CN113777293A - Method for detecting and evaluating sulfamethoxazole environmental risk by using paramecium biomarker and IBR (ion beam receptor) - Google Patents
Method for detecting and evaluating sulfamethoxazole environmental risk by using paramecium biomarker and IBR (ion beam receptor) Download PDFInfo
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Abstract
The invention belongs to the technical field of biological monitoring and evaluation of antibiotic environmental pollution, and particularly relates to a method for detecting and evaluating the environmental risk of sulfamethoxazole by using paramecium biomarkers and IBR (antibiotic resistance receptor), which is used for quickly evaluating the environmental concentration and the environmental risk of sulfamethoxazole by using a plurality of biomarkers and IBR integrated analysis of sensitive indicator organisms.
Description
Technical Field
The invention belongs to the technical field of biological monitoring and evaluation of antibiotic environmental pollution, and particularly relates to a method for evaluating the environmental risk of sulfamethoxazole by using paramecium biomarker and IBR (antibiotic resistance receptor).
Background
The integrated analysis using tested species, detection concentration range, detection parameters and whether IBR is used in the sulfamethoxazole biological detection method reported in the prior art is as follows:
for example, CN201110271631.3 is a method for evaluating soil environment safety of transgenic pest-resistant rice, which comprises culturing paramecium via soaking solution of stalk of transgenic pest-resistant rice, and determining whether transgenic rice affects growth, reproduction, etc. of paramecium via statistics of cell density of cultured paramecium; then, detecting the damage of paramecium DNA by utilizing a comet electrophoresis technology, and determining the damage condition of the paramecium DNA of the anti-pest transgenic Bt rice by counting and analyzing comet cell numbers of the experimental group, the negative control group and the positive control group; thereby judging whether the transgenic insect-resistant rice is safe to the soil environment.
CN202110224110.6 discloses a novel method for detecting environmental toxic substances by combining biological methods, which comprises the following steps: collecting an environmental sample specimen to be detected, and judging the type and content of toxic substances in the environment of the specimen to be detected according to three test results by respectively performing a seed germination test, a snail killing test and an aquatic micro-animal community killing test. Trigonella foenum-graecum and Chinese little greens seeds of two families of plant kingdom are combined with animals of three different phyla of animal kingdom, namely, the leptospira of terrestrial Olachnaceae, the rotifer of aquatic rotifer, the paramecium of ciliate and the water-bear worm of bradycardia. The method is complicated in evaluation method and increases the difficulty of analysis.
The main defects of the prior art include:
(1) the species for carrying out the sulfamethoxazole biological detection in the past are not sensitive enough or have higher cost, long period and low standardization degree;
(2) the used biomarkers are few in types, more in nature and insufficient in quantification;
(3) the detection concentration range and the actual environmental concentration have weak pertinence and low practicability;
(3) lack of IBR analysis-by-synthesis evaluation.
The IBR index method is widely applied to marine environment quality evaluation. The method has low requirements on the types and the quantity of the screened biomarkers, can be used for carrying out 'causal effect' analysis by connecting biological response with pollutants, and is widely applied.
The IBRv2 index method avoids the deviation of calculation results caused by different marker sequences when a first generation IBR index method draws a star chart, and can reflect the induction and inhibition effects of various biomarkers under different pollution conditions.
However, for the evaluation of the environmental concentration and the environmental risk of a specific substance, a large amount of research is needed to establish the evaluation, and how to quickly and accurately evaluate the environmental concentration and the environmental risk of sulfamethoxazole is the problem to be solved by the invention.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a method for evaluating the environment risk of sulfamethoxazole by using paramecium biomarker and IBR detection, and the environment concentration and the environment risk of sulfamethoxazole are quickly evaluated by using a plurality of biomarkers of sensitive indicator organisms and IBR integrated analysis.
The invention is realized by the following technical scheme:
a method for evaluating the environmental risk of Sulfamethoxazole (SMX) by using paramecium biomarker and IBR detection, comprising the following steps:
(1) culturing paramecium, selecting single cells for culturing, and taking individuals in logarithmic growth phase for sulfamethoxazole detection;
(2) selecting sulfamethoxazole solutions with different concentrations as detection samples;
(3) taking insect liquid with a certain density, after a sample to be detected is processed, washing cells by PBS, cracking the cells by cell lysate, centrifuging, collecting supernate, and detecting by biomarkers respectively;
(4) integrated biomarker analysis (IBR), calculated as follows:
firstly, standardization: all biomarker data were normalized to Y ═ log (X/X)0). Wherein X is the average of the results of each biomarker measured at each locus; x0Biomarker values for control sites;
uniformization: normalizing the normalization value of each marker at each position to be Z ═ (Y-M)/S, wherein Y is the normalization value of each biomarker; m is the total average value at all sites; s is the standard deviation of the normalized value of each biomarker at all the positions;
and 3, assignment: calculating the deviation index A ═ Z-Z of each biomarker at each site0Wherein Z is each marker in eachA normalized value of the station; z0Is the normalized value of the marker at the control site;
calculating IBRv 2: the sum of the absolute values of the deviation indices | a | of all markers at each station is first calculated and then divided by the number of biomarkers n. The calculation formula is as follows:
as a preferred embodiment of the present invention, the step (1) includes: the method can be used for collecting fresh water with rich plankton, selecting single cells, wheat grain culture solution, establishing a monoclonal culture system in a constant-temperature constant-humidity incubator at 20-25 ℃, performing starvation induced joint reproduction, performing large-scale pure culture, and taking individuals in logarithmic phase of growth for sulfamethoxazole detection.
As a preferred embodiment of the present invention, the step (2) includes: sulfamethoxazole concentrations range from 10ng to 1.7. mu.g/L, with 6 concentrations tested, 10ng, 100ng, 1. mu.g, 1.2. mu.g and 1.5. mu.g, 1.7. mu.g, set in this range.
As a preferred embodiment of the present invention, the step (3) includes: the insect solution with the density of 1000ind/ml is taken, after a sample to be detected is processed for 24 hours, cells are washed by PBS, the cells are lysed by cell lysate (20mM Tris, 150mM NaCl, 1% Triton X-100, 1mM EDTA), the cells are centrifuged for 10min at the temperature of 4 ℃ and the rpm, and the supernatant is collected.
As a preferred embodiment of the present invention, the step (3) includes: the kit is selected from SOD kit, CAT kit, MDA kit, GSH-PX kit, PGK kit, ATPase kit, CYP450 kit and RP kit, and is used for detecting biomarkers.
As a preferable technical scheme of the invention, the step (4) comprises the following steps: when A is larger than 0, the marker is induced, otherwise, the marker is inhibited.
As a preferable technical scheme of the invention, the step (4) comprises the following steps: the higher the IBRv2 value, the higher the sulfamethoxazole contamination level, the greater the biological contamination pressure effect.
As a preferable aspect of the present invention, the evaluation method includes:
a significant increase in SOD + ATPase and a significant decrease in PGK + MDA indicates that 10ng/L SMX is present;
significant elevation of CYP450 alone is indicative of SMX reaching 100 ng/L;
a significant increase in CAT and a significant decrease in MDA indicates that SMX is close to 1 μ g/L;
co-elevation of SOD + CYP450 indicates that SMX may exceed 1.2 μ g/L;
an increase in SOD alone in combination with a decrease in ATPase is indicative of an SMX of more than 1.5. mu.g/L;
a co-increase in RP and CAT indicates that SMX has reached 1.7. mu.g/L.
As a preferred embodiment of the present invention,
a significant increase in SOD + ATPase (Ai ═ 1.08, 0.53) and a significant decrease in PGK + MDA (Ai ═ 2.74, -2.39) indicated the presence of 10ng/L SMX;
a significant increase in CYP450 alone (Ai ═ 1.56) indicates an indication that SMX reached 100 ng/L;
a significant increase in CAT (Ai ═ 2.3) and a significant decrease in MDA means (Ai ═ 1.9) indicates that SMX is close to 1 μ g/L;
co-elevation of SOD + CYP450 means (Ai ═ 1.67, 1.07) indicates SMX exceeds 1.2 μ g/L;
an indication that increased SOD alone (Ai ═ 2.65) coupled with decreased ATPase (Ai ═ 1.54) indicates SMX exceeds 1.5 μ g/L;
a co-increase in RP and CAT (Ai ═ 1, 1.8) indicates that SMX has reached 1.7 μ g/L.
The beneficial effects of the invention compared with the prior art comprise:
1) a new method for detecting and evaluating the environment concentration of sulfamethoxazole by using the multiple biomarkers integrated analysis (IBR) of single-cell organisms is established;
2) the method comprises indicating standardized culture of organisms and detection and IBR calculation of 8 biomarkers;
3) the biomarker combination evaluation parameters of 6 concentrations of sulfamethoxazole are provided.
Drawings
FIG. 1 response of paramecium bifidus biomarkers to sulfamethoxazole
FIG. 2 analysis of correlation between biomarkers and sulfamethoxazole concentration
FIG. 3 analysis of 8 biomarkers IBR of paramecium caudatum
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, but the embodiments of the present invention are not limited thereto.
Example 1
1. Materials and methods
1.1 indicating organisms
Paramecium (Parameci μm ca μ dat μm), ciliate phylum, Oligomorpha, order Farinales, Paralithospermaceae, Paralithospermum. The fertilizer is widely distributed in still water with more organic matters. Is sensitive to the stimulation of environmental pollutants, and is often used for evaluation and research of the environmental pollutants.
1.2 cultivation of paramecium
The method can be used for collecting fresh water with rich plankton, selecting single cells, wheat grain culture solution, establishing a monoclonal culture system in a constant-temperature constant-humidity incubator at 20-25 ℃, performing starvation induced joint reproduction, performing large-scale pure culture, and taking individuals in logarithmic phase of growth for sulfamethoxazole detection.
1.3 setting of Sulfamethoxazole detection concentration Range
According to various literature reports and environmental monitoring reports, the sulfamethoxazole experimental concentration which can be detected in water and soil environments and organisms is comprehensively summarized and screened, the concentration range is determined to be 10 ng-1.7 mug/L, and 6 detection concentrations of 10ng, 100ng, 1 mug, 1.2 mug, 1.5 mug and 1.7 mug are set according to actual detection requirements in the range.
1.4 multiple biomarker assays
Taking insect solution with the density of 1000ind/ml, processing a sample to be detected for 24 hours, washing cells by PBS, cracking the cells by cell lysis solution (20mM Tris, 150mM NaCl, 1% Triton X-100, 1mM EDTA), centrifuging at 4 ℃ and 8000rpm for 10min, collecting supernatant, and detecting biomarkers by using an SOD kit, a CAT kit, an MDA kit, a GSH-PX kit, a PGK kit, an ATPase kit, a CYP450 kit and an RP kit respectively.
1.5 Integrated biomarker analysis (IBR)
The calculation steps are as follows:
firstly, standardization: all biomarker data were normalized to Y ═ log (X/X)0). Wherein X is the average of the results of each biomarker measured at each locus; x0Biomarker values for control sites.
Uniformization: the normalized value of each marker at each station was Z ═ Y-M)/S. Wherein Y is the normalization value of each biomarker; m is the total average value at all sites; s is the standard deviation of the normalized value for each biomarker at all positions.
And 3, assignment: calculating the deviation index A ═ Z-Z of each biomarker at each site0. Wherein Z is the normalized value of each marker at each site; z0Normalized to the marker at the control site. When A is larger than 0, the marker is induced, otherwise, the marker is inhibited.
Calculating IBRv 2: the sum of the absolute values of the deviation indices | a | of all markers at each station is first calculated and then divided by the number of biomarkers n. The calculation formula is as follows:
the higher the IBRv2 value, the higher the sulfamethoxazole contamination level, the greater the biological contamination pressure effect.
2. Results
2.1 response of Single biomarker to Sulfamethoxazole
Sulfamethoxazole treatment for 24 hours caused significant changes in multiple biomarkers in paramecium cells (fig. 1).
Wherein SOD enzyme activity (FIG. 1-A) was significantly increased under SMX stress at concentrations of 10ng, 100ng, 1.2. mu.g and 1.5. mu.g;
MDA levels (FIG. 1-B) were significantly reduced at 10ng, 1. mu.g and 1.7. mu.g concentrations;
CAT enzyme activity (FIG. 1-C), was significantly elevated at 100ng, 1. mu.g, 1.5. mu.g and 1.7. mu.g concentrations;
GSH-PX enzyme activity (fig. 1-D) was significantly higher than the control at SMX treatment concentrations of 1 μ g and 1.5 μ g, and significantly lower than the control at treatments of 10ng, 1.2 μ g and 1.7 μ g, with no significant change in the remaining concentrations;
the enzymatic activity of PGK (FIG. 1-E) was significantly reduced by SMX treatment at a concentration of 10ng, 1. mu.g, 1.2. mu.g, 1.7. mu.g;
ATPase activity (FIG. 1-F), significantly higher than control at concentrations of 10ng, 1. mu.g and 1.2. mu.g SMX, significantly lower than control at concentrations of 100ng, 1.5. mu.g and 1.7. mu.g SMX;
cellular RP content (fig. 1-G) was significantly increased with treatments at 1.5 μ G and 1.7 μ G, and significantly decreased under SMX stress except at 100ng concentration;
CYP450 activity (FIG. 1-H) was significantly elevated at SMX100ng and 1.2. mu.g concentrations, and significantly reduced at 10ng and 1.7. mu.g concentrations.
The results show that the reaction of any single biomarker to the exposure of sulfamethoxazole is not stable and uniform enough, and a comprehensive evaluation system of multiple biomarkers needs to be established.
2.2 biomarker correlation under Sulfamethoxazole stress at different concentrations
In order to construct a multi-clock biomarker comprehensive evaluation system, the correlation analysis of the biomarkers at different concentrations is analyzed (fig. 2), and the results show that: under the stress of sulfamethoxazole with different concentrations, 8 biomarkers have cross positive correlation, wherein 4 biomarkers, namely SOD, MDA, CYP450 and PGK, are most correlated with other markers.
Suggesting that oxidative stress and the tricarboxylic acid cycle may be the main biological mechanism of paramecium caudatum against sulfamethoxazole stress. Under 24hSMX stress, the response of these 4 markers can be shown to be stable as evidence of the presence of SMX contamination, especially with a significant increase in SOD and a significant decrease in MDA. However, the analysis of the correlation with concentration showed that only the increase in SOD was significantly correlated with the concentration.
Thus, the reaction of these markers is only qualitatively indicative of the presence of SMX contamination, but not quantitatively indicative of the extent of SMX contamination.
2.3 multiple biomarker integration assay (IBR)
The response of 8 paramecium biomarkers to 24h stress of sulfamethoxazole with 6 concentrations was analyzed by IBR integration, and the result is shown in FIG. 3.
A significant increase in SOD + ATPase and a significant decrease in PGK + MDA indicates that 10ng/L SMX is present;
significant elevation of CYP450 alone is indicative of SMX reaching 100 ng/L;
a significant increase in CAT and a significant decrease in MDA indicates that SMX is close to 1 μ g/L;
co-elevation of SOD + CYP450 indicates that SMX may exceed 1.2 μ g/L;
an increase in SOD alone in combination with a decrease in ATPase is indicative of an SMX of more than 1.5. mu.g/L;
whereas a co-increase in RP and CAT indicates that SMX has reached 1.7. mu.g/L.
Table 2: biomarker combination representative values
Comparative example
Selection of other species: the evaluation of zebra fish embryos, tubifex, zebra fish and zebra fish larvae is carried out according to the method, and the experimental results are shown in the following table 3, which is not as good as the effect of using paramecium.
Table 3: sulfamethoxazole marker for detecting other species and concentration range thereof
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. The method for detecting and evaluating the environmental risk of sulfamethoxazole by using the paramecium biomarker and IBR is characterized by comprising the following steps:
(1) culturing paramecium, selecting single cells for culturing, and taking individuals in logarithmic growth phase for sulfamethoxazole detection;
(2) selecting sulfamethoxazole solutions with different concentrations as detection samples;
(3) taking insect liquid with a certain density, after a sample to be detected is processed, washing cells by PBS, cracking the cells by cell lysate, centrifuging, collecting supernate, and detecting by biomarkers respectively;
(4) integrated biomarker analysis (IBR), calculated as follows:
firstly, standardization: all biomarker data were normalized to Y ═ log (X/X)0). Wherein X is the average of the results of each biomarker measured at each locus; x0Biomarker values for control sites;
uniformization: normalizing the normalization value of each marker at each position to be Z ═ (Y-M)/S, wherein Y is the normalization value of each biomarker; m is the total average value at all sites; s is the standard deviation of the normalized value of each biomarker at all the positions;
and 3, assignment: calculating the deviation index A ═ Z-Z of each biomarker at each site0Wherein Z is the normalized value of each marker at each station; z0Is the normalized value of the marker at the control site;
calculating IBRv 2: the sum of the absolute values of the deviation indices | a | of all markers at each station is first calculated and then divided by the number of biomarkers n. The calculation formula is as follows:
2. the method of claim 1, wherein step (1) comprises: the method can be used for collecting fresh water with rich plankton, selecting single cells, wheat grain culture solution, establishing a monoclonal culture system in a constant-temperature constant-humidity incubator at 20-25 ℃, performing starvation induced joint reproduction, performing large-scale pure culture, and taking individuals in logarithmic phase of growth for sulfamethoxazole detection.
3. The method of claim 1, wherein step (2) comprises: sulfamethoxazole concentrations range from 10ng to 1.7. mu.g/L, with 6 concentrations tested, 10ng, 100ng, 1. mu.g, 1.2. mu.g and 1.5. mu.g, 1.7. mu.g, set in this range.
4. The method of claim 1, wherein step (3) comprises: the insect solution with the density of 1000ind/ml is taken, after a sample to be detected is processed for 24 hours, cells are washed by PBS, the cells are lysed by cell lysate (20mM Tris, 150mM NaCl, 1% Triton X-100, 1mM EDTA), the cells are centrifuged for 10min at the temperature of 4 ℃ and the rpm, and the supernatant is collected.
5. The method of claim 1, wherein step (3) comprises: the kit is selected from SOD kit, CAT kit, MDA kit, GSH-PX kit, PGK kit, ATPase kit, CYP450 kit and RP kit, and is used for detecting biomarkers.
6. The method of claim 1, wherein step (4) comprises: when A is larger than 0, the marker is induced, otherwise, the marker is inhibited.
7. The method of claim 1, wherein step (4) comprises: the higher the IBRv2 value, the higher the sulfamethoxazole contamination level, the greater the biological contamination pressure effect.
8. The method according to claim 1, wherein the evaluation method comprises:
a significant increase in SOD + ATPase and a significant decrease in PGK + MDA indicates that 10ng/L SMX is present;
significant elevation of CYP450 alone is indicative of SMX reaching 100 ng/L;
a significant increase in CAT and a significant decrease in MDA indicates that SMX is close to 1 μ g/L;
co-elevation of SOD + CYP450 indicates that SMX may exceed 1.2 μ g/L;
an increase in SOD alone in combination with a decrease in ATPase is indicative of an SMX of more than 1.5. mu.g/L;
a co-increase in RP and CAT indicates that SMX has reached 1.7. mu.g/L.
9. The method according to claim 8, wherein a significant increase in SOD + ATPase (Ai ═ 1.08, 0.53) and a significant decrease in PGK + MDA (Ai ═ 2.74, -2.39) indicate the presence of 10ng/L SMX;
a significant increase in CYP450 alone (Ai ═ 1.56) indicates an indication that SMX reached 100 ng/L;
a significant increase in CAT (Ai ═ 2.3) and a significant decrease in MDA means (Ai ═ 1.9) indicates that SMX is close to 1 μ g/L;
co-elevation of SOD + CYP450 means (Ai ═ 1.67, 1.07) indicates SMX exceeds 1.2 μ g/L;
an indication that increased SOD alone (Ai ═ 2.65) coupled with decreased ATPase (Ai ═ 1.54) indicates SMX exceeds 1.5 μ g/L;
a co-increase in RP and CAT (Ai ═ 1, 1.8) indicates that SMX has reached 1.7 μ g/L.
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