A method for evaluating environmental risk of drugs in water environment based on fish plasma model

Abstract

The application discloses a method for evaluating environmental risks of drugs in a water environment based on a fish plasma model, and belongs to the technical field of environmental toxicology and ecological risk assessment. The fish plasma-water distribution coefficient is calculated by a drug lipophilicity parameter (Log Dow), the fish steady-state plasma concentration is obtained by combining the measured / predicted environmental concentration, the fish treatment plasma concentration is converted by the difference between the half-inhibitory concentrations of the drug on fish and human, the pharmacological effect risk of the drug on fish is quantitatively evaluated by a risk quotient, and thus high-risk drugs are screened. Compared with the traditional risk quotient method, the application has the advantages of being in line with the drug action mechanism, taking into account the in-vivo enrichment effect, being high in evaluation accuracy, and not needing to rely on a large amount of acute / chronic toxicity data, and can efficiently support the priority control and risk management of drug pollutants in the water environment.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of environmental toxicology and ecological risk assessment, specifically relating to a method for evaluating the environmental risk of drugs in aquatic environments based on a fish plasma model. Background Technology

[0002] In recent years, the production and use of various drugs have continued to grow, resulting in drugs constantly entering the aquatic environment through production emissions, clinical use, and domestic sewage discharge. Because conventional wastewater treatment processes are insufficient to completely remove biologically active compounds, some drugs persist in surface water bodies at low concentrations for extended periods. Although environmental concentrations are typically in the range of ng / L to μg / L, these drugs possess well-defined molecular targets and specific pharmacological mechanisms of action, and their biological activity does not decrease linearly with concentration. Aquatic organisms exposed to these drugs in the actual environment for extended periods at low doses may experience chronic effects such as neuroregulatory disorders, behavioral changes, and impaired reproductive function, potentially impacting population stability and ecosystem structure. Therefore, establishing environmental risk assessment methods that reflect the specific pharmacological effects of drugs is of significant theoretical and regulatory value.

[0003] Traditional methods for assessing the environmental risk of pharmaceuticals primarily rely on the Risk Quotient (RQ) method, which determines risk levels by the ratio of measured environmental concentrations to predicted no-effect concentrations (PNEC). However, PNEC is typically derived from acute toxicity endpoints (such as mortality and growth inhibition), emphasizing general toxicological effects and failing to reflect the chronic pharmacological effects of drugs at low doses through specific molecular targets, potentially underestimating ecological risks. In contrast, the Fish Plasma Model (FPM) is based on the concept of "in vivo concentration-driven effects." It predicts steady-state plasma concentrations (FssPC) in fish and compares them with therapeutic plasma concentrations (HTPC) in humans to infer the likelihood of a drug reaching its potential pharmacological effect threshold in fish. This method combines the environmental exposure level of a drug with its known pharmacodynamic information, which can, to some extent, compensate for the shortcomings of traditional acute toxicity indicators.

[0004] However, existing fish plasma models often directly use HTPC as a reference standard, failing to fully consider the differences in drug target sensitivity across different species. In reality, the target structure, binding affinity, and functional response of drugs may differ significantly between fish and humans, and directly comparing FssPC and HTPC may lead to biased risk assessment. To improve the scientific rigor and accuracy of model predictions, it is necessary to construct an improved fish plasma model that introduces correction parameters based on cross-species pharmacodynamic differences, establishes a reference value for therapeutic plasma concentration that better reflects the physiological characteristics of fish, and uses this value for risk assessment. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for evaluating the environmental risks of drugs in aquatic environments based on a fish plasma model, enabling a systematic assessment from external exposure to effective concentration in vivo and potential pharmacological effects, and providing more accurate and mechanism-oriented technical support for the ecological risk management of drug-related pollutants.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for evaluating the environmental risk of drugs in aquatic environments based on a fish plasma model, comprising the following steps: (1) Collect key parameters of the target drug: therapeutic plasma concentration (HTPC) of the drug, logarithm of the partition coefficient (LogDow) of the drug, and half-maximal inhibitory concentration (IC50) of the drug on human serotonin transporter (hSERT). 50 human And the predicted environmental concentration (PEC) of the drug; (2) Taking zebrafish as an example, the fish plasma-water partition coefficient P of the drug is calculated using the following formula. Blood:Water ; Then, the predicted environmental concentration (PEC) of the drug obtained in step (1) and the fish plasma-water partition coefficient of the drug are used to obtain the steady-state plasma concentration (FssPC) of the drug in the fish using the following formula: ; (3) The half-maximal inhibitory concentration (IC50) of the drug on zebrafish serotonin transporter (zSERT) was determined experimentally. 50 zebrafish The value is obtained using the following formula. The therapeutic plasma concentration (FTPC) in the fish was calculated. (4) The risk quotient RQ is calculated by the ratio of steady-state plasma concentration (FssPC) to therapeutic plasma concentration (FTPC) in the fish: Drugs with RQ < 0.1 are defined as low-risk drugs, drugs with RQ ≤ 1 are defined as medium-risk drugs, and drugs with RQ > 1 are defined as high-risk drugs.

[0008] Based on the above technical solution, further, the drug mentioned in step (1) includes an antidepressant.

[0009] Based on the above technical solution, the predicted environmental concentration (PEC) of the drug mentioned in step (1) is further calculated using the following formula: Where Q is the average daily drug consumption per person (μg / cap / d), E is the drug excretion rate in the human body (%), V is the average daily water consumption per person (L / cap / d), and R is the drug removal rate of the wastewater treatment plant (%).

[0010] Based on the above technical solution, further, the average daily drug consumption per person in step (1) is calculated by combining the annual sales volume of drugs with the total population.

[0011] Based on the above technical solution, further, the sales volume of the drug mentioned in step (1) is derived from the annual report of China Chemical Pharmaceutical Industry.

[0012] Based on the above technical solution, further, the logarithm of the partition coefficient of the drug in step (2) is the logarithm of the n-octanol-water partition coefficient of the drug (Log Dow), which is derived from the ChemSpider database.

[0013] Based on the above technical solution, further, the human therapeutic plasma concentration mentioned in step (3) is specifically the peak blood concentration of the drug.

[0014] Based on the above technical solution, further, in step (3), the half-maximal inhibitory concentration (IC50) of the drug on the zebrafish serotonin transporter (zSERT) is obtained through a monoamine transporter (MAT) inhibition experiment. 50 zebrafish value.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for quantitatively assessing the ecological risks of pharmaceutical pollutants in aquatic environments. Compared with the traditional risk quotient method, this invention has the advantages of accurate assessment, close alignment with drug action mechanisms, and consideration of the bioaccumulation effect of drugs in aquatic organisms. It also avoids reliance on scarce acute / chronic toxicity experimental data of drugs in aquatic organisms and eliminates the need for complex in vivo toxicology tests. It can efficiently screen drugs that pose high or medium risks to fish in aquatic environments, providing a scientific basis for environmental monitoring of pharmaceutical pollutants and the development of priority control lists, thus contributing to the precise prevention and management of ecological risks in aquatic environments. Detailed Implementation

[0016] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0017] Reagents and consumables PBS buffer (14190144, Gibco), HBSS buffer (14025-092, Gibco), complete culture medium (DMEM + 10% FBS), DMEM (C3113-0500, Viva Cell BIOSCIENCES), FBS (10099141C, made in Australia, Gibco), trypsin (15400054, Gibco), centrifuge tubes, pipettes, pipettes, electric pipette (E12-300XLS+, Rainin), 12-well plate (150628, Thermo Fisher), 96-well plate (165306, Thermo Fisher), Lipofectamine 3000 (L3000-015, Thermo Fisher), microplate reader (Infinite 200 Pro, TECAN).

[0018] Example 1 This embodiment provides a method for evaluating the environmental risk of drugs in the aquatic environment based on a fish plasma model, including the following steps: (1) Collection and organization of key parameters of target drug Annual production data for 19 antidepressants were extracted from the "2019 Annual Report of China's Chemical Pharmaceutical Industry" (Table 1). After conversion to the mass of active ingredients, the per capita daily consumption (Q) was calculated based on my country's total population (140 million to 50,000). The per capita daily water consumption (V) was determined to be 170 L / person / day with reference to the Urban and Rural Construction Yearbook. The excretion rate (E) of each drug was retrieved from the DailyMed database of the U.S. National Library of Medicine. The removal rate (R) of each drug by activated sludge wastewater treatment plants was collected from published literature. For drugs for which no data were found, the R was estimated using EPI Suite software.

[0019] (2) Calculation of Predicted Environmental Concentration (PEC) of Drugs Based on the collected baseline parameters, the predicted environmental concentration of drugs in the wastewater effluent is calculated using the following formula, reflecting the exposure level of the drugs after entering the aquatic environment: Where Q is the average daily dose of antidepressants per person (μg / cap / d), E is the drug excretion rate in the human body (%), V is the average daily water consumption per person (L / cap / d), and R is the drug removal rate at the wastewater treatment plant (%). This calculation comprehensively considers drug use, excretion, and losses during wastewater treatment to ensure the rationality of environmental concentration predictions.

[0020] (3) Calculation of fish plasma-water partition coefficient and fish steady-state plasma concentration (FssPC) Based on the drug lipophilicity parameter Log Dow, taking zebrafish as an example, the distribution capacity of the drug in fish plasma and aqueous phase is calculated using the following formula, reflecting the drug's accumulation potential in the fish: The logarithmic diffraction coefficient (Log Dow) of each drug in octanol-water was obtained from the ChemSpider database and used as the core parameter for lipophilicity. The therapeutic plasma concentration (HTPC), i.e., peak plasma concentration (Cmax), was extracted from the literature of Schulz et al. The partitioning ability of the drug in fish plasma and aqueous phase was calculated using the following formula. ; By combining the predicted environmental concentration (PEC) with the fish plasma-water partition coefficient, the steady-state plasma concentration of the drug in the fish is obtained using the following formula, thus quantifying the actual exposure level of the drug in the fish: ; (4) Calculation of therapeutic plasma concentration (FTPC) in fish Considering the differences in drug target sensitivity between fish and humans, the therapeutic plasma concentration (HTPC) in humans is converted to a more physiologically accurate therapeutic plasma concentration for fish based on the cross-species half-maximal inhibitory concentration ratio (ICP-R). The formula is as follows: ; The half-maximal inhibitory concentration (IC50) of the drug on the zebrafish serotonin transporter (zSERT) was determined using a monoamine transporter (MAT) inhibition assay. 50 zebrafish The specific process is as follows: 1) First, HEK293 cells were passaged and cultured using Mg-free medium. 2+ and Ca 2+ Wash the cells once with phosphate-buffered saline (PBS), add PBS containing 0.05% (w / v) trypsin and 0.52 mM EDTA, and after a short incubation, add DMEM medium containing 10% fetal bovine serum (to stop trypsin activity); after the cells are suspended, transfer them to centrifuge tubes, centrifuge at 190 × g for 2 minutes, seed the cells into 12-well plates, 1 mL per well, and incubate overnight in a CO2 incubator at 37°C and 5% CO2.

[0021] 2) Add 125 μL of Opti-MEM serum-depleted medium to a 1.5 ml centrifuge tube and mix it with the zSERT-expressing plasmid. Mix 125 μL of Opti-MEM serum-depleted medium with Lipofectamine 2000 transfection reagent and incubate at room temperature for 30 minutes. Then mix it with the plasmid mixture and incubate for another 20 minutes. After incubation, add it to a well plate containing HEK293 cells and incubate in a CO2 incubator at 37°C and 5% CO2 for about 40 hours for subsequent detection.

[0022] 3) First, heat the culture medium and buffer to 37°C, then use a Mg-free solution. 2+ and Ca 2+ The transfected HEK 293 cells were washed once with PBS buffer (1 mL per well), then 0.5 mL of PBS buffer containing 0.05% trypsin and 0.52 mM EDTA was added, followed by the addition of DMEM medium to stop trypsin activity. The cells were transferred to centrifuge tubes and centrifuged at 190×g for 2 minutes. The supernatant was aspirated, and 5 mM HEPES (pH 7.4) Hank's balanced salt solution (HBSS) was added. The suspended cells were added to 80 μL per well of a 96-well plate using a multichannel electronic pipette, and then incubated in a CO2 incubator at 37 ℃ and 5% CO2 for 1 hour. Using HBSS containing 5 mM HEPES buffer (pH 7.4) and 0.01% (w / v) BSA (bovine serum albumin) (to prevent compound adhesion) as a diluent, six concentration gradients of the drug standards to be tested were set from high to low and diluted (10⁻⁶ m / s). -10 -10 -5 M), then 10 μL of diluted drug standard was added to the culture medium using a multichannel electronic pipette, and the cells were mixed by tapping the plate. The mixture was then incubated at room temperature for 10 minutes. Subsequently, 10 μL of 1.0 × 10⁻⁶ HCl was added to each well using a multichannel electronic pipette. 5 M's APP fluorescent substrate (4-(4-(dimethylaminophenyl)-1-methylpyridinium) was prepared at a concentration of 1.0 × 10⁻⁶. 6 M; Cells were placed in a CO2 incubator and incubated at 37°C and 5% CO2 for 1 h to absorb APP. A control group was set up during this process, and three replicates were set up for each dilution in the experimental groups. After cell incubation, the existing culture medium was aspirated, and 50 μL of HBSS buffer containing 5 mM HEPES (pH 7.4) was added to each well for gentle washing to remove excess APP and added drug reagents. The supernatant was transferred to a new 96-well plate, and 50 μL of HBSS buffer containing 5 mM HEPES (pH 7.4) was added to each well. Finally, the fluorescence of APP in cells was measured using a microplate reader (excitation wavelength 385 nm, emission wavelength 525 nm), and the drug effect-concentration curve was plotted based on the results. IC50 was calculated. 50 zebrafish; and simultaneously obtained the half-maximal inhibitory concentration (IC50) of the drug against human serotonin transporter (hSERT) from previous studies. 50 human).

[0023] (5) Risk Quotient (RQ) Calculation and Risk Classification 1. Risk Quotient Calculation: The risk of drug pharmacological effects on fish is quantified by the ratio of steady-state plasma concentration to therapeutic plasma concentration. ; 2. Risk Classification and Target Screening: Risk classification criteria were established: RQ > 1 indicates a high-risk drug, meaning the drug concentration in the fish exceeds the therapeutic threshold and is likely to trigger pharmacological effects; 0.1 ≤ RQ ≤ 1 indicates a medium-risk drug, posing a potential risk; and RQ < 0.1 indicates a low-risk drug. Based on the RQ values, high- and medium-risk drugs were screened, and the results are shown in Tables 1, 2, 3, and 4.

[0024] Table 1. Sales volume, consumption, excretion rate, removal rate, and predicted effluent concentration of antidepressants in 2019

[0025] Table 2. Steady-state plasma concentrations of antidepressants in fish based on predicted environmental concentrations in China

[0026] Table 3. Therapeutic plasma concentrations of antidepressants

[0027] Table 4. Risk Quotient of Antidepressants Based on Predicted Environmental Concentration Values ​​in China

[0028] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention. Modifications to the technical solutions described in the foregoing embodiments, or equivalent substitutions for some or all of the technical features therein, are made. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for evaluating the environmental risk of drugs in aquatic environments based on a fish plasma model, characterized in that, Includes the following steps: (1) Collect key parameters of the target drug: therapeutic plasma concentration (HTPC) of the drug, logarithm of the partition coefficient (LogDow) of the drug, and half-maximal inhibitory concentration (IC50) of the drug on human serotonin transporter (hSERT). 50 human And the predicted environmental concentration (PEC) of the drug; (2) The fish plasma-water partition coefficient P of the drug is calculated using the following formula. Blood:Water ; ; Then, the predicted environmental concentration (PEC) of the drug obtained in step (1) and the drug's plasma-water partition coefficient are used to obtain the steady-state plasma concentration (FssPC) of the drug in the fish using the following formula: ; (3) The half-maximal inhibitory concentration (IC50) of the drug on zebrafish serotonin transporter (zSERT) was determined experimentally. 50 zebrafish The value is obtained using the following formula. The therapeutic plasma concentration (FTPC) in the fish was calculated. (4) The risk quotient RQ is calculated by the ratio of steady-state plasma concentration (FssPC) to therapeutic plasma concentration (FTPC) in the fish: Drugs with RQ < 0.1 are defined as low-risk drugs, drugs with RQ ≤ 1 are defined as medium-risk drugs, and drugs with RQ > 1 are defined as high-risk drugs.

2. The method according to claim 1, characterized in that, The medications mentioned in step (1) include antidepressants.

3. The method according to claim 1, characterized in that, The predicted environmental concentration (PEC) of the drug mentioned in step (1) is calculated using the following formula: Where Q is the average daily drug consumption per person (μg / cap / d), E is the drug excretion rate in the human body (%), V is the average daily water consumption per person (L / cap / d), and R is the drug removal rate of the wastewater treatment plant (%).

4. The method according to claim 3, characterized in that, The average daily drug consumption per person mentioned in step (1) is calculated by combining the annual sales volume of drugs with the total population.

5. The method according to claim 4, characterized in that, The sales volume of the drug mentioned in step (1) is derived from the annual report of China's chemical pharmaceutical industry.

6. The method according to claim 1, characterized in that, The logarithm of the partition coefficient of the drug mentioned in step (2) is the logarithm of the n-octanol-water partition coefficient of the drug (Log Dow), which is obtained from the ChemSpider database.

7. The method according to claim 1, characterized in that, The plasma concentration for human treatment mentioned in step (3) is specifically the peak plasma concentration of the drug.

8. The method according to claim 1, characterized in that, In step (3), the half-maximal inhibitory concentration (IC50) of the drug on the zebrafish serotonin transporter (zSERT) is obtained through a monoamine transporter (MAT) inhibition assay. 50 zebrafish value.

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