In-vitro evaluation method for metabolism and colon toxicity of composite pollutants in water

Abstract

The invention relates to an in-vitro evaluation method and system for metabolism and colon toxicity of compound pollutants in water, which are used for efficiently evaluating the compound pollutants in water. Firstly, a three-stage anaerobic reaction system formed by connecting oral cavity, stomach and intestine digestion simulation tanks in series is used for continuously simulating metabolic transformation of pollutants in an empty-belly water drinking state; secondly, enriching and concentrating intestinal digestion products by adopting a solid-phase extraction-nitrogen blowing-redissolving three-step method, and eliminating the interference of digestive enzyme residues; and finally, co-exposing the concentrate and the pre-cultured colon-like organ, and evaluating the colon toxicity effect. According to the scheme, a drinking water targeted digestion simulation system is initiated, the limitation of a food digestion model is broken through, multiple technologies are coupled to form closed-loop evaluation, the single experimental period is less than or equal to 14 days, 99% or above of digestive enzyme residues can be efficiently eliminated, large sample parallel processing is supported, and the physiological correlation and experimental efficiency of an evaluation result are remarkably improved; and the method is suitable for high-flux colon toxicity evaluation of multiple composite pollutants in water.

Description

Technical Field

[0001] The present invention relates to environmental detection and toxicity evaluation technology, in particular to an in vitro evaluation method for the metabolism and colon toxicity of composite pollutants in water. Background Art

[0002] Long-term consumption of contaminated water may have potential negative effects on human health. Epidemiological studies have shown that long-term consumption of contaminated water is significantly associated with the risk of colon cancer. Therefore, accurately evaluating the toxic effects of combined pollutants on colon tissue is crucial in water quality health risk assessment.

[0003] Traditional toxicity assessment methods have significant limitations. In vivo biological evaluations face significant animal ethics controversy, lengthy experimental cycles, and poor reproducibility. Conventional in vitro two-dimensional cell models, on the other hand, face two core issues: biological distortion and insufficient reproduction of the physiological microenvironment. Furthermore, they are unable to reproduce the toxicity characteristics of complex pollutants in water after metabolic transformation through the digestive tract. Therefore, there is an urgent need to develop a more efficient, convenient, and physiologically realistic method for studying the metabolic behavior of complex pollutants in water and evaluating colonic toxicity.

[0004] Colon organoids are a type of cell cluster with a three-dimensional structure that can highly reproduce the cellular composition and spatial structure of the colon in vivo, while also having the operational convenience of in vitro models. They have opened up a new technical path for analyzing the pathogenic factors of colon diseases and their toxicological mechanisms, and are expected to be applied to the improvement of colon toxicity evaluation technology for complex pollutants in water.

[0005] However, the pollutant toxicity assessment system based on colon organoids still has certain methodological limitations, mainly because it may overlook the important role of the digestive process in the toxic transformation of pollutants. After oral ingestion, pollutants undergo complex physicochemical processing and enzymatic hydrolysis in the digestive tract, which may significantly change their morphology, bioavailability, and toxicity. Ignoring this digestive process may lead to misjudgment of the active substances that ultimately reach the colon and their toxic effects.

[0006] Based on the above-mentioned defects, a standardized tool in the field of food digestion research, an in vitro digestion simulation model, may be able to provide a new perspective for optimizing the colon toxicity research of pollutants. Currently, relevant technologies focus on the digestion simulation of food matrices, such as CN111100787B, CN119516878A, etc. However, there are essential differences between the drinking water process and the food intake process in terms of digestive physiological parameters, including the total amount of digestive fluid secretion, digestion time, etc. Existing food digestion models are difficult to accurately simulate the dynamic digestion process of pollutants under drinking water conditions.

[0007] It should be noted that the information disclosed in the above background technology section is only used to understand the background of this application, and therefore may include information that does not constitute prior art known to ordinary technicians in this field. Summary of the Invention

[0008] The main purpose of the present invention is to overcome the defects in the above-mentioned background technology and provide an in vitro evaluation method for the metabolism and colon toxicity of complex pollutants in water.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] An in vitro evaluation method for the metabolism and colon toxicity of composite pollutants in water comprises the following steps:

[0011] (1) Digestion simulation: The water sample to be tested is continuously digested by a three-stage anaerobic reaction system consisting of an oral digestion simulation tank, a gastric digestion simulation tank, and an intestinal digestion simulation tank, simulating the metabolic conversion process under the fasting water drinking state;

[0012] (2) Pollutant enrichment and concentration: The intestinal digestion products were enriched and concentrated using a three-step method of solid phase extraction-nitrogen blowing-redissolution to eliminate the interference of residual digestive enzymes;

[0013] (3) Colon toxicity evaluation: The concentrate was co-exposed to pre-cultured colon organoids to evaluate the colon toxicity effects of the metabolites.

[0014] Furthermore, in step (1), for the digestion simulation of a single drinking of water, the digestion simulation satisfies the following conditions:

[0015] Water intake: simulated water intake of 50-500 mL;

[0016] Oral stage: simulated saliva volume 2-5 mL, pH = 6.5-7.5, temperature 37°C, reaction time ≤ 5 min, salivary amylase activity 45-75 U / mL;

[0017] Gastric stage: simulated gastric fluid volume 25-45 mL, pH = 2.5-3.5, temperature 37°C, reaction time ≤ 2 h, pepsin activity 1500-2000 U / mL;

[0018] Intestinal stage: simulated intestinal fluid volume 35-55 mL, pH = 6.5-7.5, temperature 37°C, reaction time ≤ 2 h, pancreatic enzyme activity 25-45 U / mL.

[0019] Furthermore, in step (2), the solid phase extraction uses a lipophilic equilibrium polymer column HLB, the water sample flow rate is 1 to 3 mL / min; the eluent is methanol, and the reconstitution medium is colon organoid culture medium.

[0020] Furthermore, in step (3), the colon organoids are derived from human or mouse colon tissue and are pre-cultured in matrix gel for 5 to 7 days before exposure to form a three-dimensional structure.

[0021] Furthermore, in step (3), the co-exposure time of the pollutants and the colon organoids is 5 to 7 days.

[0022] Furthermore, the method has a single experimental cycle of ≤14 days and is suitable for high-throughput toxicity evaluation of complex pollutants in drinking water, sewage and industrial wastewater.

[0023] An in vitro evaluation system for the metabolism and colonic toxicity of complex pollutants in water, comprising:

[0024] Digestion simulation module: A three-stage continuous anaerobic reaction system consisting of an oral digestion simulation tank, a gastric digestion simulation tank, and an intestinal digestion simulation tank connected in series via a peristaltic pump, with control valves installed between each tank;

[0025] Pollutant enrichment and concentration module: includes a solid phase extraction device, a nitrogen blowdown device, and a re-dissolution device, used to eliminate residual digestive enzymes and concentrate metabolites;

[0026] Colon toxicity evaluation module: includes a colon organoid culture device and a toxicity detection device, which are used to realize the co-exposure and toxicity response analysis of metabolites and organoids.

[0027] Furthermore, in the digestion simulation module, the oral digestion simulation tank, the gastric digestion simulation tank and the intestinal digestion simulation tank are respectively configured with simulated saliva electrolyte stock solution, simulated gastric fluid electrolyte stock solution and simulated intestinal fluid electrolyte stock solution, and high-purity nitrogen is introduced into the system throughout to maintain an anaerobic environment.

[0028] Furthermore, in the pollutant enrichment and concentration module, the solid phase extraction device is equipped with a lipophilic equilibrium polymer column HLB, and the re-dissolution device uses colon organoid culture medium as a solvent.

[0029] Furthermore, in the colon toxicity evaluation module, the organoid culture device supports three-dimensional culture of matrix gel and pre-culture for a predetermined period of time (such as 7 days).

[0030] The present invention has the following beneficial effects:

[0031] This invention provides a method and system for in vitro metabolic simulation and colon toxicity assessment of complex pollutants in water. By constructing an in vitro simulated digestion reaction system, combined with pollutant enrichment and concentration technology and a colon organoid culture system, this method enables high-throughput simulation of the metabolic transformation of complex pollutants in water and evaluation of their colon toxicity. The technical solution of this invention offers advantages such as high detection efficiency and good reproducibility, providing an innovative solution for studying the metabolic behavior of complex pollutants in aquatic environments and evaluating their colon toxicity effects.

[0032] As the first evaluation method of complex pollutants in water based on the drinking water rule, it innovatively couples the pollutant enrichment and concentration technology and the colon organ culture system to construct a complete simulation platform for the metabolism and transformation of complex pollutants in water and high-throughput evaluation of colon toxicity, which can more truly and efficiently evaluate the colon toxicity risk of pollutants in water. Specifically, the present application proposes a comprehensive evaluation method, which first constructs an integrated technical platform coupling the digestion simulation module, the pollutant enrichment and concentration module and the colon organ toxicity evaluation module. The platform can truly reproduce the bioavailable form of complex pollutants in water after metabolism in the digestive tract, accurately evaluate the toxicity characteristics of the pollutants on the colon tissue, and significantly improve the physiological relevance and experimental efficiency of the evaluation results.

[0033] The present application has the following significant technical advantages: first, the drinking water targeted digestion simulation system is created, a three-stage series anaerobic reaction system is constructed based on the physiological parameters of drinking water, and the applicability limitation of the food digestion model is broken through; second, by optimizing the solid phase extraction step, more than 99% of the residual interference of digestive enzymes can be eliminated, and the enrichment target of metabolic products can be fully achieved; third, the digestion metabolism simulation technology, the enrichment and concentration technology of complex pollutants in water and the colon organ technology are successfully coupled, and the closed-loop evaluation of complex pollutants in water “digestion and transformation → metabolic product enrichment → organ toxicity response” is first realized. The single experiment cycle of the method is not more than 14 days, supports large sample parallel processing, and can form a colon bionic toxicity evaluation system of complex pollutants in water with full process, high throughput and high fidelity, and further expand the application to the colon toxicity risk evaluation and classification of complex pollutants in drinking water, sewage, industrial wastewater and other complex matrices.

[0034] Other beneficial effects in the embodiments of the present application will be further described below. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The overall flowchart of the in vitro evaluation method of the metabolism and colon toxicity of complex pollutants in water of the present application.

[0036] Figure 2 The technical route diagram of the in vitro evaluation method of the metabolism and colon toxicity of complex pollutants in water.

[0037] Figure 3 The concentration change of AOCl in the simulation digestion process in an embodiment.

[0038] Figure 4 The cell toxicity dose-effect curve of water sample on colon organ and phenol equivalent toxicity in an embodiment. DETAILED DESCRIPTION

[0039] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope of the present invention and its application.

[0040] It should be noted that when an element is referred to as being "fixed to" or "disposed on" another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element. In addition, connection can be used for both fixing and coupling or communication.

[0041] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention.

[0042] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of such features. In the description of the embodiments of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0043] To address these issues, the present invention proposes a comprehensive in vitro method for the metabolism and colonic toxicity assessment of complex pollutants in water. This approach, for the first time, constructs an integrated technology platform that couples a drinking water digestion simulation module, a pollutant enrichment and concentration module, and a colon organoid toxicity assessment module. This platform can realistically reproduce the bioavailable forms of complex pollutants in water after digestive tract metabolism and accurately assess their toxicity profiles in colonic tissue, significantly improving the physiological relevance of the evaluation results and experimental efficiency.

[0044] See Figure 1 and Figure 2 The present invention provides an in vitro method for evaluating the metabolism and colon toxicity of composite pollutants in water, comprising the following steps:

[0045] (1) Digestion simulation: The water sample to be tested is continuously digested by a three-stage anaerobic reaction system consisting of an oral digestion simulation tank, a gastric digestion simulation tank, and an intestinal digestion simulation tank, simulating the metabolic conversion process under the fasting water drinking state;

[0046] (2) Pollutant enrichment and concentration: the intestinal digestion products were enriched and concentrated by solid phase extraction-nitrogen blowing-redissolving three-step method to eliminate the interference of residual digestive enzymes;

[0047] (3) Colon toxicity evaluation: the concentrated products were co-exposed with pre-cultured colon organoids to evaluate the colon toxicity effect of the metabolic products.

[0048] In some embodiments, in step (1), the digestion simulation for a single drinking water meets the following conditions:

[0049] Drinking water volume: simulated drinking water volume 50-500 mL;

[0050] Oral stage: simulated saliva volume 2-5 mL, pH = 6.5-7.5, temperature 37℃, reaction time ≤5 min, salivary amylase activity 45-75 U / mL;

[0051] Stomach stage: simulated gastric juice volume 25-45 mL, pH = 2.5-3.5, temperature 37℃, reaction time ≤2 h, pepsin activity 1500-2000 U / mL;

[0052] Intestinal stage: simulated intestinal fluid volume 35-55 mL, pH = 6.5-7.5, temperature 37℃, reaction time ≤2 h, pancreatic enzyme activity 25-45 U / mL.

[0053] In some embodiments, in step (2), the solid phase extraction uses lipophilic equilibrium polymer column HLB, and the water sample flow rate is 1-3 mL / min; the eluent is methanol, and the redissolving medium is colon organoid culture medium.

[0054] In some embodiments, in step (3), the colon organoids are derived from human or murine colon tissue, and are pre-cultured in Matrigel for 5-7 days to form a three-dimensional structure before exposure.

[0055] In some embodiments, in step (3), the co-exposure of the pollutant and the colon organoids lasts for 5-7 days.

[0056] In some embodiments, the method has a single experimental cycle ≤14 days, and is suitable for high-throughput toxicity evaluation of complex pollutants in drinking water, sewage and industrial wastewater.

[0057] An in vitro evaluation system for metabolism and colon toxicity of complex pollutants in water, comprising:

[0058] Digestion simulation module: a three-stage continuous anaerobic reaction system composed of oral digestion simulation tank, stomach digestion simulation tank and intestinal digestion simulation tank connected by peristaltic pump, and control valves are arranged between each tank;

[0059] Pollutant enrichment and concentration module: includes a solid phase extraction device, a nitrogen blowdown device, and a re-dissolution device, used to eliminate residual digestive enzymes and concentrate metabolites;

[0060] Colon toxicity evaluation module: includes a colon organoid culture device and a toxicity detection device, which are used to realize the co-exposure and toxicity response analysis of metabolites and organoids.

[0061] In some embodiments, in the digestion simulation module, the oral digestion simulation tank, the gastric digestion simulation tank and the intestinal digestion simulation tank are respectively configured with simulated saliva electrolyte stock solution, simulated gastric fluid electrolyte stock solution and simulated intestinal fluid electrolyte stock solution, and high-purity nitrogen is introduced into the system throughout to maintain an anaerobic environment.

[0062] In some embodiments, in the pollutant enrichment and concentration module, the solid phase extraction device is equipped with a lipophilic equilibrium polymer column HLB, and the re-dissolution device uses colon organoid culture medium as a solvent.

[0063] In some embodiments, in the colon toxicity evaluation module, the organoid culture device supports matrix gel three-dimensional culture and pre-culture for a predetermined time.

[0064] The present invention overcomes the limitations of the applicability of traditional food digestion models through the pioneering three-stage tandem anaerobic digestion simulation system for drinking water, accurately reproducing the metabolic transformation process of pollutants under fasting drinking water conditions; combining solid-phase extraction optimization technology to eliminate >99% of residual interference from digestive enzymes and efficiently enrich metabolites, and then coupling it with a three-dimensional culture system for colon organoids, it realizes the closed-loop evaluation of "digestion transformation → metabolite enrichment → organ toxicity response" for the first time, completing a full-process, high-throughput, high-fidelity bionic assessment of colon toxicity risk within a single experimental cycle of ≤14 days, significantly improving physiological relevance and experimental efficiency, and providing innovative technical approaches and solutions for the colon toxicity classification of complex pollutants in drinking water, sewage and industrial wastewater.

[0065] The following further describes specific embodiments of the present invention and experimental verification.

[0066] An in vitro evaluation method and system for the metabolism and colon toxicity of complex pollutants in water. This method and system replicates the metabolic transformation process of complex pollutants in water through the mouth, stomach, and intestines through a three-stage digestion simulation module connected in series, combines it with a pollutant enrichment and concentration module to achieve precise capture and purification of metabolites, and then uses a colon organoid toxicity evaluation module to complete the evaluation of the toxic effects of metabolites on colon tissue, forming a complete technical chain of "metabolic transformation-product enrichment-toxicity evaluation", which can efficiently and realistically evaluate the colon toxicity risk of complex pollutants in water.

[0067] The first module of the system is a drinking water digestion simulation module. It consists of an oral digestion simulation tank, a gastric digestion simulation tank, and an intestinal digestion simulation tank connected in series via a peristaltic pump to form a three-stage continuous reaction system. Control valves are installed between the tanks. The system is filled with high-purity nitrogen to maintain an anaerobic environment throughout the process to simulate the physiological state of the human digestive tract. The digestion process includes:

[0068] (1) Oral digestion stage: the water sample to be tested, the simulated saliva electrolyte solution and α-salivary amylase were mixed to obtain a mixed water sample 1, the pH was adjusted to 6.5-7.5, and the mixture was stirred at 37°C to obtain an oral simulated digest;

[0069] (2) Simulated gastric digestion: The oral simulated digest was transferred to a gastric digestion tank via a peristaltic pump, mixed with a simulated gastric fluid electrolyte solution and pepsin to obtain mixed water sample 2, adjusted the pH to 2.5-3.5, and reacted with stirring at 37°C to obtain a gastric simulated digest;

[0070] (3) Simulated intestinal digestion: The gastric simulated digest was transferred to an intestinal digestion tank via a peristaltic pump, mixed with a simulated gastric fluid electrolyte solution and pancreatic enzymes to obtain mixed water sample 3, adjusted the pH to 6.5-7.5, and reacted with stirring at 37°C to obtain the intestinal simulated digest;

[0071] Preferably, the simulated saliva electrolyte stock solution comprises: KCl, KH2PO4, NaHCO3, MgCl2(H2O)6, (NH4)2CO3, HCl, CaCl2, so as to restore the electrolyte environment of human saliva.

[0072] Preferably, the components of the gastric fluid electrolyte stock solution include: KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, (NH4)2CO3, CaCl2, so as to restore the electrolyte environment of human gastric fluid.

[0073] Preferably, the components of the intestinal fluid electrolyte stock solution include: KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, HCl, CaCl2, so as to restore the intestinal fluid electrolyte environment of the human body.

[0074] Preferably, the maximum duration of simulated oral digestion is 5 minutes to avoid excessive interaction time deviating from the actual oral digestion process.

[0075] Preferably, the maximum duration of simulated gastric digestion is 2 h to avoid excessive interaction time deviating from the actual gastric digestion process.

[0076] Preferably, the maximum duration of simulated intestinal digestion is 2 h to avoid excessive interaction time deviating from the actual intestinal digestion process.

[0077] In one embodiment, the salivary amylase activity in the simulated saliva is 45 U / mL to simulate the secretion of salivary amylase in a fasting state. 1 U is defined as the release of 1 mg of maltose from potato starch in 3 minutes at pH 6.9 and 20°C.

[0078] In one embodiment, the pepsin activity in the simulated saliva is 2000 U / mL to simulate the secretion of pepsin in the fasting state. 1 U is defined as the amount of ΔA produced per minute by the decomposition of a 2% (w / v) hemoglobin solution at pH 2.0 and a temperature of 37°C. 280 = 0.001 trichloroacetic acid (TCA) soluble peptide.

[0079] In one embodiment, the pancreatic enzyme activity in the simulated intestinal fluid is 25 U / mL to simulate the pancreatic enzyme secretion pattern in the fasting state. 1 U is defined as the hydrolysis of 1 μmol of p-toluenesulfonyl-L-arginine methyl ester (TAME) per minute at pH 8.1 and 25°C.

[0080] In one implementation method, the amount of digestive fluid used in the simulated digestion process is optimized based on the ratio of the normal amount of water a person drinks on an empty stomach to the volume of digestive fluid at each stage. The specific volumes are 250 mL of simulated water, 5 mL of simulated saliva, 35 mL of simulated gastric fluid, and 45 mL of simulated small intestinal fluid.

[0081] The second module of the present invention is a water pollutant enrichment and concentration module, which enriches and concentrates the intestinal digestion products through a three-step method of solid phase extraction-nitrogen blowing-redissolution.

[0082] Preferably, after solid phase extraction and enrichment, methanol is used for elution to reduce the error in toxicity evaluation caused by excessive residual digestive enzymes.

[0083] Preferably, the methanol is completely removed by nitrogen blowing, and the concentrate is reconstituted with colon organoid culture medium to eliminate toxic interference from organic solvents.

[0084] In one embodiment, the enrichment column is selected as a lipophilic balance polymer column HLB, and the water sample flow rate is set to 3 mL / min.

[0085] The third module of the present invention is a colon organoid toxicity evaluation module.

[0086] Alternatively, colon organoids are obtained by isolating human or mouse colon tissue or differentiating stem cells.

[0087] Preferably, the organoids are pre-cultured in Matrigel for 7 days before exposure to initially form a stable three-dimensional structure.

[0088] In one embodiment, the co-exposure time of the pollutant and the colon organoids is 7 days to allow for the full manifestation of the toxic effects.

[0089] Figure 2 The complete technical route for the in vitro evaluation of the metabolism and colon toxicity of composite pollutants in water is demonstrated. As shown in ①, the digestion simulation module consists of a peristaltic pump connected in series to an oral digestion simulation tank, a gastric fluid simulation digestion tank, and an intestinal fluid simulation digestion tank, forming a continuous reaction system that can achieve the gradual metabolic transformation of pollutants in artificial digestive fluid. The enrichment and concentration steps of composite pollutants in water are shown in ②-④, and the devices involved include a solid phase extractor and a nitrogen blowdown apparatus. The steps of the colon organoid toxicity evaluation module are shown in ⑤ and ⑥. Before exposure, the organoids are pre-cultured in matrix gel for 7 days and then co-exposed and cultured with the pollutants for 7 days. The colon organoids finally harvested are suitable for multi-endpoint toxicity evaluation.

[0090] 1. Drinking water digestion simulation

[0091] Prepare 1.25× digestion electrolyte stock solutions for oral, gastric, and small intestinal digestion stages, respectively, as shown in Table 1. The stock solutions can be stored at -20°C for 1 year.

[0092] Table 1 Electrolyte component ratios of digestion stock solution (1.25×)

[0093]

[0094]

[0095] Note: “-” means no addition

[0096] The amount of digestive fluid used during the simulated digestion process was optimized based on the ratio of a typical fasting human's single water intake to the volume of digestive fluid at each stage. Specifically, the volume consisted of 250 mL of simulated water, 5 mL of simulated saliva, 35 mL of simulated gastric fluid, and 45 mL of simulated small intestinal fluid.

[0097] To more reliably reflect the effects of digestive enzymes in the drinking state, the amount of digestive enzymes used in the simulated digestion process was optimized with reference to the secretion of digestive enzymes in the resting state. For specific values, refer to the following steps.

[0098] (1) Oral digestion simulation

[0099] 1) Dilute the oral digestion stock solution with ultrapure water to a concentration of 1×.

[0100] 2) Add CaCl2(H2O)2 to a total concentration of 1.5 mM in the simulated saliva.

[0101] 3) Add 45 U / mL of salivary amylase to the simulated saliva. 1 U is defined as the amount of enzyme required to catalyze the hydrolysis of potato starch to produce 1 mg of maltose within 3 minutes at pH 6.9 and 20°C.

[0102] 4) Mix the water sample to be tested with the simulated saliva.

[0103] 5) Mix at 37°C for 5 minutes.

[0104] (2) Gastric digestion simulation

[0105] 1) Dilute the gastric digestion stock solution with ultrapure water to a concentration of 1×.

[0106] 2) Add CaCl2(H2O)2 so that its total concentration in the simulated saliva reaches 0.15mM.

[0107] 3) Add 2000 U / mL of pepsin to the simulated gastric fluid. 1 U is defined as the amount of enzyme required to catalyze the hydrolysis of a 2% (w / v) hemoglobin solution per minute at pH 2.0 and 37°C, producing a trichloroacetic acid (TCA)-soluble peptide with an absorbance increase of 0.001 at 280 nm.

[0108] 4) Mixing the simulated oral digestive fluid with the simulated gastric fluid.

[0109] 5) Mix at 37°C for 2 h.

[0110] (3) Small intestinal digestion simulation

[0111] 1) Dilute the small intestinal digestion stock solution with ultrapure water to a concentration of 1×.

[0112] 2) Add CaCl2(H2O)2 so that its total concentration in the simulated saliva reaches 0.6 mM.

[0113] 3) Add 25 U / mL of pancreatic enzyme to the simulated small intestinal fluid. 1 U is defined as the amount of enzyme required to catalyze the hydrolysis of 1 μmol of p-toluenesulfonyl-L-arginine methyl ester (TAME) per minute at pH 8.1 and 25°C.

[0114] 4) Mixing the simulated gastric digestive fluid with the simulated small intestinal fluid.

[0115] 5) Mix at 37°C for 2 h.

[0116] 2. Pollutant enrichment and concentration

[0117] Water samples were adsorbed using HLB solid phase extraction cartridges. The water samples were adjusted to pH = 2 ± 0.02 with 4 mM HC1 and the HLB cartridges were activated with 10 mL of methanol and 10 mL of water, followed by the water sample at a flow rate of 3 mL / min. After the water sample passed through the cartridge, the cartridge was dried with N2 and the adsorbed organics were eluted with methanol. The eluate was finally dried with N2 and re-dissolved in organoid culture medium. The enrichment and concentration process was performed in the dark throughout.

[0118] 3. Colon organoid preparation

[0119] (1) Mouse strain

[0120] Common BALB / c mice were used for colon tissue collection, male, body weight 20-30 g, age about 2 months.

[0121] (2) Mouse CO2 euthanasia

[0122] Common BALB / c mice were placed in the euthanasia cage box, which was not filled with gas in advance. Common BALB / c mice were euthanized in a 10L volume of euthanasia cage box with an excess of CO2, with a carbon dioxide purity of 99%, and a flow rate controlled at 2L / min, so that the animals lost consciousness and died quickly within 3 min. Death was judged as the animal stopped breathing and the eyes lost color. After the mouse stopped breathing, CO2 was supplied for at least another 1 min.

[0123] (3) Mouse dissection

[0124] The dead mouse was placed on the dissection tray with its back on the ground, and the colon was dissected and taken out for organoid construction.

[0125] (4) Organoid construction

[0126] The colon contents were rinsed with medium. The intestine was cut open with surgical scissors. Then the colon surface villi were scraped off with a surgical blade, and rinsed in medium for 3 times. The rinsed colon tissue was cut into 3mm wide and transferred to 5mM EDTA for dissociation at 4°C for 30min. Then 5 times volume of medium was added to EDTA to terminate dissociation. The crypts were separated by repeated pipetting and purified by passing through a 70pm filter. The filtrate was resuspended with medium after centrifugation at 300g for 5min, and rinsed for 3 times.

[0127] The tissue fragments were wrapped with Matrigel 3D, and the mixture of Matrigel and cells was pipetted into the cell culture well plate. Further, the 96-well plate or culture dish was incubated in a 37°C incubator for 20min to completely solidify the droplets into spheres.

[0128] Add complete mouse colon culture medium to 96-well plates or culture dishes and transfer to a 37°C, 5% CO2 incubator for culture. Mouse colon culture medium contains small molecule compounds and growth factors, including a p38 MAPK kinase inhibitor (SB-202190), a TGF-β inhibitor (A 83-01), a Rho kinase inhibitor (Y-27632), a GSK inhibitor (CHIR-99021), and Jagged-1. Change the complete culture medium every two days, and passage organoids every seven days.

[0129] (5) Organoid passage

[0130] Use a pipette tip to scrape the organoids from the culture dish and transfer the suspension of organoids and culture medium to a centrifuge tube. Use a pipette tip to resuspend the organoid suspension to separate the organoids from the matrix gel. Centrifuge at 300g for 5 minutes, then discard the upper layer of culture medium and matrix gel, and repeat the wash three times. After complete washing, wrap with matrix gel and repeat the seed plate culture step. Organoids after three generations of expansion can be used for toxic exposure experiments, and excess organoids can be frozen and stored for use in the next experiment.

[0131] (6) Organoid cryopreservation

[0132] Following the organoid cleaning step for organoid passaging, add freezing solution to the organoids to be frozen, mix well by pipetting, and quickly transfer to low-temperature cryotubes. Then, quickly place the cell freezing program cooling box into a -80℃ ultra-low temperature freezer. After 24 hours, transfer the cryotubes to liquid nitrogen for long-term storage.

[0133] (7) Organoid resuscitation

[0134] Remove the cryovial from the liquid nitrogen tank and immediately place it in a 37°C water bath, shaking rapidly to thaw. Slowly add 5-10 volumes of Matrigel to dilute the cryovial suspension. Continue culturing the revived organoids for 1-2 passages before using them in toxicity exposure experiments.

[0135] 4. Toxicity exposure experiment

[0136] Before toxicity testing, organoids were seeded in sterile 96-well plates (Corning, USA) and pre-cultured for 7 days, with fresh culture medium replaced every 2 days. Subsequently, organoids were exposed to the test sample according to a set concentration gradient for 7 days, with the test sample replaced every 2 days.

[0137] Example 1

[0138] Dissolved Suwannee River natural organic matter (SRNOM) was used to prepare the original water sample. The TOC value of the dissolved SRNOM was determined to be 5.21 mg-C / L. A 2 mM sodium phosphate-citrate buffer solution was prepared and added to the original water sample at a ratio of 1:100, adjusting the pH to 7. Sodium hypochlorite solution was added to the reaction solution at a chlorine dose (mg-Cl2 / L) three times the DOC value to simulate chlorine disinfection. After vigorous shaking, the reaction was allowed to stand at room temperature in the dark for 48 hours to achieve stable water toxicity. After the reaction, residual chlorine was quenched with ammonium chloride.

[0139] Adsorbable organic chlorine (AOCl) has been shown to be the primary source of toxicity in chlorine-disinfected water samples. This study aimed to investigate the metabolic effects of a simulated continuous digestion process on AOCl levels in water samples. Example 1 utilized only the digestion simulation module.

[0140] In order to avoid the influence of water sample volume change on the measurement results, the following volume compensation measures were taken:

[0141] For water samples that have not been digested: add a volume of simulated digestive fluid equal to the oral, gastric and intestinal digestive fluid, but do not perform the stirring reaction of oral, gastric and intestinal digestion.

[0142] For water samples that have only undergone oral digestion: add a volume of simulated digestive fluid equal to that of the gastric and intestinal digestive fluid, but do not perform the stirring reaction of gastric and intestinal digestion.

[0143] For water samples that have undergone oral and gastric digestion: add a volume of simulated digestive fluid equal to that of the intestinal digestive fluid, but do not perform the stirring reaction of intestinal digestion.

[0144] AOCl concentration was determined using the method specified in the national standard "Water Quality—Determination of Adsorbable Organic Halogen (AOX)—Ion Chromatography (HJ / T83-2001)." The final AOCl concentration in the water sample was calculated based on the final sample volume.

[0145] Figure 3 The changes in the content of AOCl during the simulated digestion process are shown. S, G, and I represent the oral, gastric, and intestinal digestion stages, respectively; the numbers represent the reaction time (min) in the current stage. When no digestive enzymes are added but only electrolytes are added, the concentration of AOCl remains basically unchanged. The results show that the electrolyte changes are not sufficient to cause the removal of AOCl. When salivary amylase, pepsin, and pancreatic enzymes are added, AOCl shows a downward trend throughout the simulated digestion process. During the simulated oral digestion stage, the concentration of AOCl did not change significantly. After 2 hours of simulated gastric digestion, the concentration of AOCl dropped to 84% of the initial value. Then, after 2 hours of simulated intestinal digestion, the concentration of AOCl continued to drop to 57% of the initial value. The results show that digestion can remove AOCl from drinking water, and gastric and intestinal digestion are the main stages of AOCl decline.

[0146] Example 2

[0147] The water samples were treated using the digestion simulation module in the same manner as in Example 1. The water samples were then concentrated using the pollutant enrichment and concentration module, and subjected to a colon organoid toxicity exposure experiment using the colon organoid toxicity assessment module to determine the metabolic effects of the continuous digestion process on the cytotoxicity of the water samples.

[0148] Cytotoxicity was determined using the ATP assay. The cell viability was calculated using the following formula:

[0149]

[0150] in,

[0151] RLU 样品 = relative light units of the experimental wells, i.e., the light signal value of the wells containing cells and test compound (sample or phenol);

[0152] RLU 空白 = relative light units of the blank well, i.e., the light signal value of the well containing culture medium;

[0153] RLU 阴性对照 = Relative light units of the control wells, i.e., the light signal value of the wells containing cells and culture medium.

[0154] To further quantify the cytotoxicity of the samples, the half-lethal concentration of the positive control phenol was determined, and the cytotoxic equivalent of the samples was calculated according to the following formula:

[0155]

[0156] in,

[0157] LC 苯酚,50 = the half-lethal concentration of phenol in the positive control;

[0158] LC 样品,50 =50% lethal concentration of the sample.

[0159] The unit of cytotoxicity equivalent is mg-phenol / L.

[0160] Figure 4 The dose-effect curves of simulated digestion on the cytotoxicity of water samples and the effect of phenol toxicity equivalents are shown, including: (a) cytotoxicity dose-effect curves of water samples after each stage of digestion; (b) phenol equivalent toxicity of water samples after each stage of digestion.

[0161] Draw the cytotoxicity dose-effect curve of water sample relative to negative control. Figure 4The results show that oral digestion has almost no effect on the cytotoxicity of chlorine disinfected water, while gastric digestion and intestinal digestion reduce cytotoxicity to varying degrees. Further investigation of the effects of electrolytes and digestive enzymes on the removal of phenol equivalent toxicity is shown in Figure 2. Figure 4 (b) The changes in electrolytes did not significantly alter the cytotoxicity-inducing effect of the water sample. After the addition of digestive enzymes, gastric digestion and intestinal digestion resulted in a decrease in the cytotoxicity-inducing effect of the water sample on organoids from 6.80 mg-phenol / L to 6.02 and 3.59 mg-phenol / L, respectively.

[0162] Those skilled in the art should understand that the colon toxicity evaluation described in the present invention is not limited to cytotoxicity detection, but can also be extended to any other toxicity measurement, including but not limited to genetic toxicity indicators.

[0163] In summary, the present invention proposes a comprehensive in vitro method and system for evaluating the metabolism and colonic toxicity of complex pollutants in water. This invention, for the first time, constructs an integrated technology platform that couples a drinking water digestion simulation module, a pollutant enrichment and concentration module, and a colon organoid toxicity assessment module. This platform can realistically reproduce the bioavailable forms of complex pollutants in water after digestive tract metabolism and accurately assess their toxicity characteristics to colonic tissue, significantly improving the physiological relevance of evaluation results and experimental efficiency.

[0164] Compared with the prior art, the significant advantages of the present invention are embodied in the following aspects:

[0165] (1) The present invention pioneered a targeted drinking water digestion simulation system, which constructed a three-stage anaerobic reaction system based on the physiological parameters of drinking water, breaking through the applicability limitations of food digestion models.

[0166] (2) The present invention can eliminate >99% of the interference of residual digestive enzymes by optimizing the solid phase extraction step and can fully achieve the goal of metabolite enrichment.

[0167] (3) The present invention couples digestive tract metabolism simulation technology, water composite pollutant enrichment and concentration technology, and colon organoid technology to realize for the first time the closed-loop evaluation of "digestion transformation → metabolite enrichment → organ toxicity response" of water composite pollutants. The single experimental cycle is ≤14 days, and it supports parallel processing of large samples. It can realize a full-process, high-throughput and high-fidelity colon biomimetic toxicity assessment system for water composite pollutants, which can be expanded to be applied to the colon toxicity risk assessment and classification of composite pollutants in complex matrices such as drinking water, sewage, and industrial wastewater.

[0168] The above description further details the present invention in conjunction with specific / preferred embodiments, and the specific implementation of the present invention should not be construed as being limited to these descriptions. Persons skilled in the art will appreciate that, without departing from the spirit of the present invention, they may make various substitutions or modifications to the described embodiments, and these substitutions or modifications should be considered to fall within the scope of protection of the present invention. Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "preferred embodiments," "examples," "specific examples," or "some examples" indicates that the specific features, structures, materials, or characteristics described in conjunction with such embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Persons skilled in the art may combine and assemble the different embodiments or examples described in this specification, as well as features of different embodiments or examples, without conflicting opinions. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications may be made herein without departing from the scope of protection of the patent application.

Claims

1. An in vitro evaluation method for the metabolism and colon toxicity of complex pollutants in water, characterized in that: The following steps are involved: (1) Digestion simulation: The water sample to be tested is continuously digested by a three-stage anaerobic reaction system consisting of an oral digestion simulation tank, a gastric digestion simulation tank, and an intestinal digestion simulation tank, simulating the metabolic conversion process under the fasting water drinking state; (2) Pollutant enrichment and concentration: The intestinal digestion products were enriched and concentrated using a three-step method of solid phase extraction-nitrogen blowing-redissolution to eliminate the interference of residual digestive enzymes; (3) Colon toxicity evaluation: The concentrate was co-exposed to pre-cultured colon organoids to evaluate the colon toxicity effects of the metabolites.

2. The method according to claim 1, wherein: In step (1), for the digestion simulation of a single drinking of water, the digestion simulation satisfies the following conditions: Water intake: simulated water intake of 50-500 mL; Oral stage: simulated saliva volume 2-5 mL, pH = 6.5-7.5, temperature 37°C, reaction time ≤ 5 min, salivary amylase activity 45-75 U / mL; Gastric stage: simulated gastric fluid volume 25-45 mL, pH = 2.5-3.5, temperature 37°C, reaction time ≤ 2 h, pepsin activity 1500-2000 U / mL; Intestinal stage: simulated intestinal fluid volume 35-55 mL, pH = 6.5-7.5, temperature 37°C, reaction time ≤ 2 h, pancreatic enzyme activity 25-45 U / mL.

3. The method according to claim 1 or 2, wherein: In step (2), the solid phase extraction uses a lipophilic equilibrium polymer column HLB, the water sample flow rate is 1 to 3 mL / min; the eluent is methanol, and the reconstitution medium is colon organoid culture medium.

4. The method according to any one of claims 1 to 3, wherein: In step (3), the colon organoids are derived from human or mouse colon tissue and are pre-cultured in matrix gel for 5 to 7 days before exposure to form a three-dimensional structure.

5. The method according to any one of claims 1 to 4, characterized in that: In step (3), the co-exposure time of the pollutants and the colon organoids is 5 to 7 days.

6. The method according to any one of claims 1 to 5, characterized in that: The method has a single experimental cycle of ≤14 days and is suitable for high-throughput toxicity evaluation of complex pollutants in drinking water, sewage and industrial wastewater.

7. An in vitro evaluation system for the metabolism and colon toxicity of complex pollutants in water, characterized by: include: Digestion simulation module: A three-stage continuous anaerobic reaction system consisting of an oral digestion simulation tank, a gastric digestion simulation tank, and an intestinal digestion simulation tank connected in series via a peristaltic pump, with control valves installed between each tank; Pollutant enrichment and concentration module: includes a solid phase extraction device, a nitrogen blowdown device, and a re-dissolution device, used to eliminate residual digestive enzymes and concentrate metabolites; Colon toxicity evaluation module: includes a colon organoid culture device and a toxicity detection device, which are used to realize the co-exposure and toxicity response analysis of metabolites and organoids.

8. The system according to claim 7, wherein: In the digestion simulation module, the oral digestion simulation tank, the gastric digestion simulation tank and the intestinal digestion simulation tank are respectively configured with simulated saliva electrolyte stock solution, simulated gastric fluid electrolyte stock solution and simulated intestinal fluid electrolyte stock solution, and high-purity nitrogen is introduced into the system throughout to maintain an anaerobic environment.

9. The system according to claim 7 or 8, characterized in that: In the pollutant enrichment and concentration module, the solid phase extraction device is equipped with a lipophilic equilibrium polymer column HLB, and the re-dissolution device uses colon organoid culture medium as a solvent.

10. The system according to any one of claims 7 to 9, characterized in that: In the colon toxicity evaluation module, the organoid culture device supports matrix gel three-dimensional culture and pre-culture for a predetermined time.

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