1. Presented By Duquesne Marilyn Belgium Presented By Duquesne Marilyn Belgium 3 rd International Conference and Exhibition on Traditional & Alternative Medicine August 03-05, 2015 Birmingham, UK In Association with

2. Introduction Purpose of the research Method Results Conclusion Introduction Purpose of the research Method Results Conclusion Herbal remedies : Natural but not unhazardous Study cases of Aristolochic Acid intoxication

3. Introduction Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 2

4.  Common term  Mixture of : Aristolochic Acid I (AAI)Aristolochic Acid II (AAII) & nitrophenantrene carboxylic acid derivatives What’s Aristolochic Acid ? Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 3

5.  Produced by : Aristolochia Asarum www.fr-academic.ru Bragantia www.afleurdepau.com etc.usf.edu What’s Aristolochic Acid ? Aristolochiaceae Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 4

6.  found in : > >> What’s Aristolochic Acid ? Medicinal properties of Aristolochia Rhizomes : -Gastric stimulant -Fevers treatment -Malaria Roots: -Type 2 diabetes -Snakebites Leaves : -Skin disorders -Dyspepsia -Leishmania Stems: -Anti-inflammatory -Diuretic -Circulation stimulating Fruits : -Respiratory diseases -Skin wounds -Malaria Seeds: -Biliousness -Abdominal pain Flowers : -Diuretic -Hypoglycaemic effect Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 5

7. Aristolochic acid toxicity Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 6

8. Tubular atrophy Interstitial fibrosis Low molecular weight proteinuria I) Nephrotoxicity Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 7

9. I) Nephrotoxicity Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 8

10. II) Carcinogenicity  Induced by metabolisation of AA Aristolochic acid I/II Aristolactam nitronium ion R NQO1 CYP1A1 CYP1A2 NQO1 COX CPR R Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 9

11. II) Carcinogenicity  Properties of AA-DNA adducts Cortex Medulla Localisation:Responsible for: TP53 Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 10

12. TP53 Histochemistry Genomic Proteomic Metabonomic α-1µglob. β-2µglob. α-2 glycoprot. INVASIVE METHOD + - NON INVASIVE METHOD INVASIVE METHOD - NON INVASIVE METHOD - EARLY !! - LATE Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 11

13. Balkan Endemic Nephropathy (BEN)  Chronic Kidney disease 13 Pr. Grollman (thepoisongarden.co.uk)  Etiology:  Dietary intoxication  Diseased or suspects : 25000  At risks : >100000 Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Introduction 12

14. Purpose of research Mechanism of toxicity Exposure Absorption Distribution metabolism Absorption Distribution metabolism Primary target Target cell damage Target organ Damage to organism Herbal remedies : natural but not unhazardous. Study cases of AA intoxication; Purpose of research 13 BEN

15. Metabonomic procedure Method : Herbal remedies : natural but not unhazardous. Study cases of AA intoxication; Method 14

16. What’s Metabonomic ?  Metabolites portrait Mass spectrometry NMR  Temporal changes Herbal remedies : natural but not unhazardous. Study cases of AA intoxication; Method 15

17. Samples collection & preparation 4°C Antibacterial Phosphate buffer External Reference (TSP) Metabonomic procedure Herbal remedies : natural but not unhazardous. Study cases of AA intoxication; Method 16

18. Metabonomic procedure Spectroscopic analysis NMR Proton electron Magnetic field Z Y X O Bo RF Time Intensity Fourier transform Frequency Herbal remedies : natural but not unhazardous. Study cases of AA intoxication; Method 17

19. Spectroscopic analysis Spectrum treatment & Multivariate analysis Metabonomic procedure PC1 PC2 SCORES plot PC1 PC2 3.40 2.50 1.24 3.80 5.70 3.24 0.80 Observations Variables PC1 LOADINGS plot Herbal remedies : natural but not unhazardous. Study cases of AA intoxication; Method 18

20. Spectroscopic analysis Spectrum treatment & Multivariate analysis Biological interpretation Metabonomic procedure Herbal remedies : natural but not unhazardous. Study cases of AA intoxication; Method 19

21. Acute toxicity Chronic toxicity - Diseased - Suspects - At risks Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Results 20 Results

22. Physiological data AAIAAI/AAIIAAIAAI/AAIIAAIAAI/AAII Body Weight Food intake Water intake Urinary volume + N.D + - + N.D + - - - - N.D - - - + + - - - - - - + Clinical chemistry AAIAAI/AAIIAAIAAI/AAIIAAIAAI/AAII BUN Sr. Creatinine Albumin ≠≠≠≠≠≠ + + + ++ ≠ + ≠ + + +++ ≠ + ≠ + + +++ ≠ 50mg/kg75mg/kg100mg/kg Polyuria ↗↗ ↗ 75100 AAII - - - - - - - - - - - - - - + + +++ ≠ + + + + + ≠ Toxicity : AAI < AAI/AAII  ? AAII ? Results AA acute toxicity I) Physiological Parameters AAIAAI/AAIIAAIAAI/AAIIAAIAAI/AAII Herbal remedies : natural but not unhazardous. Study cases of AA intoxicationResults : Acute toxicity (rats) 21

23. A 50 mg/kg AAI B 75 mg/kg AAI C 100 mg/kg AAI D 50 mg/kg AAI/AAII E 75 mg/kg AAI/AAII F 100 mg/kg AAI/AAII Results AA acute toxicity I) Metabonomic results Herbal remedies : natural but not unhazardous. Study cases of AA intoxicationResults : Acute toxicity (rats) 22

24. Hippurate Glucose Betaine Osmoprotectants Lactate & acetate Results AA acute toxicity I) Metabonomic results Herbal remedies : natural but not unhazardous. Study cases of AA intoxicationResults : Acute toxicity (rats) 23

25. ↘ Hippurate ↗ Glucose Glomerulus Proximal tubule Distal tubule ↘ Cycle Krebs components ↗ Osmoprotectants ↗ Betaine ↗ Lactate & acetate Results AA acute toxicity Biological interpretation Cortex Medulla ↗↗ Bun ↗ Ser. Creat. Herbal remedies : natural but not unhazardous. Study cases of AA intoxicationResults : Acute toxicity (rats) 24

26. ↗ Nitrogenous waste in blood ↗ Osmoprotectants in urine Plasma ↘ Reabs. Glucose AA Hippurate Results AA acute toxicity Biological interpretation OAT1 ↘ Hippurate Deficient cycle Krebs Herbal remedies : natural but not unhazardous. Study cases of AA intoxicationResults : Acute toxicity (rats) 25

27.  Metabonomic profile reflecting the severity of renal damage in proximal tubules and/or interstitial tissue: Psihogios, N ; Kalaitzidis, R ; Dimou, S ; Seferiadis,K ; Siamopoulos, K ; and Bairaktari, E. Evaluation of Tubulointerstitial lesions severity in patients with glomerulonephritides : An NMR-based metabonomic study. J.Proteome Res. 2007, 6, 3760-3770 Results AA c hronic toxicity Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Results : Chronic toxicity ( Humans) 26

28. Creatinine filtration : V Hippurate secretion : V Glucose reabsorption : V Creatinine filtration : X Hippurate secretion: X Glucose reabsorption : X Metabonomic profile : Healthy Creatinine filtration : X Hippurate secretion : X Glucose reabsorption : V Metabonomic profile: Moderate renal injury Metabonomic profile: Severe renal injury Metabonomic comparison diseased, suspects, at risks, others, NEN with Belgium control samples. Metabonomics: on the Road to Detect Diagnostic Biomarkers in Endemic (Balkan) Nephropathy. Evaluation in a Retrospective Pilot Project. Duquesne et al., J Cancer Sci Ther 2012 Results AA c hronic toxicity Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Results : Chronic toxicity ( Humans) 27

29. Healthy Moderate renal injury Severe renal injury  Comparison of the different metabonomic profiles : healthy, moderale and severe renal injury Renal damage severity Results AA c hronic toxicity Herbal remedies : natural but not unhazardous. Study cases of AA intoxication Results : Chronic toxicity ( Humans) 28

30. Perspectives & Conclusions Acute toxicity model Chronic toxicity model In process Metabolites identification : early & easily biomarkers ( ↗ Glucose, betaine, osmoprotectants ; ↘ hippurate, cycle Krebs components ) New samples BEN Biological hypotheses ( renal functions, transport injuries,…)

31. Perspectives & Conclusion Metabonomic - NON INVASIVE METHOD - EARLY Biomarkers Lab. Human Biology & Toxicology UMONS (Belgium)  High possibility of human AA intoxication cases !! Preventive action

32. Duquesne Marilyn PhD student Human Biology & Toxicology laboratory University of Mons(Belgium)

33. Profile : Familial history Proteinuria : ACR, alpha- 1 Azotemia (serum creatinine) anemia 15 years in EN Diseased v v v v v v v v v v v v Suspects v v v v At risks v Others : x x x x

34. http://biology-igcse.weebly.com/dialysis.html http://www.nuhospitals.com/renal-kidney-transplant.html https://sites.google.com/a/montessori.edu.co/biology-projects/dna-vocabulary-2/_draft_post-12 http://www.medicinenet.com/kidney_infection/article.htm http://carcin.oxfordjournals.org/content/24/10/1695/F1.large.jpg http://www.google.be/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=&url=http%3A%2F%2Fww w.ayurvedindia.in%2FOthers%2FLiver- Disorders%2F961&ei=inE3VeL7GdbsaMyxgYAE&psig=AFQjCNFvJDXErveYz2GG_VmWaQIoHQKqeA&ust=1429783307048964 https://www.flickr.com/photos/14179358@N05/3678083407/ WWW.stjude.com tutocours.fr www.michigananimalhospital.com www.vcahospitals.com www.riversideonline.com www.mhhe.com hyperphysics.phy-astr.gsu.edu WWW.stjude.com www.michigananimalhospital.com www.vcahospitals.com www.riversideonline.com www.mhhe.com hyperphysics.phy-astr.gsu.edu Herbal remedies : natural but unhazardous. Study cases of AA intoxication ; Introduction

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