four of these were also given polyvinylpyridine-N-oxide (PVNO) by aerosol and four received PVNO by aerosol and injection. A prophylactic effect was demonstrated during the course of treatment, but when treatment stopped the silicosis progressed to the same degree of severity as in the untreated animals.

Determinants of pulmonary fibrosis and lipidosis in the silica model.

The conditions which might favour development of the fibrotic or the lipid component of the pulmonary reaction to inhaled quartz were examined in rats. Smaller particle size and freedom from surface contamination by amorphous silica or iron oxide, status of the animals whether specific pathogen-free or conventional, and the resistance of cell membranes to damage appeared to bear on fibrogenesis. Increased membrane stability by treatment with polyvinylpyridine-N-oxide abolished not only the fibrosis but also the response of type II cells and hence lipidosis. The rate and intensity of quartz deposition may also affect the response, a low concentration inhaled over a long period favouring nodulation. No other manipulations, environmental or pharmacological, succeeded in inhibiting lipidosis to the benefit of fibrosis. Guinea pigs, however, behaved differently, their reaction being characterized by massive alveolar accumulation of dust-bearing macrophages and type II cell hyperplasia but not by lipidosis. The species variation is unexplained but macrophage predominance may represent a phase that later transforms to lipidosis. The experimental findings may have implications for forms of pneumoconiosis other than silicosis.

Crystal-induced membrane damage: hydroxyapatite crystal-induced hemolysis of erythrocytes.

Microcrystals of hydroxyapatite cause severe membrane damage in human erythrocytes, as is evident from the strong hemolysis that is caused by these crystals. Hemolysis by hydroxyapatite crystals is time and concentration dependent, and is preceded by aggregation of erythrocytes. Polyvinylpyridine-N-oxide, a strong hydrogen acceptor, has no inhibiting effect on hydroxyapatite-induced hemolysis. This suggest that the mechanism of action of these crystals is different from that of urate crystals and silica particles, where hydrogen bonding interaction is supposed to be important. Negatively charged macromolecules, such as dextran sulfate, heparin, and polyglutamic acid, inhibit hydroxyapatite crystal-induced hemolysis, suggesting that positive charges, probably located on the crystals, play an important role in the membrane-damaging effect of these crystals. The structures with which these positive charges interact remain to be determined because removal of negative charges from the erythrocytes by treatment with neuraminidase does not affect crystal-induced hemolysis.

The cytotoxicity of kaolin towards macrophages in vitro.

The inhalation of china clay dust by man can cause pneumoconiosis. In an attempt to identify the factors responsible the cytotoxicity in vitro of china clay dust towards mouse peritoneal macrophages was examined. Respirable dusts collected at china clay drying plants were cytotoxic towards the cells. This activity was caused by kaolinite (the major mineral in china clay) and was not due to the presence of ancillary minerals. The cytotoxicity of kaolinite was not due to particle morphology and the positively charged edges of the mineral contributed only slightly to cytotoxicity. An electron microscope study showed that macrophages phagocytosed PVPNO-coated kaolinite particles indicating that the low cytotoxicity of these particles was not due to poor phagocytosis. Residence of china clay in rat lungs appeared to reduce its cytotoxicity. It was concluded that the cytotoxicity of kaolinite was probably related to the proposed amorphous silica-rich gel coating on the particles. The relevance of the findings in vitro to the effects in vivo of china clay is discussed.

[Characteristics of the morphofunctional status of lung macrophages in the phagocytosis of particles of varying cytotoxic action]. / Osobennosti morfofunktsional'nogo sostoianiia makrofagov legkikh pri fagotsitoze chastits razlichnogo tsitotoksicheskogo deistviia.

The cytotoxic effect of quartz and coal on phagocytizing lung macrophages was studied by transmissive and scanning electron microscopy and histochemically (the detection of acid phosphatase). The level of phagocytic activity, the activity and localization of acid phosphatase, the number and state of organelles, and the type of macrophage surface processes were shown to represent the objective criteria of the morphofunctional state of macrophages and the cytotoxic effect of the particles phagocytized.

Factors involved in the cytotoxicity of kaolinite towards macrophages in vitro.

The cytotoxicity of a high purity Cornish kaolinite toward mouse peritoneal macrophages in vitro was examined. The material was cytotoxic towards these cells, the activity could be decreased substantially by pretreating the dust with poly(2-vinylpyridine N-oxide). Pretreatment of the dusts with poly(acrylic acid) had a small effect on cytotoxicity, but combinations of the polymer treatments virtually abolished the material's biological activity towards macrophages. These studies indicated that the cytotoxicity of kaolinite is not due to its flakelike morphology.

Adjuvant effects of a crystalline silica on IgE and IgG1 antibody production in mice and their prevention by the macrophage stabilizer poly-2-vinylpyridine N-oxide.

A crystalline silica (standard quartz DQ12 with particle size less than 5 microns) is able to stimulate in Balb/c mice the production of IgE and IgG1 antibody to a single 1-microgram dose of ovalbumin. The adjuvant effects of silica on both IgE and IgG1 antibody production are prevented by pretreatment of animals with poly-2-vinylpyridine N-oxide, a polymer that protects macrophages from the well-documented toxic effects of silica. These results indicate that adjuvanticity of silica is, at least partly, correlated to the damage induced on macrophages.