Mass Spectrometry Imaging and Omics for Environmental Toxicology Research

Chao Zhao; Zongwei Cai

doi:10.7536/PC201240

Progress in Chemistry ›› 2021, Vol. 33 ›› Issue (4) : 503-511 DOI: 10.7536/PC201240

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Mass Spectrometry Imaging and Omics for Environmental Toxicology Research

Chao Zhao ¹^,²
, Zongwei Cai ¹^,^*

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Abstract

The spatial heterogeneity of multiple organs causes the complexity of the molecular mechanism in environmental pollutant-exposed organisms. The conventional environmental toxicology research based on chemical and biological analysis has been considered as a “homogeneous” whole, which is difficult to locate the pollutant and its metabolism in spatial level. As one of the most promising analytical method, mass spectrometry imaging(MSI) combined with mass spectrometry-based omics has been applied for the qualitative, quantitative and spatial localization analysis of pollutant and its metabolic activation pathways as well as biomolecule variation, allowing the screening of pollutant exposure-related target organs and investigation of the migration and biological effects of pollutants. Herein, we summarize the strategies and characteristics of MSI and omics in environmental toxicology. The future development and challenges are also prospected, including the single cell imaging, and the integrated microfluidic chip-MSI technique.

Contents

1 Introduction

2 MSI and omics analysis

2.1 Pollutant exposed experimental models

2.2 Pollutant exposed histological analysis

2.3 MSI and omics-based environmental toxicology research

2.4 Exploration of toxicological mechanisms

3 Toxicity research of typical pollutants

3.1 Bisphenols

3.2 PM2.5

3.3 Other applications

4 Conclusion and perspective

Graphical abstract

Keywords

mass spectrometry imaging / mass spectrometry-based omics / environmental toxicology / biomarkers / toxicological effects

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Chao Zhao, Zongwei Cai. Mass Spectrometry Imaging and Omics for Environmental Toxicology Research. Progress in Chemistry, 2021, 33(4): 503-511 DOI:10.7536/PC201240

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1 Introduction

A large number of studies have shown that different levels of environmental exposure are closely related to the occurrence and development of many human diseases^[1⇓~3]. When a variety of chemicals are re-released into the environment, the resulting toxic effects seriously endanger the human respiratory system, endocrine system and nervous system^[4⇓~6]. In order to study the complex toxic effects of pollutants, it is necessary to develop analytical techniques that can provide more information, so as to explore the various biological processes involved in the toxic mechanisms of pollutants, and to interpret the new toxic mechanisms of pollutants in time and space.

Studies on the toxicity of environmental pollutants mainly focus on the changes at the gene and protein levels^[7]. Compared with gene and protein changes, the metabolic activation characteristics of pollutants in organisms, the changes of endogenous and exogenous metabolites and metabolic pathways caused by exposure can more accurately reflect the dynamic changes of external stimuli on the physiology of organisms, which is expected to become an important way to study the toxic mechanism of environmental pollutants^[8]. Obviously, the traditional environmental toxicology based on analytical chemistry and molecular biology methods can not meet the requirements. Extending genes and proteins to the metabolic level and developing related analytical techniques are the key to solving this problem. Based on the strategy of mass spectrometry imaging (MSI) and omics analysis, the toxicity mechanism of pollutants is explored by studying the characteristic changes of the whole metabolome, lipidome and proteome of organisms, so as to realize the accurate analysis of qualitative, quantitative and positioning of pollutant metabolism and endogenous and exogenous metabolites in organisms, with the characteristics of high throughput, high sensitivity and label-free. More importantly, this research strategy can obtain the spatial location of biomarkers and the information of toxic target organs^[9,10].

It is particularly important to design and construct the MSI and omics research protocols suitable for environmental toxicology research, so as to screen new toxicological mechanisms. In this paper, the MSI research strategies, toxicological hotspots and difficulties in environmental toxicology in recent years were reviewed, and the development prospects of integrated MSI technology in environmental toxicology were prospected, which will provide an important reference for the development of this field.

2 MSI and omics analysis

The strategy of environmental toxicology research based on MSI and omics analysis includes the following four parts (Figure 1): 2.1 Model construction of pollutant exposure; 2.2 Histomorphological analysis of pollutant exposure; 2.3 MSI and omics analysis of environmental toxicology; 2.4 Exploration of new mechanism of toxicity. Among them, 2.1, 2.2 and 2.4 are mainly based on environmental toxicology and molecular biology. 2.3 Mass spectrometric analysis is the main method.

2.1 Model construction of pollutant exposure

cell,animal and human specimens are common models for environmental toxicology research.Among them,the cell model is widely used because of its easy access to materials and short growth cycle.It is difficult to accurately simulate the microenvironment of cell growth and the interaction between cells by traditional monolayer cell culture,which may lead to the loss of cell signal transmission,cell structure and function.Mass spectrometry imaging strategy based on three-dimensional(3D)cell microspheres can accurately simulate the real state of organisms,including cell proliferation,cell-cell interactions and changes in the cell microenvironment^{[11⇓⇓⇓~15]}。 This model can not only simulate the signal transmission between cells and extracellular matrix before and after exposure to environmental pollutants,but also reproduce important biological behaviors such as cell migration and infiltration,which has important application value in rapid screening of environmental pollutant toxicity,evaluation of drug toxicity and resistance,and drug metabolism^{[11⇓⇓⇓~15]}。

Contaminant exposure animal models for MSI and omics research include two categories:(1)exposure of pollutants to common animal models to investigate their effects on animal organs,animal behavior and other indicators,so as to screen toxicity-related target organs;(2)Exposing pollutants to special animal models,including tumor transplantation models and disease-related animal models,to investigate the effects of pollutant exposure on tumor progression and disease development,and to explore the molecular mechanism and susceptible organs of pollution-induced disease development。

Among them,the animal model of tumor transplantation and the culture of tumor cells related to pollutant exposure have attracted much attention.Continuous exposure to environmental pollutants is closely related to the formation and metastasis of tumors^[16]。 In fact,the formation and metastasis of tumors are not only determined by the phenotypic changes of tumor cells,but also by the co-evolution of tumor cells with extracellular matrix,tumor vasculature,microenvironment,immune cells and external environmental stimuli.Spatial heterogeneity among and within tumors is a significant feature of malignant lesions,and its complexity directly leads to the diversity of tumor-related animal models^[17,18]。 tumor cell culture in vitro is the most commonly used exposure model.Cytokines and chemokines related to tumor growth and a variety of environmental pollutants can be added to the culture medium respectively,and a co-culture system of tumor cells and stromal cells can also be constructed to evaluate the toxicity of pollutants.It is difficult to simulate the tumor microenvironment,blood vessels and immune system in vitro^[19]。 Animal models of xenotransplantation of human tumor cells can better simulate the characteristics of tumor microenvironment and vasculature,but the immune system is absent.If primary cells or tumor tissues from tumor patients are used for xenotransplantation in mice,not only the characteristics of tumor microenvironment can be preserved,but also the heterogeneity of tumor can be studied,and the exposure of pollutants can be carried out after successful tumor-bearing^[19]。 Dairkee et al used minimally invasive surgery to collect biopsy tissues from patients with primary breast cancer,including non-malignant,fibroma,invasive ductal carcinoma,ductal carcinoma in situ and atypical ductal papilloma,and cultured human cells.to investigate the effect of bisphenol A(BPA)exposure on human tumor proliferation,the co-culture system of tumor cells and fibroblasts from non-malignant lesions was established and the cells were exposed to BPA^[20]。 In addition,some genetic techniques can be used to change the immune system of mice to construct tumor animal models。

in addition,the choice of exposure concentration and time of environmental pollutants is very important,and relevant cell and animal pre-experiments must be carried out,and the final experimental value must be determined by referring to the concentration and time related to exposure In the real environment.Taking bisphenol S(BPS)and bisphenol F(BPF),the typical substitutes of BPA,as examples,Rochester and others comprehensively expounded the selection of exposure conditions,which provided an important reference value for the development of toxicity screening of bisphenol chemicals^[21]。

2.2 Histomorphological analysis of exposure to pollutants

histomorphology and MSI analysis complement each other and verify each other,neither of them is dispensable.Accurate interpretation of Histomorphology is the key to disease diagnosis.Studies have confirmed that exposure to environmental pollutants,especially long-term low-dose exposure at the early stage of organ formation,is associated with poor organ development,tumor formation and malignant lesions in adulthood^[22,23]。 Take the development of female mammary gland as an example,the mammary gland has developed after birth and presents a fully differentiated form in the late pregnancy.Studies have shown that exposure to environmental endocrine disrupting chemicals(EDCs)significantly affects the development of this process,especially during pregnancy,adolescence and lactation,which is mainly manifested by the development of mammary branch ducts,the exponential growth of epithelial cells during adolescence,and the rapid transition from pregnancy to lactation^[23,24]。 At the same time,the persistent exposure of EDCs leads to the transformation of breast tissue morphology from normal epithelial cells to invasive and even cancerous,thus increasing the susceptibility of breast cancer(Fig.2)^[23,25]。

Histomorphological methods commonly used in MSI include HE staining and immunohistochemistry.Among them,the preparation of frozen tissue sections is the most common.For formalin soaked and formalin fixed paraffin-embedded(FFPE)tissue specimens,specific dewaxing procedures were used according to the different purposes of the study.For example,to detect the distribution of protein in situ,FFPE samples need to be deacetylated and trypsinized in situ^[26]。 When detecting small metabolites,FFPE samples need to be dewaxed directly by organic solvents such as xylene^[27]。

2.3 MSI and omics analysis of environmental toxicology

the spatial resolution,operability and repeatability of instruments are still important indicators for the study of Environmental toxicology and omics.environmental pollutants and their induced changes At the molecular level show many"details"with the improvement of resolution.at present,the commonly used MSI technology has achieved the spatial resolution of micron level(1~100μm).Compared with conventional matrix-assisted laser desorption ionization(MALDI)and desorption electrospray ionization(DESI),secondary ion mass spectrometry(SIMS)has stronger matrix effect and background interference,based on which TOF-SIMS and Nano-SIMS can achieve Nano-scale resolution(Fig.3A)^[28]。 With the introduction of femtosecond laser,new optical focusing system and near-field optics,the spatial resolution of laser-based MSI technology can reach less than 5μm(Fig.3 B):for example,laser ablation-inductively coupled plasma mass spectrometry(LA-ICP-MS)can be used to image breast cancer tissue,and subcellular(~1μm)resolution can be obtained^[28]^[29]。 At the same time,it can be combined with mass spectrometry flow cytometry to draw imaging maps of 32 proteins and their derivatives^[30]。 The subcellular spatial resolution(400~500 nm)can be obtained in HeLa cells by combining VUV with MSI^[31]。 in addition,the Abliz team developed an air flow-assisted desorption electrospray ionization mass spectrometry imaging(AFADESI-MSI),and constructed a new method for the qualitative,quantitative and spatial localization of metabolites in biological samples.the method can be used for the analysis and detection of more than 1500 metabolites of more than 10 types,including some metabolites with large differences in physicochemical properties and contents,and the metabolic reprogramming of tumor cells related to esophageal cancer is successfully expounded by using the research method,thereby providing a potential new target for cancer diagnosis and treatment^[32,33]^[34]。

Due to The improvement of spatial resolution of image analysis,a large number of high-dimensional original data are generated,and accurate visualization analysis based on images has become a hot issue to be solved urgently.the calculation of MSI data includes many steps,such as mass spectrometry data preprocessing,format conversion,dimensionality reduction,image segmentation,spatial labeling,clustering and multivariate statistical analysis,that is,to extract effective imaging data from high-dimensional data^[35]。 Currently,the commonly used MSI software includes SCiLS Lab,Metaspace,MSiReader,etc.In addition,Abliz has developed MSI workstation software MassImager,which has the functions of data processing and information mining^[33]。 The team also integrated artificial neural network algorithm into MSI data mining,constructed a virtual correction quantitative imaging analysis method,and achieved accurate quantification of drugs in tissue sections^[36]。

In addition,the"fusion"of MSI and microscopic imaging can achieve high spatial resolution and high chemical specificity,taking full advantage of the rich molecular information of MSI and the high resolution of microscopic imaging(Fig.4).Through this image"fusion"technology,the spatial distribution of target molecules can be predicted at a higher resolution,which is more than 10 times higher than that of MSI technology alone^[37]。

2.4 Exploration of new mechanism of toxicity

Combining the results based on MSI and omics analysis with the methods based on molecular biology is an important way to explore new mechanisms of pollutant toxicity.Generally,there are two approaches:the first is to screen the target molecules and related pathways disturbed by pollutants Through omics and imaging techniques,and then to detect the activity of metabolic synthetic enzymes of the targets by conventional molecular biology techniques to verify the changes in molecular weight and spatial distribution of the targets before and after exposure to pollutants(Figure 5).the second is to design targeted toxicological experiments on cells or animals(such as immunotoxicity),then screen biomolecules with quantitative and spatial distribution changes according to the results,and finally integrate the results of environmental toxicology,omics,MSI and molecular biology experiments to draw conclusions.For example,through cytotoxicity experiments,our research group found that environmental low-dose BPF induced apoptosis,polarization,abnormal expression and secretion of immune-related cytokines in macrophages.through omics experiments,we further identified the abnormal Lipid metabolism pathway caused by BPF exposure at the molecular level:BPF induces apoptosis by activating sphingomyelin-ceramide signaling pathway and oxidative stress.Both endogenous(Bax and Caspase-9)and exogenous apoptotic pathways(tumor necrosis factor receptor 1,Caspase-8,and Caspase-3)are involved.lipid metabolism plays an important role in cellular signaling regulation of BPF induced immunotoxicity^[38]。

3 Study on the Toxicity of Typical Pollutants

3.1 Bisphenol pollutant

BPA and its typical substitute chemicals BPS and BPF are one of the most important raw materials,which are widely used in the production of industrial products and daily necessities,and are also the most studied EDCs^[39,40]。 Their presence has been detected in environmental and human samples,such as water,soil,sediment,human urine and blood^[41]。 Environmental exposure to these bisphenol chemicals is strongly associated with organ damage and the development of chronic diseases,including kidney and liver disease,diabetes,and cardiovascular disease^[42,43]。 In renal tissue,BPA upregulates cytochrome P450 enzyme activity by altering steroid production,thereby increasing its estrogen-like activity and inducing nephrotoxicity^[44]。 Similar to BPA,its substitute BPS also has estrogen-like effects,induces cellular DNA damage,and affects signaling pathways related to lipid metabolism^[21]^[45,46]。

In addition,BPA and its substitutes are immunotoxic^[47,48]。 BPA exposure can induce the release of proinflammatory cytokines,the dysfunction of innate immune regulation,the dysfunction of T cell function,and the proliferation of lymphocytes^[49,50]。 the spleen is considered to be an important site for the initiation of the primary immune response,and is also a susceptible organ to exposure to environmental pollutants,playing a key role in the immune response^[51]。 Dong et al.Found that BPA caused mitochondrial dysfunction in mouse spleen cells by increasing oxidative stress^[52]。 Gear et al.Found that BPA exposure during pregnancy can change the histomorphological structure of the spleen of mice,and cause immune regulation and hematopoietic dysfunction^[53]。

the above studies based on molecular biology usually regard the organ as a"uniform"whole to study the toxicity of pollutants.In fact,organs or tissues have 3D spatial structure and heterogeneity within tissues,so sub-organ level methods and strategies are more suitable for environmental toxicology research^[10]。

Metabolic and lipid molecular analysis can provide the overall disturbance of biological systems in the face of external stimuli,and combine mass spectrometry-based omics and MSI studies with graphical data processing to explore the toxic effects of pollutants.Alterations in metabolism and lipid homeostasis are associated with the development of common diseases and can be used as biomarkers to indicate the occurrence of human diseases^[54,55]。 lipid molecules can act as second messengers in cell signal regulation and transmission,and play an important role in cell membrane shaping,membrane permeability and Lipid biosynthesis^{[54⇓⇓⇓⇓~59]}。 Liu et al.Used MSI to study the mechanisms of renal fibrosis,an important process in chronic kidney disease,in a mouse model of unilateral ureteral obstruction^[58]。 They found that ATP metabolism,TCA cycle,fatty acid metabolism and glycolysis pathway were disturbed in the process of fibrosis,which provided important ideas for clarifying the mechanism of chronic kidney disease.Rao et al.used MSI-based lipidomics to elucidate the mechanism of renal injury in a mouse model of early ischemia-reperfusion,and screened two biomarkers,phosphatidylcholine(38:1)and phosphatidylethanolamine(42:3)^[59]。 Phosphatidylcholine(38:1)was up-regulated in the proximal tubules of the kidney after ischemia-reperfusion。

Different from the study on the pathological changes of the organs themselves,our group used the techniques based on MSI and omics combined with molecular biology to elaborate the process of kidney and spleen damage induced by BPS at environmentally relevant doses(10 and 100μg-BPS/kg bw/day)at the sub-organ level^[60,61]。 Compared with the toxicity of intact kidney and spleen in mice,BPS may cause organ damage by inducing histomorphological lesions and inflammatory reactions in susceptible sub-organs(renal cortex and splenic white pulp,etc.),which is the first time that MSI technology has been used to precisely locate the target organs related to the toxic effects of environmental pollutants(Fig.6)。

3.2 Study on the Toxicity of Atmospheric Fine Particles

There are several results in the study of the toxicity of atmospheric fine particles(aerodynamic diameter≤2.5μm,PM_2.5)based on molecular biology methods.The toxic components of PM_2.5mainly include free radicals,organic chemicals and transition metals,which affect the normal physiological processes of cells by inducing oxidative stress,inflammation,genotoxicity and other mechanisms to produce reactive oxygen species and a variety of cytokines,while changing the physiological functions of target cells,leading to organ damage^[62]。 Short-term exposure to PM_2.5induces apoptosis in human lung epithelial cells through activation of tumor necrosis factor and mitochondrial pathways^[63]。 Haberzettl et al.Found that exposure to PM_2.5increases the proliferation of endothelial progenitor cells in bone marrow and further leads to defects in angiogenesis or repair^[64]。

Many studies have shown that PM_2.5exposure not only affects the function of human organs such as heart and lung,but also has intergenerational toxicity,and even causes a variety of adverse birth outcomes in the next generation^[65,66]。 Using high-resolution imaging technology,Bove and others first found the existence of carbon black particles,the main component of PM_2.5,on the side of human placenta near the fetus^[67]。 They found higher concentrations of soot particles in the placentas of 10 women exposed to high levels of soot particles during pregnancy and 10 women exposed to low levels.In addition,PM_2.5can cause neuroinflammation and neurotoxicity,induce diseases related to the central nervous system,and induce abnormal animal behavior through oxidative stress and inflammatory response^[68,69]^[70,71]。 PM_2.5can also cross the placental barrier and enter fetal blood vessels,causing brain damage in offspring^[72,73]。

Due to the complexity of the composition of PM_2.5,the mechanism of its toxicity is very unclear.Traditional toxicological studies can only examine the internal changes of cells and animal tissues at the overall level,lacking detailed toxicological information at the molecular level of PM_2.5induction.Xiao et al.Found that the proteome of macrophages treated with diesel exhaust particulate extract changed significantly,in which antioxidant enzymes(such as metallothionein,catalase and heme oxygenase-1)were activated at low extract doses,while pro-inflammatory enzymes(such as p38 MAPK)required high extract doses to be activated^[74]。 Through mass spectrometry-based MSI and omics studies,new toxic mechanisms can be obtained from multiple perspectives such as metabolism and protein,and MSI technology can be used to locate potential target organs or substructural regions of toxic effects,which is essential to comprehensively measure the process of human disease development induced by PM_2.5exposure.Our research team selected animal models exposed to PM_2.5during pregnancy to investigate the effects of such exposure on the behavior and mood of offspring mice,and preliminarily clarified the molecular mechanism of multiple organ damage and metabolic remodeling caused by PM_2.5exposure,which provided an important basis for revealing the possible intergenerational toxicity of air pollution(Figure 7 )^[75]。

3.3 Other application

MSI has also been used to study the spatial localization of pesticides and herbicides in animal or plant tissues.Lagarrigue et al.Applied MSI technique to the quantitative study of the organochlorine pesticide chlordecone in mouse liver as a model,and the sensitivity of the quantitative method was sufficient for in situ localization analysis of structurally similar pesticides in animal tissues^[76]。 Anderson et al.analyzed the uptake of nicosulfuron,a sulfonylurea herbicide widely used in cereal cultivation,using sunflower root and stem slices^[77,78]。 In addition,they examined the absorption characteristics of four other commonly used herbicides,chlorimuron-ethyl,chlorimuron-ethyl,imazosulfuron and pyrazolon ethyl,using sunflower root,stem and leaf slices as research objects.the study found that the diffusion from the application point to the plant growth tip to the root was quite different according to the different herbicide varieties。

4 Conclusion and prospect

In order to explore the quantitative changes of environmental pollutants in animal and plant tissues and the spatial distribution changes of biomolecules induced by them, researchers used MSI-based and omics analysis techniques to provide high-throughput molecular information and visualize it. On this basis, combined with molecular biology methods, the samples induced by pollutants were analyzed based on 2D and 3D spatial structure in time and space. At present, this research strategy has been applied to the study of bisphenols, PM_2.5, pesticides and herbicides, which opens up a new way to solve the problem of the location and diffusion of pollutants in tissues and the discovery of new toxic mechanisms^[10]. It should be pointed out that although this research has developed rapidly in recent years, it is still in the initial stage of development, and both basic research and applied research need continuous improvement and development. The current research focuses on the following aspects: (1) developing more simplified sample preparation techniques, especially FFPE samples, to improve the species and abundance of detected molecules, as well as the stability and reproducibility of the method; (2) Develop a more universal MSI technology based on laser sampling to visualize and locate endogenous and exogenous molecules in single cells, with a resolution of nanometer level; (3) Explore new technologies based on the integration of microfluidic chips and mass spectrometry/MSI to simulate real-time exposure scenarios of pollutants to cells or animals. Biomimetic Microsystems representing different organs (lung, heart, intestine, liver, kidney and bone) can be integrated into a miniature device and simulate the dynamic process of real-time exposure of pollutants through microfluidic systems.Examples include absorption of contaminants from the lungs into the microcirculatory system, cardiotoxicity, transport and clearance in the kidneys, metabolism in the liver, and toxicity to immune cells. In addition, drugs or contaminants can also be introduced into the intestinal system to study the interactions between foreign substances, molecular transporters, and metabolic enzymes in different organs^[13]. At the same time, mass spectrometry and MSI technology were used to qualitatively, quantitatively and locationally monitor the molecular changes during real-time exposure.

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