Bisphenol substance exposed exogenous marker and application thereof in monitoring load in human body

Abstract

The invention discloses a bisphenol substance exposed exogenous marker and an application thereof in human body load monitoring. Energy required by different site reactions of bisphenol substances is calculated through metabolic simulation so as to determine potential targets of metabolic reactions; a fragment ion search algorithm is used for automatically simulating a characteristic mass spectrum fragment map of a compound so as to calculate the similarity between actually collected fragment ions and theoretically generated fragment ions, the structural formula of the exposed marker is determined according to the matching degree, and finally accurate structural analysis of the exogenous marker exposed by the bisphenol substances is achieved. According to I-phase and II-phase metabolic conversion products, a BPs in-vivo metabolic conversion rule is clarified, semi-quantitative analysis is carried out according to peak area intensity of the exposure marker, and the high-sensitivity exposure marker capable of being used for monitoring loads in a human body is determined.

Description

Technical Field

[0001] The present invention belongs to the field of environmental toxicology and risk assessment of chemicals, and specifically relates to exogenous markers of exposure to bisphenols and their application in monitoring the body load in the human body. Background Art

[0002] Bisphenol A is a widely used synthetic chemical that is widely used in the synthesis of materials such as polycarbonate and epoxy resin, and is used to manufacture plastic bottles, sippy cups for children, and coatings on the inside of food and beverage cans. However, in recent years, more and more studies have shown that low-dose exposure to bisphenol A is associated with numerous endocrine and metabolic disruption effects, and is an important risk factor for breast cancer, neurodegenerative diseases, etc. Therefore, bisphenol A has been gradually banned and replaced, resulting in a large class of bisphenol substitutes (BPs), including bisphenol AF (BPAF), bisphenol B (BPB), and bisphenol AP (BPAP).

[0003] In vitro cell and animal experimental studies have shown that these BPs may be more toxic than bisphenol A, which has caused widespread health concerns. However, the health effects of human exposure to these BPs have rarely been paid attention to, mainly because their detection rates and concentrations in human biological samples such as serum and urine are not high, making it difficult to conduct statistical association analysis.

[0004] It is worth noting that in recent years, domestic and foreign lake and river water monitoring studies have shown that the BPs content in water is comparable to or even higher than the concentration of bisphenol A. However, human exposure monitoring studies have shown that the detection rate and concentration of most BPs are lower than bisphenol A. The reason for the inconsistency between the external environment and human exposure monitoring data may be that bisphenols can be rapidly metabolized and transformed after entering the body, and only measuring the parent compound may cause huge exposure assessment bias.

[0005] Bisphenols are not persistent organic pollutants in terms of structure. After entering the body, they can undergo phase I metabolic transformation under the action of cytochrome P450 oxidase and other factors. Active phase I metabolic transformation can form adducts with proteins and nucleic acids to cause cytotoxicity, and can also be further directly transformed into water-soluble conjugates through phase II metabolism to reduce toxicity and promote excretion. The metabolic transformation products formed in these processes and the parent compounds can be used as exposure markers.

[0006] Exposure markers are the key to accurately reconstructing the exposure level of pollutants and are the bridge between environmental pollutants and health effects. At present, the quantitative assessment of human serum BPs exposure level is usually carried out by comparing the concentration of maternal BPs in serum. This assessment method assumes that BPs mainly exist in the form of maternal BPs after entering the body. However, if BPs can be rapidly metabolized and transformed after entering the body, the concentration of the parent compound will undoubtedly be much lower than the concentration of its metabolic transformation products. Then, assessing the exposure only by measuring maternal BPs will cause the assessment results to be seriously underestimated. Therefore, it is urgent to conduct research to establish a method for identifying BPs exposure markers and clarify the metabolic transformation rules of BPs in the body, so as to provide exposure markers for monitoring the internal load of BPs in the human body. Summary of the invention

[0007] Current studies mostly use maternal BPs in serum or urine to assess the internal load of bisphenols, ignoring the metabolic transformation process of BPs in vivo, resulting in high uncertainty in exposure assessment. To this end, the present invention aims to provide exogenous markers of bisphenol exposure and their application in human internal load monitoring, clarify the metabolic transformation rules of BPs in vivo, and quickly provide exposure markers for population internal load monitoring.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for screening exogenous markers of bisphenol exposure comprises the following steps:

[0010] (1) Animal exposure, serum acquisition and data collection: Experimental animals were gavaged with bisphenols at an exposure (dosing) concentration of 50 to 400 mg / kg / day for more than 15 days. After the exposure, the serum of the experimental animals was obtained, purified, and analytical samples were obtained. The samples were analyzed and measured using an ultra-high performance liquid chromatography tandem quadrupole / orbitrap high-resolution mass spectrometer, and several fragment ion spectra were collected.

[0011] The experimental animals include rodents or non-rodents;

[0012] The rodents are preferably rats and mice;

[0013] The non-rodent animal is preferably a rabbit, dog, monkey, or others;

[0014] The bisphenol substances include bisphenol A and its substitutes;

[0015] The bisphenol A substitutes include bisphenol AF (BPAF), bisphenol B (BPB) and bisphenol AP (BPAP);

[0016] The method of obtaining serum from experimental animals is that after the exposure, the experimental animals are fasted for more than 12 hours, anesthetized, and subjected to abdominal incision to collect blood from the abdominal aorta, which is then centrifuged to obtain serum;

[0017] (2) Screening of potential biomarkers: Based on the possible phase I and phase II metabolic transformation reactions of bisphenols, a list of potential exposure biomarkers (Generate Expected Compounds) is generated; Compound Discoverer software is used to perform an automated screening workflow on the fragment ion spectra collected in step (1), including: peak extraction (Select Spectra), peak alignment (Align Retention Times), expected compound screening (Find Expected Compounds), expected compound grouping (Group Expected Compounds), compound identification and annotation, and fragment ion search scoring (FISH Scoring); parent ions with high fragment ion scores are matched with the list of exposure biomarkers to screen out the molecular formulas of potential bisphenol exposure biomarkers;

[0018] There are 7 phase I metabolic conversion reactions, including oxidation, reduction, hydrolysis, reductive defluorination, oxidative defluorination, etc.; there are 17 phase II metabolic conversion reactions, including acetylation, methylation, glucuronide conjugation, sulfation, glutathione conjugation, glycine binding, cysteine ​​coupling, etc.; the maximum number of reactions for phase I and phase II metabolic reactions is set to 2, respectively, and the total number of reactions is 3. Through the combination of metabolic reactions, a list of potential exposure markers (Generate Expected Compounds) is formed (including thousands of potential exposure markers);

[0019] (3) Structural analysis: Use metabolic simulation software to calculate the energy required for reactions at different sites of bisphenols, thereby identifying potential targets of metabolic reactions, and preliminarily deduce their structural formula based on the molecular formula obtained in step (2); then, input the deduced structural formula into the Compound Discoverer software, and use the fragment ion search algorithm to automatically simulate the characteristic mass spectrum fragmentation pattern of the compound, calculate the similarity between the fragment ions actually collected and the theoretically generated (calculated by the software) fragment ions, and determine the structural formula of the exposure marker if the matching degree is greater than 20%; in addition, perform semi-quantitative analysis based on the peak area intensity of the exposure marker to identify the sensitive exposure marker;

[0020] The metabolic simulation software includes ADMET Predictor TM and BioTransformer 3.0.

[0021] The structural formula of the exogenous markers of bisphenol exposure is shown in the following table:

[0022]

[0023]

[0024]

[0025]

[0026] The exogenous markers of bisphenol exposure can be used to monitor the internal load of bisphenols in the human body, to evaluate the toxic effects of bisphenol exposure, and so on.

[0027] Compared with the prior art, the present invention has the following advantages and effects:

[0028] 1. The present invention systematically constructs a list of serum exposure markers for bisphenols based on the phase I and phase II metabolic transformation rules of bisphenols, and combines high-resolution information acquisition and fragment ion search scoring results to achieve full coverage screening of exogenous markers of bisphenol exposure.

[0029] 2. The present invention calculates the energy required for reactions at different sites of bisphenols through metabolic simulation to identify potential targets of metabolic reactions, and uses a fragment ion search algorithm to automatically simulate the characteristic mass spectrum fragmentation pattern of the compound to calculate the similarity between the fragment ions actually collected and the fragment ions theoretically generated, and determines the structural formula of the exposure marker based on the matching degree, ultimately achieving accurate structural analysis of exogenous markers of exposure to bisphenols.

[0030] 3. The present invention clarifies the metabolic transformation rules of BPs in vivo based on the phase I and phase II metabolic transformation products, and performs semi-quantitative analysis based on the peak area intensity of exposure markers, thereby establishing highly sensitive exposure markers for monitoring body loads in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAF_M1.

[0032] Figure 2 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAF_M2.

[0033] Figure 3 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAF_M3.

[0034] Figure 4 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAF_M4.

[0035] Figure 5 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAF_M5.

[0036] Figure 6 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAF_M6.

[0037] Figure 7 This is a diagram of the metabolic transformation pathway of BPAF in vivo.

[0038] Figure 8 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAP_M1.

[0039] Fig. 9 It is the parent ion (MS1) and fragment ion (MS2) spectrum of BPAP_M2-1.

[0040] Fig.10 It is the parent ion (MS1) and fragment ion (MS2) spectrum of BPAP_M2-2.

[0041] Fig.11 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAP_M3.

[0042] Fig.12 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAP_M4-1.

[0043] Fig.13 It is the parent ion (MS1) and fragment ion (MS2) spectrum of BPAP_M4-2.

[0044] Fig.14 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPAP_M5.

[0045] Fig.15 It is a diagram of the metabolic conversion pathway of BPAP in the body.

[0046] Fig.16 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPB_M1.

[0047] Fig.17 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPB_M2.

[0048] Fig.18 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPB_M3.

[0049] Fig.19 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPB_M4.

[0050] Fig. 20 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPB_M5.

[0051] Fig.21 It is the parent ion (MS1) and fragment ion (MS2) spectra of BPB_M6.

[0052] Fig. 22 This is a diagram of the metabolic transformation pathway of BPB in vivo.

[0053] Fig.23 is the concentration distribution of different exposure markers of BPAF, BPB and BPAP. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0055] Example

[0056] A method for screening exogenous markers of bisphenol exposure comprises the following steps:

[0057] (1) Animal exposure: Given that dietary intake is the main route of human exposure to BPs, this study used oral administration for exposure, with an exposure interval of 15 days. Rats were randomly divided into a control group and an exposure group, with 5 females and 5 males in each group, weighing 180 to 220 grams. Among them, the exposure group was exposed to 400 mg / kg-day of BPs, while the control group was only given an equal amount of corn oil. After the exposure, the rats were fasted for 12 hours, anesthetized with isoflurane, positioned dorsally, and underwent abdominal incision to collect blood from the abdominal aorta. 2 ml of blood was collected using lithium heparin anticoagulant tubes and centrifuged at 3500 rpm for 5 minutes to obtain serum.

[0058] (2) Serum purification and enrichment: Take 400 μL of blood supernatant and add 1.2 mL of methanol precooled at -80°C. Vortex the sample for 30 seconds, place it at -80°C for 60 minutes, and centrifuge it at 14,000 rpm for 15 minutes. Take 800 μL of supernatant, vacuum concentrate to dryness, add 200 μL of 1% formic acid methanol solution to reconstitute, centrifuge it at 14,000 rpm for 15 minutes, and take 120 μL of supernatant for analysis.

[0059] (3) Data acquisition: Ultra-high performance liquid chromatography tandem quadrupole / orbitrap high-resolution mass spectrometer (OrbitrapExploris 240) was used for analysis and determination. The specific conditions were as follows: the chromatographic separation column was a HypersilGOLD AQ C18 column (150×2.1 mm, 1.9 um, Thermo Fisher Scientific, Germany), the organic phase (B) was methanol, the aqueous phase (B) was deionized water, the column temperature was 35°C, and the injection volume was 2 μL. The gradient elution program was as follows: 0-2 min 2% B, flow rate 0.25 mL / min; 2-8 min 2-98% B, flow rate 0.25 mL / min; 8.01-11 min 98% B, flow rate 0.40 mL / min; 11.01-14 min 2% B, flow rate 0.25 mL / min. Mass spectrometry detection was performed in electrospray ionization (ESI) mode, and the spray voltage in negative ion mode was -2.5 kV. The ESI source parameters were as follows: sheath gas, auxiliary gas, and sweep gas were set to 45, 8, and 1 arbitrary units, respectively. The ion transfer tube and vaporizer temperatures were 320 and 350 °C, respectively. The data acquisition process included full mass spectrometry scan, dynamic exclusion, target mass exclusion, peak detection, and fragment ion scan. In full scan mode, the resolution was 120,000 and the scan range was 70–1000 m / z. Fragment ion scan: isolation window: 2 m / z; collision energy type: standardized; HCD collision energy: 20%, 40%, 60%, 80%; resolution: 15,000; scan range mode: automatic; AGC target: standard; number of fragment ion spectra collected: 5.

[0060] (4) Suspect screening: Based on the possible phase I and phase II metabolic transformation reactions of BPs (Table 1), a list of suspected BPs exposure markers was constructed. Among them, there are 7 phase I metabolic processes, including oxidation, reduction, hydrolysis, reductive defluorination, oxidative defluorination, etc.; there are 17 phase II metabolic processes, including acetylation, methylation, glucuronide conjugation, sulfation, glutathione conjugation, glycine binding, cysteine ​​coupling, etc. The maximum number of reactions for phase I and phase II metabolic reactions was set to 2, and the total number of reactions was set to 3. Through the combination of metabolic reactions, a list of potential exposure markers (Generate Expected Compounds) (including thousands of potential exposure markers) was formed. Compound Discoverer software was used to establish an automated screening workflow for suspected BPs exposure markers, including: peak extraction (Select Spectra), peak alignment (Align Retention Times), expected compound screening (Find Expected Compounds), expected compound combination (Group Expected Compounds), compound identification and annotation, and fragment ion search scoring (FISH Scoring). By matching the list of suspected BPs exposure markers with high-resolution spectral information, a list of molecular formulas of potential BPs exposure markers was screened out.

[0061] Table 1. List of phase I and phase II metabolic transformation reactions used to construct a list of suspected BP exposure markers

[0062]

[0063] (5) Structural analysis: using ADMET Predictor TM The energy required for the reaction of different sites of BPs was calculated using the BioTransformer 3.0 metabolic simulation software. Active reaction sites were identified based on quantum chemical models and density functional methods, thereby clarifying the potential targets of metabolic reactions. Based on this, the structural formula of BPs exposure markers was preliminarily deduced based on their molecular formulas. Subsequently, the deduced structural formula was input into the Compound Annotation Editor in the Compound Discoverer software, and the characteristic mass spectrum fragmentation pattern of the compound was automatically simulated using the fragment ion search algorithm, thereby calculating the similarity between the actual collected fragment ions and the theoretically generated fragment ions, and finally determining the structure of the exposure marker based on the matching degree (>20%). According to the metabolic transformation process, a metabolic pathway diagram of BPs was drawn. In addition, a semi-quantitative analysis was performed based on the peak area intensity of the BPs exposure marker to clarify the sensitive exposure marker.

[0064] (6) Experimental results:

[0065] Through the above steps, we identified a total of 19 BPs exposure markers, including 6 BPAF metabolic products, 7 BPAP metabolic products, and 6 BPB metabolic products. The structural confidence of most exposure markers can reach the L2 level, with FISh coverage >20%, and at least two fragment ions matched in the database with a mass deviation of <5ppm (Table 2).

[0066] Isotope distribution of parent ions and fragment ions of six BPAF exposure markers Figures 1 to 7 As shown. Among them, BPAF_M1 is the oxidation product of BPAF. In the identification analysis, its parent ion isotope peak matches 100% ( Figure 1 ) ; BPAF_M2 is the glucuronide binding product of BPAF. In the identification analysis, its parent ion isotope peak matched 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions was 36.9% ( Figure 2 ) ; BPAF_M3 is the glucuronide binding product of BPAF_M1. In the identification analysis, its parent ion isotope peak matched 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions was 33.3% ( Figure 3 ) ; BPAF_M4 is the methylation product of BPAF_M1. In the identification analysis, its parent ion isotope peak matches 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions is 20% ( Figure 4 ) BPAF_M5 is the methylation product of BPAF_M2. In the identification analysis, its parent ion isotope peak matches 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions is 18% ( Figure 5 ) BPAF_M6 is the methylation product of BPAF_M3. In the identification analysis, its parent ion isotope peak matched 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions was 45.2% ( Figure 6 Finally, based on the metabolic reaction rules of phase I and phase II, a metabolic transformation pathway diagram of BPAF in vivo was drawn ( Figure 7 )

[0067] Isotope distribution of parent ions and fragment ions of seven BPAP exposure markers Figures 8 to 15 As shown. Among them, BPAP_M1 is the glucuronide binding product of BPAP. In the identification analysis, its parent ion isotope peak matches 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions is 71.9% ( Figure 8) ; BPAP_M2-1 and BPAP_M2-2 are the glucuronide conjugation products of BPAF after oxidation. In the identification analysis, their parent ion isotope peaks matched 100%, and the matching degrees between the collected fragment ions and the theoretically generated fragment ions were 64.1% and 65.7%, respectively ( Fig. 9 and Fig.10 ) ; BPAP_M3 is the methylation product of BPAF_M1. In the identification analysis, its parent ion isotope peak matches 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions is 50.0% ( Fig.11 );BPAP_M4-1 and BPAP_M4-2 are the methylation products of BPAP_M2-1 and BPAP_M2-2, respectively. In the identification analysis, the parent ion isotope peaks matched 100%, and the matching degrees between the collected fragment ions and the theoretically generated fragment ions were 54.6% and 53.3%, respectively ( Fig.12 and Fig.13 ) ; BPAP_M5 is the product of BPAF after secondary oxidation, glucuronide binding and methylation. In the identification analysis, its parent ion isotope peak matched 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions was 61.6% ( Fig.14 ); Finally, based on the metabolic reaction rules of phase I and phase II, a metabolic transformation pathway diagram of BPAP in vivo was drawn ( Fig.15 ).

[0068] Isotope distribution spectra of parent ions and fragment ions of six BPB exposure markers Figures 16 to 22 Among them, BPB_M1 is the oxidation product of BPB. In the identification analysis, its parent ion isotope peak matches 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions is 16.7% ( Fig.16 ); BPB_M2 is the glucuronide binding product of BPB. In the identification analysis, its parent ion isotope peak matched 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions was 80.7% ( Fig.17 ); BPB_M3 is the methylation product of BPB_M2. In the identification analysis, its parent ion isotope peak matches 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions is 57.9% ( Fig.18 ); BPB_M4 is the glucuronide binding product of BPB_M1. In the identification analysis, its parent ion isotope peak matched 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions was 62.5% ( Fig.19); BPB_M5 is the methylation product of BPB_M4. In the identification analysis, its parent ion isotope peak matches 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions is 35.7% ( Fig. 20 ); BPB_M6 is the combination product of glucoside and glucuronide of BPB. In the identification analysis, its parent ion isotope peak matched 100%, and the matching degree between the collected fragment ions and the theoretically generated fragment ions was 33.3% ( Fig.21 ); Finally, based on the laws of phase I and phase II metabolic reactions, a metabolic transformation pathway diagram of BPB in vivo was drawn ( Fig. 22 ).

[0069] In summary, the 19 BPs exposure markers are shown in Table 3:

[0070] Table 3: Structural formulas of 19 BPs exposure markers

[0071]

[0072]

[0073]

[0074]

[0075] Maternal BPAF in serum accounted for 3.6% of the total exposure marker concentration ( Fig.23 ), while its phase I and II conjugates accounted for 0.4% (BPAF_M1) and 96.0%, respectively. In serum, maternal BPB accounted for 0.5% of the total exposure marker concentration, while its phase I and II conjugates accounted for 0.2% (BPB_M1) and 99.2%, respectively. In contrast, only maternal BPAP (0.9%) and II conjugates (99.1%) were found in the serum after BPAP exposure, and no phase I oxidation products were found. This shows that after BPs compounds enter the body, they can be rapidly metabolized and transformed to form II conjugates, and only a small part of the serum is maternal BPs. Therefore, if the exposure amount of BPs is assessed only by directly measuring the BPs content in serum, it may cause a deviation of 1 to 2 orders of magnitude in the exposure assessment amount. In this study, we propose that by monitoring II conjugates such as BPAF_M2, BPAP_M1, and BPB_M2, the exposure amount of BPAF, BPAP, and BPB can be better reflected.

[0076] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0077]

Claims

1. A method for screening exogenous markers of exposure to bisphenols, characterized in that The following steps are involved: (1) Animal exposure, serum acquisition and data collection: Experimental animals were gavaged with bisphenols at an exposure (dosing) concentration of 50 to 400 mg / kg / day for more than 15 days. After the exposure, the serum of the experimental animals was obtained, purified, and analytical samples were obtained. The samples were analyzed and measured using an ultra-high performance liquid chromatography tandem quadrupole / orbitrap high-resolution mass spectrometer, and several fragment ion spectra were collected. (2) Screening of potential biomarkers: Based on the possible phase I and phase II metabolic transformation reactions of bisphenols, a list of potential exposure biomarkers is formed; Compound Discoverer software is used to perform an automated screening workflow on the fragment ion spectra collected in step (1), including: peak extraction, peak alignment, expected compound screening, expected compound combination, compound identification and annotation, and fragment ion search scoring; parent ions with high fragment ion scores are matched with the list of exposure biomarkers to screen out the molecular formulas of potential bisphenol exposure biomarkers; (3) Structural analysis: Use metabolic simulation software to calculate the energy required for reactions at different sites of bisphenols, thereby identifying potential targets of metabolic reactions, and preliminarily deduce their structural formula based on the molecular formula obtained in step (2). Subsequently, input the deduced structural formula into the Compound Discoverer software, and use the fragment ion search algorithm to automatically simulate the characteristic mass spectrum fragmentation pattern of the compound, calculate the similarity between the fragment ions actually collected and the theoretically generated fragment ions, and determine the structural formula of the exposure marker if the match is greater than 20%. In addition, perform semi-quantitative analysis based on the peak area intensity of the exposure marker to identify the sensitive exposure marker.

2. The method according to claim 1, characterized in that: The bisphenol substances described in step (1) include bisphenol A and its substitutes.

3. The method according to claim 2, characterized in that: The bisphenol A substitutes include bisphenol AF, bisphenol B and bisphenol AP.

4. The method according to claim 1, characterized in that: There are 7 phase I metabolic conversion reactions in step (2), and 17 phase II metabolic conversion reactions in total; the maximum reaction times of phase I and phase II metabolic reactions are set to 2, respectively, and the total reaction times are set to 3, and a list of potential exposure markers is formed through the combination of metabolic reactions.

5. The method according to claim 1, characterized in that: The metabolic simulation software described in step (3) includes ADMET Predictor TM and BioTransformer 3.

0.

6. The method according to claim 1, characterized in that: The experimental animals described in step (1) include rodents or non-rodents.

7. The method according to claim 6, characterized in that: The rodents include rats and mice.

8. The method according to claim 1, characterized in that: The step (1) of obtaining serum from experimental animals is that after the exposure, the experimental animals are fasted for more than 12 hours, anesthetized, and subjected to abdominal incision to collect blood from the abdominal aorta, which is then centrifuged to obtain serum.

9. An exogenous marker of exposure to bisphenols, characterized in that: The structural formula is shown in the following table: The bisphenol substances include bisphenol A and its substitutes.

10. Use of the exogenous marker of bisphenol exposure according to claim 9 in monitoring body load in the human body.

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