Method for evaluating combined toxicity of nano plastic and tamoxifen based on caenorhabditis elegans and method for resisting drug toxicity caused by nano plastic

Abstract

The invention discloses a method for evaluating combined toxicity of nano plastic and tamoxifen based on caenorhabditis elegans and a method for resisting drug toxicity caused by the nano plastic, a sample to be detected is an NGM solid culture medium of a mixture of the nano plastic and tamoxifen, and the caenorhabditis elegans is inoculated into the sample to be detected. The toxic effect of the joint exposure of the nano plastic and the tamoxifen on nematode movement, nerve, development and reproduction under the actual environment condition can be quickly and accurately evaluated, and a basis is provided for evaluation of human health risks. The invention provides a combined toxicity detection method which is suitable for laboratories, simple and convenient to operate and high in applicability by fully considering the problems that the toxicity is changed due to the chemical effect generated by nano-plastics and medicines and the in-vivo conditions are complicated and are difficult to observe, and provides a combined toxicity detection method which is suitable for laboratories and can be used for detecting the toxicity by supplementing N-acetylcysteine through diet. The effect of N-acetylcysteine in relieving tamoxifen toxicity caused by nano-plastics is further evaluated.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental toxicology, and specifically relates to an evaluation system for the combined toxicity of nanoplastics and tamoxifen and an application of a method for resisting drug toxicity caused by nanoplastics. Background Art

[0002] Tamoxifen is a widely used selective estrogen receptor modulator (SERM) used in more than 120 countries around the world to treat early and advanced estrogen receptor-positive breast cancer in premenopausal and postmenopausal women and men. It has also become the first breast cancer prevention drug approved by the US Food and Drug Administration (FDA). Similar to many anti-tumor drugs, tamoxifen has a series of side effects in clinical practice, such as stroke, pulmonary embolism, vasomotor symptoms, endometrial abnormalities and vaginal symptoms, gastrointestinal irritation (nausea, diarrhea) and hepatotoxicity. However, these side effects do not seem to be related to the patient's genetic background. We consider whether the side effects of tamoxifen are related to the environmental exposure factors to which the patient is exposed.

[0003] As the most important class of materials in the 21st century, polymers (commonly referred to as "plastics") play a huge role in our daily lives. While bringing great convenience to people's lives, they also lead to a large amount of plastic waste being discharged into the environment and decomposed into (NPs; <0.1 μm in diameter) through heating, photochemical reactions, oxidation and other processes. Nanoplastics are stable and difficult to degrade. They can migrate and accumulate in various ecosystems for a long time, so they are widely distributed in oceans, soils and other ecosystems. Therefore, humans can be directly or indirectly exposed to plastic particles through ingestion, inhalation or skin contact. In recent years, there have been continuous reports of nanoplastics being detected in human faces, hair, saliva, blood, breast milk, placenta and feces. In addition, based on the stability and porosity of MPs / NPs, their ability to adsorb environmental pollutants, such as organic pollutants, heavy metals, and halogenated antibiotics, is enhanced. Nanoplastics interact with these coexisting toxic substances, exacerbating the original toxicity of these pollutants. Therefore, the difference in tamoxifen side effects may be related to the environmental concentration of nanoplastics.

[0004] As an important model organism, C. elegans is very sensitive to environmental exposure and has been widely shown to be helpful for toxicity assessment and toxicology studies of pollutants at environmentally relevant concentrations. Even at environmentally relevant concentrations, the toxicity of some poisons can be successfully detected in nematodes, and some sensitive environmental pollutant assessment endpoints, such as motor behavior and pharyngeal pumping (indicators of basic nervous system functions), reproductive rate (reflecting reproductive capacity damage), and area (reflecting developmental damage) can be used for toxicological assessment. In addition, about 40% of the genes in the C. elegans genome are homologous to the human genome, suggesting that toxicity assessments conducted on C. elegans will largely reflect similar toxic effects on humans.

[0005] At present, no article has proposed using Caenorhabditis elegans as a research model to comprehensively evaluate the combined toxic effects of nanoplastics and tamoxifen in terms of movement behavior, pharyngeal pumping frequency, reproduction, and development. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for evaluating the combined toxicity of nanoplastics and tamoxifen, using the fact that Caenorhabditis elegans is very sensitive to environmental exposure, which enables us to determine the in vivo toxicological manifestations of different environmental toxins or stresses. And by supplementing N-acetylcysteine ​​with food, we further evaluate the role of N-acetylcysteine ​​in alleviating the tamoxifen toxicity caused by nanoplastics.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] A method for evaluating the combined toxicity of plastic and tamoxifen based on Caenorhabditis elegans:

[0009] (1) The nanoplastics were polystyrene nanoplastics (PS-NPs). Nanoplastic solutions of different concentrations were prepared with OP50 bacterial liquid and evenly coated on NGM plates. L1-stage Caenorhabditis elegans were placed on the NGM plates containing nanoplastics for exposure experiments and cultured in a constant temperature incubator at 20°C. The development, motility, and fertility of the Caenorhabditis elegans were recorded.

[0010] (2) Tamoxifen was dissolved in a sterile solvent and added to NGM agar heated to 55°C at a final volume of 0.1% to obtain an NGM plate containing tamoxifen at a final concentration of 10 μM. OP50 was evenly spread on the NGM plate, and L1-stage Caenorhabditis elegans was placed on the NGM plate containing tamoxifen for exposure experiments. The plate was cultured in a constant temperature incubator at 20°C, and the development, motility, and fertility of the Caenorhabditis elegans were recorded.

[0011] (3) Nanoplastic solutions of different concentrations were prepared using OP50 bacterial liquid and evenly coated on NGM plates containing tamoxifen at a final concentration of 10 μM. L1 stage Caenorhabditis elegans were inoculated into the solutions and cultured in a constant temperature incubator at 20°C. The development, motility, and fertility of the Caenorhabditis elegans were recorded.

[0012] (4) Data processing: Body bending, head swing and number of eggs laid are directly plotted based on the data collected within a specified time. The area data displayed in developmental toxicity are additionally normalized.

[0013] The method for obtaining L1 stage Caenorhabditis elegans includes: using a lysis solution to lyse the Caenorhabditis elegans in the egg-laying stage to obtain sufficient eggs, collecting the nematodes hatched to the L1 stage with K buffer after culturing for 18 to 22 hours, centrifuging and discarding the supernatant, adding K buffer, and inoculating into NGM culture medium for culturing.

[0014] The NGM solid culture medium of the mixture of nanoplastics and tamoxifen contains 0.1-10 g / L of plastic solution and 10 μM of tamoxifen.

[0015] A method for using N-acetylcysteine ​​to resist drug toxicity caused by nanoplastics, wherein the drug toxicity caused by nanoplastics includes drug toxicity caused by the combination of nanoplastics and tamoxifen. N-acetylcysteine ​​supplementation at a concentration of 10 μM has a resisting effect on toxicity caused by nanoplastics in a concentration range of 0.1-10 ug / L.

[0016] Furthermore, N-acetylcysteine ​​supplementation at a concentration of 10 μM alleviated the toxic aggravation effect of low-concentration tamoxifen (0.1-10 μM) caused by a nanoplastic concentration range of 0.1-10 ug / L.

[0017] The Caenorhabditis elegans is the L1 stage Caenorhabditis elegans.

[0018] The K buffer solution was prepared as follows: 1.4902 g NaCl, 1.1928 g KCl, and the volume was fixed to 500 mL using ddH2O; the lysate solution was prepared as follows: NaClO solution: 1 mol / L NaOH: water = 1:1:3.

[0019] NGM solid culture medium: weigh 3g sodium chloride (NaCl), 2.5g peptone and 17g agar respectively, add double distilled water to 1L; sterilize at 121℃ for 20min, wait until cooled to 55℃, add 1mL 1M CaCl2, 1mL 1M MgSO4, 1mL 5M cholesterol solution, 25mL 1M KHPO4 buffer respectively, mix well and pour into the culture dish.

[0020] In the present invention, a lysis solution is used to lyse the ovipositing Caenorhabditis elegans to obtain sufficient eggs, and the nematodes hatched to the L1 stage are transferred to the culture medium of the blank control group, the culture medium of the single exposure experimental group, and the culture medium of the combined exposure experimental group for toxicity experiments. After the exposure period, the nematodes are evaluated for the combined toxicity of nanoplastics and tamoxifen based on at least one of the head swing frequency, body bending frequency, pharyngeal pumping frequency, reproductive rate and area of ​​the Caenorhabditis elegans.

[0021] Based on experimental results, the toxicity of combined exposure to nanoplastics and tamoxifen on the nervous system was evaluated as the body bending frequency of Caenorhabditis elegans was less than 13.4 times / 20 seconds.

[0022] According to the experimental results, the toxicity of combined exposure to nanoplastics and tamoxifen on the nervous system was evaluated as the head swing frequency of Caenorhabditis elegans was less than 155.7 times / min.

[0023] Based on the experimental results, the toxicity of the combined exposure of nanoplastics and tamoxifen to the feeding ability was evaluated as the pharyngeal pump frequency of Caenorhabditis elegans was less than 182.1 times / min.

[0024] Based on the experimental results, the toxicity of combined exposure to nanoplastics and tamoxifen on reproductive capacity was evaluated as the number of eggs laid by Caenorhabditis elegans was less than 281.8 per egg.

[0025] According to the experimental results, the toxicity of the combined exposure of nanoplastics and tamoxifen to the growth and development of the Caenorhabditis elegans was evaluated to be less than 82% by area.

[0026] In the present invention, the body bending frequency of Caenorhabditis elegans is based on the central axis of the nematode's body, and is the number of times the nematode moves forward through a sinusoidal wavelength within 20 seconds.

[0027] In the present invention, the head swing frequency of the Caenorhabditis elegans refers to the number of times the nematode swings from one side to the other side and then back to the original position within 1 minute.

[0028] In the present invention, the pharyngeal pump frequency refers to the frequency at which the pharyngeal pump of the nematode beats within 1 minute.

[0029] In the present invention, the step of determining the number of eggs laid by the Caenorhabditis elegans comprises: inoculating the Caenorhabditis elegans mother worms after the exposure into the NGM culture dish, culturing, transferring the mother worms to the NGM culture dish every day during the egg-laying period of the mother worms, and counting the number of Caenorhabditis elegans in the old culture dish containing the eggs after culturing, and counting the number of Caenorhabditis elegans in all the culture dishes every day to obtain the number of offspring during the entire egg-laying period.

[0030] In the present invention, the area determination step of the Caenorhabditis elegans comprises: after the L1 stage Caenorhabditis elegans is exposed for 3 days, a portion of the nematodes is collected and washed, the nematodes are placed in a water bath at 60°C for 10 minutes, the nematodes are taken out and transferred to a solid culture medium, and the flat surface area of ​​the nematodes is measured using Image-ProH Express software. The calculation formula is:

[0031]

[0032] The evaluation of the present invention is specifically:

[0033] When there was no significant difference in the measured indicators between the combined exposure group and the single exposure group (P>0.05), nanoplastics had no effect on the toxic effects of tamoxifen;

[0034] When the measured indicators in the combined exposure group were significantly different from those in the single exposure group (P<0.05), nanoplastics had an impact on the toxic effects of tamoxifen.

[0035] When there was no significant difference in the measured indicators between the NAC supplementation group and the combined exposure group (P>0.05), N-acetylcysteine ​​had no effect on the toxic effects of nanoplastics and tamoxifen;

[0036] When the measured indicators in the NAC supplementation group were significantly different from those in the combined exposure group (P<0.05), N-acetylcysteine ​​had an effect on the toxic effects of nanoplastics and tamoxifen.

[0037] Compared with the prior art, the present invention has the following significant advantages:

[0038] 1. This paper provides an efficient and economical system and method for evaluating the combined toxicity of environmental toxicants and drugs. The research results of this method have good generalizability and can be easily applied to the study of other organisms and human disease models, thereby improving the wide applicability and practical value of the research.

[0039] 2. The present invention uses Caenorhabditis elegans as a model organism to evaluate the combined toxicity of nanoplastics and tamoxifen. Since Caenorhabditis elegans has a completely resolved genome and a clear genetic background, it provides a reliable experimental idea and technical platform for exploring the chronic exposure toxicity of environmental toxins and drugs.

[0040] 3. Through multi-dimensional toxicity-related index detection, the present invention can quickly and accurately evaluate the toxic effects of combined exposure to nanoplastics and tamoxifen on nematode movement, nervous system, development and reproduction under actual environmental conditions. These research results can provide a scientific basis for further analysis of human health risks and lay an important research foundation.

[0041] 4. The present invention innovatively discovered that dietary supplementation with N-acetylcysteine ​​(NAC) can significantly alleviate the toxic effects of combined exposure to nanoplastics and tamoxifen on the movement, nervous system, development and reproduction of Caenorhabditis elegans, revealing the potential role of NAC in anti-oxidation and toxicity mitigation. This research result not only provides new ideas for toxic response to combined exposure to environmental toxins and drugs, but also proposes a new application direction for NAC as a drug in toxicity intervention, providing a scientific basis for the development of new health protection measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 (A) Transmission electron microscope image and (BC) Field emission scanning electron microscope image of the PS-NPs polystyrene nanoplastic used in the present invention.

[0043] Figure 2 (A) Particle size distribution and (B) Zeta potential of PS-NPs polystyrene nanoplastics used in the present invention.

[0044] Figure 3 The effect of combined exposure of PS-NPs polystyrene nanoplastics used in the present invention and tamoxifen on the movement behavior of nematodes.

[0045] Figure 4 The effect of combined exposure of PS-NPs polystyrene nanoplastics used in the present invention and tamoxifen on the pharyngeal pumping rate of nematodes.

[0046] Figure 5 The effect of combined exposure to PS-NPs polystyrene nanoplastics used in the present invention and tamoxifen on the reproductive rate of nematodes.

[0047] Figure 6 The effect of combined exposure to PS-NPs polystyrene nanoplastics used in the present invention and tamoxifen on the development of nematodes.

[0048] Figure 7 is the structural formula of N-acetylcysteine ​​used in the present invention.

[0049] Figure 8 This is the toxic effect of N-acetylcysteine ​​supplementation on the body bending of nematodes under combined exposure to polystyrene nanoplastics and tamoxifen in the present invention.

[0050] Fig. 9 This is the toxic effect of supplementing N-acetylcysteine ​​in the present invention on the head swing of nematodes under combined exposure to polystyrene nanoplastics and tamoxifen.

[0051] Fig.10 This is the toxic effect of supplementing N-acetylcysteine ​​on the pharyngeal pumping rate of nematodes under combined exposure to polystyrene nanoplastics and tamoxifen in the present invention.

[0052] Fig.11 This is the toxic effect of supplementing N-acetylcysteine ​​on the development of nematodes under combined exposure to polystyrene nanoplastics and tamoxifen in the present invention.

[0053] Fig.12 This is the toxic effect of supplementing N-acetylcysteine ​​in the present invention on the oxidative stress of nematodes under combined exposure to polystyrene nanoplastics and tamoxifen. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0055] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0056] Example 1

[0057] Experimental Materials

[0058] 20 nm monodisperse polystyrene microspheres (PS-NPs) were purchased from Nanjing Genesis New Materials Co., Ltd. Tamoxifen citrate was purchased from Selleck (China)

[0059] 1M CaCl2: Weigh 11.10 g of anhydrous CaCl2, add deionized water to 100 mL, shake until the granules are dissolved, put it in an autoclave at 121°C for 20 min, wait for the solution to cool to room temperature, and store it in a 4°C refrigerator.

[0060] 1M MgSO4: Weigh 24.65 g MgSO4·7H2O, add deionized water to 100 mL, shake until the granules are dissolved, put into an autoclave at 121°C for 20 min, wait for the solution to cool to room temperature, and store in a 4°C refrigerator.

[0061] 1M KHPO4 buffer (pH = 6.0): weigh 10.83g KH2PO4, 4.66g K2HPO4·3H2O, add double distilled water to 100mL, shake until the particles are dissolved, put into an autoclave at 121℃ for 20min, wait for the solution to cool to room temperature, and store in a 4℃ refrigerator.

[0062] 5M cholesterol: weigh 0.5 g cholesterol and add 100 mL anhydrous ethanol. After fully dissolving, filter and sterilize, and store in a 4°C refrigerator.

[0063] NGM solid culture medium: weigh 3g sodium chloride (NaCl), 2.5g peptone and 17g agar respectively, add double distilled water to 1L; sterilize at 121℃ for 20min, wait until cooled to 55℃, add 1mL 1M CaCl2, 1mL 1M MgSO4, 1mL 5M cholesterol solution, 25mL 1M KHPO4 buffer respectively, mix well and pour into the culture dish.

[0064] LB liquid culture medium: Weigh 10 g of tryptone, 5 g of sodium chloride (NaCl), and 5 g of yeast extract, add double distilled water to 1 L, and sterilize at 121°C for 20 min.

[0065] Solution K: 1.4902 g sodium chloride, 1.1928 g potassium chloride, 0.4 g sodium acetate, dilute to 500 mL with ddH2O, sterilize with high temperature and high pressure, and store at room temperature.

[0066] Experimental steps:

[0067] Nanoplastic characterization: The initial concentration of 20nm PS-NPs aqueous solution was 5% w / v. The morphology, size and dispersion were characterized by TEM and SEM. PS-NPs were diluted with ultrapure water, properly ultrasonically mixed, then absorbed, dried and tested; the Zeta potential of water-diluted and LB-diluted PS-NPs was measured using a Malvern laser particle size analyzer.

[0068] Cultivation of Caenorhabditis elegans: Caenorhabditis elegans was cultured and propagated using nematode growth medium (NGM). A layer of OP50 (Escherichia coli strain OP50) colonies was spread on the surface of the culture medium and cultured in a constant temperature incubator at 20°C.

[0069] Synchronization of Caenorhabditis elegans: Rinse the NGM culture plate with egg-laying nematodes repeatedly with K-buffer, collect the egg-laying nematodes in an EP tube, wash the nematodes repeatedly with K solution, and discard the supernatant. Add the lysis solution to the EP tube, shake and lyse repeatedly to obtain enough eggs, and collect the nematodes that have hatched to the L1 stage with K solution after 18-22 hours of culture, centrifuge and discard the supernatant, add K solution, and inoculate into NGM culture medium for culture.

[0070] Cultivation of OP50: Two bottles of LB liquid culture medium sterilized with high temperature and high pressure, one bottle as blank control, add 100 μL of E. coli OP50 culture medium to the other bottle, and culture on a shaker at 37°C and 170 rpm for 4 hours until the culture medium becomes turbid. If the blank LB culture medium is clear, it can be placed at 4°C for use.

[0071] PS-NPs single exposure: The nanoplastic exposure concentration was divided into three dose groups: 0.1 μg / L, 1 μg / L and 10 μg / L. PS-NPs solutions of different concentrations were prepared with OP50 bacterial liquid and evenly spread on the NGM plate. L1-stage Caenorhabditis elegans were randomly placed in the exposure plate, and three replicates were set up and cultured in a constant temperature incubator at 20°C.

[0072] Tamoxifen single exposure: The exposure concentration of tamoxifen was 10 μM. Tamoxifen citrate (Selleckchem, S1972) was dissolved in sterile dimethyl sulfoxide (DMSO) and added to NGM agar heated to 55°C at a final volume of 0.1% to obtain an NGM plate containing tamoxifen at a final concentration of 10 μM. OP50 was evenly spread on the NGM plate, and L1-stage Caenorhabditis elegans was randomly placed in the exposure plate, and 3 replicates were set up and cultured in a constant temperature incubator at 20°C.

[0073] Combined exposure of PS-NPs and tamoxifen: The exposure concentration of nanoplastics was divided into three dose groups: 0.1 μg / L, 1 μg / L and 10 μg / L. PS-NPs solutions of different concentrations were prepared with OP50 bacterial liquid and evenly coated on NGM plates containing tamoxifen at a final concentration of 10 μM. L1-stage Caenorhabditis elegans were randomly placed in the exposure plate, and 3 replicates were set up and cultured in a constant temperature incubator at 20°C. Head thrashes test of Caenorhabditis elegans: The nematodes that have been treated were collected into EP tubes and rinsed with K solution, and then the supernatant was discarded. The clean nematodes were placed in a small dish containing K solution, and after standing for 1 minute, the head thrashes were counted and counted for 1 minute. The center of the body is the X-axis, and the Y-axis is perpendicular to the X-axis. The head thrashes of the nematode deviate from the X-axis and approach the Y-axis, and each completed round is recorded as a head thrashes.

[0074] Body bends test of C. elegans: Take a clean NGM plate, use a platinum wire pick to pick up the nematodes and place them on the clean NGM plate, wait for the nematodes to adapt for 1 minute, and then start counting for 20 seconds. A complete S-shaped curve of the nematode is considered a body bend.

[0075] Pharyngeal pumping rate detection of C. elegans: Collect the exposed nematodes, wash them with K solution, move the clean nematodes to an empty NGM plate, and use a high-power microscope to observe the beating rate of the pharyngeal pump of the nematodes within 1 minute.

[0076] Brood Size test of C. elegans: C. elegans mother worms after exposure were inoculated into NGM culture dishes and cultured. During the egg-laying period of the mother worms, the mother worms were transferred to NGM culture dishes every day. After the old culture dishes containing eggs were cultured, the number of C. elegans was counted. The number of C. elegans in all culture dishes every day was counted to obtain the number of offspring during the entire egg-laying period.

[0077] Body Size Detection of Caenorhabditis elegans: After the L1 stage Caenorhabditis elegans were exposed for 3 days, some nematodes were collected and washed, and the nematodes were placed in a water bath at 60°C for 10 minutes. The nematodes were taken out and transferred to solid culture medium, and the flat surface area of ​​the nematodes was measured using Image-ProH Express software. The calculation formula is:

[0078]

[0079] All data in the present invention are expressed as mean ± standard deviation (SD). Graphs were drawn using GraphPad Prism10. Data analysis was performed using Microsoft Excel software for statistical analysis, and variance analysis was used to compare the differences between the two groups of data. Lifespan analysis was performed using a two-tailed two-sample T-test. All P values ​​less than 0.05 were statistically significant, *P<0.05, **P<0.01.

[0080] result:

[0081] like Figure 1-2 As shown, scanning electron microscopy (SEM), transmission electron microscopy (TEM) images and particle size analysis show that the PS-NPs used in the experiment are smooth spheres with an average particle size of about 26nm. In PS-NPs (Water), the Zeta potential is -2.44mV, and in PS-NPs (LB), the Zeta potential of PS-NPs increases slightly to 0.11mV.

[0082] like Figure 3 As shown, when exposed to 10 μM Tamoxifen alone and 0.1-10 μg / LPS-NPs alone, the body was bent ( Figure 3 A), head bobbing ( Figure 3 B) There was no significant change compared with the control group. However, when 10 μM Tamoxifen was co-exposed with 0.1-10 μg / L PS-NPs, body bending and head shaking were significantly reduced, and the higher the concentration of PS-NPs, the greater the toxicity.

[0083] like Figure 4As shown in the figure, when exposed to 10 μM Tamoxifen and 0.1-10 μg / L PS-NPs, the pharyngeal pumping rate did not change significantly compared with the control group. However, when 10 μM Tamoxifen was co-exposed with 0.1-10 μg / L PS-NPs, the pharyngeal pumping rate was significantly reduced.

[0084] like Figure 5 As shown in the figure, when exposed to 10 μM Tamoxifen and 0.1-10 μg / LPS-NPs, the number of eggs laid did not change significantly compared with the control group. However, when 10 μM Tamoxifen was co-exposed with 0.1-10 μg / LPS-NPs, the number of eggs laid was significantly reduced.

[0085] like Figure 6 As shown in the figure, when exposed to 10 μM Tamoxifen and 0.1-10 μg / LPS-NPs, the area did not change significantly compared with the control group. However, when 10 μM Tamoxifen was co-exposed with 0.1-10 μg / LPS-NPs, the area was significantly reduced.

[0086] The above results revealed that exposure of Caenorhabditis elegans alone to predicted environmentally relevant concentrations of PS-NPs did not have adverse effects on nematodes, while exposure to PS-NPs (1-10 μg / L) increased the toxicity of tamoxifen-treated nematodes and adversely affected several toxic endpoints, including growth, brood size, and movement behavior. Therefore, the adverse effects of clinical drugs caused by widespread and persistent PS-NP contamination in the environment may be an underestimated ecological consequence.

[0087] Example 2

[0088] The effect of N-acetylcysteine ​​supplementation on nematode toxicity under combined exposure to polystyrene nanoplastics and tamoxifen was investigated.

[0089] The difference between Example 2 and Example 1 is that the Caenorhabditis elegans inoculated with PS-NPs and tamoxifen was supplemented with NAC from the L1 larval stage to the third day of adulthood, and after this supplementation period, the indicators were tested to evaluate the treatment effect. The other operations were the same as in Example 1.

[0090] Combined exposure of PS-NPs and tamoxifen: The nanoplastic exposure concentration was divided into three dose groups: 0.1 μg / L, 1 μg / L and 10 μg / L. PS-NPs solutions of different concentrations were prepared with OP50 bacterial liquid and evenly coated on NGM plates containing tamoxifen at a final concentration of 10 μM. L1-stage Caenorhabditis elegans were randomly placed on the exposure plates, and three replicates were set up and cultured in a constant temperature incubator at 20°C.

[0091] NAC supplementation: A 0.5 M N-acetylcysteine ​​stock solution was prepared in ddH2O and stored at -20 °C. NAC stock solution was diluted to a final concentration of 10 μM in NGM agar before pouring into plates. C. elegans exposed to tamoxifen, PS-NPs, and the mixture (tamoxifen + PS-NPs) were supplemented with NAC from the L1 larval stage to the third day of adulthood. After this supplementation period, indicators were tested to evaluate the treatment effect.

[0092] The evaluation treatment methods are: head thrashes test of C. elegans, body bends test of C. elegans, pharyngeal pumping rate test of C. elegans, and body size test of C. elegans. The detection methods are the same as those in Example 1.

[0093] Free radical (ROS) detection in Caenorhabditis elegans: After 3 days of exposure, some nematodes were collected and washed, and the nematodes to be tested were transferred to 1 mL of M9 buffer containing CM-H2DCFDA (10 μM), treated for 1 h in the dark, and then fixed on a 2% agar pad containing 10 mM levamisole. Fluorescence imaging was performed at 488 nm (excitation wavelength) / 510 nm (emission filter) under a laser confocal microscope (Leica, TCS SP2, Bensheim, Germany). Semi-quantification was performed using intestinal autofluorescence as a control to determine the relative fluorescence intensity of intestinal ROS signals.

[0094] All data in the present invention are expressed as mean ± standard deviation (SD). Graphs were drawn using GraphPad Prism10. Data analysis was performed using Microsoft Excel software for statistical analysis, and variance analysis was used to compare the differences between the two groups of data. Lifespan analysis was performed using a two-tailed two-sample T-test. All P values ​​less than 0.05 were statistically significant, *P<0.05, **P<0.01.

[0095] result:

[0096] like Figure 8 As shown, NAC supplementation did not affect the body curvature of the control nematodes. In contrast, NAC supplementation significantly alleviated the effect of PS-NPs exposure on the enhanced tamoxifen toxicity on the reduction of body curvature in nematodes.

[0097] like Fig. 9 As shown, NAC supplementation did not affect the head swing of control nematodes, while NAC supplementation significantly alleviated the effect of PS-NPs exposure on the enhanced tamoxifen toxicity on the reduction of head swing in nematodes.

[0098] like Fig.10As shown, NAC supplementation did not affect the pharyngeal pumping rate of the control nematodes, while NAC supplementation significantly alleviated the effect of PS-NPs exposure on the enhanced tamoxifen toxicity on the reduction of pharyngeal pumping rate in nematodes.

[0099] like Fig.11 As shown, NAC supplementation did not affect the development of the control group of nematodes, while NAC supplementation significantly alleviated the effect of PS-NPs exposure on the enhanced tamoxifen toxicity on the delayed development of nematodes.

[0100] like Fig.12 As shown, NAC supplementation did not stimulate the production of ROS in the control nematodes, while NAC supplementation significantly alleviated the effect of PS-NPs exposure on enhanced tamoxifen toxicity on ROS in nematodes, reflecting the protective effect of NAC supplementation on oxidative stress in Caenorhabditis elegans.

[0101] The above results revealed that exposure of C. elegans to predicted environmentally relevant concentrations of PS-NPs alone did not adversely affect the nematodes, while exposure to PS-NPs (1-10 μg / L) increased the toxicity of tamoxifen-treated nematodes and adversely affected several toxic endpoints, including growth, ROS production, and locomotor behavior. However, dietary supplementation with N-acetylcysteine ​​induced protective effects on nematode growth, ROS production, and locomotor behavior, indicating that N-acetylcysteine ​​can alleviate the toxic effects of PS-NP exposure-enhanced tamoxifen, suggesting a new pharmaceutical use for N-acetylcysteine.

[0102] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for evaluating the combined toxicity of nanoplastics and tamoxifen based on Caenorhabditis elegans, characterized in that: First, Caenorhabditis elegans was inoculated into NGM culture medium containing nanoplastics and tamoxifen, respectively, for single exposure experiments on nanoplastics and tamoxifen. Then, it was inoculated into NGM solid culture medium containing a mixture of nanoplastics and tamoxifen, for a combined exposure experiment on nanoplastics and tamoxifen. The development, motility, and fertility of Caenorhabditis elegans in single and combined exposure experiments were recorded to analyze the combined toxicity of nanoplastics and tamoxifen.

2. The method for evaluating the combined toxicity of nanoplastics and tamoxifen based on Caenorhabditis elegans according to claim 1, characterized in that: (1) Nanoplastics are polystyrene nanoplastics (PS-NPs) (2) Nanoplastic solutions of different concentrations were prepared with OP50 bacterial liquid and evenly coated on NGM plates. L1-stage Caenorhabditis elegans were placed on the NGM plates containing nanoplastics for exposure experiments and cultured in a constant temperature incubator at 20°C. The development, motility, and fertility of the Caenorhabditis elegans were recorded. (3) Tamoxifen was dissolved in a sterile solvent and added to NGM agar heated to 55°C at a final volume of 0.1% to obtain an NGM plate containing tamoxifen at a final concentration of 10 μM. OP50 was evenly spread on the NGM plate, and L1-stage Caenorhabditis elegans was placed on the NGM plate containing tamoxifen for exposure experiments. The plate was cultured in a constant temperature incubator at 20°C, and the development, motility, and fertility of the Caenorhabditis elegans were recorded. (4) Nanoplastic solutions of different concentrations were prepared using OP50 bacterial liquid and evenly coated on NGM plates containing tamoxifen at a final concentration of 10 μM. L1 stage Caenorhabditis elegans were inoculated into the solutions and cultured in a constant temperature incubator at 20°C. The development, motility, and fertility of the Caenorhabditis elegans were recorded.

3. The method for evaluating the combined toxicity of nanoplastics and tamoxifen based on Caenorhabditis elegans according to claim 1, characterized in that: The method for obtaining L1 Caenorhabditis elegans comprises: using a lysis solution to lyse Caenorhabditis elegans in the egg-laying stage to obtain sufficient eggs, collecting the nematodes hatched to the L1 stage with a K buffer solution after culturing for 18 to 22 hours, centrifuging and discarding the supernatant, adding a K buffer solution, and inoculating into an NGM culture medium for culturing.

4. The method for evaluating the combined toxicity of plastic and tamoxifen based on Caenorhabditis elegans according to claim 1, characterized in that: The NGM solid culture medium of the mixture of nanoplastics and tamoxifen contains 0.1-10 g / L of plastic solution and 10 μM of tamoxifen.

5. The method for evaluating the combined toxicity of plastic and tamoxifen based on Caenorhabditis elegans according to claim 1, characterized in that: The K buffer solution was prepared as follows: 1.4902 g NaCl, 1.1928 g KCl, fixed to 500 mL using ddH2O; the lysate solution was prepared as follows: NaClO solution: 1 mol / L NaOH: water volume ratio = 1:1:

3.

6. A method for using N-acetylcysteine ​​to resist drug toxicity caused by nanoplastics, characterized in that: The drug toxicity caused by nanoplastics includes the drug toxicity caused by the combination of the nanoplastics described in claim 1 and tamoxifen.

7. The method of using N-acetylcysteine ​​to resist drug toxicity caused by nanoplastics according to claim 6, characterized in that: N-acetylcysteine ​​supplementation at a concentration of 10 μM has a protective effect against the toxicity caused by nanoplastics in the concentration range of 0.1-10 ug / L.

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