Mutagenesis by man-made mineral fibres in the lung of rats.

The potential of two asbestos substitute mineral fibres--rock (stone) wool RW1 and glass wool MMVF10--to induce gene mutations, DNA strand breaks, inflammation and oxidative stress has been studied in rats. Male homozygous lamda-lacI transgenic F344 rats were intratracheally instilled with single doses of 1 and 2 mg/animal of fibres or with multiple doses of 2 mg/animal administered weekly on four consecutive weeks (8 mg in total). Exposure to RW1 fibres for 16 weeks significantly increased mutant frequency (MF) in the lung in a dose-dependent manner, while MMVF10 fibres did not exhibit any increase of MF at any dose. RW1 fibres gave a significant increase of MF at a dose of 1 mg. Four weeks after instillation, neither the single nor the multiple doses significantly increased MF for both fibre types. To investigate mechanisms for induction of mutations, other genotoxicity markers and parameters of inflammatory and oxidative damage were determined in relation to MF. A weak correlation of mutagenicity data with other genotoxicity parameters studied was observed. DNA strand breaks as measured by comet assay were increased in alveolar macrophages and lung epithelial cells of RW1 and MMVF10 treated rats. RWl fibres caused more extensive lung inflammation as measured by release of neutrophils into broncho-alveolar lavage fluid than MMVF10 fibres. The effects were observed 16 weeks post-exposure, indicating a persistence of the pathogenic process during the exposure period. Only minor differences in the extent of inflammatory processes were observed between the doses of 2 mg and 4 x 2 mg, suggesting that any threshold for inflammation lies below the dose of 2 mg. With the exception of the highest dose of MMVF10 fibres after 16 weeks of exposure, no significant increase of oxidative damage as measured by levels of malondialdehyde in lung tissue was observed. MMVF10 fibres caused weaker inflammation in the lung of rats and did not exhibit any mutagenic effect. We conclude that a weak but chronic inflammation (more likely than acute inflammation or direct oxidative damage) in the lung tissue of fibre treated rats characterized by moderate influx of inflammatory cells into BAL is probably responsible for the observed mutagenic effect of RW1 fibres.

Effects of vinyl chloride exposure alone and in combination with trypan blue--applied systematically during all thirds of pregnancy on the fetuses of CFY rats.

Vinyl chloride (VC) has been shown to be present in the fetal and maternal blood as well as in the amniotic fluid after the exposition of pregnant CFY rats to VC at an atmospheric concentration of 5500, 18 000 or 33 000 mg/m3 (approximately 2000, 7000 or 12 000 ppm) for 2.5 h on the 18th day of pregnancy, indicating the permeability of the placenta to the agent. Teratological investigation of the offspring of pregnant rats exposed continuously to VC at an atmospheric concentration of 4000 mg/m3 air (1500 ppm) during the first, second or last third of pregnancy has shown that VC has no teratological effect in the rat and has no embryotoxic effects either, when applied during the second or last third of pregnancy in the above concentration. Exposition to VC during the first third of pregnancy resulted in an increased fetal mortality and in the manifestation of embryotoxic effects. Fetal losses and induction of central nervous system malformation due to trypan blue administration were not potentiated by a combined exposure of pregnant rats to VC and the dye.

Study on the role of maternal sex steroid production and metabolism in the embryotoxicity of para-xylene.

CFY rats were exposed to inhalation of clean air or air containing para-xylene (3000 mg/m3) on the 10th, and 9th and 10th days of gestation. Uterine and ovarian venous blood flow, fetal weight, ovarian progesterone and 17 beta-oestradiol secretion, and the progesterone and 17 beta-oestradiol level of peripheral blood (uterine and femoral veins) were measured on the 11th day of gestation. Exposure to para-xylene decreased the weight of the fetuses and the progesterone and 17 beta-oestradiol levels of peripheral blood, but it did not influence the uterine and ovarian venous outflow and the ovarian progesterone and 17 beta-oestradiol secretion rate. It is concluded that para-xylene, by inducing the hepatic monooxygenase system, facilitates the biotransformation of progesterone and 17 beta-oestradiol, which is metabolized by this enzyme system. The decrease in the sex hormone level of peripheral blood is supposed to play a role in the embryotoxicity (retarding and lethal effects) of para-xylene.

Acute effects of cadmium on preovulatory serum FSH, LH, and prolactin levels and on ovulation and ovarian hormone secretion in estrous rats.

On the day of diestrus II CFY rats were given 5, 10, or 15 mg/kg cadmium chloride (CdCl2) or 1.0 mL/kg of 0.9% NaCl. On the next day a group of animals was anesthetized with pentobarbital and blood was collected from the aorta at 13:00, 15:00, 16:30, or 18:00 h. for FSH, LH, prolactin (PRL), progesterone (P), and estradiol-17 beta (E2) determination. On the day of the expected estrus, the second group of animals was anesthetized with pentobarbital and cannulas were inserted in one of the femoral arteries and veins, and in one of the utero-ovarian veins. Five-minute blood fractions were collected from the ovary for 40 min, and following the first blood samples, 10 IU hCG was injected iv. Ovarian venous outflow and blood pressure were continuously recorded. From the blood fractions, P and E2 were determined, and their secretion rates were calculated. In a third group of treated animals, the ovaries were excised for histological examination, and oviducts were flushed for counting oocytes. CdCl2 in the dose of 10 or 15 mg/kg increased the PRL serum levels at 13:00 h; it diminished FSH serum levels in the dose of 10 mg/kg and LH serum levels in the doses of 10 and 15 mg/kg at 15:00 h. The decrease in LH levels continued until 16:30 h in the dose of 10 mg/kg CdCl2. In estrous animals, CdCl2 did not influence the blood pressure and ovarian blood flow. In animals receiving 10 or 15 mg/kg CdCl2, a decrease in basal secretion of P occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenesis by asbestos in the lung of lambda-lacI transgenic rats.

In order to get more insight into the mechanism of asbestos-related lung cancer, the mutagenic potential of asbestos was examined in vivo in rat lung. Groups of five transgenic lambda-lacI (Big Blue) rats were intratracheally instilled with single doses of 1 or 2mg, or with four weekly doses of 2mg, per animal of the amosite asbestos. Sixteen weeks after instillation, the mutation frequency was found to be increased in lung DNA by 2-fold at doses of 2 mg (P = 0.035) and of 4 x 2 mg (P = 0.007) amosite. No significant changes were observed after 4 weeks of exposure. In separate experiments, wild-type F344 rats were treated by the same regimen as described above and markers of inflammation, genotoxicity, cell proliferation and lung tissue damage were analysed. Our results indicate a weak but persistent inflammation and cell proliferation which possibly plays a major role in the observed mutagenic effect.

The effect of prenatal indium chloride exposure on chondrogenic ossification.

Daily indium chloride doses of control (0) or 400 mg/kg were administered orally to pregnant Sprague-Dawley (SD) rats by gavage, on d 20 of gestation. Indium concentration was determined in the maternal and fetal blood, livers, kidneys, skulls, and femurs by atomic absorption spectrometry. Further groups of pregnant rats were treated with control (0) or 400 mg/kg indium chloride orally, during the whole gestation period. The fetuses were examined on d 21 of gestation, using histological and histochemical methods. Four hours after the administration indium concentration was found to be significant in the blood, liver, and kidneys of the dams. Twenty-four hours later it increased in the blood but not in the liver and kidney. Fetal indium concentrations were 40-50% of the maternal levels due to a barrier of the placenta. In the skull and the femur, indium was already detectable at 4 h after the administration, and by the end of 24 h, metal concentration was several times higher than that at 4 h, indicating accumulation. Furthermore, it was found that the birefringency of collagen detectable by picrosirius red staining in polarized light around the chondrocytes disappeared and became irregular. In the matrix of the epiphyseal cartilage, the regular, birefringent network demonstrable by Rivanol reaction became irregular and hardly recognizable. In the cytoplasm of the chondrocytes, the diffuse, evenly distributed positive Ricinus communis agglutinin reaction became irregular or disappeared. Similar but much weaker changes were observed with concanavalin A and wheat germ agglutinin stainings. It was concluded that the missing femur and micromelia diagnosed by alizarin staining is the consequence of a specific toxic effect of indium that inhibits chondrogenic ossification. No similar histochemical changes were observed in the bones of the skull developing by desmogenic ossification, despite the presence of indium. Data indicate that the mechanisms of the effects of indium causing retardation and/or malformation differ in the bones developing through desmogenic or chondrogenic ossification.

Effect of Ni2+ on the testosterone production of mouse primary Leydig cell culture.

This study evaluated the effects of Ni2 on testosterone (T) production of mouse Leydig cells in vitro following an in vivo or in vitro exposure. CFLP mice were subjected to repeated exposure (4 treatments, subcutaneously, every 3 d) to 10, 20 or 40 mg/kg body weight of NiSO4 or 1.0 ml of 0.9% NaCl solution. Depressed human chorionic gonadotropin (hCG)-stimulated T response was seen over a 48-h culture of testicular interstitial cells obtained from the animals exposed to 20 mg/kg or higher dose of NiSO4, while the basal T production remained unaltered. There were no Ni2+-related changes in the body weights or in the weights of testes, epididymides, adrenals, and kidneys. No histopathological alteration was found in the examined organs of NiSO4-treated groups except the dose-dependent tubular lesions in kidney as a result of a specific rather than a general cytotoxic action. To assess the direct effect of Ni2+ on Leydig-cell T production, testicular interstitial cells were cultured with Ni2+ (62.5 to 1000 microM) for 48 h in the presence or absence of maximally stimulating concentration of hCG. Dose-dependent depression in hCG-stimulated T production was seen at 125 microM or higher dose of Ni2+, while basal T production was unaffected. In order to evaluate the time dependency of this effect the cells were cultured for various times in the presence or absence of 250 and 1000 microM Ni2+. Decreased hCG-stimulated T production was found in the cultures maintained at least for 4 h in the presence of 1000 microM Ni2+, whereas at 250 microM at least 16 h was required to elicit the depression. Cell viability was assessed by a metabolic activity (MTT) assay. The viability of cells was unaltered by 250 microM Ni2+, and only a slight decrease was found even at the end of the 48-h culture period in the presence of 1000 microM Ni2+. Our results show a dose-related depression in stimulated T production of mouse Leydig cells in culture following either in vivo or in vitro Ni2+ treatment at a dose that does not induce any general toxic or significant cytotoxic action. The data of the time-course study indicate that the effect of Ni2+ on Leydig-cell T production is both time and concentration dependent, and not due to cytotoxicity.

Effects of cobalt sulfate on prenatal development of mice, rats, and rabbits, and on early postnatal development of rats.

The effects of cobalt sulfate administered to pregnant C57BI mice, OFA-SD rats, and New Zealand rabbits was studied on fetal and postnatal offspring. Cobalt concentration in the maternal blood was increased in proportion to the administered doses. Cobalt crossed the placenta and appeared in the fetal blood and amniotic fluid. Regardless of the administered dose of cobalt sulfate, cobalt concentration in the blood peaked 2 h after administration. Cobalt produced dose-dependent maternal toxicity and was found to be embryotoxic in all three species, as evidenced by elevated frequency of fetuses with body weight or skeletal retardation and embryolethality. Cobalt increased the frequency of major anomalies significantly in mice and rats, with anomalies of the eyes, kidneys, skull, spine, and sternum in mice, and anomalies of the urogenital system in rats. Cobalt sulfate was not teratogenic in rabbits. Intra-amnial administration of cobalt sulfate produced a dose-dependent increase of the frequency of dead fetuses, and weight retardation of the live fetuses. The direct cytotoxic effect probably plays a role in the embryotoxic and teratogenic effects of cobalt. The postnatal examinations revealed a decrease of the perinatal index in the treated group. The body weight of the pups in the treated group was lower during wk 1 of life, but no difference was found between the control and treated by the end of wk 2. Eye opening was completed in the usual time period in both groups, while time of appearance of the teeth, descending of the testes, shaping of ears, and development of hearing was delayed in the treated group. The development of muscle strength and of the locomotor system was delayed. All the functions studied (forward movement, swimming, righting reflex) normalized by postnatal d 21, with the exception of muscle strength. It was concluded that cobalt sulfate exposure decreases the perinatal viability of the fetuses, but the functions of the surviving fetuses with perinatal retardation become compensated by postnatal wk 2-3. The development of fetuses is undisturbed thereafter.

Study of inflammatory responses to crocidolite and basalt wool in the rat lung.

The subacute effects of crocidolite and basalt wool dusts were studied by nmeans of biochemical, morphological. and histological methods 1 and .3 mo after intrabronchial instillation. The cell count, protein and phospholipid contents, and lactate dehydrogenase (LDH) activity were determined in the bronchoalveolar lavage (BAL). Both types of fibers induced a prolonged inflammatory reaction in the lung. All the parameters studied in the experimental groups were more markedly elevated after 3 mo. Relative to the control, the protein and LDH values were increased three- to fivefold, the phospholipid content twofold, and the number of free cells in the BAL exceeded the control level up to ninefold. The inflammatory responses to crocidolite and basalt wool in the lung did not differ significantly. In spite of this, basalt wool is recoinmended as an asbestos substitute, as the use of this man-nade fiber may result in a significantly lower release of dust than that from crocidolite.

Effects of sodium diethyldithiocarbamate on type II pulmonary epithelial cells in vitro.

Dithiocarbamates (DDTC) are chemicals widely used in the form of pesticides, therapeutic and chelating agents, and scavengers. Since DDTC interfere with SH, Cu, and Zn enzymes due to chelating properties, it was of interest to clarify, in primary culture of type II alveolar pneumocytes, the effect of this compound upon enzymes of glutathione cycle, Cu, Zn-superoxide dismutase, and the membrane structure of cells. DDTC significantly inhibited the activity of superoxide dismutase and the activity of gamma-glutamyl transpeptidase, glutathione reductase, and alkaline phosphatase, whereas an increase in the activity of glutathione peroxidase was found. The membranes of pneumocytes type II were injured. Data show that DDTC adversely affected type II pneumocyte function and structure.

Embryotoxic and teratogenic effects of indium chloride in rats and rabbits.

Daily indium chloride doses of control (0), 50, 100, 200, or 400 mg/kg were administered orally to Sprague-Dawley rats by gavage, on d 6-15 of gestation, and daily metal doses of control (0), 50, 100, or 200 mg/kg were administered to New Zealand rabbits on d 6-20 of gestation. Further groups of pregnant rats were treated with control (0) or 400 mg/kg indium chloride orally on one of d 8, 9, 10, 11, 12, 13, 14, or 15 of gestation. The dams and fetuses were examined on d 21 (rats) and 30 (rabbits) of gestation, using standard teratological methods. Indium concentration was determined in the maternal and fetal blood, as well as in the amniotic fluid, by atomic absorption spectrometry. Indium was found to cross the placenta and appeared in fetal blood in proportion to the metal concentration of the maternal blood. In the amniotic fluid, indium concentrations remained below the detection limit. In rats, indium chloride produced dose-dependent maternal toxic effects, with a dose of 400 mg/kg inducing embryotoxicity (embryolethality) and teratogenicity. Doses of 200 and 100 mg/kg were embryotoxic (retarding) and teratogenic, causing skeletal and visceral anomalies in addition to external anomalies (rudimentary or missing tail, syndactylia, clubfoot, exencephalia) in rats. In rabbits, 200 mg/kg indium chloride was lethal for the dams and the embryos (some of the animals died, and the number of abortions and full resorptions increased). This dose was found to be teratogenic (caused gross renal anomalies) and increased the frequency of fetuses with skeletal retardation. In rats, the effects of indium chloride causing fetal retardation was found to be independent of exposure time. The teratogenic effects were the highest on d 11 and 12 of gestation, when indium chloride caused gross external malformations. Data suggest that the teratogenic effects of indium chloride can be attributed primarily to a direct cytotoxic action of indium resulting from placental transfer, but the effect is not a selective one, as it appears only in the presence of maternal toxic effects.

The pulmonary toxicity of cinnamon dust in rats.

The histopathological effect of a single intratracheal dose of respirable cinnamon dust, cinnamon dust extract, and cellulose dust on the lungs of rats was studied sequentially one, seven days and one month after treatment. Exposure to respirable cinnamon and cellulose dusts resulted in alveobronchiolitis at the end of the first and seventh day, and fibrotic changes by the end of the first month. As the extract of cinnamon dust caused no histopathological alterations, it is assumed that the cellulose content of cinnamon dust was responsible for the histological reactions.

Surface changes in type II pneumocytes isolated from rats during the cultivation.

Type II cells isolated from the rat lung were maintained in culture for 8 days. The activity of alkaline phosphatase and lectin binding properties were studied. The alkaline phosphatase activity and the number of lamellar bodies were continually decreasing during the studied time period. The profile of lectin binding (Maclura pomifera and Ricinus communis) did not change during the cultivation.

Ozone-induced micronuclei frequency in rat alveolar Type II cells.

The effect of ozone, a ubiquitous air pollutant, was tested on cultured pulmonary epithelial type II cells isolated from rats. After 40-hour culture, the cells were exposed for 6 h to 400 ppb of ozone or air. The number of micronucleated cells was counted after the exposure. In each group, 17000 cells were evaluated. The number of micronucleated cells was significantly increased in the ozone-exposed group (12.24 per 1000 cells) compared to the control group (5.00 per 1000 cells). The results showed the mutagenic effect of ozone exposure on alveolar type II cells, manifested in the increased frequency of their micronuclei.

On the structural differences in the membranes between ciliated bronchial and non-ciliated lymphoepithelium in rats.

The membrane structure of bronchial ciliated epithelium and lymphoepithelium was studied in rats by lectin histochemistry. The lymphoepithelium, contrary to bronchial ciliated epithelium, did not contain terminal beta-D-galactose residues. Moreover L-fucose and beta-D-Gal (1-3)-D-GalNAc residues, being masked, could be visualized only after enzymatic digestion of terminal sialic acid. These structural differences in membranes provide a basis for the different functions of bronchial lymphoepithelium.

Isolation and cultivation of lung epithelial type 2 cells. The effect of cadmium.

The effects of cadmium exposure on rat lung type 2 cells were evaluated by morphological and biochemical examinations. The results showed dose dependent reduction in the marker enzyme (alkaline phosphatase), changes in cell membranes and in the antioxidant status.

The effect of cardiac lymphostasis on the microcirculation of the heart: effect of cardiac lymphoedema on the development of an arteriovenous shunt circulation.

The effect of cardiac lymphostasis on the microcirculation of the heart was studied in 18 dogs. By ligation of the main lymph trunks and regional lymph nodes of the heart, cardiac lymphoedema--lymphogenic cardiomyopathy--was induced in 9 dogs, while 9 served as control animals. For microcirculatory investigations: 1) gelatin Indian ink injection, 2 benzidine reaction, 3) PVC injection corrosion preparation was applied. Characteristic changes were demonstrated in the microcirculation and capillary circulation of the heart in cardiac lymphostasis. In some capillaries-mainly where the interstitial oedema exists--the capillary circulation decreased: inhomogeneously vessel-free spots were formed in the heart. Around the vessel-free capillaries, elongated capillaries were found including very dilated pre- and post-capillary vessel sections. Arteriovenous shunts can be revealed in the heart in consequence of lymphoedema. The pathogenesis of the microcirculatory circulation changes caused by cardiac lymphostasis was discussed.

The effect of fibrous dusts on lung cells. In vitro study.

The mechanism of toxicity of selected asbestos substitute mineral fibres was examined and compared to that of asbestos. Alveolar macrophages and type II cells were isolated from Fischer 344 rats and after 20 h cultivation various concentration of fibres alone (amosite, wollastonite, rockwool or glass fibres) or in combination with cigarette smoke were added to cells and the cultivation continued for another 24 h. After finishing the exposure the number of alkaline phosphatase positive type II cells was counted, the comet assay was used to detect DNA damage (strand breaks) in both cell types and ultrastructural changes were evaluated by transmission electron microscopy. The decrease of the number of alkaline positive type II cells was dose dependent in all cases. The number of DNA strand breaks (SBs) in both cell types was enhanced after exposure to all types of fiber, the enhancement was dose dependent, the highest level of SBs was observed after amosite exposure. The combined exposure to mineral fibres and cigarette smoke showed synergic effect on the level of SBs. Transmission electron microscopy showed that already 1 microg x cm(-2) amosite caused destruction of AM while other fibres were phagocytized.

Patchy myocardial pattern of virus sequence persistence in heart transplant recipients--possible role of sampling error in the etiology.

BACKGROUND: The pathway from viral myocarditis to end-stage heart failure is commonly accepted, but diagnosis of virus-mediated myocardial injury remains challenging. Virus persistency in the myocardium may accelerate ventricular failure; thus, a precise diagnosis of virus persistency may prevent the development of end-stage heart failure. METHODS: We performed a systematic investigation on the sampling error of viral diagnostics in heart transplant recipients: Transmural samples from 5 regions of the explanted hearts from recipients during heart transplantation were amplified using entero-, adeno-, and herpesvirus sequences and histologic examinations performed. RESULTS: We examined 175 myocardial samples from dilated cardiomyopathy and 100 samples from 20 forensic medicine patients. Seven patients were positive for the examined viruses: 10 positive regions for adenovirus, and 1 positive region for herpes virus DNA, but none for enterovirus. A focal myocardial pattern was detected for adenovirus. CONCLUSION: Our results with the patchy myocardial viral persistence may explain possible false-negative results related to virus-mediated etiology among end-stage dilated cardiomyopathy patients. Therefore, repeated endomyocardal biopsies, and multiple cardiac samples are recommended to be obtained to evaluate the etiology of heart failure, thus reducing the occurrence of end-stage heart failure and decreasing the number of patients requiring heart transplantation.