馬兜鈴酸具腎毒性，過去傳統中藥裏常使用的馬兜鈴酸屬的植物含有此物質。本實驗研究馬兜鈴酸標準品、馬兜鈴草果實及含馬兜鈴草的中藥複方補肺阿膠湯3種物質在囓齒類動物上的腎毒性，並以代謝體學方法分析尿液核磁共振氫譜在馬兜鈴酸腎病變中的變化。實驗目的在初步探討如何以容易取得之尿液來建立腎毒性之診斷模式。實驗共分4部份進行，一為大鼠以胃灌食馬兜鈴酸標準品測試。二為小鼠以胃灌食馬兜鈴酸標準品測試。三為小鼠以胃灌食馬兜鈴草果粉末測試。四為小鼠以胃灌食補肺阿膠湯粉末測試。實驗收集每日尿液樣本並冷凍保存以備分析，實驗結束後犧牲鼠隻取其肝腎做病理分析。果：大鼠馬兜鈴酸標準品給藥5劑後腎病理檢查顯示高劑量組有1-3度的急性腎小管間質病變。小鼠馬兜鈴酸標準品給藥10劑後，高低劑量組皆有3-4度的急性腎小管間質病變。小鼠馬兜鈴草給藥21劑後，低及中劑量組有1-2度而高劑量組有3-4度之急性腎小管間質病變。小鼠補肺阿膠湯給藥20劑後，低劑量組有2度而高劑量組有3-4度之急性腎小管間質病變。尿液核磁共振氫譜以主成份分析法分析各實驗中各個組別在同一天之分數圖，結果發現大鼠馬兜鈴酸標準品給藥後2天3組皆能分群。小鼠馬兜鈴酸標準品給藥後8天高劑量組能與其他2組分群，10天時給藥的2組能與對照組分群。小鼠馬兜鈴草給藥後8天高劑量組能與其他3組分群，10天時高劑量與中劑量組能與對照及低劑量組分群。小鼠補肺阿膠湯各組則至給藥後16天仍未見分群。以t檢定比對給藥組與對照組尿液中內生代謝物隨時間相對濃度變化發現大鼠馬兜鈴酸標準品實驗早期時甘氨酸、琥珀酸、氧化三甲胺及2-酮戊二酸有下降的現象，而到晚期時糖類、尿囊素、肌酐、二甲基甘胺酸、2-酮戊二酸及氧化三甲胺有上升現象(p < 0.05)。小鼠馬兜鈴酸酸標準品實驗之代謝物濃度有明顯變化者相對較少，但在實驗後期可見給藥組有丙氨酸、乳酸及甲酸平均濃度相對增加之現象。論：馬兜鈴酸標準品及含有此物質的中草藥馬兜鈴和其複方補肺阿膠湯在鼠類是有腎毒性的。以主成份分析尿液的核磁共振氫譜可以分辨出有發生腎病變的組別，其代謝體的濃度變化也可進一步分析找出相關的生物標誌。但在含低劑量馬兜鈴酸的複方補肺阿膠湯在本實驗中則未能以主成份分析法從尿液分析出小鼠發生的腎病變，這有待進一步的代謝體學分析研究。Aristolochic acid (AA) is a potent nephrotoxic agent that can be found in several herbs of the genus Aristolochia. It was once commonly used in traditional Chinese medicine remedy. In this study, we applied our metabolomics platform of the 1H NMR spectroscopy on urine with principal component analysis (PCA) to detect and further dissect the nephrotoxicity in rodent of four AA containing materials, AA standard, Madouling, an AA containing herb Aristolochia contorta and Bu-Fei-A-Jiau-Tang (BFAJT), a herb compound containing Aristolochia contorta. The aim was to use the easy available urine samples to establish a model for early diagnosis of nephrotoxicity. The experiment was divided into four parts. The first was rat AA experiment. The second was mouse AA experiment. The third was mouse Madouling experiment. The fourth was mouse BFAJT experiment. Urine samples were collected daily and freeze-dried for storage. All animals were euthanized after experiment and their kidneys and livers procured for pathological studies.esults: In the rat AA experiment, pathology showed grade (gr.) 1-3 acute renal tubulointerstitial necrotic change (ATIN) after 5 doses in the high dose group. In the mouse AA experiment, 10 doses were given. Both high and low dose group showed gr. 3-4 ATIN. In the mouse Madouling experiment, 21 doses were given. It was gr. 1-2 for low and moderate groups, and gr. 3-4 ATIN for the high dose group. In the BFAJT experiment, 20 doses were given. It was gr. 2 ATIN in the low dose group and gr. 3-4 in the high dose group. PCA scoring plots for the urine NMR spectral chemical shifts variables showed early cluster at 2 day and later for each group in the rat AA experiment. In the mouse AA experiment, the high dose group was clustered from the other 2 groups at day 8. It was at day 10 that the 2 dose groups were clustered from the control group. In the mouse Madouling experiment, the high dose group clustered from all other 3 groups. It was at day 10 that the high dose and the moderate dose groups clustered together from the other 2 groups. In the BFAJT experiment, PCA scoring plots failed to classify across control and dose groups even at day 16.he endogenous metabolites assigned from NMR spectroscopy and their integral concentration among dose and control groups were tested by t-test. In the rat AA experiment glycine, succinate, 2-oxoglutarate and trimethylamine-N-oxide (TMAO) were decreased in the dose groups in earlier days of the experiment. At later days, sugar, allantoin, creatine, dimethylglycine, 2-oxoglutarate and TMAO were increased in the dose groups (p < 0.05). In the mouse AA experiment no significant metabolite difference was noted, only alanine, lactate and formate had more than two fold change at day 10.n conclusion, AA standard, Madouling and BFAJT were all nephrotoxic. PCA scoring plots for the urine NMR spectroscopy collected from living animals showed the ability to classify the dose and control groups days before confirming the renal pathology. But toxicity classifying failed in the mouse BFAJT experiment by PCA scoring plot. Further metabolomics study is expected to resolve this issue.致謝 ii要 iiibstract vontents viiiist of Figures xiist of Tables xxiihapter 1. Background and Literature Review 1.1 Introduction 1.2 Literature Review 1.2.1 Aristolochic acid nephropathy 1.2.1.1 Aristolochia herbs 1.2.1.2 Toxicology Related to Aristolochic Acids 3.2.1.3 Evaluation of Aristolochic Acid Nephropathy 5.2.2 Metabolomics 7.2.2.1 Definition and Technologies used 7.2.2.1.1 NMR Spectroscopy 9.2.2.2 Metabolomics in Toxicological Studies 11.2.3 Chemometrics and Pattern Recognition Technology 13.2.3.1 Data Processing of NMR Spectroscopy 13.2.3.2 Unsupervised Pattern Recognition 15.2.3.2.1 Principal Component Analysis 15.2.3.3 Metabolites Assignment for NMR Spectroscopy 16.3 Aim 17hapter 2. Materials and Methods 19.1 Aristolochic Acid Sources 19.2 In vivo Experiments and Sample Preparation 19.2.1 Rat 20.2.2 Mouse 21.3 NMR Measurement and Pattern Recognition Investigation 23.3.1 NMR Spectra Acquisition 23.3.2 Spectral Data Processing 23.3.3 Statistical Methods 24.3.3.1 PCA 24.4 Metabolite Assignment 25hapter 3. Results 26.1 In vivo Experiment 26.1.1 Rat on AA 26.1.1.1 Clinical Signs and Biochemical Parameters 26.1.1.2 Pathology 26.1.1.3 Metabolomic Profiling 26.1.2 Mouse on AA 27.1.2.1 Clinical Signs and Biochemical Parameters 28.1.2.2 Pathology 28.1.2.3 Metabolomic Profiling 28.1.3 Mouse on Madouling 29.1.3.1 Clinical Signs and Biochemical Parameters 30.1.3.2 Pathology 30.1.3.3 Metabolomic Profiling 30.1.4 Mouse on Bu-Fei-A-Jiau-Tang 31.1.4.1 Clinical Signs and Biochemical Parameters 31.1.4.2 Pathology 32.1.4.3 Metabolomic Profiling 32hapter 4. Discussion 34hapter 5. Conclusion and Future Work 43ppendix 83ibliography 88ist of Figuresigure 1. Chemical structure of aristolochic acid I (AA-I) and II (AA-II). 44igure 2. Cascade of "-omics". Metabolomics studies the end point of biological complex system. 44igure 3. Illustrations of principles of nuclear magnetic resonance. The moment μ comes from the momentum of a proton spin (a). When there is an external magnetic field B0, a Larmor precession (ωL) occurs (b). Energy status of protons are divided into two energy level under external magnetic field (c). A radio frequency pulse (RF+) along the y axis will rotate the internal magnetic field produced by Larmor precession M0, which is directed along the z axis to x axis (d). After the radio frequency pulse ceased (RF-), the rotated magnetic field will relax to two magnetic vectors MZ and MXY (e). 45igure 4. Mean body weight (BW) of three groups of rat in AA experiment. The mean BW of dosed groups was tested with the same group at day 1 (D1). Mean BW of high AA dosed group was significantly different from that at D1 at all other days. It was day 0 (D0) and day 5 for low dose group. * p < 0.05, paired t-test. Error bars are the standard deviation of group means. Groups were C: control (n=3), L: low (n=3), H: high (n=3). 47igure 5. Mean urine volume (U-vol) for three groups of rat during AA dosing. Daily group mean urine volume was compared with the mean urine volume of control group at the same day. The mean U-vol of the two dosed groups was not significantly different from the control group (p > 0.05, paired t-test). Error bars are the standard deviation of group means. 48igure 6. Mean serum urea nitrogen (UN) for three groups of rat in AA experiment. UN of high dose groups on day 0, 2, 4 (D0, D2 and D4) was significant different from their respective group mean at day 1 (D1). Error bars are the standard deviation of group means. (* p < 0.05, paired t test). 49igure 7. Renal histopathological findings in the rat AA experiment. Normal architecture of kidney in No. 1 rat of control group (A and B. 400x). A pattern of acute proximal renal tubular necrosis (arrow) was note in high dose AA group rats (C. 100x, D. 200x, E. 400x). No morphological change was noted in the glomerulus (arrow) (F. 400x). H&E stain. 50igure 8. PCA scoring plots of rat urine NMR spectra showed daily change before and after AA standard exposure. All 3 groups clustered together on day 0 under fasting status. Control, low dose (4 mg/day) and high dose (8 mg/day) groups were clustered separately from day 2 (D2) to day 5 (D5). The labels in day 5 was the individual urine sample, 1-3: control, 4-6: low dose and 7-9: high dose group. The x-axis of all plots was the value of PC1 and y-axis PC2. The sum of PC1 and PC2 is 26.2%. 51igure 9. Loading plot of rat urine NMR spectra. Several variables were significantly deviated from the center with Euclidean distance (p<0.05). Their binned ppm values were 1.38, 3.06, 3.18, 3.22, 3.34, 3.38, 3.50, 3.54, 3.62, 3.66, 3.70, 3.74, 3.78, 3.98, 4.50 and 7.58. 52igure 10. Representative urine NMR spectra of a rat in the high dose AA group. 53igure 11. Relative concentration ratio of assigned metabolites to creatinine in rat AA experiment. Each standard deviation bar is the mean of metabolite concentration of the same dosed group (C: control, L: low and H: high) on the same day from day 1 (D1) to day 5 (D5). Student paired t-test was used for estimating significant difference between control and either low or high dosed group at the same day. Parenthesis below the name of metabolites denotes the chemical shifts range for integration. Here shows several metabolites within 1.32 to 3.03 ppm. * p < 0.05. 54igure 11 (continued). Metabolite concentration changes for assigned endogenous metabolites with 3.26 and 8.45 ppm. 55igure 12. Mean body weight (BW) changes in three groups of mouse in AA experiment. The group mean BW of each day was tested with that at day 0 (D0) of the same group. * p < 0.05, t-test. C: control (n=3), L: low (n=3), H: high (n=4 D0-D8, n=3 at D10 and D11 ) dose group. 56igure 13. Mean serum urea nitrogen (UN) for three groups of mouse in AA experiment. Mean UN of each group at Day 4, 8 and 11 was compared with day 2 of the same group. * p < 0.05, t-test. Mouse groups were C: control (n=3), L: low (n=3), H: high (n=4 D2, 4 and 8, n=3 at D11 ). 57igure 14. Mean serum creatinine (Cr) for three groups of mouse in AA experiment. Cr of the high dose group increased more from day 4 (D4) to day 11 (D11) than other groups. Mouse groups were C: control (n=3), L: low (n=3), H: high (n=4 D2, 4 and 8, n=3 at D11 ). 58igure 15. Histopathological findings of mouse kidneys dosed with AA standard. No significant change of renal tubules and glomeruli of kidney in a mouse of control group (A. 200x, B. 400x). A mouse of low dose AA (5 mg/kg/day) group showed focal moderate (3) to severe acute proximal tubular necrosis and dilation with hyaline casts (C. 200x, D. 400x). A mouse of high dose AA (7.5 mg/kg/day) group showed focal moderately severe (4) acute proximal tubular necrosis with hyaline casts (E. 200x, F. 400x). No morphological change was noted in the glomeruli. H&E stain. 59igure 16. Representative urine NMR spectra of a mouse from the high dose AA group. 60igure 17. PCA scoring plots of mouse urine NMR spectra showed clustering before and after AA standard exposure in the mouse AA experiment. The chemical shifts range selected was from 1 to 4.5 ppm. The control group and the dose groups were clustered separately at day 3 and later except a mouse of the low dose group labeled "L2" at day 8. There was no urine sample at D10 for a mouse of high dosed group labeled "H1". The x-axis of all plots was the value of PC1 and y-axis PC2. The sum of PC1 and PC2 is 43.7%. 61igure 18. Loading plot of mouse urine NMR spectra of the AA experiment. Several variables were significantly deviated from the center with Euclidean distance (p < 0.1, green; p < 0.05, red). Their binned ppm values were 0.66, 0.98, 1.02, 1.06, 1.74, 2.66, 3.46, 3.50, 3.54, 3.58, 3.74, 3.78, 3.86, 3.94, 4.30, 7.42, 7.66, 8.34, 8.38 and 8.98. 62igure 19. Major endogenous metabolites profile in the mouse AA experiment. Relative concentration ratio of assigned metabolites to creatinine. Each standard deviation bar is the mean of metabolite concentration of the same dosed group (C: control, L: low (5 mg/kg/day) and H: high (7.5 mg/kg/day)) on the same day from day 1 (D1) to day 10 (D10). Parenthesis below the name of metabolites denotes the chemical shifts range for integration. * p < 0.05. 63igure 19 (continued). Metabolite concentration changes among three groups of mouse dosed with AA. 64igure 20. Mean body weight (BW) changes in groups of mouse dosed with Madouling. C: control, L: low (559 mg/kg/day), M: moderate (1118 mg/kg/day), H: high (2236 mg/kg/day). Each mean BW of the same group at different day was compared with its mean BW at day 0 (D0). 65igure 21. Mean serum urea nitrogen (UN) for groups of mouse in Madouling experiment. Each mean BW of the same group at different day was compared with its mean UN at day 0 (D0). UN was not increased in all group until day 20 (D20) in the high dose group. There were 3 mice for each group and UN of one mouse in the high dose group was not detected at 9 and day 20 due to mortality. * p < 0.05, t-test. 66igure 22. Mean serum creatinine (Cr) for four groups of mouse in Madouling experiment. No difference of mean Cr change for each day creatinine as compared to day 0 of the same group. Mouse groups were C: control (n=3), M: moderate (n=3),L: low (n=3), H: high (n=3 D2, 4 and 6, n=2 at D9 and D20). 67igure 23. Histopathological findings of mouse kidneys dosed with Madouling at day 20. A mouse of low dose (559 mg/kg/day) group showed focal slight acute proximal tubular degeneration with cellular swelling (A. 200x, B. 400x). The moderate dose (1118 mg/kg/day) group showed focal moderate acute proximal tubular necrosis with dilation (C. 200x, D. 400x). The high dose (2236 mg/kg/day) group showed focal moderate/serve acute proximal tubular necrosis with dilation (E. 200x, F. 400x). No morphological change was noted in the glomerulus. H&E stain. 68igure 24. Representative urine NMR spectra of a mouse of high dose (2236 mg/kg/day) group in the Madouling experiment. 69igure 25. PCA scoring plots of mouse urine NMR spectra showed daily change before (D0) and after Madouling (AC) exposure. At day 8 (D8), the high dose group showed separation from other groups. At day 10 (D10) the cluster of two dose groups were separated from the control group. Scoring points labeled M1-3 were individual mouse of the moderate dose group. The sum of PC1 and PC2 is 22.6%. 70igure 26. Loading plot of mouse urine NMR spectra in the Madouling experiment. Several variables were significantly deviated from the center with Euclidean distance (p < 0.05, red). Their binned ppm values were 0.94, 1.02, 1.06, 1.34, 1.70, 1.74, 1.90, 2.14, 2.62, 2.78, 3.50, 3.74, 3.78, 4.10, 4.14, 6.90, 6.94, 7.14 and 8.02. 71igure 27. Relative concentration ratio of assigned metabolites to creatinine in the mouse Madouling experiment. Each standard deviation bar is the mean of metabolite concentration of the same dosed group (C: control, L: low (559 mg/kg/day), M: moderate (1118 mg/kg/day) and H: high (2236 mg/kg/day)) at day 0, 1, 3 and 8. Parenthesis below the name of metabolites denotes the chemical shifts range for integration. * p < 0.05. 72igure 27 (continued). Metabolite concentration changes among four groups of mouse dosed with Madouling. 73igure 28. Mean body weight (BW) changes in groups of mouse dosed with Bu-Fei-A-Jiau-Tang (BFAJT). C: control (n=3), L: low (n=3), H: high (n=3). Each group mean BW at different day was compared with day 0 (D0) of the same group. The mean BW was significantly different at day 10 (D10) and day 20 (D20) for high dose (4 g/kg/day) group and at D20 for low dose (2 g/kg/day) group. * p < 0.05, paired t-test. 74igure 29. Mean serum urea nitrogen (UN) for groups of mouse during Bu-Fei-A-Jiau-Tang dosing. The mean UN of high dose group at day 20 (D20) was significantly higher than its mean UN at day 0 (D0). The standard deviation of group means are shown in stick arrows. * p < 0.05 as comparing with control group at the same day. 75igure 30. Histopathological findings of mouse kidneys dosed with BFAJT after dosing for 20 days. A mouse of low dose (2 g/kg/day) group showed focal slight acute proximal tubular degeneration with cellular swelling (A. 200x, B. 400x). The high dose (4 g/kg/day) group showed focal moderate acute proximal tubular necrosis with dilation (C. 200x, D. 400x). No morphological change was noted in the glomerulus. H&E stain. 76igure 31. Representative urine NMR spectra of a mouse of high dose (2236 mg/kg/day) group in the BFAJT experiment. 77igure 32. PCA scoring plots of mouse urine NMR spectra in the Bu-Fei-A-Jiau-Tang (BF) experiment. No group clustering from day 0 to day 16 (D16). The sum of PC1 and PC2 is 24%. 78igure 33. Metabolites in their metabolic pathways and concentration changes. Comparison of our rat AA experiment with two other experiments usign rats are shown in the left lower panels of metabolites detected in our urine NMR spectroscopy. The most left panel indicated metabolite fold change between the high dose group and the control group at the time-point of day 5. The right next panel indicated changes in an rat AA experiment reported by Zhang et al. with data collected at day 10 with 10 mg/kg/day AA in the initial 5 days [1]. The right most panel column indicated changes in an gentamicin experiment with a dose of 60 mg/kg/day for the upper panel and 120 mg/kg/day for the lower panel [2]. The chemicals of both experiments were given subcutaneously . 81igure 34. Metabolites in their metabolic pathways and concentration changes. Metabolites detected from urine NMR spectroscopy were compared among three mouse experiments. The upper row showed metabolites changes relative to the control group at day 5 of the mouse AA experiment. The left panel indicated low and the right high dose group. The middle row showed metabolites changes relative to the control group at day 10 of the mouse Madouling experiment. The left panel indicated low, the middle moderate and the right high dose group. The lower row showed metabolites changes relative to the control group at day 10 (left panel) and 16 (right panel) of the high dose group in the mouse BFAJT experiment. 82igure A.1. The raw data contained 16384 measurement and the approximated data interpolated the measurement width to 0.0001 ppm. 83igure A.2. A spectrum before and after baseline correction. OS: original spectrum, CL: correction baseline, CS: corrected spectrum 85ist of Tablesable 1. Summary of the four experiments for aristolochic acid nephrotoxicity 46able 2. Metabolomics studies on AA nephrotoxicity and their PCA results 79able 3. Pathological grading of reanl injury in all four experiments 8
