CN-121971464-A - Reproductive development toxicity evaluation method based on combined contamination of greenhouse pesticides in plastic greenhouse

Abstract

The invention relates to the field of environmental toxicology and developmental biology, in particular to a reproductive developmental toxicity evaluation method based on combined contamination of greenhouse pesticides in a plastic greenhouse. By combining in-vivo model and in-vitro cell verification, starting from hormone level, ovarian tissue structure, placenta function, offspring development, cell activity and mitochondrial state multidimensional degree, the system evaluates the combined action toxicity of the beta-cypermethrin and emamectin benzoate, reveals the potential hazard mechanism of the beta-cypermethrin in the reproductive development process, and provides scientific basis for pesticide toxicology risk evaluation and safety limit formulation.

Inventors

• ZHAI QINGFENG
• Bai Menxin
• MENG JIAN

Assignees

• 山东第二医科大学

Dates

Publication Date

20260505

Application Date

20260205

Claims (10)

1. 1. The reproductive development toxicity evaluation method based on the combined contamination of the greenhouse pesticides in the plastic greenhouse is characterized by comprising the following steps of: Exposing animals to a combined environment containing different concentrations of beta-cypermethrin and emamectin benzoate to obtain an experimental group, and taking normal animals as a control group; Randomly dividing the animal model into 2 groups, namely a body level group and a cell level group; Detecting estrogen, endocrine hormone and ovary morphology of a part of female animals in the body level group and comparing the detected estrogen, endocrine hormone and ovary morphology with a part of female animals in the control group, mating the other part of female animals in the body level group with adult male animals, selecting a part of female animals to detect placenta tissue injury degree, embryotoxicity, placenta oxidative damage, estrogen and progesterone synthesis during pregnancy and comparing the detected placenta tissue injury degree, embryotoxicity, placenta oxidative damage, estrogen and progesterone synthesis with the control group, naturally delivering the other part of female animals, recording newborn survival rate, body mass and body surface deformity, and comparing the obtained result with the control group; The cell level group is treated by female animal serum, and the survival rate, invasion capacity, progesterone content and progesterone key enzyme expression, mitochondrial injury degree and activation condition of a mitochondrial autophagy related pathway of HTR8/SVneo cells are judged and compared with a control group; And according to the comparison result, obtaining the toxicity concentration of the combined beta-cypermethrin and emamectin benzoate.
2. 2. The method for evaluating reproductive development toxicity based on combined exposure to pesticides in plastic greenhouses according to claim 1, wherein the toxic concentration of beta-cypermethrin is not less than 55mg/cm 2 , and the toxic concentration of emamectin benzoate is not less than 7.8mg/cm 2 .
3. 3. The method for evaluating reproductive developmental toxicity based on combined exposure to greenhouse pesticides of claim 1, wherein the degree of placenta tissue damage refers to the thickness and mitochondrial activity of placenta tight junction region, labyrinthine region.
4. 4. The method for evaluating reproductive developmental toxicity based on combined exposure to pesticides in a greenhouse of a plastic house according to claim 1, wherein the oxidative damage of placenta is the activity of hydrogenase, atpase and lactate dehydrogenase; The estrogen is estradiol.
5. 5. The method for evaluating reproductive developmental toxicity based on combined exposure to pesticides in greenhouses according to claim 4, wherein mitochondrial activity refers to ATP content, TOMM20, PGC-la, LC3, PINK1, parkin.
6. 6. The evaluation method for reproductive developmental toxicity based on combined exposure to pesticides in greenhouses according to claim 5, wherein the key enzymes secreted by progesterone are CYP11A1, HSD3B1, pgR.
7. 7. The reproductive developmental toxicity evaluation method based on combined exposure to pesticides in plastic greenhouses according to claim 6, wherein each evaluation index in the body level group is determined as follows: The content of the estradiol is obviously lower than that of a control group; The expression quantity of CYP11A1, HSD3B1 and PgR synthesized by the progesterone is obviously lower than that of a control group; The placenta tight junction area is thicker than the control group, and the labyrinth area is thinner than the control group; The weight of the fetus and the newborn is lower than that of the control group, and the occurrence rate of the intrauterine growth restriction of the fetus is higher than that of the control group; the activity of catalase and the content of the apyrase in the placenta are obviously lower than those of a control group, and the activity of lactic dehydrogenase is obviously higher than that of the control group; The relative expression quantity of mRNA and protein of TOMM20 in placenta is obviously lower than that of a control group, and the relative expression quantity of mRNA of LC3 and PINK1 and protein of PGC-1a, PINK1 and Parkin is obviously higher than that of the control group.
8. 8. The method for evaluating reproductive developmental toxicity based on combined exposure to pesticides in plastic greenhouses according to claim 6, wherein each evaluation index in the cell level group is determined as follows: HTR8/SVneo cell viability was significantly lower than control; group HTR8/SVneo had significantly lower cell invasiveness than the control group; The progesterone content in the cell culture solution and the expression level of progesterone key enzymes CYP11A1, HSD3B1 and PgR are all lower than those of a control group; the active oxygen expression of the mitochondrial injury degree is obviously higher than that of a control group, and the ATP content and mitochondrial membrane potential are obviously lower than those of the control group; The relative expression quantity of mRNA of TOMM20 in the activation condition of the mitochondrial autophagy related pathway is lower than that of a control group, the relative expression quantity of mRNA of LC3, PINK1 and Parkin is higher than that of the control group, the relative expression quantity of TOMM20 protein is lower than that of the control group, the relative expression quantity of LC3 protein I/I is higher than that of the control group, and the relative expression quantity of PINK1 and Parkin proteins is higher than that of the control group.
9. 9. The method for evaluating reproductive developmental toxicity based on combined exposure to pesticides in greenhouses according to claim 1 wherein the number ratio of female animals to adult male animals is 2:1.
10. 10. The reproductive developmental toxicity evaluation method based on combined exposure of greenhouse pesticides in plastic greenhouses according to claim 2, wherein the exposure process is specifically as follows: Exposing adult females to a combination of different concentrations of beta-cypermethrin and emamectin benzoate for 1 time per day for 2 hours for 90 days; the temperature in the environment is 27-29 ℃, and the humidity is 72-82%.

Description

Reproductive development toxicity evaluation method based on combined contamination of greenhouse pesticides in plastic greenhouse Technical Field The invention relates to the field of environmental toxicology and developmental biology, in particular to a reproductive developmental toxicity evaluation method based on combined contamination of greenhouse pesticides in a plastic greenhouse. Background The pesticide plays an important role in the production process of the greenhouse. The greenhouse staff uses high-toxicity and high-residue pesticides to prevent diseases and insect pests, and the pesticide effect is increased by arbitrarily mixing the pesticides, wherein the high-efficiency cypermethrin and the emamectin benzoate are the common pesticide mixing mode for the greenhouse staff, so that the influence of the high-efficiency cypermethrin and the emamectin benzoate on the health and the environment is more and more paid attention to scientists. Beta-CYP, which can influence the secretion of reproductive hormone through hypothalamus-pituitary-ovary axis to further interfere the estrus cycle, obstruct the ovulation process and influence fertility of female mammals. Emamectin benzoate, which is called EMB for short, is a novel high-efficiency antibiotic pesticide synthesized by abamectin. Avermectin is used as a sixteen-membered macrolide compound and has an inhibiting effect on the reproductive endocrine function of rats. However, the related indexes of adverse effects of β -CYP and EMB on reproduction and development are generally determined based on a single index, such as embryo malformation and mortality or sperm malformation rate, however, the determination of the single index cannot accurately determine the adverse effects of β -CYP and EMB on reproduction and evaluation of toxicity concentration of β -CYP and EMB in the case of combination. So that sometimes the concentration of beta-CYP and EMB has had an adverse effect on reproduction and development, but single index results could not be detected. Disclosure of Invention In order to solve the technical problems, the invention provides a reproductive development toxicity evaluation method based on combined contamination of greenhouse pesticides in a plastic greenhouse. The invention aims to construct a biological feeling model combined with the cell level in vivo, define the developmental toxicity effect of the combined exposure of the high-efficiency cypermethrin and the emamectin benzoate, and detect the developmental toxicity effect from a multi-dimensional index, thereby realizing the accurate judgment of the toxicity of the combined situation of the high-efficiency cypermethrin and the emamectin benzoate. The technical scheme of the invention is as follows. The reproductive development toxicity evaluation method based on the combined contamination of the greenhouse pesticides in the plastic greenhouse comprises the following steps: Exposing animals to a combined environment containing different concentrations of beta-cypermethrin and emamectin benzoate to obtain an experimental group, and taking normal animals as a control group; Randomly dividing the animal model into 2 groups, namely a body level group and a cell level group; Detecting estrogen, endocrine hormone and ovary morphology of a part of female animals in the body level group and comparing the detected estrogen, endocrine hormone and ovary morphology with a part of female animals in the control group, mating the other part of female animals in the body level group with adult male animals, selecting a part of female animals to detect placenta tissue injury degree, embryotoxicity detection, placenta oxidative damage, estrogen and progesterone synthesis during pregnancy and comparing the detected placenta tissue injury degree, embryotoxicity detection, placenta oxidative damage, estrogen and progesterone synthesis with the control group, naturally delivering the other part of female animals, recording newborn survival rate, body mass and body surface deformity, and comparing the newborn survival rate, the body mass and body surface deformity with the control group; The cell level group is treated by female animal serum, and the survival rate, invasion capacity, progesterone content and progesterone key enzyme expression, mitochondrial injury degree and activation condition of a mitochondrial autophagy related pathway of HTR8/SVneo cells are judged and compared with a control group; And according to the comparison result, obtaining the toxicity concentration of the combined beta-cypermethrin and emamectin benzoate. In another preferred embodiment, the toxic concentration of beta-cypermethrin is equal to or greater than 55mg/cm 2 and the toxic concentration of emamectin benzoate is equal to or greater than 7.8mg/cm 2. In another preferred embodiment, the degree of placental tissue damage refers to the thickness of the tight junction region, the labyrinthine region, and mitochondrial