| Objective:To explore the potential developmental toxicity and possible toxicity mechanism of Pinellia Rhizoma (BX) and its processed product Pinelliae Rhizoma Praeparatum Cum Zingibere et Alumina (JBX), an metabonomics approach was used to evaluate the metabolic perturbation in pregnant rats and Be Wo cells caused by BX and JBX.Methords:In vivo study to evaluate the potential developmental toxicity of BX and JBX:Maternal plasma, placenta and amniotic fluid samples were collected from control Sprague-Dawley pregnant rats and exposed to BX suspension and JBX decoction. Samples were analyzed using UPLC-LTQ-Orbitrap MS and GC-MS. The acquired MS data of above samples were further subjected to multivariate data analysis such as OPLS-DA, and the significantly altered metabolites were identified. The associated pathways were constructed using MetaboAnalyst 3.0.In vitro study to evaluate the potential developmental toxicity of BX and JBX:Be Wo cell lines were selected to establish the in vitro placenta model. The intracellular metabolites were extracted after exposed with normal saline, BX and JBX solution for 0,6,12,24 and 36 h, respectively. The intracellular metabolic profiling was acquired by GC-MS and further subjected to multivariate data analysis such as OPLS-DA. The significantly altered metabolites were identified and the associated pathways were constructed using Metabo Analyst 3.0.Results:In vivo study to evaluate the potential developmental toxicity of BX and JBX:1. The maternal weight gain were significantly decreased on GD 9,12,15 in BX group and on GD 15 in JBX group when compared with control group. However, the food consumption, weight of fetuses and placentas were not significantly altered.2. Serum biochemical parameters including ALT, AST, CK, BUN and sCr were not significanltly altered when compared with controm group.3. The histopathology of the placenta, liver, kidney from each group of rats had no definite difference. On the other hand, BX induced myocardial degeneration with mild vacuolation of cardiomyocytes in some rats and JBX induced a minimal vacuolation change.4. Upper and lower phases extract of maternal plasma metabolic profiling pattern analysis using OPLS-DA models exhibited clear separation for each group, which indicated the metabolic profiling in BX and JBX were significantly changed when compared with control group. Thirty-three significantly altered metabolites were screened and thirteen of them were identified as potential biomarkers. Ptnitic acid, sphinganine, phytanic acid, Cer(dl 8:0/18:0) in JBX group, LysoPC(0:0/18:0), PE(20:0/182) in BX group and LysoPE(20:0/0:0), glycine, lysine, myo-inositol in two experimental group were significalty decreased when compared with control group. On ther other hand, SM(d18:0/18:1) in JBX group and tryptophan in BX group were significantly increased.5. AF metabolic profiling pattern analysis using OPLS-DA model exhibited clear separation between each group, which indicated the metabolic profiling in BX and JBX were significantly changed when compared with control group. Nine significantly altered metabolites were screened and seven of them were identified as potential biomarkers. β-hydroxybutyric acid, succinic acid in JBX group, orthinine, glutamine in BX group and glycine, lysine in two experimental group were significalty decreased when compared with control group. On ther other hand, tryptophan in JBX group was significantly increased.6. Upper and bwer phases extract of placenta metabolic profiling pattern analysis using OPLS-DA models exhibited clear separation for each group, which indicated the metabolic profiling in BX and JBX were significantly changed when compared with control group. Thirty-four significantly altered metabolites were screened and fourteen of them were identified as potential biomarkers. Cer(dl8:0/18:0) in JBX group, PE(16:0/182), PE(P-18:1/183), PE(20:4/P-18:1), PC(36:5), PC(363), PC(O-160/182), PC(32:1), PC(42:1) in BX group and DG(38:4), lysine in two experimental group were significalty decreased when compared with control group. On ther other hand, N-acetylglutamine in two experimental group were significantly increased.7. The alterations of above identified metabolites indicated a perturbation in Glyoxylate and dicarboxylate metabolism, Glycine, serine and threonine metabolism, Glycerophospholipid metabolism, Alanine, aspartate and glutamate metabolism, Sphingolipid metabolism, Inositol phosphate metabolism, Arginine and proline metabolism in pregnant rats and fetuses exposed to BX and JBX.In vitro study to evaluate the potential developmental toxicity of BX and JBX:1. Be Wo cells metabolic profiling pattern analysis using OPLS-DA models exhibited similar time-dependent changes in all three groups. In general, the samples in JBX group were closer to that of control group when compared with BX group. As in each time point, JBX group was more clearly separated with control group at 6 h and 36 h while BX group was more clearly separated wih control group at 24 h. The separation indicated the metabolic profiling in BX and JBX were significantly changed when compared with control group. Twenty-six significantly altered metabolites were screened and nine of them were identified as potential biomarkers. Glycine, glucose, galactose, stearic acid, myo-inositol were significantly decreased and citric acid, cholesterol were significantly increased at most of above time points. Interestingly, amino malonic acid and proline were only significantly alterd (decreased) at 6h. Among all these potential biomarkers, most of them were significantly altered in BX group when compared with that of JBX group. The alterations of above identified metabolites indicated a perturbation in Glycine, serine and threonine metabolism, Inositol phosphate metabolism, Arginine and proline metabolism in BeWo cells exposed to BX and JBX.Conclusion:1. LC-MS and GC-MS platform adopted by this research can well profiling the samples of plasma, amniotic fluid, placenta and Be Wo cells, which also capable of differentiate and explaining the metabolic characterization after exposed to BX and JBX.2. Perturbation of amino acids metabolism and lipids metabolism are mainly induced in pregnant rats and fetuses after exposed to BX and JBX.3. BeWo cells intervented with BX and JBX are characterized by amino acids metabolism disorder.4. The metabonomics approach based on GC-MS and LC-MS reveal the potential developmental toxicity of BX and JBX at an earlier time than the general observation, serum biochemistry and histophathology methods. Moreover, it preliminary reveals BX and JBX may induce developmental toxicity through the purterbation of intermediary metabolism of amino acids and lipids.5. The processed product, JBX, has lesser developmental risk than BX in the in vivo SD rat model and in vitro BeWo cell model. |
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