Investigating palygorskite's role in the development of mesothelioma in southern Nevada: Insights into fiber-induced carcinogenicity

Abstract

Similar to asbestos fibers, nonregulated mineral fibers can cause malignant mesothelioma (MM). Recently, increased proportions of women and young individuals with MM were identified in southern Nevada, suggesting that environmental exposure to carcinogenic fibers was causing the development of MM. Palygorskite, a fibrous silicate mineral with a history of possible carcinogenicity, is abundant in southern Nevada. In this study, our aim was to determine whether palygorskite was contributing to the development of MM in southern Nevada. While palygorskite, in vitro, displayed some cytotoxicity toward primary human mesothelial (HM) cells and reduced their viability, the effects were roughly half of those observed when using similar amounts of crocidolite asbestos. No Balb/c (0/19) or MexTAg (0/18) mice injected with palygorskite developed MM, while 3/16 Balb/c and 13/14 MexTAg mice injected with crocidolite did. Lack of MM development was associated with a decreased acute inflammatory response, as injection of palygorskite resulted in lower percentages of macrophages (p = .006) and neutrophils (p = .02) in the peritoneal cavity 3 d after exposure compared to injection of crocidolite. Additionally, compared to mice injected with crocidolite, palygorskite-injected mice had lower percentages of M2 (tumor-promoting) macrophages (p = .008) in their peritoneal cavities when exposed to fiber for several weeks. Our study indicates that palygorskite found in the environment in southern Nevada does not cause MM in mice, seemingly because palygorskite, in vivo, fails to elicit inflammation that is associated with MM development. Therefore, palygorskite is not a likely contributor to the MM cases observed in southern Nevada.

Figure 1.

Palygorskite deposits are localized in regions of southern Nevada, where environmental MM cases are suggested. Map of Nevada shows (A) four sample locations for pedogenic palygorskite analyzed in this study (pink diamonds), published locations for pedogenic palygorskite/sepiolote (yellow stars) (Brock and Buck ; Brock-Hon, Robins, and Buck ; Jube et al. ; Reheis et al. ; Robins, Brock-Hon, and Buck 2012), published locations for nonpedogenic palygorskite/sepiolite (green stars) (Miles ; Papke ; Post 1978), and surface units predicted to be permissive for pedogenic palygorskite to be present (gray shading) using PRISM climatological data (PRISM Climate Group n.d.). SEM photographs of palygorskite samples collected from Mormon Mesa Old Highway site 1 (B), Mormon Mesa Old Highway site 2 (C), southwest Las Vegas petrocalcic region (D), Ivanpah ooid region (E), and Source Clays Repository palygorskite standard (F).

Figure 2.

Palygorskite is cytotoxic and reduces cell viability in human mesothelial cells. Lactate dehydrogenase (LDH) assay performed by exposing HM cells to increasing concentrations of palygorskite for 48 h (A). Reductions in the viability of HM cells 48 h after adding palygorskite demonstrated by using the Alamar blue assay (B) and MTS assay (C). HM cells from at least 2 different patients and 12 or more total replicates. Mean ± SEM values shown (*p < .05, **p < .01, ****p < .0001).

Figure 3.

Palygorskite does not cause MM in vivo. MM was diagnosed in tissues taken from Balb/c mice after they were injected twice, 1 wk apart, with 1 mg glass, palygorskite, or crocidolite, or from MexTAg mice after they were injected twice, 1 mo apart, with 6 mg glass or palygorskite, or 3 mg crocidolite. MM visible in hematoxylin and eosin (H&E)-stained diaphragm tissue from Balb/c mouse exposed to crocidolite for approximately 8 mo (A). Presence of MM was confirmed by positive nuclear WT1 staining in a serial tissue section (B). Under higher magnification, aberrant mitosis (green arrows) was observed in MM cells infiltrating the diaphragm of MexTAg mouse exposed to crocidolite for 6 mo or more (C). Magnification 400× for (A) and (B), and 1000× for (C).

Figure 4.

Palygorskite induces significantly less inflammation in mice compared to crocidolite. Representative flow cytometry dot plots showing the identification of macrophages (F4/80 + cells) and neutrophils (Ly6G + cells) from the peritoneal cavity of Balb/c mice (A). Percentages of macrophages (B) and neutrophils (C) in the peritoneal cavity of mice 3 d after intraperitoneal injection of 1 mg of glass, crocidolite, or palygorskite. Percentage of neutrophils in the peritoneal cavity 5 wk after injecting Balb/c mice twice (1 wk apart) with 1 mg of glass, crocidolite, or palygorskite (D). Sustainability of inflammation determined by dividing the percentage of neutrophils in the peritoneal cavity of Balb/c mice at 5 wk by the average percentage of neutrophils measured in the peritoneal cavity 3 d after fiber exposure (E). Mean ± SEM values displayed from 4 to 8 mice per group (*p < .05, **p < .01, ****p < .0001).

Figure 5.

Sustained exposure to palygorskite does not lead to the development of hyperplasia in mice. Diaphragm tissue obtained from Balb/c mice 5 wk after receiving 5 biweekly injections with 0.5 mg of glass (A), palygorskite (B), or crocidolite (C, D) and stained with H&E. Normal mesothelium was observed after glass and palygorskite exposure (A, B, black arrows). Mesothelial hyperplasia observed after exposure to crocidolite (C, D, black arrows). Magnification 200×.

Figure 6.

M2 macrophages increase in the peritoneal cavity of mice after sustained exposure to crocidolite but not to glass and palygorskite. After excluding doublet cells (A, left panel) and debris (A, middle panel), peritoneal macrophages were identified using flow cytometry by gating on CD11b⁺ and F4/80⁺ cells (A, right panel). Representative flow cytometry dot plots demonstrating changes in peritoneal macrophage CD206 expression in Balb/c mice 5 wk after receiving 5 biweekly injections of 0.5 mg glass (B, left panel), crocidolite (B, middle panel), or palygorskite (B, right panel). Peritoneal macrophage CD206 mean fluorescence intensity (MFI) from Balb/c mice exposed to glass, crocidolite, or palygorskite (C). Percentage of peritoneal macrophages from Balb/c mice expressing CD206, but not CD86 and identified as M2 macrophages (D). Mean ± SEM values displayed from five mice per group (**p < .01).