Rosalinda Sepulveda

The pathological response and fate in the lung and pleura of chrysotile in combination with fine particles compared to amosite asbestos following short-term inhalation exposure: interim results.

The pathological response and translocation of a commercial chrysotile product similar to that which was used through the mid-1970s in a joint compound intended for sealing the interface between adjacent wall boards was evaluated in comparison to amosite asbestos. This study was unique in that it presents a combined real-world exposure and was the first study to investigate whether there were differences between chrysotile and amosite asbestos fibers in time course, size distribution, and pathological response in the pleural cavity. Rats were exposed by inhalation 6 h/day for 5 days to either sanded joint compound consisting of both chrysotile fibers and sanded joint compound particles (CSP) or amosite asbestos. Subgroups were examined through 1-year postexposure. No pathological response was observed at any time point in the CSP-exposure group. The long chrysotile fibers (L > 20 µm) cleared rapidly (T1/2 of 4.5 days) and were not observed in the pleural cavity. In contrast, a rapid inflammatory response occurred in the lung following exposure to amosite resulting in Wagner grade 4 interstitial fibrosis within 28 days. Long amosite fibers had a T1/2 > 1000 days and were observed in the pleural cavity within 7 days postexposure. By 90 days the long amosite fibers were associated with a marked inflammatory response on the parietal pleural. This study provides support that CSP following inhalation would not initiate an inflammatory response in the lung, and that the chrysotile fibers present do not migrate to, or cause an inflammatory response in the pleural cavity, the site of mesothelioma formation.

Quantification of the pathological response and fate in the lung and pleura of chrysotile in combination with fine particles compared to amosite-asbestos following short-term inhalation exposure

The marked difference in biopersistence and pathological response between chrysotile and amphibole asbestos has been well documented. This study is unique in that it has examined a commercial chrysotile product that was used as a joint compound. The pathological response was quantified in the lung and translocation of fibers to and pathological response in the pleural cavity determined. This paper presents the final results from the study. Rats were exposed by inhalation 6 h/day for 5 days to a well-defined fiber aerosol. Subgroups were examined through 1 year. The translocation to and pathological response in the pleura was examined by scanning electron microscopy and confocal microscopy (CM) using noninvasive methods. The number and size of fibers was quantified using transmission electron microscopy and CM. This is the first study to use such techniques to characterize fiber translocation to and the response of the pleural cavity. Amosite fibers were found to remain partly or fully imbedded in the interstitial space through 1 year and quickly produced granulomas (0 days) and interstitial fibrosis (28 days). Amosite fibers were observed penetrating the visceral pleural wall and were found on the parietal pleural within 7 days postexposure with a concomitant inflammatory response seen by 14 days. Pleural fibrin deposition, fibrosis, and adhesions were observed, similar to that reported in humans in response to amphibole asbestos. No cellular or inflammatory response was observed in the lung or the pleural cavity in response to the chrysotile and sanded particles (CSP) exposure. These results provide confirmation of the important differences between CSP and amphibole asbestos.The marked difference in biopersistence and pathological response between chrysotile and amphibole asbestos has been well documented. This study is unique in that it has examined a commercial chrysotile product that was used as a joint compound. The pathological response was quantified in the lung and translocation of fibers to and pathological response in the pleural cavity determined. This paper presents the final results from the study. Rats were exposed by inhalation 6 h/day for 5 days to a well-defined fiber aerosol. Subgroups were examined through 1 year. The translocation to and pathological response in the pleura was examined by scanning electron microscopy and confocal microscopy (CM) using noninvasive methods. The number and size of fibers was quantified using transmission electron microscopy and CM. This is the first study to use such techniques to characterize fiber translocation to and the response of the pleural cavity. Amosite fibers were found to remain partly or fully imbedded in the interstitial space through 1 year and quickly produced granulomas (0 days) and interstitial fibrosis (28 days). Amosite fibers were observed penetrating the visceral pleural wall and were found on the parietal pleural within 7 days postexposure with a concomitant inflammatory response seen by 14 days. Pleural fibrin deposition, fibrosis, and adhesions were observed, similar to that reported in humans in response to amphibole asbestos. No cellular or inflammatory response was observed in the lung or the pleural cavity in response to the chrysotile and sanded particles (CSP) exposure. These results provide confirmation of the important differences between CSP and amphibole asbestos.

Evaluation of the fate and pathological response in the lung and pleura of brake dust alone and in combination with added chrysotile compared to crocidolite asbestos following short-term inhalation exposure.

This study was designed to provide an understanding of the biokinetics and potential toxicology in the lung and pleura following inhalation of brake dust following short term exposure in rats. The deposition, translocation and pathological response of brake-dust derived from brake pads manufactured with chrysotile were evaluated in comparison to the amphibole, crocidolite asbestos. Rats were exposed by inhalation 6h/day for 5 days to either brake-dust obtained by sanding of brake-drums manufactured with chrysotile, a mixture of chrysotile and the brake-dust or crocidolite asbestos. The chrysotile fibers were relatively biosoluble whereas the crocidolite asbestos fibers persisted through the life-time of the animal. This was reflected in the lung and the pleura where no significant pathological response was observed at any time point in the brake dust or chrysotile/brake dust exposure groups through 365 days post exposure. In contrast, crocidolite asbestos produced a rapid inflammatory response in the lung parenchyma and the pleura, inducing a significant increase in fibrotic response in both of these compartments. Crocidolite fibers were observed embedded in the diaphragm with activated mesothelial cells immediately after cessation of exposure. While no chrysotile fibers were found in the mediastinal lymph nodes, crocidolite fibers of up to 35 μm were observed. These results provide support that brake-dust derived from chrysotile containing brake drums would not initiate a pathological response in the lung or the pleural cavity following short term inhalation.

Genetic Polymorphisms Associated with Hearing Threshold Shift in Subjects during First Encounter with Occupational Impulse Noise

Noise-induced hearing loss (NIHL) is the most significant occupational health issue worldwide. We conducted a genome-wide association study to identify single-nucleotide polymorphisms (SNPs) associated with hearing threshold shift in young males undergoing their first encounter with occupational impulse noise. We report a significant association of SNP rs7598759 (p < 5 x 10-7; p = 0.01 after permutation and correction; Odds Ratio = 12.75) in the gene coding for nucleolin, a multifunctional phosphoprotein involved in the control of senescence and protection against apoptosis. Interestingly, nucleolin has been shown to mediate the anti-apoptotic effect of HSP70, a protein found to prevent ototoxicity and whose polymorphisms have been associated with susceptibility to NIHL. Increase in nucleolin expression has also been associated with the prevention of apoptosis in cells undergoing oxidative stress, a well-known metabolic sequela of noise exposure. To assess the potential role of nucleolin in hearing loss, we tested down-regulation of nucleolin in cochlear sensory cells HEI-OC1 under oxidative stress conditions and report increased sensitivity to cisplatin, a chemotherapeutic drug with ototoxic side effects. Additional SNPs were found with suggestive association (p < 5 x 10-4), of which 7 SNPs were located in genes previously reported to be related to NIHL and 43 of them were observed in 36 other genes previously not reported to be associated with NIHL. Taken together, our GWAS data and in vitro studies reported herein suggest that nucleolin is a potential candidate associated with NIHL in this population.

Host-Derived Pericytes and Sca-1+ Cells Predominate in the MART-1− Stroma Fraction of Experimentally Induced Melanoma

Identification of cell types in tumor-associated stroma that are involved in the development of melanoma is hampered by their heterogeneity. The authors used flow cytometry and immunohistochemistry to demonstrate that anti–MART-1 antibodies can discriminate between melanoma and stroma cells. They investigated the cellular composition of the MART-1−, non-hematopoietic melanoma-associated stroma, finding it consisted mainly of Sca-1+ and CD146+ cells. These cell types were also observed in the skin and muscle adjacent to developing melanomas. The Sca-1+ cell population was observed distributed in the epidermis, hair follicle bulges, and tumor capsule. The CD146+ population was found distributed within the tumor, mainly associated with blood vessels in a perivascular location. In addition to a perivascular distribution, CD146+ cells expressed α-smooth muscle actin, lacked expression of endothelial markers CD31 and CD34, and were therefore identified as pericytes. Pericytes were found to be associated with CD31+ endothelial cells; however, some pericytes were also observed associated with CD31−, MART-1+ B16 melanoma cells that appeared to form blood vessel structures. Furthermore, the authors observed extensive nuclear expression of HIF-1α in melanoma and stroma cells, suggesting hypoxia is an important factor associated with the melanoma microenvironment and vascularization. The results suggest that pericytes and Sca-1+ stroma cells are important contributors to melanoma development.

Pulmonary delivery of d-methionine is associated with an increase in ALCAR and glutathione in cochlear fluids

In animals, hearing loss resulting from cochlear mechanosensory cell damage can be mitigated by antioxidants such as d-methionine (d-met) and acetyl-l-carnitine (ALCAR). The systemic routes of administration of these compounds, that must of necessity transit trough the cochlear fluids, may affect the antioxidant levels in the cochlea and the resulting oto-protective effect. In this study, we analyzed the pharmacokinetics of [(14)C]d-met in the cochlea and four other tissues after intratracheal (IT), intranasal (IN), and oral by gavage (OG) administration and compared it to intravenous administration (IV). We then analyzed the effect of these four routes on the antioxidant content of the cochlear fluids after d-met or ALCAR administration, by liquid chromatography/mass spectrometry. Our results showed that the concentration of methionine and ALCAR in cochlear fluids significantly increased after their respective systemic administration. Interestingly, d-met administration also contributed to an increase of ALCAR. Our results also showed that the delivery routes differently affected the bioavailability of administered [(14)C]d-met as well as the concentrations of methionine, ALCAR and the ratio of oxidized to reduced glutathione. Overall, pulmonary delivery via IT administration achieved high concentrations of methionine, ALCAR, and oxidative-related metabolites in cochlear fluids, in some cases surpassing IV administration, while IN route appeared to be the least efficacious. To our knowledge, this is the first report of the direct measurements of antioxidant levels in cochlear fluids after their systemic administration. This report also demonstrates the validity of the pulmonary administration of antioxidants and highlights the different contributions of d-met and ALCAR allowing to further investigate their impact on oxidative stress in the cochlear microenvironment.

Effect of pericytes on epithelial-to-mesenchymal transition in melanoma.

84 Background: Melanoma-associated stroma cells play an important role in supporting cancer cell proliferation, invasion, and dissemination. Increased invasiveness has been observed in cancer cells undergoing epithelial-to-mesenchymal transition (EMT); however, it is unknown whether EMT is facilitated by stroma cells. We showed that pericytes constitute a predominant stroma subpopulation that promotes melanoma growth by interacting with cancer cells. This study investigates the mechanism by which pericytes facilitate melanoma development. METHODS GFP+ pericytes isolated from tumors and adipose tissue were co-cultured in vitro with B16 melanoma cells at a 3:1 ratio. After 4 days, proliferation of B16 cells and pericytes was assessed. Also, co-cultures were labeled for several markers and analyzed by flow cytometry (FC). Tumorigenicity was investigated by isolating B16 cells from co-cultures by FACS sorting and then injected into SCID mice to measure tumor growth. RESULTS B16 cells induced pericyte differentiation into FAP+ myofibroblasts. However, no change in the pericyte proliferation rate was observed. B16 cells co-cultured with pericytes, and later separated by FACS sorting, displayed faster proliferation rates in vitro, and induced increased tumor growth rates when injected into SCID mice, compared with naive B16 cells. Pericytes increased B16 cell proliferation rate and induced a change in cell morphology, from cobblestone to fibroblast-like colonies. Interaction between B16 cells and pericytes in co-culture increased pericyte production of TGF-β and increased the expression of the stem cell markers SSEA-1, OCT3/4 and CD271 in B16 cells. In addition, cadherin switching, demonstrated by loss of E-cadherin expression and an up-regulation of the mesenchymal markers N-cadherin and vimentin was observed in B16 cells. CONCLUSIONS Pericyte production of TGF-β promotes B16 cell development of EMT, as shown by loss of E-cadherin expression and up-regulation of the mesenchymal markers N-cadherin and vimentin, resulting in an increase in melanoma cell tumorigenicity. These results suggest that therapies targeting stromal pericytes may be a promising approach for melanoma treatment.

Effect of pericytes on melanoma development.

83 Background: Melanoma-associated stroma cells regulate tumor establishment, invasion, and dissemination through paracrine and heterotypic cell-cell interactions with cancer cells. Pericytes constitute a major noninflammatory component of the melanoma-associated stroma. Our study is designed to evaluate the role of pericytes in melanoma development. METHODS Tumors induced in GFP+/SCID mice with GFP-B16 cells were harvested after 14 days, enzyme digested, labeled against mesenchymal markers, and analyzed by flow cytometry (FC). The distribution of stromal subpopulations in tumors was investigated by immunofluorescence microscopy (IFM). Stromal pericytes from tumors and adipose tissue were FACS sorted, mixed with B16 cells at a 3:1 ratio, and injected into SCID mice. Tumor diameters were taken periodically with a scientific caliper, and used to determine tumor volumes using the ellipsoid formula. RESULTS FC analysis showed that the melanoma stroma was composed of CD45+ inflammatory cells, CD31+ endothelial cells, FAP+ myofibroblasts and CD146+ cells. IFM analysis showed that the CD146+ cells are perivascular CD31-/CD34-/α-SMA+ stromal pericytes that associated with TGF-β immunoreactive CD271+/CD146+B16 cells in perivascular tumor niches. Co-induction experiments were performed to investigate the effect of pericytes on melanoma development. We observed faster growth rates in the size of tumors co-induced with pericytes, compared with tumors induced with B16 cells alone. These co-induced tumors had bigger masses, more volume, were hypercellular, and had increase vascularization compared with control tumors. In addition, pericytes increased the B16 cell proliferation rate, as determined by higher expression of Ki-67 in the tumor fraction of co-induced tumors. CONCLUSIONS Pericytes promote melanoma development by inducing angiogenesis and melanoma cell proliferation. Our results suggest that pericytes could be utilized as a therapeutic target for melanoma treatment.

Evaluation of the dose-response and fate in the lung and pleura of chrysotile-containing brake dust compared to chrysotile or crocidolite asbestos in a 28-day quantitative inhalation toxicology study

ABSTRACT This study provides an understanding of the biokinetics and potential toxicology in the lung and pleura following inhalation of brake‐dust (brakes manufactured with chrysotile). The design included a 28‐day repeated multi‐dose inhalation exposure (6h/d, 5 d/wk, 4 wks) followed by 28‐days without exposure. Fiber control groups included a similar grade chrysotile as used in the brakes and a commercial crocidolite asbestos. Aerosol fiber distributions of the chrysotile and crocidolite were similar (fiber‐length>20&mgr;m/cm3: Chrysotile‐low/high 42/62; Crocidolite‐low/high 36/55; WHO‐fibers/cm3: Chrysotile‐low/high 192/219; Crocidolite‐low/high 211/255). The total number of aerosol particles/cm3 in the brake‐dust was similar to that in the chrysotile (Brake‐dust 710–1065; Chrysotile 532–1442). Brake‐dust at particle exposure levels equal to or greater than chrysotile or crocidolite caused no indication of microgranulomas, epithelial hyperplasia, or fibrosis (Wagner score<1.7) or changes in bronchoalveolar lavage (BAL) indices from the air control. Chrysotile BAL indices did not differ from the air control. Pathologically, there was low level of inflammation and epithelial hyperplasia, but no fibrosis (Wagner score≤3). Crocidolite induced elevated neutrophils and cell damage (BAL), persistent inflammation, microgranulomas, and fibrosis (Wagner scores 4) which persisted through the post exposure period. Confocal microscopy of snap‐frozen chestwalls showed no difference between control, brake‐dust and chrysotile‐HD groups or in thickness of visceral or parietal pleural. The crocidolite exposure resulted in extensive inflammatory response, collagen development and adhesions between the visceral and parietal surfaces with double the surface thickness. These results provide essential information for the design of a subsequent subchronic study. HIGHLIGHTSEvaluated brake dust from brake pads manufactured with chrysotileIncluded comparative chrysotile and crocidolite asbestos exposure groupsNo significant pathology observed at any time point in the brake‐dust groupsChrysotile produced low level of inflammation lung, but no fibrosis.Crocidolite produced pathological response (fibrosis) in lung & pleura