| Acetaminophen(APAP) is an effective and widely used over-the-counter analgesic-antipyretic. The arbitrary and excessive use of APAP drugs induced hepatotoxicity is the main reason of acute liver failure(ALF) in many countries including the United States. In this paper, we used mice and silkworms as a model, representing mammal and invertebrate model animal.We found the phenomenon of liver melanization which occurred before APAP-induced ALF in mice. A melanic surface induced by APAP which was time- and dose-dependent in the silkworms was observed. In addition, an APAP-induced ALF was established using a metabolic detoxification tissue fat body which simulated mouse liver. Systematic research and parallel comparison of APAP-induced melanization biochemical mechanisms in the two animal models had done. Related results are as follows:1 APAP induced silkworms fat body and mice liver similar acute injuryThe 48 h old of the 5th instar of Bombyx mori Haoyue strain and body weight 20 g of ICR mice were orally exposed to APAP, at 0.5 h- 2 h after oral administration, the HPLC results showed that the presence of original APAP was found in circulatory blood(plasma) and the liver of mice and Similarly, the presence of original APAP was also found in the circulatory hemolymph(plasma) of silkworms and their fat bodies. And they all existed typical time- and dose- response. The contents in silkworm plasma and fat body were significantly higher than mice plasma and liver.At 16 h after oral administration of APAP, the results of tissue HE staining showed that at 150 mg/kg or more doses could induce liver cells acute necrotic injury in mice and had a significant dose response; and at 3600mg/kg dose could induce fat body cells acute necrotic injury in silkworms. Measurement of enzyme activities representing acute liver failure in plasma showed that alanine aminotransferase(ALT), aspartate aminotransferase(AST) and alkaline phosphatase(ALP) activity had a significantly increased along with liver / fat body cells acute necrotic injury in the two animal models. It demonstrated that high concentration of APAP exposure could induce silkworm fat body and mice liver similar acute necrotic injury. It indicated that silkworms fat body can be used to study the the mechanism of tissue damage which can replace the mice liver.2 Melanization is a reliable indicator of liver / fat body injuryIn order to explore mice liver and silkworms fat body injury occurred simultaneously, if there is a more convenient method of appearance inspection. The results showed that melanization occurred in mouse liver at the 600 mg/kg dose-treated group, melanization was severer within 4 h after APAP administration, after which the degree of melanization started to diminish, and at 32 h after administration the liver tissue recovered to the level of the control group, which showed a tipical time- and dose-response. No significant changes were observed in its surface color. After oral administration of APAP, silkworms fat body color did not change significantly, but the colored spots were observed on the surface, the greater the dose of APAP, the greater the degree of melanization and the earlier the melanization appeared, which showed a tipical time- and dose-response.At 1 h- 32 h after APAP administration, the melanin content of the plasma and liver in mice were investigated. The results showed that the melanin content of treatment group were significantly higher than control group, showed a time- and dose-response; After oral administration of APAP in silkworms, the activity of the rate-limiting enzyme, phenoloxidase(PO), in high dose treatment group were higher than control group at 0.5 h- 4 h after APAP administration, then gradually restored. It showed that, accompanying the liver/fat body damage, melanin accumulated in the circulatory blood and liver of mice and liver melanization was observed. Melanin synthesis increased in the circulatory hemolymph of silkworms, and melanization of silkworm body surface indicated synchronous injury to fat bodies following APAP exposure.3 Comparison of the metabolic pathways of melanin biosynthesis in mice and silkwormsAt 1 h- 8 h after APAP administration, we used HPLC method to determine the contents of Phenylalanine(Phe) and Tyrosine(Tyr) in plasma which are the melanin synthesized initial substrates. The results showed that APAP administration induced a decrease in Phe both in mice and silkworms. However, when the animals recovered, Phe levels increased, and the recovery rate of mice was faster than that of silkworms. Tyr contents first increased and then declined, however, the time to decline and the degree of decline in silkworms were significantly earlier / greater than those in mice. These results indicated that APAP induced an increase in Tyr synthesis from Phe.Analysis of Tyr downstream metabolic pathways showed that epinephrine(EPI) significantly higher than the control group in mouse plasma after 4 h of APAP treatment, indicated that APAP-induced Tyr both use to form the melanin and use to synthesis EPI. In silkworms the formation of dopa is Tyr, dopa is further metabolized to dopamine and dopa quinone, dopa content decreased, while dopamine levels increased significantly in silkworms plasma after APAP-treated, indicated that the metabolic activity of dopamine pathway which is induced by APAP was also significantly enhanced in silkworms, and finally to form melanin; while mice dopamine branch formed EPI, only dopaquinone branch ultimate synthesis of melanin. Therefore, it suggested that the use of silkworm animal model has a higher sensitivity than mice to judge the APAP-induced melanin metabolism.In conclusion, before high dose APAP induced acute liver injury in mammals, APAP significantly increased the level of melanin biosynthesis and liver melanization was accompanied occured. Melanic surface area in this invertebrate model was expected to be a sensitive appearance indicator for detecting the metabolic tissue toxicity of APAP drugs. |
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