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Study on the connection between mycotoxins and Kashin-Beck Disease in China

par Sun, Danlei
Président du jury Stévigny, Caroline
Promoteur Fontaine, Véronique
Co-Promoteur Delporte, Cédric
Publication Non publié, 2024-02-15

Thèse de doctorat

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Résumé :

Kashin-Beck disease (KBD) is an endemic disease in Asia, from the southeast of Siberia in Russia to Tibet, affecting the northern and central provinces of China. It is a chronic, disabling disease characterized by multiple deformed bones and joints. However, the etiology of this disease remains unknown. Mycotoxin contamination, especially from the Fusarium spp. in the north of China, has been considered, during the last decades, as one of the main risk factors within a multifactorial KBD context. However, Fusarium were seldom detected in Tibet in previous studies. The objective of my doctoral research was therefore to investigate whether other mycotoxins could be specifically detected or more abundantly detected in KBD Tibetan-related areas. We searched for mycotoxins in two distinct types of samples. Initially, our focus was on the mycotoxins identified in cereal samples collected from regions associated with KBD. Subsequently, we analyzed fungi isolated from cereals in KBD-related areas and cultivated them under various conditions to assess mycotoxin production.For the cereal samples, thanks to the previous fungal analysis in Tibet from both endemic areas (EA) and non-endemic areas (NEA), 292 barley samples were previously collected from Tibet KBD EA and NEA during 2009-2016. Meanwhile, 19 wheat samples collected from Inner Mongolia, the northern part of China, in 2006, were also analyzed as control. We took advantage of recent analytical and data treatment tools to identify mycotoxins present in the samples. Metabolomics analysis of the data obtained by liquid chromatography coupled to a high-resolution tandem mass spectrometer (LC-HRMS(/MS)) was performed to analyze and compare the compound profiles in endemic versus non-endemic KBD areas. Enniatin B (ENN B) was observed specifically or more abundantly in the KBD-related regions. ENN B is a mycotoxin produced by some Fusarium spp. It was reported to exhibit cytotoxic effects within a limited range in both human and animal cells, inducing apoptosis and necrosis in vitro. However, in vivo toxicity of ENN B was found to be low. As previously mentioned, the production of ENN B was exclusively attributed to Fusarium, a genus rarely isolated from KBD-related regions. We therefore search for the presence of Fusarium DNA in cereals from KBD-endemic areas, potentially establishing a connection with the detection of ENN B in these cereal samples. Notably, Fusarium DNA was only detected in barley samples from KBD-related EA, offering insights into the occurrence of ENN B and other Fusarium mycotoxins in this region. Further analyses are needed to monitor ENN B in recent cereal samples from KBD-related regions and to isolate Fusarium from cereals affected by KBD.Additionally, we searched for mycotoxin production from fungi isolated from cereals collected or not from KBD-related areas. As a previous mycological study suggested that Alternaria spp. were the most abundant fungi isolated from collected KBD EA-related barleys, our study focused on thirteen Alternaria isolates from China, including 9 isolates from KBD EA barley samples of TAR in 1998 and 2004, one isolate from the maize sample from a nearby TAR KBD NEA in 1998, and three isolates from the former EA in Gansu. Additionally, eleven Alternaria isolates from regions outside of China, primarily from Belgium, were included as a control group. These isolates were cultivated under various conditions to investigate their mycotoxin production. Unfortunately, no KBD-related mycotoxins, including ENN, T-2 toxin and others, were detected under the chosen culture conditions. However, LC-HRMS(/MS) analyses, along with untargeted metabolomics and a molecular network approach, allowed us to identify a compound produced by Alternaria cantlous (previously named Ulocladium cantlous) as a Destruxin B analog. Destruxin B is typically produced by Alternaria brassicae, another Alternaria spp. Further analyses of the production of the Destruxin B analog are necessary to investigate the mycotoxin synthesis from Alternaria caulous. Destruxin B is a cyclic depsipeptide, showing a similar structure to Enniatin. Destruxin B shows versatile properties, such as an insecticidal and anti-cancerous activities.In this study, the in vitro findings indicate that both ENN B and Destruxin B exhibit no cytotoxicity on human chondrocytes up to concentrations of 15.6 μM and 16.9 μM, respectively. The involvement of these probable mycotoxins in KBD should be further investigated both in vitro and in vivo.