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

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. K E Y W O R D S animal model, anthophyllite, Cdkn2a/2b, malignant mesothelioma, tremolite 1 | INTRODUC TI ON Asbestos, which are naturally occurring fibrous minerals, are classified as carcinogens on the basis of sufficient evidence in humans; thus, many countries have banned the use of all asbestos. 1 The WHO estimates that more than 107 000 people die each year from asbestos-related lung cancer, malignant mesothelioma (MM), and asbestosis from occupational exposure. 2 Malignant | 1181 OKAZAKI et Al.

Asbestos‐induced mesothelial injury and carcinogenesis: Involvement of iron and reactive oxygen species

“…64 The coexistence of HD (5/8) and loss of heterozygosity (LOH) (3/8) of Cdkn2a/2b was detected in tremolite-induced MM cases. 21 Furthermore, the coexistence of HD (10/12) and LOH (2/12) of Cdkn2a/2b was observed in MWCNTs-induced MM cases. 26 In an intrapleurally injected mouse model, long CNTs and asbestos elicited prolonged inflammation and injured mesothelial cells with a hypermethylated CpG island of Cdkn2a that caused transcriptional silencing; subsequently, LOH to HD of this region, finally, induced MM.…”

“…While tremolite in Kumamoto, whose fiber number, mean length and width with standard deviation were 11.76 × 10 7 fibers/mg, 11.42 ± 11.36 and 0.993 ± 0.682 μm, respectively, induced cytotoxicity, diffuse fibrous peritoneal thickening and carcinogenicity in rats. 20,21 Recently, nanosized materials, which are equal to or smaller than 100 nm in one or more dimensions, have been developed for electric engineering or drug delivery applications. 22 Diverse applications of nanomaterials are commercially available in titanium oxides for cosmetics, nano Si cluster composite materials for rechargeable batteries and carbon nanotubes (CNTs) for aircraft and space vehicles.…”

“…65 Unfortunately, we did not detect a significant difference in lifespan between the LOH and HD groups of Cdkn2a/2b in tremolite-and MWCNTs-induced MM. 21 To investigate the tumor suppressive role of Cdkn2a, Nf2, and Bap1 in mesothelial carcinogenesis, single Cdkn2a/Arf, Nf2 and Bap1 heteroKO ( +/− ) mice were treated with intraperitoneal injection of 3.2 mg crocidolite. In this experimental setting, each of the single heteroKO mice developed MM significantly faster than their wild-type (Wt) littermates.…”

Iron as spirit of life to share under monopoly

“…There are many reports, including our own, on the direct effect of asbestos. (62,93,(102)(103)(104)(105)(106)(107) We recently found an indirect effect of asbestos mediating macrophages to mesothelial cells. When asbestos comes to the mesothelium, submesothelial macrophages intrinsically collect most of the exposed asbestos fibers.…”

Bridging the gap between toxicity and carcinogenicity of mineral fibres by connecting the fibre crystal-chemical and physical parameters to the key characteristics of cancer

“…This proviso is not universally shared. For example, animal experiments using anthophyllite and tremolite fibres suggest that the fibre diameter (width) is a more critical factor than the fibre length in the asbestos-induced carcinogenic process by intraperitoneal administration ( Aierken et al, 2014 , Okazaki et al, 2020 ). Nevertheless, width is of paramount importance to indicate if a fibre is respirable (when the width < 3 µm and length/width (aspect) ratio > 3) and whether it will reach the alveolar space and pleural/peritoneal surface ( Wylie et al, 2020 and references therein).…”