Uncle Hu
Editor|Uncle Hu
preface
Cadmium is not only a trace element of great significance to human health, but also has important application prospects in metal smelting, plastic processing, coatings, batteries and other fields.
CD is the world's most important environmental pollutant, and as a non-essential element for human beings, it has been listed as the world's first CD pollutant.
In the Yangtze River and Southeast Coast PM10, the average concentration of Cd was 0.52mg/kg, which exceeded the limit value of GB15618-2018 "Agricultural Land Soil Pollution Risk Management and Control Standards (Trial)" (0.30mg/kg).
CD is difficult to biodegrade, and will continue to release wastewater and waste gas into the atmosphere, causing more and more ecological problems.
Investigations have shown that cadmium exposure will not only adversely affect the developmental function of the placenta, but also enter the fetus through the placenta, causing serious effects on the normal growth and cognitive ability of the fetus.
In a lower concentration of cadmium environment, it can cause changes in the intestinal flora of mice, accelerate the fat metabolism of the liver, and lead to lifelong metabolic disorders.
Long-term exposure to cadmium solution activates the metallothionein gene Mtn expression in corn borer larvae, and also inhibits the expression of antioxidant genes Cat and GPx.
During this process, the lipid peroxidation reaction in the fruit fly will also increase significantly, resulting in a decrease in its growth rate and survival rate.
Heavy metal pollution can make fruit flies shorter in body length, lighter in weight, and inhibit the expression of growth genes such as BAZ and TJ, and this effect will pass it on to the offspring without pollution.
Because it can be developed in vitro, the development rate is fast, and the embryo is transparent, so it is widely used in the environment.
Zebrafish embryos are sensitive to heavy metals, and in an environment of 3.00mg/L, 96h will cause delayed embryo hatching, small heads, reduced heart rate, missing body segments, and are insensitive to the external environment.
In addition, heavy metals can also inhibit the expression of genes related to Ca2+ regulation in embryos ATP2B1A, PTH1, STC1, etc., thereby affecting the development of otolith of embryos.
Materials and methods
Under the circulating water rearing system of the China Institute of Marine Biobiology, a wild zebrafish seed bank of class AB was introduced from the China International Zebrafish Fingerling Bank, with a pH value between 7±0.5.
The light-dark cycle is 14 hours/10 hours; Feeding twice a day in the morning and evening, the day before birth, males and females are placed in spawning boxes in a 2:1 ratio to collect fertilized eggs, while embryos are grown in ISO15088 prescribed water, divided according to the stage of embryonic development of zebrafish.
In this project, Beijing Jinweizhi Sequencing Co., Ltd. is planned to amplify and recover the above genes, compare them with T-type genes, screen and culture positive pathogenic bacteria, isolate the corresponding genes, and sequence determine and analyze the target genes.
According to the OECD236 standard method, 5 concentrations were set, and then the semi-lethal dose of cadmium was calculated.
Based on LC50 data, the concentration of CdCl2 in the exposure group was set to 7.50mg/L.
After the fetus was cultured at 28.5 °C in a biological incubator for 2~4h, the fetus with normal development was selected for exposure testing, and the exposure time was 4~48hpf.
Following the method established by the laboratory, the in situ hybridization test was carried out, that is, the gene of interest was recovered with restriction enzyme digestion, and the Gaoxin-labeled RNA probe was inserted into the Gaoxin-labeled RNA probe with SP6/T7 transcriptase synthesis, dissolved in 50 μL of hybridization solution, and stored in reserve.
A 48hpf control group and 60 Cd-infected zebrafish were used as the research subjects, total RNA was extracted with TRLzol, reverse transcribed into cDNA, repeated three times for three times, and fluorescent PCR was performed at 95°C for 2 minutes.
30 cycles at 95°C, 30 sec at 55°C, 1 min at 72°C; Hold at 72 °C for 5 min and 4 °C for 30 sec, the sequence of these primers is given in Table 1.
The LC50 and 95% confidence intervals of cadmium were calculated using IBMSPSS20 software, and then the expression differences of the target gene were analyzed using GraphPadPrism8 and One-wayANOVA software.
Results and discussion
Using the exposure test method, the semi-lethal concentration of cadmium exposure on zebrafish embryos of 120hpf can be calculated, and the toxic effect of zebrafish embryonic development can also be observed with stereo microscopy.
Based on the results of three repeated experiments, the dose-effect curve of cadmium was constructed, and Figure 1(a) could be seen, and when the embryonic mortality rate reached 50%, the corresponding cadmium concentration was 15.20mg/L.
Zebrafish embryos exposed to 20.00mg/L cadmium 4~48hpf will have deformities such as smaller head and eyes, shortened body length, curvature of the trunk, abnormal yolk sac and cardiac dysplasia.
Studies have shown significant differences in the Prdx1 hypothalamus, nasal cavity, pericardial area, and epithelial cells in a normal fetus, as well as in the jaw, nasal cavity, lateral line, and blood.
The prdx1 gene in the control group embryos was mainly expressed in the hypothalamus [see Figures 2 and 3(a)], and cadmium exposure led to abnormal expression of the PRDX1 gene in olfactory bulbs, lateral line organs, and epithelial cells (see Figures 2 and 3(a′)).
Under cadmium irradiation, the relative expression levels of PRDx1, GSTP 1.2, ATF3, JDP2B, HSP70L, EIF4A1B and FABP7A genes were significantly upregulated, and their levels increased by 2.31, 3.58, 2.08, 3.42, 5.27, 1.42 and 2.06 times, respectively (see Figure 4).
The HSP70L gene is mainly involved in stress and response to metal ions, the GSTP1.2 and PRDx1 genes are involved in antioxidant, and the ATF3 and JDP2b genes are DNA-binding transcription factors.
From the apparent upregulation of these genes, cadmium has an impact on processes such as oxidative stress, transcriptional translation, and early neurodevelopment in zebrafish embryos.
It has been reported that cadmium mainly replaces metal ions with Fenton reaction, thereby causing oxidative stress, and causing oxidative stress and calcium salt transport in the body, which adversely affects the normal physiological function of proteins.
The applicant conducted a preliminary analysis of 8 genes in the early stage, found that 5 genes had abnormal expression on the olfactory bulb of zebrafish larvae after cadmium treatment, and preliminary analysis confirmed that the expression of 4 genes in zebrafish larvae increased significantly after cadmium treatment.
In 5 of these 5 genes, zebrafish larvae showed abnormal expression on the olfactory bulb after cadmium treatment, and played a key role in cell metabolism, detoxification and anti-cancer.
The experimental results showed that Cd could inhibit the endothelial growth of the eye and the proliferation of retinal neurons.
Cadmium affects the development of zebrafish embryonic eyes by blocking the formation of neural crests, inhibiting the differentiation of retinal neurons, and destroying the axonal formation of retinal ganglion cells.
Based on the previous work, this project intends to further explore the influence of Cd on the crystal structure by taking eif4a1b and hsp90aa11 in zebrafish lenses treated with Cd as the starting point.
FABP7a has high levels of expression in the center and retina, regulating eye development.
Therefore, Cd treatment can cause significant increases in FabP7a protein levels, but it is unclear whether Cd treatment can cause changes in CNS and RNA-seq.
conclusion
After 20mg/LCd infection, zebrafish fetus developed malformation, small head, small eyes, trunk curvature, pericardial edema, yolk sac malformation, and it was determined that the LC50 content was 15.20mg/L after 120h of infection.
7.50mg/L cadmium induced significant increases in the expression of PRDx1, GSTP1.2, ATF3, JDP2B, HSP70L, EIF4A1B and FABP7A genes, indicating that cadmium may affect oxidative stress, transcriptional translation and early neurodevelopment in zebrafish embryos.
In nerve tissue, retina, lens, olfactory bulb, lateral line, heart, skin and other tissues and organs, 7.50mg/LCd can cause abnormal expression of this gene, which indicates that Cd has a certain effect on the expression area of this gene.
These all have an impact on a variety of tissues and organs of zebrafish, resulting in disorders in their sense of smell, vision, movement, etc.
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