Expression of heme oxygenase-1 in the lungs of rats exposed to potassium octatitanate whiskers.

OBJECTIVES: Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by asbestos, and heme oxygenase-1 (HO-1) protects lung tissue against oxidative stress. We hypothesized that HO-1 is also associated with oxidative lung injury caused by exposure to potassium octatitanate whiskers (PT1), which is one of the asbestos substitutes. METHODS: Male Wistar rats were administered 1 mg or 2 mg PT1 suspended in saline by a single intratracheal instillation and were sacrificed after recovery for 3 days, 1 wk, 1 mo, 3 mo or 6 mo. Gene expression of HO-1 protein and mRNA and immunostaining were investigated in rat lungs. RESULTS: HO-1 protein expression was increased from 3 days to 1 mo and at 6 mo in the 1 or 2 mg PT1-exposed groups, and the gene expression of HO-1 mRNA was also increased at 3 days and from 1 mo to 6 mo. HO-1-positive cells were mainly found in the alveolar macrophages and the bronchial epithelial cells in immunostaining. CONCLUSIONS: These findings suggest that HO-1 is involved in lung damage caused by PT1.

Expression of inflammation-related cytokines following intratracheal instillation of nickel oxide nanoparticles.

The objective of this study was to examine what kinds of cytokines are related to lung disorder by well-dispersed nanoparticles. The mass median diameter of nickel oxide in distilled water was 26 nm. Rats intratracheally received 0.2 mg of nickel oxide suspended in distilled water, and were sacrificed from three days to six months. The concentrations of 21 cytokines including inflammation, fibrosis and allergy-related ones were measured in the lung. Infiltration of alveolar macrophages was observed persistently in the nickel oxide-exposed group. Expression of macrophage inflammatory protein-1alpha showed a continued increase in lung tissue and broncho-alveolar lavage fluid (BALF) while interleukin-1alpha (IL-1alpha), IL-1beta in lung tissue and monocyte chemotactic protein-1 in BALF showed transient increases. Taken together, it was suggested that nano-agglomerates of nickel oxide nanoparticles have a persistent inflammatory effect, and the transient increase in cytokine expression and persistent increases in CC chemokine were involved in the persistent pulmonary inflammation.

Differential expression of EC-SOD, Mn-SOD and CuZn-SOD in rat lung exposed to crystalline silica.

Superoxide dismutases (SODs) are antioxidant enzymes that catalyze the dismutation of superoxide into hydrogen peroxide. There are 3 kinds of isozymes: extracellular superoxide dismutase (EC-SOD), manganese-containing superoxide dismutase (Mn-SOD) and copper- and zinc-containing superoxide dismutase (CuZn-SOD). To examine the expression of SOD isozymes in lungs injured by crystalline silica, we intratracheally instilled male Wistar rats with 2 mg (8 mg/kg) of crystalline silica and investigated the mRNA, protein level and distribution of SOD isozymes in the rat lungs using RT-PCR, western blot analysis and immunostaining, respectively at from 3 d to 180 d of recovery following the exposure. EC-SOD mRNA levels significantly increased from 3 d to 90 d and the EC-SOD protein level was significantly higher after 90 and 180 d recovery in the crystalline silica exposed groups than in the control groups. Mn-SOD increased in silica treated rat lungs at both mRNA and protein levels, peaking at 30 d post-exposure. CuZn-SOD mRNA levels were decreased at 3, 7 and 30 d, and CuZn-SOD protein levels were also significantly lower than the control group at 90 and 180 d recovery. There was prominent EC-SOD immunostaining mainly in the plasma and alveolar macrophages and strong Mn-SOD staining in alveolar macrophages and interstitial cells of the proximal and distal portions of the alveolar duct following crystalline silica exposure. There was less CuZn-SOD staining in epithelial cells at terminal bronchioles in the crystalline silica-exposed group. These findings suggest that these SOD isozymes may be related to lung injury induced by crystalline silica.

Biological marker of furfural, chemicals without administrative control level.

Furfural, a colorless liquid used in solvent-extraction processes, petroleum refining and as a rubber additive, has been assigned an occupational exposure limit of 2.5 ppm by the Japan Society for Occupational Health, but an administrative control level for furfural has not been established. In order to conduct effective occupational health management in workplaces where furfural is used, we measured furfural concentrations in working environments and collected urine samples to measure furoic acid levels (one of the principal metabolites), which act as a biomarker of exposure to furfural. The measurements of airborne concentrations in a working environment where furfural or a solution containing furfural was handled were made in 2004. Workers answered a questionnaire on working conditions, urine samples were collected at the end of the workshift, and furoic acid in the urine was measured by gas chromatography/flame ionization detector (GC/FID). The ambient concentrations of furfural during the period were 2.1 ppm in a mixer room and 1.6 ppm in a filling room. The mean concentrations of furoic acid in the workers' urine were 7.7 +/- 7.8 mg/g-creatinine in summer and winter, respectively (normal range: 3 - 60 mg/g-creatinine). The average exposure to furfural per month calculated by multiplying the concentration in the working environment by working hours for a month was 86.4 +/- 108.6 ppm hours/months (mean +/- standard deviation) (range; 0 - 336 ppm hours/month). The relationship between average exposure to furfural and furoic acid in the urine was analyzed by simple linear regression analysis and a positive correlation was found. These findings suggest that furoic acid in urine is useful for biological monitoring of exposure to furfural, and that the measurement of both furfural in the environment and furoic acid in the urine are beneficial in occupational health management of furfural.

Calcitonin gene-related peptide (CGRP) as hazard marker for lung injury induced by dusts.

Calcitonin gene-related peptide (CGRP), which has a function as a growth factor of epithelial cells, is thought to play a role in pulmonary epithelium repair. In order to establish whether or not CGRP is associated with repair in lung damaged by dust, we examined gene expression of CGRP in the lungs of animal models exposed to different dusts. Male Wistar rats were administered 2 mg of crystalline silica, crocidolite, potassium octatitanate whisker (PT-1), and silicon carbide whisker (SiCW) suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 mo, 3 mo, and 6 mo of recovery time. Pathological findings of advanced pulmonary fibrosis were present in the rats exposed to crystalline silica and crocidolite through the experiment, whereas findings of mild or reversible pulmonary fibrosis were present in those exposed to SiCW and PT-1. The expression of CGRP in rat lung was observed by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme immunometric assay (EIA). In RT-PCR, CGRP gene expression was decreased at the interval of 3 d and 1 wk in the case of crystalline silica and crocidolite; on the other hand, it was increased at 3 d and 1 wk in SiCW and at 3 d, 1 wk, and 3 mo in PT-1-exposed rats. CGRP protein level in lungs exposed to PT-1 and SiCW was also higher than that to silica and crocidolite at 3 d of recovery time. These data suggest that CGRP is associated with repair in lung damaged by different dusts, and that CGRP could be used as a sensitive biomarker to indicate the pathogenicity of dusts.

Change of heme oxygenase-1 expression in lung injury induced by chrysotile asbestos in vivo and in vitro.

Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by asbestos, and heme oxygenase-1 (HO-1) protects lung tissue against oxidative stress. We hypothesized that HO-1 is also associated with oxidative lung injury caused by exposure to chrysotile asbestos. This study was conducted to investigate the HO-1 expression of lungs in lung injury by chrysotile asbestos in vivo and in vitro. Male Wistar rats were administered 1 mg or 2 mg chrysotile suspended in saline by a single intratracheal instillation and were sacrificed at 3 days, 1 wk, 1 mo, 3 mo, and 6 mo of recovery time. The expression of HO-1 was observed by Western blot analysis, reverse-transcription polymerase chain reaction, and immunostaining. Protein levels of HO-1 increased at from 3 days to 6 mo following intratracheal instillation of 1 or 2 mg chrysotile. The mRNA levels of HO-1 increased at 3 mo and 6 mo following intratracheal instillation of 1 or 2 mg chrysotile. HO-1-positive cells were mainly found in the alveolar macrophages during immunostaining. We then examined HO-1 protein expression in human alveolar epithelial cells (A549). A549 cells were incubated with chrysotile at concentrations of 0, 12.5, 25, 50, and 100 microg/ml over 24 h. Increased expression of HO-1 protein was found following exposure to 25 or 50 microg/ml of chrysotile. Increased expression of HO-1 was also found at 6, 12, 24, and 48 h after exposure to 50 microg/ml of chrysotile with a peak at 24 h. These findings suggest that HO-1 is related to lung injury arising from exposure to chrysotile asbestos in vivo and in vitro.

Expression of heme oxygenase-1 in the lungs of rats exposed to crystalline silica.

Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by particles, and heme oxygenase-1 (HO-1) protects lung tissue against oxidative stress. We hypothesized that HO-1 is also associated with oxidative lung injury caused by exposure to particles. The present study was conducted to investigate the time course of HO-1 expression of lungs exposed to crystalline silica in vivo. Male Wistar rats were administered 1 mg or 2 mg of crystalline silica suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 month, 3 months and 6 months of recovery time. The expression of HO-1 was observed by western blot analysis and immunostaining. Protein levels of HO-1 were increased compared to the controls at 3 d, and from 1 month to 6 months following intratracheal instillation of 2 mg of crystalline silica. The levels of HO-1 were increased compared to the controls from 1 month to 6 months following intratracheal instillation of 1 mg of crystalline silica. Many HO-1 positive cells were found particularly in the alveolar macrophages during immunostaining. These findings suggest that HO-1 is related to lung injury arising from exposure to crystalline silica.

Phospholipid concentration in lung lavage fluid as biomarker for pulmonary fibrosis.

Pulmonary surfactant comprised primarily of phospholipids is a phospholipid-protein complex synthesized by type II alveolar epithelial cells or Clara cells and secreted to the pulmonary alveoli. As changes have been found in phospholipid concentrations in the bronchoalveolar lavage fluid (BALF) of patients with pulmonary fibrosis, phospholipid is considered to be involved in the process of fibrois/fibrotic process. Therefore, we made a crystalline silica rat model and measured phospholipid concentrations in lung lavage fluid in order to study the relationship of phospholipid to particle-induced pulmonary fibrosis. Eight-week-old Wistar male rats (n = 35) were injected with 2 mg crystalline silica particles suspended in 0.4 ml physiological saline. Rats in the control group (n = 35) were injected with physiological saline only. There were 7 rats in each of the ten subgroups. Rats were sacrificed and dissected at 3 days, 1 wk, 1 mo, 3 mo, and 6 mo after injection. Bronchoalveolar lavage was conducted on bronchoalveoli recovered from the right lung of each rat, the lavage fluid was centrifuged, and the supernatant was used to measure phospholipid concentration. The results were compared with previously reported inflammation scores. Phospholipid concentrations in lung lavage fluid for the exposed group showed a statistically significant increase compared to the control group throughout the observation period. Moreover, when compared to histopathologically examined inflammation scores, a positive correlation was found between the two. Judging from the facts that phospholipid concentrations in lung lavage fluid increased and that this increase correlated with the severity of inflammation, this experiment indicated that phospholipids are involved in particle-induced lung disorders.

Expression of clara cell secretory protein in the lungs of rats exposed to crystalline silica in vivo.

It has been theorized that Clara cell secretion protein (CCSP) plays a critical role in regulating the acute inflammatory response in the lung. We hypothesized that CCSP is also related to lung injury induced by occupational dust. The present study was conducted to investigate the time course of the expression of CCSP in lungs exposed to crystalline silica in vivo. Male Wistar rats were administered 1 mg or 2 mg of silica suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 month, 3 months and 6 months of recovery time. The expression of CCSP was observed by RT-PCR and western blot analysis. Exposure to 2 mg of silica decreased in levels of CCSP mRNA at 3 d, 1 wk, 1 month and 6 months following intratracheal instillation. The protein level of CCSP in silica-exposed rats was decreased at 3 d, 7 d and 1 month after a single instillation of 2 mg. The decreases in CCSP at the acute phase in this experiment suggest that CCSP may regulate the acute injury of the lung exposed to silica.

Negative effect of long-term inhalation of toner on formation of 8-hydroxydeoxyguanosine in DNA in the lungs of rats in vivo.

We assessed the effects of long-term inhalation of toner on the pathological changes and formation of 8-hydroxydeoxyguanosine (8-OH-Gua) in DNA in a rat model. Female Wistar rats (10 wk old) were divided evenly into a high concentration exposure group (H: 15.2 mg/m(3)), a low concentration exposure group (L: 5.5 mg/m(3)), and a control group. The mass median aerodynamic diameter of the toner was 4.5 microm. The rats were sacrificed at the termination of a 1-yr or 2-yr inhalation period. Pathological examination was performed on the left lung, and the level of 8-OH-Gua in DNA from the right lung was measured using a high-performance liquid chromatography (HPLC) column. The pathological findings showed that lung cancer was not observed in any of the exposed or control groups, though pleural thickening and small foci of collagen were observed in toner-exposed rat lungs. Inhalation of the toner for 1 and even 2 yr did not induce the formation of 8-OH-Gua in DNA in rat lungs. These data suggest that long-term inhalation of toner may not induce lung tumors.

Expression of heme oxygenase-1 in the lungs of rats exposed to crocidolite asbestos.

Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by asbestos, and heme oxygenase-1 (HO-1) protects lung tissue against oxidative stress. We hypothesized that HO-1 is associated with oxidative lung injury caused by exposure to asbestos. This study was conducted to investigate the time course of HO-1 expression of lungs exposed to crocidolite asbestos in vivo. Male Wistar rats were administered 1 mg or 2 mg crocidolite asbestos suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 mo, 3 mo, and 6 mo of recovery time. The expression of HO-1 was observed by Western blot analysis and immunostaining. Protein levels of HO-1 increased at from 3 d to 6 mo following intratracheal instillation of 2 mg crocidolite asbestos. The levels of HO-1 increased at 1 wk and 1 mo following intratracheal instillation of 1 mg crocidolite asbestos. Many HO-1-positive cells were found, particularly in the alveolar macrophages, during immunostaining. These findings suggest that HO-1 may be related to lung disorder induced by dust and therefore can act as a biomarker of lung injury due to dust exposure.

Effect of long-term inhalation of toner on extracellular matrix in the lungs of rats in vivo.

We assessed the effects of long-term inhalation of toner on the pathological changes and gene expression with the synthesis and degradation of collagenous extracellular matrix in a rat model. Female Wistar rats (10 wk old) were divided evenly into a high concentration exposure group (H: 15.2 mg/m3), a low concentration exposure group (L: 5.5 mg/m3), and a control group. The mass median aerodynamic diameter of toner was 4.5 microm. The rats were sacrificed at the termination of a 1-yr or 2-yr inhalation period. Pathological examination was performed from the left lung, and transcriptional levels of mRNA extracted from the right lung were assessed by semiquantitative reverse-transcription polymerase chain polymerase (RT-PCR). The pathological findings showed mild pulmonary fibrosis in 20% (L, 1 yr), 40% (H, 1 yr), 56% (L, 2 yr) and 62% (H, 2 yr), while lung cancer was not observed in any of the exposed groups. In the 1-yr high-concentration group, gene expression of matrix metalloproteinase-2 (MMP-2) and type I collagen mRNA in the rat lungs increased, while tissue inhibitors of metalloproteinase-2 (TIMP-2) decreased. The 2-yr high-concentration group increased in message level of type I collagen and TIMP-2 but not that of MMP-2. These data suggested that results of gene expression of MMP, TIMP, and collagen in the 2-yr exposure may lead to accumulation of collagen compared to the 1-yr exposure, and that the imbalance of the expression of MMPs, TIMPs, and extracellular matrix might be associated with pulmonary fibrosis induced by toner.

Gene expression of surfactant protein-A and thyroid transcription factor-1 in lungs of rats exposed to silicon-carbide whisker in vivo.

Intratracheal instillation studies have shown that exposure to silicon carbide whisker (SiCW), an asbestos substitute, produces pulmonary fibrotic changes, suggesting that SICW might have a fibrogenic potential. It is thought that surfactant protein is a good biomarker of lung injury and pulmonary fibrotic activity. In order to explore whether or not surfactant protein is associated with lung disorder through exposure to SiCW, we examined the expression of SP-A, SP-C and thyroid transcription factor-1 (TTF-1), a common transcription factor of SP-A and SP-C mRNA in lungs exposed to SiCW. Male Wistar rats were administered 2 mg or 10 mg of SiCW suspended in saline by a single intratracheal instillation, and were sacrificed at 3 d, 1 wk, 1 month, 3 months and 6 months after the intratracheal instillation. RNA was subsequently extracted from the lungs, and expression of SP-A, SP-C and TTF-1 mRNA from the lungs was observed by reverse transcription-polymerase chain reaction (RT-PCR). Exposure to 2 mg of SiCW showed a decrease in mRNA of SP-A and TTF-1 at 6 months, but exposure to 10 mg of SiCW showed decreased levels of SP-A and TTF-1 mRNA at 3 d and 6 months. On the other hand, 2 mg of SiCW increased the level of SP-C mRNA from 3 d to 3 months, and 10 mg of SiCW decreased the levels of SP-C mRNA in the rat lungs at 3 d, 1 month and 6 months. No clear tendency to the expression of SP-C was observed, but the patterns of expression of TTF-1 and SP-A were similar. These data suggest that SP-A and TTF-1 are associated with not only the acute phase but also the chronic phase in lungs exposed to SiCW.