Frequent homozygous deletion of Cdkn2a/2b in tremolite-induced malignant mesothelioma in rats

Our plain-English summary of Frequent homozygous deletion of Cdkn2a/2b in tremolite-induced malignant mesothelioma in rats, by Y. Okazaki, N. Misawa, S. Akatsuka, et al (Cancer Science, 2020).

The study

• Authors: Y. Okazaki, N. Misawa, S. Akatsuka, N. Kohyama, Y. Sekido, T. Takahashi, S. Toyokuni
• Journal: Cancer Science
• Published: 2020;111(4):1180-1192
• Cited by: 7 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The onset of malignant mesothelioma (MM) is linked to exposure to asbestos fibers. Asbestos fibers are classified as serpentine (chrysotile) or amphibole, which includes the crocidolite, amosite, anthophyllite, tremolite, and actinolite types. Although few studies have been undertaken, anthophyllite has been shown to be associated with mesothelioma, and tremolite, a contaminant in talc and chrysotile, is a risk factor for carcinogenicity. Here, after characterizing the length and width of these fibers by scanning electron microscopy, we explored the cytotoxicity induced by tremolite and anthophyllite in cells from an immortalized human mesothelial cell line (MeT5A), murine macrophages (RAW264.7), and in a rat model. Tremolite and short anthophyllite fibers were phagocytosed and localized to vacuoles, whereas the long anthophyllite fibers were caught on the pseudopod of the MeT5A and Raw 264.7 cells, according to transmission electron microscopy. The results from a 2-day time-lapse study revealed that tremolite was engulfed and damaged the MeT5A and RAW264.7 cells, but anthophyllite was not cytotoxic to these cells. Intraperitoneal injection of tremolite in rats induced diffuse serosal thickening, whereas anthophyllite formed focal fibrosis and granulomas on peritoneal serosal surfaces. Furthermore, the loss of Cdkn2a/2b, which are the most frequently lost foci in human MM, were observed in 8 cases of rat MM (homozygous deletion [5/8] and loss of heterozygosity [3/8]) by array-based comparative genomic hybridization techniques. These results indicate that tremolite initiates mesothelial injury and persistently frustrates phagocytes, causing subsequent peritoneal fibrosis and MM. The possible mechanisms of carcinogenicity based on fiber diameter/length are discussed.

© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

In plain English

This laboratory study compared two amphibole asbestos types, tremolite and anthophyllite, in cultured cells and in rats. Under electron microscopy, the tremolite fibers were shorter and thinner (median length 7.76 μm, median width 0.787 μm) than the anthophyllite fibers (median length 23.55 μm, median width 2.94 μm). In cell cultures that included human mesothelial cells and mouse macrophages, tremolite suppressed cell growth even at the lowest dose tested, 1 μg/cm2. Anthophyllite did not affect the growth of these two cell types at doses up to 10 μg/cm2. Rats were given an injection of 1 mg of fibers into the abdominal (peritoneal) cavity and examined 4 weeks later. Tremolite caused diffuse fibrous thickening of the peritoneal lining, while anthophyllite caused only focal fibrosis and granulomas. The authors also analyzed DNA from 8 peritoneal mesotheliomas that tremolite had produced in rats in an earlier study. The Cdkn2a/2b genes were deleted on both copies in 5 of these tumors and showed loss of one copy in the other 3. The authors concluded that tremolite, unlike anthophyllite, damaged mesothelial cells and macrophages, leading to chronic inflammation and mesothelioma.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study combining cell culture experiments, a short-term rat experiment with intraperitoneal injection, and genomic (array CGH) analysis of rat mesothelioma tumors from an earlier experiment
• Who was studied: Cell lines: MeT5A (immortalized human mesothelial cells), RAW264.7 (mouse macrophages), HeLa, and three human mesothelioma cell lines (Meso8A, Meso8D, Meso12A). Animals: six-week-old F1 hybrid rats (Fischer 344 x Brown Norway), in small groups of 4 to 7 rats per treatment (Table 1). Also 8 tremolite-induced rat peritoneal mesotheliomas produced in a previous study.
• Exposure: Tremolite (from Japan) and anthophyllite (from Afghanistan), with UICC crocidolite and chrysotile as comparisons. Cells received 1, 5 or 10 μg/cm2 of tremolite or anthophyllite, or 10 μg/cm2 of crocidolite. Rats received a single intraperitoneal injection of 1 mg of chrysotile, anthophyllite or tremolite, some followed by nitrilotriacetate (NTA, 80 mg/kg).
• Main results:
  • Tremolite fibers (median length 7.76 μm, median diameter 787 nm) were significantly thinner and shorter than anthophyllite fibers (median length 23.55 μm, median diameter 2940 nm).
  • Mesothelial cells and macrophages took up short anthophyllite and tremolite fibers into internal compartments (phagosomes). Long anthophyllite fibers stayed attached to the cells' projections (pseudopods).
  • In MeT5A and RAW264.7 cells, tremolite suppressed proliferation even at 1 μg/cm2, and the effect increased with dose. Anthophyllite did not affect proliferation of these cells at doses up to 10 μg/cm2.
  • After 24 hours, tremolite significantly increased release of LDH (a sign of cell damage) in MeT5A mesothelial cells but not in RAW264.7 macrophages. Anthophyllite did not increase LDH release.
  • In rats, tremolite caused diffuse fibrous thickening of the peritoneum. Anthophyllite caused granulomatous inflammation and only focal fibrosis of the serosal surface.
  • In rats, NTA treatment did not affect serosal fibrosis.
  • Among the 8 tremolite-induced rat mesotheliomas, the Cdkn2a/2b loci were the most frequently altered. Homozygous deletion was found in 5 cases and loss of heterozygosity in the remaining 3.
  • This analysis used the tremolite tumors supplemented with 12 previously published carbon nanotube-induced rat mesotheliomas. Loss of heterozygosity of Cdkn2a/2b was not significantly related to earlier development of mesothelioma (Fisher's exact test, P greater than .999; combined data P = .319).
• The authors' conclusion: The authors concluded that anthophyllite did not cause mesothelial injury, whereas tremolite was toxic to mesothelial cells and macrophages, leading to chronic inflammation and mesothelial carcinogenesis, with homozygous deletion of Cdkn2a/2b again the major genetic change.
• Limitations the authors state:
  • The authors state that animal models have difficulties: fibers may be encapsulated in granulomas or fibrous tissue, small fibers are carried to lymph nodes, and adhesive ileus can cause death before cancer develops.
  • The combined effect of tremolite and NTA was unclear and requires further study.
  • Why the Cdkn2a/2b loci are preferentially damaged remains unclear.
  • Ultrathin specimens for transmission electron microscopy broke because of the rigid asbestos fibers.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Anthophyllite, Chrysotile, Crocidolite, Lung cancer, Mesothelioma, Tremolite.

Medical terms in this study, explained in our Lexicon: Homozygous Deletion, CDKN2A, Tremolite, Malignant Mesothelioma, Serpentine Group, Chrysotile, Amphibole Group, Crocidolite.

Related studies

• Homozygous deletion of CDKN2A/2B is a hallmark of iron-induced high-grade rat mesothelioma — Q. Hu, S. Akatsuka, et al. (2010)
• Homozygous deletion of CDKN2A in malignant mesothelioma: Diagnostic utility, patient characteristics and survival in a UK mesothelioma centre — K. Marshall, S. Jackson, et al. (2020)
• Frequent Homozygous Deletions of Type I Interferon Genes in Pleural Mesothelioma Confer Sensitivity to Oncolytic Measles Virus — T. Delaunay, C. Achard, et al. (2020)
• Fluorescence in situ hybridization (FISH) provides estimates of minute and interstitial BAP1, CDKN2A, and NF2 gene deletions in peritoneal mesothelioma — S. Brich, F. Bozzi, et al. (2020)
• Malignant mesothelioma in situ diagnosed by methylthioadenosine phosphorylase loss and homozygous deletion of CDKN2A: a case report — K. Minami, N. Jimbo, et al. (2020)
• Homozygous deletion of p16INK4a and tobacco carcinogen exposure in nonsmall cell lung cancer — K.S. Kraunz, H.H. Nelson, et al. (2006)

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