Studies On The Invertebrate Bombyx Mori As Alternatives To Hepatotoxic Animal Model

Alternative methods of experimental animal(AMEA) have been widely applied in the field of life science research. The silkworm(Bombyx mori), a lepidopteran model insect, has been domesticated for thousands of years. We have accumulated systematic data on silkworm physiology, pathology and toxicology. Due to the innovation in genetic mutation resources of silkworm, as well as their similarities with mammals in basic life system, metabolism, energy metabolism and inheritance patterns,silkworm has been gradually developed as an experimental animal model. However, there are still several bottlenecks needed to be overcome before silkworm can be used as a standard laboratory animal:1) How to establish standard operating procedures and facilities, systematically control genetic factors and growth environment, and finally obtain silkworm that can be used as laboratory animals? 2) How to choose the preferred alternative methods according to the biological characteristics of silkworm? In addition, systematic studies are still not available in the world to compare the commonalities and distinctions in the pharmacokinetics of exogenous compounds between silkworm and classical model animals such as mice.In order to establish standardization for experimental animalization of silkworm, we summarized the operating procedures for silkworm breeding, raising management and facilities management, and designed a standardized Micro-Barrier System for silkworm rearing. The acute toxicity grading and pharmacokinetic properties of hepatotoxic drug Acetaminophen(N-Acetyl-p-Aminophenol; APAP) was also systematically compared between silkworm and mammals. To revealing APAP’s biological effects in vivo, we studied the roles of APAP in genes expression and endogenous metabolite levels in silkworm by high-throughput RNA-Seq and metabolomics analysis. We then analyzed the activity of critical enzymes catalyzing phases I and II reaction after APAP exposure, and further studied the differences of their evolutionary and active sites between silkworm and mammals such as mice by bioinformatics method. The main results are given as follows:1. The acute toxicity grading and pharmacokinetic properties of hepatotoxic drug APAP in silkworm could establish parallel data for alternatives of experimental animal.Acute toxicity assay revealed that the toxic sensitivity of the silkworms to APAP is different, depending on the strain and gender: the Dazao strain is more sensitive than Haoyue, while female Dazao silkworm is more sensitive to APAP than males. The LD50 of APAP in female and male Dazao silkworms is2017±254 μg/g and 2169±300 μg/g, respectively. The grading of acute toxicity of APAP in silkworm is among low- to mid- level, which is similar to that in mammals.Pharmacokinetic analysis suggested that the pharmacokinetics of APAP at lower dose(600μg/g)and higher dose(3600 μg/g) presented an open one compartment model in silkworm: 1) their elimination half-life(t1/2) were 1.06 h and 2.77 h, respectively; 2) the time-to-peak concentrations(tmax)of original drugs were 0.50 h and 1.00 h, respectively; 3) the peak concentrations(Cmax) of original drugs were 67.69 μg /ml and 568.73 μg /ml, respectively. Their relative bioavailabilities(AUC0-t) were222.63 h?μg /ml and 1976.32 h?μg /ml, respectively. N-acetyl-p-benzoquinone imine(NAPQI) was presented in the metabolic intermediates. Compared with mammals, the drug absorption and distribution,metabolic intermediates, as well as overall kinetics(PK) were similar in silkworm, suggesting that they are suitable for establishing parallel data for alternatives of experimental animal.2. The biological effects of APAP to endogenous metabolites were similar between silkworm and mammals.Metabolomic analysis was performed 8 h after oral administration of 600 μg/g of APAP, and revealed that the metabolites in the circulating blood enriched in the citric acid cycle-, glycolysis-, amino acid metabolism-, melanogenesis-related pathways. These results suggested that APAP-induced toxicity pathways resulted from the drug metabolism-produced oxidative stress, which further affected the energy supply, intracellular transport and signal transduction.Through analysis of metabolite variance, we found APAP induced a change of amino acid metabolism in silkworm hemolymph. We deduced that the abnormal metabolisms of endogenous metabolites such as animo acid, malic acid, succinic acid and fumaric acid are associated with oxidative stress due to APAP exposure. In particular, significant increased tyrosine, dopa and dopamine confirmed that overdose of APAP would lead to the generation of oxidative stress. APAP exposure also induced an increase in trehalose, suggesting that trehalose may play a role under adversity stress. The level of cholesterol decreased after APAP treatment, while β-β-hydroxy methyl glutaric acid was induced by APAP. These results suggested that APAP exposure to silkworm led to abnormal cholesterol synthesis,disturbed the material supply for steroid hormone synthesis, and finally resulted in body’s hormonal disorder.3. The pattern drug of hepatotoxicity APAP induced comparable pharmacological effects and toxicity mechanisms in silkworm and mammals.RNA-Seq revealed abundant differentially expressed genes between APAP–administrated and control groups. Based on the KEGG analysis, we found that a number of differentially expressed genes involved in metabolism were highly enriched in pathways producing high energy such as the citric acid cycle, oxidative phosphorylation, fatty acid oxidation, glycolysis and gluconeogenesis.These results further confirmed, from the m RNA expression level, that APAP-induced injuries to silkworm were mainly mediated through oxidative stress and energy metabolism, material transport, signal transduction actions, which is consistent with the mechanisms of APAP-induced liver injury in mammals.KEGG analysis also revealed an enrichment of differentially expressed genes in pathways involved in the circulatory system such as cardiac muscle contraction, vascular smooth muscle contraction and regulation of the actin cytoskeleton, and some pathways at synapses. These analysis results are consistent with that obtained by metabolomics assay. Thus, we speculated APAP processes consistent pharmacological effects with other non-steroidal anti-inflammatory drugs, through inhibiting arachidonic acid pathway, in both silkworm and mammals.4. The key enymes for detoxification have common active sites and mechanisms of protein-protein interaction in both Silkworms and mice.By the method of bioinformatics, we found that: 1) most members of glutathione S-transferase(GSTs)family in both silkworms and mice have GST active regions and thioredoxin fold patterns; 2) the catalytic subunits(GCLC) of both silkworm and mouse glutamate cysteine ligases have glutathione synthase domains, while both glutamate cysteine ligases(GCLM) have NADP-dependent oxidoreductase domains; 3) the silkworm peroxidase-1 protein encoded by Jafrac1 gene, a member of thioredoxin peroxidase family(TPXs), shares a highly conserved structure with mouse Pedx1- and Prdx2-encoded peroxidases, while the predicated silkworm proteins LOC733003, LOC101735759 and LOC732921 have the conserved domains of mouse Prdx3-, Prdx4- and Prdx5-encoded proteins,respectively; 4) two members of silkworm glucuronosyltransferase family(UGTs) contain the uridine diphosphate- glucuronide and uridine diphosphate- glucuronosyltransferase enzyme- functional domains, which are conserved in 21 mouse UGTs; 5) Cytochrome P450 enzymes(CYP450) in both silkworms and mice also share the conserved structural and functional domains. The conservation of structural and functional domains of critical enzymes involved in drug metabolism suggested that there may be common features, between silkworms and mice, in the main detoxification process and functional mechanism.5. Silkworm midgut is the “first pass effect” organ for establishing the liver toxicity model through oral administration.According to the distribution of original drug(APAP) and intermediate product(NAPQI) in silkworm tissues, as well as changes of the activity of critical enzymes(GST, GCL, TPX, UGT and CYP450)involved in phase I and II reaction after oral administration, we found that: 1) intermediate products were significantly increased in the midgut; 2) the intestinal absorption was obviously inhibited by high dose of APAP; 3) CYP450 enzyme activity was significantly changed in the midgut from silkworm,while the background activity of GST, GCL, TPX and UGT was noticeably higher in midgut than that in fat body. The obvious changes of APAP-related metabolic enzymes in midgut suggested that midgut is the organ of “first pass effect” after oral administration in silkworm. In the oral route of administration,silkworm midgut, as the “first pass effect” organ of exogenous drugs, is more accurate than the fat body when performing analogy to the detoxification organ liver in mammals. Silkworm gut is more reasonable target organ(tissue) model to study the toxicity mechanism of mammalian hepatotoxic drugs.6. ConclusionsThe basic conditions for silkworm to be used as laboratory animals have matured, and are sufficient to establish a systematic and standardized operating procedure for laboratory animal breeding and management, as well as the environmental control. Silkworm has comparable dynamic characteristics of drug metabolism with mouse to the pattern drug APAP. The metabolic activity changes of key enzymes in phase I and II detoxification reaction as well as the mechanisms are similar in both silkworms and mice. The midgut injury model using silkworm as experimental animal can replace the liver injury model in mammal, and could be used to study mechanisms of mammalian liver damage and detoxification.