Asbestos exposure and malignant mesothelioma: the role of inorganic fiber burden and disruption of iron homeostasis in lung microenvironment. A postmortem study on human lungs

Asbestos-related diseases still represents a major public health problem all over the world. Among them, malignant mesothelioma (MM) is a highly aggressive, poor-prognosis cancer, arising from the serosal lining of pleura, pericardium and peritoneum, triggered by asbestos exposure. Asbestos is the collective name of six kinds of naturally occurring minerals, namely chrysotile (the only serpentine asbestos), and the amphiboles crocidolite, amosite, tremolite asbestos, actinolite asbestos, anthophyllite asbestos. The response of human lungs to asbestos inhalation and the molecular mechanisms which lead to MM development several decades after exposure are still largely unknown. One of the most debated issues is the formation of asbestos bodies, that are asbestos fibers covered by an iron-rich amorphous substance. Literature data suggest the key role of iron metabolism in the coating process leading to the formation of asbestos bodies, that has been regarded as both protective and harmful. This study aims to understand more about the reaction of the human organisms to asbestos inhalation and the individual susceptibility to MM. First, the lung inorganic fiber burden has been characterized in lungs of individuals who were previously exposed to asbestos (occupationally or environmentally) using electron scanning microscopy with energy dispersive spectroscopy (SEM-EDS). All the subjects used to work at an asbestos-cement industry, which was active between 1932 and 1993 in Broni, a small town in Lombardy. Unexpectedly, a significantly lower concentration of asbestos fibers has been found in MM compared to asbestosis patients. Chrysotile was not detected at all in any of the examined samples, despite it was largely used at the plant, suggesting a complete clearance of this type of asbestos from lungs. Crocidolite was the most represented asbestos, followed by amosite, tremolite/actinolite asbestos and anthophyllite asbestos, consistently with the data about the industry production. The ratio between asbestos fibers and asbestos bodies was widely different from subject to subject. Based on the well-known role of iron in asbestos-induced pulmonary toxicity, the second part of the study investigated the frequency of a group of single nucleotide polymorphisms (SNPs) in genes involved in iron homeostasis in individuals who died from MM compared to controls. Despite the successful DNA extraction from formalin-fixed paraffin-embedded samples (FFPE), we failed to identify any genotype associated with a protective or predisposing effect in relation to MM development as a consequence of asbestos exposure. Finally, on the basis of the established role of BAP1 in MM pathogenesis and its association with ferroptosis impairment observed in various kinds of cancers, the expression of BAP1, transferrin receptor 1 (TRF1), ferritin heavy chain 1 (FTH1) and ferroportin (FPN) has been investigated using immunohistochemistry and rtPCR, finding that asbestos affects the expression of the mentioned proteins in lungs differently in MM patients compared to subjects exposed to asbestos but died of other causes. These findings suggest that a different biological response to asbestos inhalation and to the consequent iron overload in lungs may play an important role in cancer initiation. The formation of asbestos bodies appears to be a key mechanism in the formation of a pro-neoplastic microenvironment, as well as ferroptosis impairment. These results are important from a prevention point of view, as iron metabolism, as well as the consequent oxidative stress, chronic inflammation and cancerogenic stimuli might be targets for therapeutic strategies aiming to delay or prevent MM onset in individuals previously exposed to asbestos. Moreover, knowing the mechanism that can make an individual vulnerable to asbestos can be of crucial importance for prevention