[B-CG07] Frontier in Biology and Paleobiology of Fossilized Micro-organisms

[BCG07-12] The breakdown of foraminiferal detoxification in response to TiO₂ nanoparticles exposure

*Inagaki Yuka¹, Yurika Ujiie², Yoshiyuki Ishitani³, Caterina Ciacci⁴, Fabrizio Frontalini⁵, Federica Rebecchi⁵, Mattia Tiboni⁴ (1.Kochi University, Graduate School of Integrated Arts and Sciences, Science Program, Biological Science Course, 2. Marine Core Research Institute, Kochi University , 3.Japan Agency for Marine-Earth Science and Technology, 4.University of Urbino, Department of Biomolecular Science, 5.University of Urbino, Department of Pure and Applied Sciences)

Keywords:Titanium dioxide nanoparticle, marine pollution, benthic foraminifera, toxicity, Transcriptome Analysis, metabolism

Titanium dioxide nanoparticles (TiO₂ NPs) are used in various industrial products, but their accumulation as waste in marine environments raises concerns for their toxicity to aquatic organisms. Previous studies have successfully revealed the mechanisms of cytotoxicity of TiO₂ NPs through confocal laser scanning microscopy (CLSM) observations, transcriptome analyses, and culture experiments with the benthic foraminifer Ammonia veneta (Ishitani et al., 2023; Inagaki et al., 2024). Metabolic pathway predictions based on exposure of A. veneta to 1 ppm TiO₂ NPs for 24h showed that foraminifers produce reactive oxygen species (ROS) as stress indicators after uptaking TiO₂ NPs in cells, but they survived by enabling detoxification mechanism: suppression of ROS and extracellular excretion of TiO₂ NPs (Ishitani et al., 2023). Long-term cytotoxicity and lethal concentration of TiO₂ NPs for A. veneta were examined by exposure experiments with 1, 5, 10, and 50 ppm TiO₂ NPs for five weeks (Inagaki et al., 2024). Foraminifers have grown in TiO₂ NPs medium higher than 5 ppm for the first two weeks, but all of them died by the third week. TiO₂ NPs were found in both foraminiferal vesicles and extracellular wastes from 5 ppm exposure individuals by the transmission electron microscopy (TEM) and TEM equipped with an energy dispersive X-ray spectrometer (TEM-EDS) observations. These results suggested that the detoxification system of A. veneta could be disrupted at concentrations higher than 5 ppm TiO₂ NPs. This could be ascribed to acceleration of ROS production due to gene regulation related to stress or toxic substance release, or by overexpression of genes that inhibit cell growth, leading to cell death. Here, we investigate the metabolic change of A. veneta exposed to 5 ppm TiO₂ NPs based on the CLSM observation and transcriptome analyses. We examined physiological changes of A. veneta with molecular probes (i.e., CellROX Green and Acridine Orange) through time-course experiments at 30 min, 1, 6, 24, and 48 h. Both ROS and acidic vesicles were not detected at any time. The five individuals for each of three conditions (1, 6, and 24 h exposure to 5 ppm TiO₂ NPs) were used to cDNA amplification for transcriptome analyses. Open reading frames (ORFs) with altered expression levels in the 5 ppm TiO₂ samples were extracted by comparison with control samples of Ishitani et al. (2023) and the corresponding metabolic pathway was searched through the KEGG database. Among 165,801 ORFs annotated in this study, 24,554 ORFs were only expressed in the 5 ppm exposure group and 122,722 ORFs were highly expressed compared to the controls. The expression of 21,546 ORFs were significantly (p<0.05) increased. The expression levels of 8,415 ORFs, which were identical in both 1 and 5 ppm TiO₂ NPs exposures, showed higher in 5 ppm than in 1 ppm. The proportion of ORFs, highly expressed in 5 ppm TiO₂ NPs, was much greater than that of the previous study in 1 ppm TiO₂ NPs, and many more different genes were detected. The genes, which were annotated as ROS production by 1 ppm TiO₂ NPs exposure, showed increased expression in 5 ppm TiO₂ NPs exposure. Moreover, this study newly found that NOX genes involved in ROS production showed high expression, and SOD1/CAT genes involved in ROS quenching were expressed as well. Significant expression of these genes were consistent with the CLSM observations, which revealed no detection of ROS. Thus, our results indicate that different metabolic processes occur at 5 ppm, in particular, many genes were overexpressed in over-stressed organisms.

Ishitani Y, et al. (2023) Environ Pollut. doi: 10.1016/j.envpol.2023.121538
Inagaki Y, et al. (2024) Front. Mar. Sci. doi: 10.3389/fmars.2024.1381247