Bringing Artificial Intelligence (AI) into Environmental Toxicology Studies: A Perspective of AI-Enabled Zebrafish High-Throughput Screening.

The booming development of artificial intelligence (AI) has brought excitement to many research fields that could benefit from its big data analysis capability for causative relationship establishment and knowledge generation. In toxicology studies using zebrafish, the microscopic images and videos that illustrate the developmental stages, phenotypic morphologies, and animal behaviors possess great potential to facilitate rapid hazard assessment and dissection of the toxicity mechanism of environmental pollutants. However, the traditional manual observation approach is both labor-intensive and time-consuming. In this Perspective, we aim to summarize the current AI-enabled image and video analysis tools to realize the full potential of AI. For image analysis, AI-based tools allow fast and objective determination of morphological features and extraction of quantitative information from images of various sorts. The advantages of providing accurate and reproducible results while avoiding human intervention play a critical role in speeding up the screening process. For video analysis, AI-based tools enable the tracking of dynamic changes in both microscopic cellular events and macroscopic animal behaviors. The subtle changes revealed by video analysis could serve as sensitive indicators of adverse outcomes. With AI-based toxicity analysis in its infancy, exciting developments and applications are expected to appear in the years to come.

Deep Learning-Enabled Morphometric Analysis for Toxicity Screening Using Zebrafish Larvae.

Toxicology studies heavily rely on morphometric analysis to detect abnormalities and diagnose disease processes. The emergence of ever-increasing varieties of environmental pollutants makes it difficult to perform timely assessments, especially using in vivo models. Herein, we propose a deep learning-based morphometric analysis (DLMA) to quantitatively identify eight abnormal phenotypes (head hemorrhage, jaw malformation, uninflated swim bladder, pericardial edema, yolk edema, bent spine, dead, unhatched) and eight vital organ features (eye, head, jaw, heart, yolk, swim bladder, body length, and curvature) of zebrafish larvae. A data set composed of 2532 bright-field micrographs of zebrafish larvae at 120 h post fertilization was generated from toxicity screening of three categories of chemicals, i.e., endocrine disruptors (perfluorooctanesulfonate and bisphenol A), heavy metals (CdCl2 and PbI2), and emerging organic pollutants (acetaminophen, 2,7-dibromocarbazole, 3-monobromocarbazo, 3,6-dibromocarbazole, and 1,3,6,8-tetrabromocarbazo). Two typical deep learning models, one-stage and two-stage models (TensorMask, Mask R-CNN), were trained to implement phenotypic feature classification and segmentation. The accuracy was statistically validated with a mean average precision >0.93 in unlabeled data sets and a mean accuracy >0.86 in previously published data sets. Such a method effectively enables subjective morphometric analysis of zebrafish larvae to achieve efficient hazard identification of both chemicals and environmental pollutants.

Hazard of polystyrene micro-and nanospheres to selected aquatic and terrestrial organisms.

Plastics contamination in the environment is a major concern. Risk assessment of micro- and nanoplastics (MPL and NPL) poses significant challenges due to MPL and NPL heterogeneity regarding compositional polymers, particle sizes and morphologies in the environment. Yet, there exists considerable toxicological literature on commercial polystyrene (PS) micro- and nanospheres. Although such particles do not directly represent the environmental MPL and NPL, their toxicity data should be used to advance the hazard assessment of plastics. Here, toxicity data of PS micro- and nanospheres for microorganisms, aquatic and terrestrial invertebrates, fish, and higher plants was collected and analyzed. The evaluation of 294 papers revealed that aquatic invertebrates were the most studied organisms, nanosized PS was studied more often than microsized PS, acute exposures prevailed over chronic exposures, the toxicity of PS suspension additives was rarely addressed, and ∼40 % of data indicated no organismal effects of PS. Toxicity mechanisms were mainly studied in fish and nematode Caenorhabditis elegans, providing guidance for relevant studies in higher organisms. Future studies should focus on environmentally relevant plastics concentrations, wide range of organisms, co-exposures with other pollutants, and method development for plastics identification and quantification to fill the gap of bioaccumulation assessment of plastics.

"Fishing" nano-bio interactions at the key biological barriers.

Understanding nano-bio interactions is pivotal to the safe implementation of nanotechnology for both biological and environmental applications. Zebrafish as a model organism provides unique opportunities to dissect nano-bio interactions occurring at different biological barriers. In this review, we focus on four key biological barriers, namely cell membrane, blood-brain barrier (BBB), skin and gill epithelia, and gastrointestinal tract (GIT), and highlight recent advancement achieved by using zebrafish to conduct both visualized observations and mechanistic investigations on a diversity of nano-bio interactions.

Metabolomic profiling reveals the intestinal toxicity of different length of microplastic fibers on zebrafish (Danio rerio).

To explore the intestinal toxicity of microplastic fibers, zebrafish larvae and adults were exposed to different length of microplastic fibers (50 ± 26 µm and 200 ± 90 µm). After exposure, microplastic fibers were observed in the gut of zebrafish even at the early life stage, causing length-dependent intestinal damage and toxicities manifested by histopathological changes and biomarker responses. Long microplastic fibers induced more serious effects. They significantly decreased the food intake of zebrafish by 54 %-67 % compared with short microplastic fibers. Metabolomics was conducted to further reveal the metabolic alterations induced by microplastic fibers in zebrafish. A total of 124 and 123 metabolites were significantly changed by short and long microplastic fibers. At the meanwhile, 41 significantly changed metabolites were shared between short and long fibers treatment groups and were further investigated to reveal the influence of fiber length on the toxicity. The results demonstrate that microplastic fibers can up-regulate glycerophospholipids metabolism which exacerbates oxidative damage and inflammation and down-regulate fatty acyls metabolism related to nutritional deficiency. These novel findings enhance our understanding of the intestinal toxicity of microplastic fibers and demonstrate that metabolomics is powerful to unravel the underlying mechanisms of microplastics (MPs) toxicity.

An efficient method for extracting microplastics from feces of different species.

Concerns have been raised regarding the ingestion of microplastics (MPs) by numerous organisms including humans. However, no efficient and standardized methods are available for extracting MPs from feces. In this study, we introduce a novel approach with high digestion efficiency that involves using Fenton's reagent and nitric acid to remove feces solids. Firstly, Fenton's reagent was used to degrade small solids and decompose large solids into small pieces. Secondly, nitric acid was used to digest the remaining solids and filters. Furthermore, absolute ethyl alcohol was used to remove the mineral residues wrapped on the plastic surfaces and disperse MPs. By using this method, 97.78 % MPs can be recovered from human and chicken feces, and no significant changes were observed in the physical and Raman spectral properties of different polymer types of MPs. This method has also been verified by extracting MPs from field feces. Overall, the proposed method can efficiently digest feces solids and extract MPs with higher recovery rate, less intermediate steps and less damage, which can serve as an economical and feasible method for the detection of MPs in the feces of different species.

Accumulation of different shapes of microplastics initiates intestinal injury and gut microbiota dysbiosis in the gut of zebrafish.

Different shapes of microplastics are widely detected in the environment and organisms and most of them remain in the gut. However, the influences of shapes on the bioaccumulation and toxicity of microplastics in the gut are largely unknown. Three shapes (bead, fragment, and fiber) of microplastics of comparable size in one dimension were prepared to exposure to zebrafish. The accumulation and toxicities of microplastics in the gut were detected. Shape-dependent accumulation in the gut was observed with the order of fibers (8.0 µg/mg) > fragments (1.7 µg/mg) > beads (0.5 µg/mg). The accumulation of microplastics caused multiple toxic effects in fish intestine, including mucosal damage, and increased permeability, inflammation and metabolism disruption. Based on these toxic effects, microplastic fibers resulted in more severe intestinal toxicity than microplastic fragments and beads did. Furthermore, microplastics also induced gut microbiota dysbiosis and specific bacteria alterations, which will provide novel insights into the potential mechanism of microplastics causing intestinal toxicities in fish. Our results also suggested that shape-depended effects should not be ignored in the health risk assessment of microplastics.

Combined effects of polystyrene microplastics and natural organic matter on the accumulation and toxicity of copper in zebrafish.

As emerging contaminants, microplastics (MPs) are predicted to act as vectors for other contaminants and their combined effects are largely unknown. In this study, the combined effects of MPs and natural organic matter (NOM) on the accumulation and toxicity of copper (Cu) in zebrafish (Danio rerio) were investigated. As a result, small-size MPs could absorb more Cu than large-size MPs. The presence of NOM promoted Cu adsorption on MPs in the pH range of 6-8. Our results demonstrate that the combination of MPs and NOM increased Cu accumulation in the livers and guts in a size-depended manner. Correspondingly, the results of biochemical test showed that MPs and NOM could aggravate Cu-toxicity in the livers and guts, which is manifested in the increased levels of malonaldehyde (MDA) and metallothionein (MT) and decreased levels of superoxide dismutase (SOD). Furthermore, the results of transcriptomic analysis suggested that such aggravation of toxicity was mainly attributed to the inhibition of Cu-ion transport and the enhanced oxidative stress. Since the co-existence of MPs and NOM in the environment is inevitable, their enhancement effects on the bioaccumulation and toxicity of other pollutants such as heavy metals deserve more attention.

Microplastics induce intestinal inflammation, oxidative stress, and disorders of metabolome and microbiome in zebrafish.

Microplastics (MPs) can be ingested by a variety of species and mainly accumulate in the gut. However, the consequences of MPs exposure in the gut are largely unknown. Here we evaluated the impacts of MPs exposure in zebrafish gut. Animals were experimentally exposed to polystyrene MPs (5-µm beads; 50 µg/L and 500 µg/L) for 21 days and monitored for alterations in tissue histology, enzymatic biomarkers, gut microbiome and metabolomic responses. Inflammation and oxidative stress were observed in the zebrafish gut after exposed to MPs. Furthermore, significant alterations in the gut microbiome and tissue metabolic profiles were observed, with most of these were associated with oxidative stress, inflammation and lipid metabolism. This study provides evidence that MPs exposure causes gut damage as well as alterations in gut metabolome and microbiome, yielding novel insights into the consequences of MPs exposure.

Evidence that microplastics aggravate the toxicity of organophosphorus flame retardants in mice (Mus musculus).

This study was performed to reveal the health risks of co-exposure to organophosphorus flame retardants (OPFRs) and microplastics (MPs). We exposed mice to polyethylene (PE) and polystyrene (PS) MPs and OPFRs [tris (2-chloroethy) phosphate (TCEP) and tris (1,3-dichloro-2-propyl) phosphate (TDCPP)] for 90 days. Biochemical markers and metabolomics were used to determine whether MPs could enhance the toxicity of OPFRs. Superoxide dismutase (SOD) and catalase (CAT) increased (p < 0.05) by 21% and 26% respectively in 10 µg/L TDCPP + PE group compared to TDCPP group. Lactate dehydrogenase (LDH) in TDCPP + MPs groups were higher (18%-30%) than that in TDCPP groups (p < 0.05). Acetylcholinesterase (AChE) in TCEP + PE groups were lower (10%-19%) than those in TCEP groups (p < 0.05). These results suggested that OPFR co-exposure with MPs induced more toxicity than OPFR exposure alone. Finally, in comparison to controls we observed that 29, 41, 41, 26, 40 and 37 metabolites changed significantly (p < 0.05; fold-change > 1.2) in TCEP, TCEP + PS, TCEP + PE, TDCPP, TDCPP + PS and TDCPP + PE groups, respectively. Most of these metabolites are related to pathways of amino acid and energy metabolism. Our results indicate that MPs aggravate the toxicity of OPFRs and highlight the health risks of MP co-exposure with other pollutants.

Influence of microplastics on the accumulation and chronic toxic effects of cadmium in zebrafish (Danio rerio).

As the accumulation of microplastics (MPs) in the environment continues to rise, more concerns focus on the health risk of combined exposure to MPs and other contaminants. The aim of this study is to investigate the influences of MPs on the tissue-accumulation of cadmium (Cd) in zebrafish and explore the related chronic toxic effects induced by combined exposure of Cd and MPs. After co-exposure to MPs and Cd for 3 weeks, 20 and 200 µg/L MPs increased the accumulation of Cd in zebrafish livers (46% and 184%), guts (10% and 25%) and gills (9% and 46%). The Cd accumulation was gill > gut > liver. Comprehensive analyzes of biochemical biomarkers, histopathological observation and functional gene expression firstly demonstrated that the presence of MPs enhanced the toxicity of Cd on zebrafish and the combined exposure caused oxidative damage and inflammation in zebrafish tissues. Collectively, our results highlight the chronic effects of combined exposure to MPs and heavy metals.