Exposures to silica and inducers of xenobiotic metabolism in the rat lung

CYP1A1 metabolizes polycyclic aromatic hydrocarbons (PAHs), such as those in cigarettes, to reactive intermediates which interact with DNA and lead to cancer. Silica is a lung carcinogen. Epidemiology studies of silica and lung cancer are not all positive. A possible explanation for these inconsistencies may be that silica is a modifier of PAH metabolism and, thus, cigarette smoke carcinogenesis. We hypothesize that crystalline silica exposure alters CYP1A1 expression, thereby modifying lung cancer risk. Rats exposed to both crystalline silica and the model PAH, beta-naphthoflavone (NF), a CYP1A1 inducer have significantly decreased CYP1A1 enzymatic activity and CYP1A1 protein expression. In the proximal alveolar region of NF-exposed rats, silica exposure increases markers of alveolar type II cells but decreases proportional CYP1A1 expression in type II cells as detected by immunofluorescence. Our experiments support the hypothesis that silica is a negative modifier of CYP1A1 induction by PAH

Coal dust alters β-naphthoflavone-induced aryl hydrocarbon receptor nuclear translocation in alveolar type II cells

<p>Abstract</p> <p>Background</p> <p>Many polycyclic aromatic hydrocarbons (PAHs) can cause DNA adducts and initiate carcinogenesis. Mixed exposures to coal dust (CD) and PAHs are common in occupational settings. In the CD and PAH-exposed lung, CD increases apoptosis and causes alveolar type II (AT-II) cell hyperplasia but reduces CYP1A1 induction. Inflammation, but not apoptosis, appears etiologically associated with reduced CYP1A1 induction in this mixed exposure model. Many AT-II cells in the CD-exposed lungs have no detectable CYP1A1 induction after PAH exposure. Although AT-II cells are a small subfraction of lung cells, they are believed to be a potential progenitor cell for some lung cancers. Because CYP1A1 is induced via ligand-mediated nuclear translocation of the aryl hydrocarbon receptor (AhR), we investigated the effect of CD on PAH-induced nuclear translocation of AhR in AT-II cells isolated from <it>in vivo</it>-exposed rats. Rats received CD or vehicle (saline) by intratracheal (IT) instillation. Three days before sacrifice, half of the rats in each group started daily intraperitoneal injections of the PAH, β-naphthoflavone (BNF).</p> <p>Results</p> <p>Fourteen days after IT CD exposure and 1 day after the last intraperitoneal BNF injection, AhR immunofluorescence indicated that proportional AhR nuclear expression and the percentage of cells with nuclear AhR were significantly increased in rats receiving IT saline and BNF injections compared to vehicle controls. However, in CD-exposed rats, BNF did not significantly alter the nuclear localization or cytosolic expression of AhR compared to rats receiving CD and oil.</p> <p>Conclusion</p> <p>Our findings suggest that during particle and PAH mixed exposures, CD alters the BNF-induced nuclear translocation of AhR in AT-II cells. This provides an explanation for the modification of CYP1A1 induction in these cells. Thus, this study suggests that mechanisms for reduced PAH-induced CYP1A1 activity in the CD exposed lung include not only the effects of inflammation on the lung as a whole, but also reduced PAH-associated nuclear translocation of AhR in an expanded population of AT-II cells.</p

Extrapulmonary transport of MWCNT following inhalation exposure

Background Inhalation exposure studies of mice were conducted to determine if multi-walled carbon nanotubes (MWCNT) distribute to the tracheobronchial lymphatics, parietal pleura, respiratory musculature and/or extrapulmonary organs. Male C57BL/6 J mice were exposed in a whole-body inhalation system to a 5 mg/m3 MWCNT aerosol for 5 hours/day for 12 days (4 times/week for 3 weeks, lung burden 28.1 ug/lung). At 1 day and 336 days after the 12 day exposure period, mice were anesthetized and lungs, lymph nodes and extrapulmonary tissues were preserved by whole body vascular perfusion of paraformaldehyde while the lungs were inflated with air. Separate, clean-air control groups were studied at 1 day and 336 days post-exposure. Sirius Red stained sections from lung, tracheobronchial lymph nodes, diaphragm, chest wall, heart, brain, kidney and liver were analyzed. Enhanced darkfield microscopy and morphometric methods were used to detect and count MWCNT in tissue sections. Counts in tissue sections were expressed as number of MWCNT per g of tissue and as a percentage of total lung burden (Mean ± S.E., N = 8 mice per group). MWCNT burden in tracheobronchial lymph nodes was determined separately based on the volume density in the lymph nodes relative to the volume density in the lungs. Field emission scanning electron microscopy (FESEM) was used to examine MWCNT structure in the various tissues. Results Tracheobronchial lymph nodes were found to contain 1.08 and 7.34 percent of the lung burden at 1 day and 336 days post-exposure, respectively. Although agglomerates account for approximately 54% of lung burden, only singlet MWCNT were observed in the diaphragm, chest wall, liver, kidney, heart and brain. At one day post exposure, the average length of singlet MWCNT in liver and kidney, was comparable to that of singlet MWCNT in the lungs 8.2 ± 0.3 versus 7.5 ± 0.4 um, respectively. On average, there were 15,371 and 109,885 fibers per gram in liver, kidney, heart and brain at 1 day and 336 days post-exposure, respectively. The burden of singlet MWCNT in the lymph nodes, diaphragm, chest wall and extrapulmonary organs at 336 days post-exposure was significantly higher than at 1 day post-exposure. Conclusions Inhaled MWCNT, which deposit in the lungs, are transported to the parietal pleura, the respiratory musculature, liver, kidney, heart and brain in a singlet form and accumulate with time following exposure. The tracheobronchial lymph nodes contain high levels of MWCNT following exposure and further accumulate over nearly a year to levels that are a significant fraction of the lung burden 1 day post-exposure

Pulmonary fibrotic response to aspiration of multi-walled carbon nanotubes

<p>Abstract</p> <p>Background</p> <p>Multi-walled carbon nanotubes (MWCNTs) are new manufactured nanomaterials with a wide spectrum of commercial applications. To address the hypothesis that MWCNTs cause persistent pulmonary pathology, C57BL/6J mice were exposed by pharyngeal aspiration to 10, 20, 40 or 80 μg of MWCNTs (mean dimensions of 3.9 μm × 49 nm) or vehicle. Lungs were preserved at 1, 7, 28 and 56 days post- exposure to determine the potential regions and target cells for impact by MWCNT lung burden. Morphometric measurement of Sirius Red staining was used to assess the connective tissue response.</p> <p>Results</p> <p>At 56 days post-exposure, 68.7 ± 3.9, 7.5 ± 1.9 and 22.0 ± 5.1 percent (mean ± SE, N = 8) of the MWCNT lung burden were in alveolar macrophages, alveolar tissue and granulomatous lesions, respectively. The subpleural tissues contained 1.6% of the MWCNT lung burden. No MWCNTs were found in the airways at 7, 28 or 56 days after aspiration The connective tissue in the alveolar interstitium demonstrated a progressive increase in thickness over time in the 80 μg exposure group (0.12 ± 0.01, 0.12 ± 0.01, 0.16 ± 0.01 and 0.19 ± 0.01 μm for 1, 7, 28 and 56 days post-exposure (mean ± SE, N = 8)). Dose-response determined at 56 days post-exposure for the average thickness of connective tissue in alveolar septa was 0.11 ± 0.01, 0.14 ± .02, 0.14 ± 0.01, 0.16 ± 0.01 and 0.19 ± 0.01 μm (mean ± SE, N = 8) for vehicle, 10, 20, 40 and 80 μg dose groups, respectively.</p> <p>Conclusions</p> <p>The distribution of lung burden was predominately within alveolar macrophages with approximately 8% delivery to the alveolar septa, and a smaller but potentially significant burden to the subpleural tissues. Despite the relatively low fraction of the lung burden being delivered to the alveolar tissue, the average thickness of connective tissue in the alveolar septa was increased over vehicle control by 45% in the 40 μg and 73% in the 80 μg exposure groups. The results demonstrate that MWCNTs have the potential to produce a progressive, fibrotic response in the alveolar tissues of the lungs. However, the increases in connective tissue per μg dose of MWCNTs to the interstitium are significantly less than those previously found for single-walled carbon nanotubes (SWCNTs).</p

Distribution and persistence of pleural penetrations by multi-walled carbon nanotubes

<p>Abstract</p> <p>Background</p> <p>Multi-walled carbon nanotubes (MWCNT) are new manufactured nanomaterials with a wide spectrum of commercial applications. The durability and fiber-like dimensions (mean length 3.9 μm long × 49 nm diameter) of MWCNT suggest that these fibers may migrate to and have toxicity within the pleural region. To address whether the pleura received a significant and persistent exposure, C57BL/6J mice were exposed by pharyngeal aspiration to 10, 20, 40 and 80 μg MWCNT or vehicle and the distribution of MWCNT penetrations determined at 1, 7, 28 and 56 days after exposure. Following lung fixation and sectioning, morphometric methods were used to determine the distribution of MWCNT and the number of MWCNT fiber penetrations of three barriers: alveolar epithelium (alveolar penetrations), the alveolar epithelium immediately adjacent to the pleura (subpleural tissue), and visceral pleural surface (intrapleural space).</p> <p>Results</p> <p>At 1 day 18%, 81.6% and 0.6% of the MWCNT lung burden was in the airway, the alveolar, and the subpleural regions, respectively. There was an initial, high density of penetrations into the subpleural tissue and the intrapleural space one day following aspiration which appeared to decrease due to clearance by alveolar macrophages and/or lymphatics by day 7. However, the density of penetrations increased to steady state levels in the subpleural tissue and intrapleural from day 28 - 56. At day 56 approximately 1 in every 400 fiber penetrations was in either the subpleural tissue or intrapleural space. Numerous penetrations into macrophages in the alveolar airspaces throughout the lungs were demonstrated at all times but are not included in the counts presented.</p> <p>Conclusions</p> <p>The results document that MWCNT penetrations of alveolar macrophages, the alveolar wall, and visceral pleura are both frequent and sustained. In addition, the findings demonstrate the need to investigate the chronic toxicity of MWCNT at these sites.</p

Apoptosis and Bax Expression are Increased by Coal Dust in the Polycyclic Aromatic Hydrocarbon-Exposed Lung

BACKGROUND: Miners inhaling respirable coal dust (CD) frequently develop coal workers’ pneumoconiosis, a dust-associated pneumoconiosis characterized by lung inflammation and variable fibrosis. Many coal miners are also exposed to polycyclic aromatic hydrocarbon (PAH) components of diesel engine exhaust and cigarette smoke, which may contribute to lung disease in these workers. Recently, apoptosis was reported to play a critical role in the development of another pneumoconiosis of miners, silicosis. In addition, CD was reported to suppress cytochrome P450 1A1 (CYP1A1) induction by PAHs. METHODS: We investigated the hypothesis that apoptosis plays a critical role in lung injury and down-regulation of CYP1A1 induction in mixed exposures to CD and PAHs. We exposed rats intratracheally to 0.0, 2.5, 10.0, 20.0, or 40.0 mg/rat CD and, 11 days later, to intraperitoneal β-naphthoflavone (BNF), a PAH. In another group of rats exposed to CD and BNF, caspase activity was inhibited by injection of the pan-caspase inhibitor Q-VD-OPH [quinoline-Val-Asp (OMe)-CH(2)-OPH]. RESULTS: In rats exposed to BNF, CD exposure increased alveolar expression of the proapoptotic mediator Bax but decreased CYP1A1 induction relative to BNF exposure alone. Pan-caspase inhibition decreased CD-associated Bax expression and apoptosis but did not restore CYP1A1 activity. Further, CD-induced lung inflammation and alveolar epithelial cell hypertrophy and hyperplasia were not suppressed by caspase inhibition. CONCLUSIONS: Combined BNF and CD exposure increased Bax expression and apoptosis in the lung, but Bax and apoptosis were not the major determinants of early lung injury in this model

Effect of Fiber Length on Carbon Nanotube-Induced Fibrogenesis

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-β) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-β release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivolung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-β activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in viv

Pulmonary toxicity and lung tumorigenic potential of surrogate metal oxides in gas metal arc welding–stainless steel fume: Iron as a primary mediator versus chromium and nickel

In 2017, the International Agency for Research on Cancer classified welding fumes as “car- cinogenic to humans” (Group 1). Both mild steel (MS) welding, where fumes lack carcino- genic chromium and nickel, and stainless steel (SS) increase lung cancer risk in welders; therefore, further research to better understand the toxicity of the individual metals is needed. The objectives were to (1) compare the pulmonary toxicity of chromium (as Cr(III) oxide [Cr2O3] and Cr (VI) calcium chromate [CaCrO4]), nickel [II] oxide (NiO), iron [III] oxide (Fe2O3), and gas metal arc welding-SS (GMAW-SS) fume; and (2) determine if these metal oxides can promote lung tumors. Lung tumor susceptible A/J mice (male, 4–5 weeks old) were exposed by oropharyngeal aspiration to vehicle, GMAW-SS fume (1.7 mg), or a low or high dose of surrogate metal oxides based on the respective weight percent of each metal in the fume: Cr2O3 + CaCrO4 (366 + 5 μg and 731 + 11 μg), NiO (141 and 281 μg), or Fe2O3 (1 and 2 mg). Bronchoalveolar lavage, histopathology, and lung/liver qPCR were done at 1, 7, 28, and 84 days post-aspiration. In a two-stage lung carcinogenesis model, mice were initi- ated with 3-methylcholanthrene (10 μg/g; intraperitoneal; 1x) or corn oil then exposed to metal oxides or vehicle (1 x/week for 5 weeks) by oropharyngeal aspiration. Lung tumors were counted at 30 weeks post-initiation. Results indicate the inflammatory potential of the metal oxides was Fe2O3 \u3e Cr2O3 + CaCrO4 \u3e NiO. Overall, the pneumotoxic effects were negligible for NiO, acute but not persistent for Cr2O3 + CaCrO4, and persistent for the Fe2O3 exposures. Fe2O3, but not Cr2O3 + CaCrO4 or NiO significantly promoted lung tumors. These results provide experimental evidence that Fe2O3 is an important mediator of welding fume toxicity and support epidemiological findings and the IARC classification

Differential Pulmonary Effects of CoO and La2O3 Metal Oxide Nanoparticle Responses During Aerosolized Inhalation in Mice

Background: Although classified as metal oxides, cobalt monoxide (CoO) and lanthanum oxide (La2O3) nanoparticles, as representative transition and rare earth oxides, exhibit distinct material properties that may result in different hazardous potential in the lung. The current study was undertaken to compare the pulmonary effects of aerosolized whole body inhalation of these nanoparticles in mice. Results: Mice were exposed to filtered air (control) and 10 or 30 mg/m3 of each particle type for 4 days and then examined at 1 h, 1, 7 and 56 days post-exposure. The whole lung burden 1 h after the 4 day inhalation of CoO nanoparticles was 25 % of that for La2O3 nanoparticles. At 56 days post exposure, \u3c 1 % of CoO nanoparticles remained in the lungs; however, 22–50 % of the La2O3 nanoparticles lung burden 1 h post exposure was retained at 56 days post exposure for low and high exposures. Significant accumulation of La2O3 nanoparticles in the tracheobronchial lymph nodes was noted at 56 days post exposure. When exposed to phagolysosomal simulated fluid, La nanoparticles formed urchin-shaped LaPO4 structures, suggesting that retention of this rare earth oxide nanoparticle may be due to complexation of cellular phosphates within lysosomes. CoO nanoparticles caused greater lactate dehydrogenase release in the bronchoalveolar fluid (BALF) compared to La2O3 nanoparticles at 1 day post exposure, while BAL cell differentials indicate that La2O3 nanoparticles generated more inflammatory cell infiltration at all doses and exposure points. Histopathological analysis showed acute inflammatory changes at 1 day after inhalation of either CoO or La2O3 nanoparticles. Only the 30 mg/m3 La2O3 nanoparticles exposure caused chronic inflammatory changes and minimal fibrosis at day 56 post exposure. This is in agreement with activation of the NRLP3 inflammasome after in vitro exposure of differentiated THP-1 macrophages to La2O3 but not after CoO nanoparticles exposure. Conclusion: Taken together, the inhalation studies confirmed the trend of our previous sub-acute aspiration study, which reported that CoO nanoparticles induced more acute pulmonary toxicity, while La2O3 nanoparticles caused chronic inflammatory changes and minimal fibrosis