Species Differences of Bakuchiol-induced Liver Injury in Mice Based on Transcriptomics / 中国实验方剂学杂志

ObjectiveTo explore the differences in response to bakuchiol-induced hepatotoxicity between Institute of Cancer Research （ICR） mice and Kunming （KM） mice. MethodThe objective manifestations of bakuchiol-induced hepatotoxicity in mice were confirmed by acute and subacute toxicity animal experiments， and enrichment pathways of differential genes between normal ICR mice and KM mice were compared by transcriptomics. The real-time quantitative polymerase chain reaction （real-time qPCR） assay was used to verify the mRNA expression of key genes in the related pathways to confirm the species differences of bakuchiol-induced liver injury. ResultIn the subacute toxicity experiment， compared with the normal mice， the ICR mice showed increased serum content of alkaline phosphatase （ALP）， and 5′-nucleotidase （5′-NT）， without significant difference， and no manifest change was observed in KM mice. Pathological results showed that hepatocyte hypertrophy was the main pathological feature in ICR mice and hepatocyte steatosis in KM mice. In the acute toxicity experiment， KM mice showed erect hair， mental malaise， and near-death 3 days after administration. The levels of serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in KM mice （400 mg·kg-1） significantly increased（P<0.01）， and the activity of total reactive oxygen species （SOD） in liver significantly decreased（P<0.01）compared with those in normal mice， while the serum content of 5′-NT and cholinesterase （CHE） in ICR mice （400 mg·kg-1） were significantly elevated （P<0.01）. The liver/brain ratio in ICR mice increased by 20.34% and that in KM mice increased by 29.14% （P<0.01）. The main pathological manifestation of the liver in ICR mice was hepatocyte hypertrophy， while those in KM mice were focal inflammation， hepatocyte hypertrophy， and hepatocyte steatosis. Kyoto Encyclopedia of Genes and Genomes（KEGG）and Reactome pathway enrichment analyses showed that the differential gene expression between ICR mice and KM mice was mainly involved in oxidative phosphorylation， bile secretion， bile acid and bile salts synthesis， and metabolism pathway. CYP7A1 was up-regulated in all groups with drug intervention （P<0.01） and MRP2 was reduced in all groups with drug intervention of KM mice （P<0.01） and elevated in all groups with drug intervention of ICR mice （P<0.01） compared with those in the normal group. The expression of BSEP was lowered in ICR mice with acute liver injury （400 mg·kg-1） （P<0.05）. SHP1 was highly expressed in KM mice with acute liver injury （400 mg·kg-1）. The expression of FXR was diminished in ICR mice with subacute liver injury （200 mg·kg-1） （P<0.01）. SOD1， CAT， and NFR2 significantly decreased in KM mice with acute liver injury （400 mg·kg-1）， and CAT dwindled in KM mice with subacute liver injury （200 mg·kg-1） （P<0.01）. GSTA1 and GPX1 significantly increased in KM mice with acute liver injury （400 mg·kg-1） （P<0.01） and SOD1， CAT， NRF2， and GSTA1 significantly increased in ICR mice with subacute liver injury （200 mg·kg-1） （P<0.01）. CAT and NRF2 significantly increased in ICR mice with acute liver injury （400 mg·kg-1） （P<0.01）. ConclusionWith the increase in the dosage of bakuchiol， the liver injury induced by oxidative stress in KM mice was gradually aggravated， and ICR mice showed stronger antioxidant capacity. The comparison of responses to bakuchiol-induced hepatotoxicity between ICR mice and KM mice reveals that ICR mice are more suitable for the investigation of the mechanisms related to bile secretion and bile acid metabolism in the research on bakuchiol-induced hepatotoxicity in mice. KM mice are more prone to liver injury caused by oxidative stress.

Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin / 中国中药杂志

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 μL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 μL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 μL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 μL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 μL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 μL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 μL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Comparative Study on Hepatotoxicity of Psoraleae Fructus in Rats and Mice / 中国实验方剂学杂志

Objective:Arrange long-term toxicity experiments by a uniform design method， so as to explore the effect of different extracts of Psoraleae Fructus on liver toxicity in rats and mice， and find the drug factors that cause psoralen liver toxicity. Method:Based on the factors of processing， extraction technology， dosage and treatment course， each experimental group was arranged by uniform design method. A total of 220 SD rats and 220 Kunming mice with half male and half female were divided into normal groups and drug groups 1 to 8. The corresponding drugs （50% alcohol extract of salt Psoraleae Fructus in rats 2.57 g·kg-1， mice 5.14 g·kg-1， 95% alcohol extract of Psoraleae Fructus in rats 0.51 g·kg-1， mice 1.02 g·kg-1， 70% alcohol extract of salt Psoraleae Fructus in rats 1.71 g·kg-1， mice 3.42 g·kg-1， water extract of Psoraleae Fructus in rats 1.03 g·kg-1， mice 2.06 g·kg-1， water extract of salt Psoraleae Fructus in rats 1.03 g·kg-1， mice 2.06 g·kg-1， 70% alcohol extract of Psoraleae Fructus in rats 1.71 g·kg-1， mice 3.42 g·kg-1， 95% alcohol extract of salt Psoraleae Fructus in rats 0.51 g·kg-1， mice 1.02 g·kg-1， 50% alcohol extract of Psoraleae Fructus in rats 2.57 g·kg-1， mice 5.14 g·kg-1） were administered by gavage daily. The body weight and food intake of the rats and mice were measured once a week. After the treatment course， the rats were anesthetized with sodium pentobarbital， and blood was taken from the abdominal aorta， and the mice were sacrificed by removing the eyeballs， and the liver and brain were taken to calculate the organ coefficients. Serum was taken to determine liver function-related indicators， and the liver was taken for histopathological examination by hematoxylin-eosin （HE） staining. Result:The liver visceral-brain ratio of female rats in group 3 were significantly increased （P<0.05）. The liver quality， visceral-body ratio and visceral-brain ratio of male mice in groups 1 to 3 were significantly increased （P<0.05， P<0.01）. Histopathological manifestations in mice were more obvious than those in rats. Histopathology showed hepatocyte hypertrophy in the central area of liver lobules in mice， in particular in group 3. According to the multiple regression equation， there were interactions between extraction technology， processing， dosage and treatment course， and the extraction technology was positively correlated with the pathological score of liver injury. Based on the results of visual analysis and other indicators， it is concluded that the extraction technology factor is most relevant to psoralen liver toxicity of Psoraleae Fructus. Conclusion:Psoraleae Fructus has the hepatotoxicity， which is related to ethanol extraction technology； alcohol extraction is more toxic than water extraction， and 70% ethanol extraction is the most toxic. Besides， there are species differences， with a more significant hepatotoxicity in mice than that in rats.

The effect of species difference in carboxylesterase on the drug metabolism / 国际药学研究杂志

Carboxylesterase(CES), the most abundant hydrolase in humans and animals, participates in the metabolism of various endogenous and exogenous compounds and drugs. The distribution and activity of CES have obvious species differences in hu mans and animals, which are closely related to the pharmacokinetics(PK), pharmacodynamics(PD)and toxic side effect of drugs. Therefore, this paper summarizes the effects of the difference in the classification, distribution, induction and inhibition of CES as well as the effects of species difference in plasma, liver, small intestine and skin on the metabolism of ester prodrugs, so as to provide a reference for the correct selection of experimental animals in preclinical studies of new drugs to accurately predict the clinical PK, PD and toxicological effects.

Role of carboxylesterase in drug metabolism / 药学学报

Carboxylesterases (CESs) belong to the esterase family, which are mainly responsible for catalyzing metabolism of a variety of drug as well as endogenous and exogenous compounds. CESs are widely distributed in the body, mainly expressed in lung, liver, intestine, kidney, skin epithelial cells, etc. There are significant species differences in the expression of CESs, which results in the difference on the drug metabolism with genetic polymorphism. In this paper, an overview of the classification and distribution, physiological function and mechanism, species differences and gene polymorphism of CESs are provided for the research of CESs and drug design.

Pharmacokinetic characteristics of YZG-331, a promising sedative-hypnotic candidate agent / 药学学报

The purpose of this article was to study the pharmacokinetic characteristics of YZG-331, plasma protein binding and metabolic stability in vivo and in vitro. Plasma and tissue concentrations of YZG-331 were determined in mice and rats after administration by LC-MS/MS analysis orally or intravenously. The plasma protein binding of YZG-331 with human, dog, monkey, rat and mouse were measured by ultrafiltration method. The stability of YZG-331 in animal and human plasma, liver microsomes, intestinal bacteria and artificial gastrointestinal fluid was also investigated in vitro. The results show that YZG-331 was absorbed rapidly in both mice and rats after oral administration, while the absorption and elimination saturation YZG-331 were also observed. The bioavailability of YZG-331 was much higher in male mice (51.2%) than that in female mice (27.7%), however, the bioavailability in male rats (27.1%) was lower than that in female rats (78.7%). YZG-331 was widely distributed in different tissues of mice, especially in certain regains of brain, including thalamus, hippocampi, cortical and striatal. YZG-331 was found to bind to human, dog, monkey, rat and mouse plasma protein in vitro (93.3%-98.9%) without significant concentration dependences and species differences. YZG-331 was stable in animal and human plasma, simulated gastric/intestinal fluid and liver microsomal incubations, except rat liver microsomes and intestinal flora. Therefore, we concluded that:the pharmacokinetics of YZG-331 in mice and rats have gender and species differences; YZG-331 was widely distributed in vivo including brain, the targets of the agent; YZG-331 had a high affinity to plasma protein and was metabolized by rat liver microsomes and intestinal flora.

Species difference of T-2 toxin metabolism in liver microsomes by high performance liquid chromatography-tandem mass spectrometry / 中国药理学与毒理学杂志

OBJECTIVE To compare the species difference of T-2 toxin metabolism in liver micro-somes of different animals. METHODS T-2 toxin was incubated with liver microsomes from mice, rats,Beagle dogs, monkeys and humans, respectively, at 37℃ for some time. Then, the incubation liquid was detected by high liquid chromatography-mass spectrometry method after albumen precipitation. RESULTS The half-life (t1/2) of T-2 toxin was less than 1 min, 2-4 min in mouse and monkey liver microsomes, 13 min in dog liver microsomes, and 39 min in rat liver microsomes. The hepatic clear-ance (Clh) of T-2 toxin was divided into three groups among the five species of animals:humans, dogs and rats were in one group, monkeys a second group, and mice in another group. The Clh of mouse group was 3-4 times that of the human, dog and rat group. The affinity to T-2 toxin was different between the liver microsomes of these five species. The affinity of mouse liver microsomes was the strongest, followed by that of humans, dogs, rats and monkeys. The enzyme transfer rate of T-2 toxin was the highest in monkey liver microsomes followed by that of rats and dogs. It was one million times higher in monkey liver microsomes than in human and mouse liver microsomes. The major metabolites were 3′-hydroxyl-T-2 toxin and neosolaniol. T-2 triol and HT-2 toxins were the major metabolites in human and rat liver microsomes. HT-2 toxin and 3′-OH-T-2 toxin were the dominating metabolites in dog liver microsomes and T-2 triol and 3′-OH-T-2 toxin in mouse liver microsomes. T-2 toxin metabolited by hydrolysis effect in mouse, rat, dog and human liver microsomes, but through hydroxylation in monkey liver microsomes. CONCLUSION There are species differences in metabolic parameters, metabolites, amounts of metabolites, metabolic pathways of T-2 toxin in mouse, rat, dog, monkey and human liver microsomes.

In vitro metabolism of liquiritigenin in liver microsomes of different species / 中草药

Objective: To compare in vitro metabolic differences of liquiritigenin, an aglycone of liquiritin, among liver microsomes of different species, which would provide reference for further research and development of liquiritin. Methods: Metabolic stability and metabolic biotransformation were investigated after liquiritigenin was incubated with rat, mouse, human, dog, and monkey liver microsomes. Metabolic stability was evaluated using a substrate depletion approach, and the results were reported as "% liquiritigenin remaining", which was then used to calculate the in vitro half-life (t1/2). Metabolic biotransformation was characterized by metabolite profiling. Results: In liver microsomal incubation systems of five species, the t1/2 values of liquiritigenin for phase I were as follows: rat < mouse < human < monkey < dog, whereas for phase II, the metabolic rates were all fast and the remaining of liquiritigenin were all below 50% in 5 min except mouse. In addition, phase II metabolite profiling in monkey liver microsomes was identical to that of human, but marked differences were found between other species and human. Conclusion: The metabolic characteristics of liquiritigenin in monkey liver microsomes is most similar to that of human, then followed by dog, and marked differences existed between rat, mouse and human. Therefore, monkey or dog could be the animal model for further preclinical pharmacokinetic and toxicological studies.

In vitro comparison of thienorphine metabolism in liver microsomes of human,Beagle dog and rat / 药学学报

The inter-species differences of thienorphine metabolism were investigated in human, Beagle dog and rat liver microsomes, by comparing enzyme kinetics of the parent drug and the formation of its major metabolites. The incubation systems of thienorphine with liver microsomes of the three species were optimized in terms of thienorphine concentration, microsomal protein content and incubation time. The concentrations of thienorphine and its metabolites in incubates were measured by a LC-MS/MS method. The biotransformation of thienorphine by human liver microsomes was the lowest among the three species. The Km, Vmax, CLint and T1/2 of thienorphine obtained from human liver microsomes were (4.00 ? 0.59) ?mol?L-1, (0.21 ? 0.06) ?mol?L-1?min-1, (117 ? 3.19) mL?min-1?kg-1 and (223 ? 6.10) min, respectively. The corresponding kinetic parameters for dog and rat liver microsomes were (3.57 ? 0.69) and (3.28 ? 0.50) ?mol?L-1, (0.18 ? 0.04) and (0.14 ? 0.04) ?mol?L-1?min-1, (213 ? 1.06) and (527 ? 7.79) mL?min-1?kg-1, (244 ? 1.21) and (70.7 ? 1.05) min, respectively. A total of six phase I metabolites were observed in liver microsomes, including one N-dealkylated metabolite, three oxidative metabolites and two N-dealkylated oxidation metabolites. All these six metabolites were detected in the liver microsomes of the three species. However, the relative amounts of the metabolites generated were different in three species. The results indicated that the major phase I metabolic pathway of thienorphine was similar in the liver microsomes from all three species. However, the inter-species differencesobserved were relative amounts of the metabolites as well as the metabolic characteristics of thienorphine in liver microsomal incubates.