CN103276042B - Method for detecting genetic toxicity of organic pollutants in water - Google Patents
Method for detecting genetic toxicity of organic pollutants in water Download PDFInfo
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- CN103276042B CN103276042B CN201310200244.XA CN201310200244A CN103276042B CN 103276042 B CN103276042 B CN 103276042B CN 201310200244 A CN201310200244 A CN 201310200244A CN 103276042 B CN103276042 B CN 103276042B
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- gobiocypris rarus
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Abstract
The invention provides a method for detecting the genetic toxicity of organic pollutants. The method comprises the following steps of: (1) treating gobiocypris rarus by a water solution of an organic-pollutant-containing sample to be detected in an exposed way; and (2) detecting the peripheral blood erythrocyte of the gobiocypris rarus which is treated in the exposed way, carrying out statistics on cell micronucleus rate and drawing a dosage-effect curve. The detection method is sensitive, simple, convenient, rapid, more visual and accurate, has good result repeatability, and has important significance for the research of genetic toxicity of water quality.
Description
Technical field
The invention belongs to Study of Water Environment field, relate to a kind of genotoxic method of detection organic pollutant.
Background technology
Environmental pollution increases the weight of, and brings threat to the health of people.Various research method has been carried out and has been evaluated genetoxic, proposes micronucleus test so far first from Heddle etc., micronucleus test because it is economical, simple and easy to do, confidence level advantages of higher is more and more widely used in karyomit(e)/detection of spindle body damaging effect.
Hereditary poisonous substance in water surrounding can bring out fish body and produce micronucleus, by the change of monitoring Erythrocyte Micronucleus of Fishes rate can Study of Exogenous thing genetoxic and evaluate ambient water quality.At present, investigator discusses to the validity that the micronucleus in erythrocytes of multiple fish is tested, domestic scholars application Chinese tradition fish crucian, carp, silver carp and bighead etc. successfully take in situ test to evaluate ambient water quality, confirm the feasibility of fish peripheral red blood cells as the biomarker of the hereditary poisonous substance chromosome damage of monitoring.And along with Economic development and process of industrialization aggravation, water body is polluted by a large amount of organic pollutant, wherein manyly belong to carcinogenic, teratogenesis, mutagenic matter.Organic Pollutants In Water is not yet carried out Gobiocypris rarus peripheral red blood cells genotoxicity testing method detect delay.
Summary of the invention
An object of the present invention is to provide a kind of genotoxic detection method detecting organic pollutant in sample, comprise following steps:
(1) with the aqueous solution of testing sample, process Gobiocypris rarus is exposed;
(2) peripheral red blood cells of the Gobiocypris rarus after exposing process is detected, statistics cell micronucleus rate.
Described detection method is the genetoxic judging organic pollutant in testing sample according to cell micronucleus rate.
In described step (1), before described exposure process, the peripheral red blood cells micronuclear rates of Gobiocypris rarus is 0.1 ‰.
In described step (1), the described method exposing process is: Gobiocypris rarus is placed in the described aqueous solution containing testing sample, and between resting period, every 2-3d changes and exposes treatment solution once, replaces half at every turn.
In described step (1), lucifuge in described exposure treating processes.
In described step (1), the described time exposing process is 21-28d; Be specially 28d.
In described step (1), the compound method of the aqueous solution of described testing sample is: dilute with water and get final product again after determinand being dissolved in organic solvent.
In described step (2), the described method of peripheral red blood cells detecting the Gobiocypris rarus after exposing process is: after exposing process from step (1), put to death after Gobiocypris rarus get blood, smear, fix, dry, the dyeing of Giemsa application liquid, clean dye liquor, dry, microscopy.
In described method, described organic pollutant is benzo [a] pyrene or 4-nitroquinoline-1-oxide compound.
Another object of the present invention is to provide Gobiocypris rarus and is detecting the application in the genetoxic that causes of organic pollutants.
In described application, described organic pollutant is benzo [a] pyrene or 4-nitroquinoline-1-oxide compound.
Also object of the present invention is to provide a kind of method of auxiliary detection organic pollutants content, comprises following steps:
(1) production standard curve:
A, the determinand preparing a series of concentration known are dissolved in the solution storing solution of organic solvent, and after being diluted in activated carbon treatment, water obtains the determinand aqueous solution of various different concns;
B, expose process Gobiocypris rarus with the determinand aqueous solution of various concentration respectively;
The peripheral red blood cells of the Gobiocypris rarus after C, detection exposure process, statistics cell micronucleus rate;
D, with cell micronucleus rate for ordinate zou, with the concentration of determinand in the described determinand aqueous solution for X-coordinate, production standard curve;
(2) get water sample to be measured, according to method identical in step B and C, exposure process and statistics cell micronucleus rate are carried out to Gobiocypris rarus; Cell micronucleus rate is substituted into the typical curve in above-mentioned steps D, namely obtains the content of determinand in water sample to be measured.
In described step B, before described exposure process, the peripheral red blood cells micronuclear rates of Gobiocypris rarus is 0.1 ‰.
In described step B, the described method exposing process is: Gobiocypris rarus is placed in the described aqueous solution containing testing sample, places, and period every 2-3d changes and exposes treatment solution once, replaces half at every turn.
In described step B, lucifuge in described exposure treating processes.
In described step B, the described time exposing process is 21-28d; Be specially 28d.
In described step C, the described method of peripheral red blood cells detecting the Gobiocypris rarus after exposing process is: after exposing process from step B, put to death after Gobiocypris rarus get blood, smear, fix, dry, the dyeing of Giemsa application liquid, clean dye liquor, dry, microscopy.
In described method, described organic pollutant is benzo [a] pyrene or 4-nitroquinoline-1-oxide compound.
Compared with prior art, tool has the following advantages in the present invention:
1. Gobiocypris rarus is Chinese peculiar small-scale test fish, is easy to standardized study, and reduce the impact of other experimental factors, its peripheral red blood cells micronucleus test can detect organic pollutants genetoxic hazard conditions well.
2. exploitation is for organism exposure chamber and the micronucleus test technology of open-assembly time, and draws dose-effect curve, realizes the quantitative evaluation to hereditary poisonous substance toxicity.
Therefore, the inventive method is sensitive, easy, fast, more directly perceived, accurately, result favorable reproducibility, studies significant for water quality genetoxic.
Accompanying drawing explanation
Fig. 1 is benzo [a] pyrene dose-effect curve.
Fig. 2 is 4-nitroquinoline-1-oxide compound dose-effect curve.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Below in conjunction with specific embodiment, the invention will be further described, but the present invention is not limited to following examples.
The genotoxic detection of embodiment 1, dimethyl sulfoxide (DMSO)
(1) detection method and step
(1) the raising and train of Gobiocypris rarus: raise and train same batch, Gobiocypris rarus 14d that body weight gain is close, period, its Micronucleus Rate of Peripheral Lymphocytes rate reached about 0.1 ‰ without natural death fish phenomenon, namely can be used for test;
(2) trial test selects the dimethyl sulfoxide (DMSO) of appropriate concentration range to process, without dead fish phenomenon under plan adopts the highest exposure concentrations;
(3) preparation for the treatment of solution is exposed: dimethyl sulfoxide (DMSO) is dissolved in activated carbon treatment tap water, is mixed with the aqueous solution of Volume fraction 0.03%, be the exposure treatment solution prepared;
(4) process is exposed: establish the blank not adding dimethyl sulfoxide (DMSO); Experiment exposure group selects 5 tail Gobiocypris rarus at random, and lucifuge exposes 28d, every 2-3d and changes exposure treatment solution half;
(5) blood smear of Gobiocypris rarus erythrocyte is made: get blood by exposing after the fish after processing is put to death, methyl alcohol fixes 10min, dries, and through Giemsa application liquid dyeing 15min, cleans dye liquor, dries and get final product immediately with the phosphate buffered saline buffer of pH=6.8;
(6) detection of cell micronucleus rate: blood smear is placed in basis of microscopic observation, 10*100 doubly observes, count, choose complete in the visual field, be uniformly dispersed and painted suitable cell, double-blind method diagosis, often open the erythrocytic micronuclear rates of fish of smear counting about 5000 endochylema complete displays, result represents with permillage (‰).The cell micronucleus rate that dimethyl sulfoxide (DMSO) exposed volume mark is corresponding when being 0%, 0.03% is respectively 0.29 ± 0.04 ‰, 0.37 ± 0.06 ‰, and there was no significant difference, shows that dimethyl sulfoxide (DMSO) does not show genetic toxic effect under this test condition.
Embodiment 2, the genotoxic detection of benzo [a] pyrene
(1) detection method and step
(1) the raising and train of Gobiocypris rarus: raise and train same batch, Gobiocypris rarus 14d that body weight gain is close, period, its Micronucleus Rate of Peripheral Lymphocytes rate reached about 0.1 ‰ without natural death fish phenomenon, namely can be used for test;
(2) trial test selects appropriate concentration range to process; Do not observe dead fish phenomenon to intend adopting the highest exposure concentrations to expose process, show to can be used for follow-up micronucleus exposure test.
(3) preparation for the treatment of solution is exposed: be dissolved in dimethyl sulfoxide (DMSO) by benzo [a] pyrene, be mixed with the mother liquor that concentration is respectively 0mg/L, 4/3mg/L, 20/3mg/L, 100/3mg/L, 500/3mg/L, then the mother liquor getting same volume product is respectively dissolved in the tap water of activated carbon filtration, be mixed with the solution of benzo [a] pyrene concentration difference 0 μ g/L, 0.4 μ g/L, 2.0 μ g/L, 10.0 μ g/L, 50.0 μ g/L, be the 5 groups of exposure treatment solutions prepared;
(4) process is exposed: set the dimethyl sulfoxide (DMSO) blank solution that do not add benzo [a] pyrene as control group; Often group selects 10 tail Gobiocypris rarus; Adopt semi-static exposure chamber, namely every 2-3d changes and exposes treatment solution once, a half; Lucifuge exposes 28d;
(5) blood smear of Gobiocypris rarus erythrocyte is made: get blood after being put to death by the fish after exposing process, smear, methyl alcohol fixes 10min, dry, through Giemsa application liquid dyeing 15min, clean dye liquor with the phosphate buffered saline buffer of pH=6.8, dry and get final product immediately;
(6) detection of cell micronucleus rate: blood smear is placed in basis of microscopic observation, 10*100 doubly observes, count, choose complete in the visual field, be uniformly dispersed and painted suitable cell, double-blind method diagosis, often open the erythrocytic micronuclear rates of fish of smear counting about 5000 endochylema complete displays, result represents with permillage (‰).The cell micronucleus rate that exposure concentrations increases successively is respectively 0.37 ± 0.06 ‰, 1.53 ± 0.11 ‰, 4.19 ± 0.08 ‰, 10.29 ± 0.93 ‰, 17.93 ± 0.43 ‰.
Result shows, and when benzo [a] pyrene concentration is 0.4 μ g/L, cell micronucleus rate and blank have significant difference.The detection method of existing bibliographical information is as shown in table 1.
Table 1
Table 1 result shows, compared with existing detection method, detection method sensitivity provided by the invention is higher.
(2) dose-effect curve
With benzo [a] pyrene concentration for X-coordinate, micronucleus permillage is ordinate zou, draws dose-effect curve, as shown in Figure 2.Quantizing dose-effect relationship by dose-effect curve is following formula:
y＝2.14(1-e
-1.76x)+16.30(1-e
-0.069x)
Benzo [a] pyrene 28d exposes fit curve equation change: R
2=0.9994; Adjusted R
2=0.9974
Multiple comparisons shows and to there are differences extremely remarkable between each concentration group, illustrates that benzo [a] pyrene has Genotoxic Effect to Gobiocypris rarus peripheral red blood cells, and has obvious dose-effect relationship.
Embodiment 3,4-nitroquinoline-1-oxide compound (4-NQO) genotoxic detection
(1) detection method and step
(1) the raising and train of Gobiocypris rarus: raise and train same batch, Gobiocypris rarus 14d that body weight gain is close, period, its Micronucleus Rate of Peripheral Lymphocytes rate reached about 0.1 ‰ without natural death fish phenomenon, namely can be used for test;
(2) trial test selects appropriate concentration range to process; Do not observe dead fish phenomenon to intend adopting the highest exposure concentrations to expose process, show to can be used for follow-up micronucleus exposure test;
(3) preparation for the treatment of solution is exposed: be dissolved in dimethyl sulfoxide (DMSO) by 4-nitroquinoline-1-oxide compound (4-NQO), be mixed with the mother liquor that concentration is respectively 0mg/L, 1/3mg/L, 5/3mg/L, 25/3mg/L, 125/3mg/L, then the mother liquor getting same volume product is respectively dissolved in the tap water of activated carbon filtration, be mixed with the solution of 4-NQO concentration difference 0 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 2.5 μ g/L, 12.5 μ g/L, be the 5 groups of exposure treatment solutions prepared;
(4) process is exposed: set the dimethyl sulfoxide (DMSO) blank solution that do not add 4-nitroquinoline-1-oxide compound as control group; Often group selects 10 tail Gobiocypris rarus; Adopt semi-static exposure chamber, namely every 2-3d changes and exposes treatment solution once, a half; Lucifuge exposes 28d;
(5) blood smear of Gobiocypris rarus erythrocyte is made: get blood after being put to death by the fish after exposing process, smear, methyl alcohol fixes 10min, dry, through Giemsa application liquid dyeing 15min, clean dye liquor with the phosphate buffered saline buffer of pH=6.8, dry and get final product immediately;
(6) detection of cell micronucleus rate: blood smear is placed in basis of microscopic observation, 10*100 doubly observes, count, choose complete in the visual field, be uniformly dispersed and painted suitable cell, double-blind method diagosis, often open the erythrocytic micronuclear rates of fish of smear counting about 5000 endochylema complete displays, result represents with permillage (‰).The cell micronucleus rate that exposure concentrations increases successively is respectively 0.37 ± 0.06 ‰, 1.36 ± 0.08 ‰, 4.35 ± 0.40 ‰, 8.89 ± 0.26 ‰, 22.69 ± 0.56 ‰.
Result shows, and when 4-nitroquinoline-1-oxide concentration is 0.1 μ g/L, has significant difference with blank.Document Valentin-Severin, I.et al.Mutation Research-Genetic Toxicology andEnvironmental Mutagenesis [J] .536 (1-2): the genetoxic being detected 4-nitroquinoline-1-oxide compound in 79-90. by human liver cancer cell HepG2, experimental result shows, when cell micronucleus rate reaches two times of negative control micronuclear rates, the concentration of 4-nitroquinoline-1-oxide compound is 22.8 μ g/L.This shows compared with existing detection method, and detection method sensitivity provided by the invention is higher.
(2) dose-effect curve
With 4-nitroquinoline-1-oxide concentration for X-coordinate, micronucleus permillage is ordinate zou, draws dose-effect curve, as shown in Figure 2.Quantizing dose-effect relationship by dose-effect curve is following formula:
y＝4.19(1-e
-3.24x)+39.26(1-e
-0.05x)
4-nitroquinoline-1-oxide compound (4-NQO) 28d exposes fit curve equation change: R
2=0.9996; AdjustedR
2=0.9983
Multiple comparisons shows and to there are differences extremely remarkable between each concentration group, illustrates that 4-nitroquinoline-1-oxide compound has Genotoxic Effect to Gobiocypris rarus peripheral red blood cells, and has obvious dose-effect relationship.
Claims (5)
1. detect a genotoxic detection method for organic pollutant in sample, comprise following steps:
(1) with the aqueous solution of testing sample, process Gobiocypris rarus is exposed;
(2) peripheral red blood cells of the Gobiocypris rarus after exposing process is detected, statistics cell micronucleus rate;
In step (1), the described method exposing process is: Gobiocypris rarus is placed in the described aqueous solution containing testing sample, places, and period every 2-3d changes and exposes treatment solution once, replaces half at every turn;
In described step (1), lucifuge in described exposure treating processes;
In described step (1), the described time exposing process is 21-28d;
In described method, described organic pollutant is benzo [a] pyrene or 4-nitroquinoline-1-oxide compound.
2. method according to claim 1, is characterized in that: in step (1), and before described exposure process, the peripheral red blood cells micronuclear rates of Gobiocypris rarus is 0.1 ‰.
3. Gobiocypris rarus is detecting the application in the genetoxic that causes of organic pollutants.
4. a method for auxiliary detection organic pollutants content, comprises following steps:
(1) production standard curve:
A, the determinand preparing a series of concentration known are dissolved in the solution storing solution of organic solvent, and after being diluted in activated carbon treatment, water obtains the determinand aqueous solution of various different concns;
B, expose process Gobiocypris rarus with the determinand aqueous solution of various concentration respectively;
The peripheral red blood cells of the Gobiocypris rarus after C, detection exposure process, statistics cell micronucleus rate;
D, with cell micronucleus rate for ordinate zou, with the concentration of determinand in the described determinand aqueous solution for X-coordinate, production standard curve;
(2) get water sample to be measured, according to method identical in step B and C, exposure process and statistics cell micronucleus rate are carried out to Gobiocypris rarus; Cell micronucleus rate is substituted into the typical curve in above-mentioned steps D, namely obtains the content of determinand in water sample to be measured;
In step B, the described method exposing process is: Gobiocypris rarus is placed in the described aqueous solution containing testing sample, places, and period every 2-3d changes and exposes treatment solution once, replaces half at every turn;
In described step B, lucifuge in described exposure treating processes;
In described step B, the described time exposing process is 21-28d;
In described method, described organic pollutant is benzo [a] pyrene or 4-nitroquinoline-1-oxide compound.
5. method according to claim 4, is characterized in that: in step B, and before described exposure process, the peripheral red blood cells micronuclear rates of Gobiocypris rarus is 0.1 ‰.
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| CN104830946B (en) * | 2015-05-13 | 2018-07-17 | 中国环境科学研究院 | Environment water causes TK6 cell chromosome damages genetic toxicology assays and application |
| CN105548514A (en) * | 2015-12-25 | 2016-05-04 | 哈尔滨工业大学 | Multi-genetic terminal biological group test method for evaluating genetic toxicity of industrial wastewater reuse of water treatment plant |
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