Metabolic Transformation And Detection Of Aflatoxin B₁ And Cyclopiazonic Acid Toxin Based On Liver Microsome Models

In recent years,there have been numerous food safety problems caused by mycotoxin contamination,posing a great threat to human health.Cyclopiazonic acid（CPA）,an emerging mycotoxin,has been of interest and studied because of its co-exposure pathway with the highly toxic aflatoxin B₁（AFB₁）.Their combined action has been reported to cause synergistic toxicity to the human liver.Therefore,it is necessary to systematically investigate the toxicological mechanisms of AFB₁ and CPA in the liver,of which the metabolic transformation of AFB₁ and CPA in the liver is an important part to clarify the toxicological mechanisms of both.Currently,the main models for studying the metabolic transformation of fungal toxins in the liver include animal models and cellular models,each of which has its own advantages as well as its own shortcomings.As an in vitro model of liver metabolism,liver microsomes have shown advantages over other models in terms of simplicity of operation and short experimental cycle time.Therefore,in this thesis,various liver microsomal models were constructed and optimized,and their reliability and advantages were investigated for the study of the metabolic transformation mechanism of fungal toxins using AFB₁ as the target;subsequently,they were used to study the metabolic transformation and toxicological mechanism of the emerging toxin CPA.Finally,based on the mechanism that liver microsomal models require external electrons for metabolic reactions,we combined liver microsomes with electrochemistry to investigate and construct an electrochemical bioreactor that can be used for the metabolic detection of AFB₁and CPA.The main studies include:1.Liver microsomal models were constructed and applied to the metabolism of the fungal toxin AFB₁.Four liver microsomal models were constructed using human,rat,mouse and chicken liver microsomes by optimizing the metabolic incubation time and the amount of liver microsomes incubated.The reliability and advantages of the constructed liver microsomal models were investigated in terms of metabolites,metabolic pathways and differences in metabolic species,using AFB₁ as the object of study.The results showed that AFB₁ was metabolized by the four liver microsomal models to produce six metabolites,namely,the reduction product M-1(C₁₇H₁₄O₆),the dehydrogenation product M-2(C₁₇H₁₀O₆),the hydroxylation product M-3(C₁₇H₁₂O₇),the hydroxylation product M-4(C₁₇H₁₄O₇),the hydroxylation product M-5(C₁₆H₁₀O₆)and the glucuronide-binding product M-6(C₂₃H₂₀O₁₃),of which products M-2 and M-6 were found for the first time.In addition,rat liver microsomes were found to be the most efficient metabolizers of AFB₁,while mouse liver microsomes were found to be less efficient in metabolizing AFB₁.In conclusion,the liver microsomal model better simulated the physiological environment of human and animal livers and realized the multi-pathway metabolism of AFB₁,while its simple operation and short experimental cycle can be used for subsequent metabolic transformation studies of mycotoxins.2.The metabolic transformation of the emerging toxin CPA in human and animal livers was investigated based on the four liver microsomal models constructed above.The results showed that CPA produced four metabolites after incubation in each of the four liver microsomal models,namely,dehydrogenation product C-1(C₂₀H₁₈N₂O₃),hydroxylation product C-2(C₂₀H₂₀N₂O₄),methylation product C-3(C₂₁H₂₂N₂O₃)and glucuronide-binding product C-4(C₂₆H₂₈N₂O₁₀).Species metabolic differences analysis showed that rat liver microsomes metabolized CPA most strongly,followed by human liver microsomes,and mouse and chicken liver microsomes were the weakest.On this basis,seven major cytochrome P450enzymes（CYP450）were selected from human liver microsomes to further investigate the specific enzyme types of the emerging toxin CPA in human liver microsomes.The results showed that the metabolites produced by the different enzyme types differed significantly.The dehydrogenation product C-1 was only detected in the incubation system with CYP3A4 enzyme.The hydroxylation product C-2 and the methylation product C-3 were detected in the CYP3A4,1A2,2D6,2E1,2A6 and 2C19 incubation systems,with the CYP3A4 enzyme being the most metabolizable.Therefore,on reflection,it is hypothesized that CYP3A4 enzymes are the main enzyme types that metabolize CPA in human liver microsomes.3.A"Cocktail"probe drug method based on human liver microsomes was developed to investigate the toxicological mechanisms of the emerging toxin CPA.This section investigates the effect of CPA on the activity of four major CYP450 enzymes（CYP3A4,1A2,2D6,2A6）in human liver microsomes from the perspective of metabolic interactions.Firstly,a"Cocktail"drug-probe assay was constructed to evaluate the activity of CYP450 enzymes,and the accuracy and reliability of the assay was confirmed by various methods such as specificity and precision.The effect of CPA on the activity of four CYP450 enzymes was then investigated based on the constructed probe drug method.The results showed that CPA had a moderate inhibitory effect on CYP3A4 enzymes in human liver microsomes(IC₅₀ value of 8.658μM)and no inhibitory effect on CYP2D6,CYP1A2 and CYP2A6 enzymes.As CYP3A4 enzymes are one of the major metabolic enzyme types in human liver,the moderate inhibition of CYP3A4 enzyme activity by CPA may lead to metabolic disturbances in the liver,which may be one of the important toxicological mechanisms of CPA.4.Exploring the construction of a liver microsomal electrochemical bioreactor for the metabolic detection of the fungal toxins AFB₁ and CPA.This section combines liver microsomes with electrochemistry based on the mechanism that the liver microsome model requires external electrons for metabolic reactions to occur.Firstly,Au@MXene nanocomposites were investigated and used to adsorb rat liver microsomes（RLM）to construct a liver microsomal electrochemical bioreactor.The electrodes were used to provide electrons for the metabolism of the liver microsomal model at a negative potential.The results showed that good electron transfer between the liver microsomes and the electrode was achieved and that the Au@MXene nanocomposite significantly improved the catalytic performance of the electrochemical bioreactor.The constructed liver microsomes electrochemical bioreactor successfully catalyzed the hydroxylation of the fungal toxins AFB₁ and CPA to their hydroxylated products under the amperometric current method（I-t）.Based on this metabolic pathway,we established a proportional relationship between the catalytic current and the substrate concentration to achieve the metabolic detection of the substrates:AFB₁ was linear in the range of 0.01μM to 50μM with a lower line of detection of 2.8 n M;CPA was linear in the range of 0.1μM to 100μM with a lower line of detection of 0.02μM.In addition,the constructed liver microsomal electrochemical bioreactor showed good stability and accuracy.In summary,this thesis constructed and optimized four liver microsomal models,systematically investigated the metabolic transformation of AFB₁ and CPA in human and animal livers,and explored the effects of CPA on the activities of the four major CYP450enzymes from the perspective of metabolic interactions,providing data to support the study of the toxicological mechanisms of CPA.In addition,the constructed electrochemical bioreactor for liver microsomes provides a new method for rapid and accurate metabolic detection of AFB₁and CPA.