Phd in ENVIRONMENTAL AND EVOLUTIONARY BIOLOGY

Thesis title: Development of new cost-effective assays for monitoring oxidative stress in model organisms exposed to particulate matter in in-vivo controlled conditions

This study aims to contribute to the development of a novel method to assess the oxidative stress induced by atmospheric Particulate Matter (PM) using in vivo model organisms under both controlled laboratory and real-world conditions. (A) Given the innovative approach of this project, an extensive literature search was conducted during the PhD research, resulting in a scientific review of methods for assessing PM-induced oxidative stress (paper A1). Methods for the assessment of OP by acellular assays were summarized and their limitations highlighted. Subsequently, methods based on the in vitro application of cellular techniques were described by mentioning the main cellular models used to study PM-induced adverse effects and describing the main assays and markers of oxidative stress. Finally, a special focus and systematic literature review was placed on works involving the use of model/experimental organisms to study PM-induced oxidative stress effects highlighting the possibility of applying such techniques under both laboratory and real exposure conditions proposing this type of application as a future prospect for the study of PM-induced oxidative stress effects. (B) To date, the oxidative potential of PM is mainly evaluated through the application of acellular oxidative potential assays. In general, the most applied OP assays on PM are: Ascorbic Acid (OPAA), dithiothreitol (OPDTT) and 2,7-dichlorofluorescein (OPDCFH). The association between these three assays and PM constituents was studied (paper B1) through the application of a sampling campaign to obtain samples from a mixed urban-industrial area where an iron and manganese processing industry was present. Samples were also obtained from a second urban-type site, and an ICP-MS analysis was applied for chemical characterization of the soluble and insoluble fractions of PM. Finally, the soluble fraction of PM was associated with the results of OP assays and local emission sources through the application of multivariate statistical analysis (i.e. principal component analysis PCA). (C) Given the emerging importance and perspectives offered by the use of in vivo model organisms, the possibility of using the model plant organism Arabidopsis thaliana to study PM-induced oxidative stress effects was explored (paper C1). Seedlings of A. thaliana were exposed to aqueous suspensions containing two types of dust (certified NIST1648a urban dust material and real PM2.5 samples) at different concentrations, and both qualitative and quantitative analysis was carried out to assess the production of super oxide anion (·O2-) as a proxy for oxidative stress using the nitroblue tetrazole (NBT) assay. In addition, on the seedlings exposed to NIST1648a, an analysis was carried out to assess the total chlorophyll content and a bioaccumulation analysis was performed to assess which elements were most bioaccumulated and induced stress. (D) Oxidative stress induced by selected PM components was also evaluated on the experimental animal species Aedes albopictus (paper D1). Brake dust (BD), one of the most dangerous contributions to PM in urban areas, was extracted by sonication and was added at different concentrations in an aqueous medium, Aedes albopictus eggs were hatched in the aqueous medium containing the BD, and larval survival, presence of behavioral alterations (e.g., cannibalism phenomena), and development were evaluated. For the assessment of oxidative stress, gene expression of two key enzymes in the oxidative stress response (superoxide dismutase - SOD - and catalase) were evaluated, and elements bioaccumulation by Ae. albopictus larvae was measured. The results of bioaccumulation were combined with those of oxidative stress through the application of multivariate statistical techniques (PCA) to identify the elements most correlated with oxidative stress in the experimental species Ae. albopictus. Finally, morphological analyses (wing shape and size) were performed on individuals that reached the adult stage to identify possible alterations induced by the presence of BD during the growth and development of individuals. (E) In the last part of the experimental work, with the aim of moving from an exposure under laboratory conditions to a field exposure, were carried out a series of activities that are proposed as ongoing activities and future perspectives. For these activities we selected the animal model organism Drosophila melanogaster, given the greater representative power of model species compared to experimental species (e.g. Ae. Albopictus). D. melanogaster individuals were exposed under laboratory conditions to two different concentrations of BD with the aim of identifying the sensitivity of this species to PM and optimizing a method to assess PM-induced oxidative stress on living organisms (paper E1, in preparation). BD was added to the food and placed in 50 mL centrifuge tubes, and a pair of individuals (one male and one female) was placed in each tube and left to incubate for six days with the goal of obtaining offspring. After 6 days, the pair of adult individuals was removed from the tubes, the tubes were left in incubation for a total of 10 days to allow any eggs produced to hatch and the offspring produced to develop into adults. At the end of the 10 days, the resulting adult offspring were removed from the tubes and stored at -80o C to prevent reactive oxygen species (ROS) degradation. Oxidative stress was assessed by the application of the NBT assay for quantification of ·O2- production and metabolic alterations were evaluated by non-targeted Nuclear Magnetic Resonance (NMR) analysis. Bioaccumulation analysis was also conducted to assess the elements most bioaccumulated by individuals exposed to BD. Further analysis will regard SOD and catalase determination and statistical analysis of the results. Finally with the goal of moving from the laboratory to the field individuals of D. melanogaster were exposed in situ in two severely impacted workplaces such as a metal smelting and processing plant and an art ceramics manufacturing company to assess the oxidative stress induced by PM produced in these particular workplaces. The use of model organisms to estimate the worker exposure may be of great impact in the field of work safety. For the first exposure (paper E2, in preparation) four sites were selected within metal smelting and processing plant, each representative of a different production process (meeting room, hot rolling, Dust recovery, and polishing); at each site, approximately one thousand adult individuals were exposed and exposure lasted 24 h. For assessment of oxidative stress, NBT assay was conducted and gene expression of antioxidant enzymes SOD and catalase will be evaluated. In addition, a bioaccumulation analysis was performed to assess the ability of this species to bioaccumulate elements from PM and NMR metabolomic analysis evidenced significant metabolic alteration due to the exposure at each site. In addition, PM10 sampling was carried out during the exposure period, and the samples obtained were subjected to chemical characterization for their element content, and the OP was evaluated. Also in this case, cause-effect relations were studied by advanced multivariate statistical methods. For the second exposure (paper E3, in preparation) four sites were selected within the art ceramics manufacturing company, also in this case approximately one thousand adult individuals were exposed, and the exposure time was 7 days. Analyses to assess PM-induced oxidative stress and elements bioaccumulation on the individuals exposed within the factory were performed. Also in this case, PM10 was sampled for the evaluation of its elemental content and to assess the OP. Further analyses for the assessment of antioxidant enzyme expression and metabolomic profiles will be conducted.