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Identification of Biomarkers for Toxicity by NMR-Based Metabonomics

Identification of Biomarkers for Toxicity by NMR-Based Metabonomics. Nelly Aranibar SMASH Conference Sep 23, 2009. Nelly Aranibar Karl-Heinz Ott Example I: Muscle lipidosis Lindsay Tomlinson Mark Tirmenstein Michael Bennett Joseph Horvath Greg Cosma. Example II: Enzyme induction

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Identification of Biomarkers for Toxicity by NMR-Based Metabonomics

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  1. Identification of Biomarkers for Toxicity by NMR-Based Metabonomics Nelly Aranibar SMASH Conference Sep 23, 2009

  2. Nelly Aranibar Karl-Heinz Ott Example I: Muscle lipidosis Lindsay Tomlinson Mark Tirmenstein Michael Bennett Joseph Horvath Greg Cosma Example II: Enzyme induction Vasanthi Bhaskaran Jeff Vassallo Lloyd Lecureux Lois Lehman-McKeeman Co-authors

  3. Overview • Urine metabonomics of dogs in a toxicology study: biomarker discovery in the case of drug-related skeletal muscle lipid accumulation in dogs • Urine metabolomics analysis of a CRF R1 antagonist compound: potential biomarker identification • Enzyme induction study with four known enzyme inducers: metabolomics analysis • Transcriptomics and RT PCR results and data integration

  4. I. Biomarker discovery in skeletal muscle lipid accumulation

  5. Myofiber necrosis with inflammatory cell infiltrate (arrows) Day 15; 10x obj Skeletal muscle toxicity in dogs after daily drug treatment for 3 months • Compound in high doses caused degeneration and lipid accumulation in the skeletal muscle and heart of dogs. • Muscle degeneration could be monitored with the muscle enzymes (CK, AST, ALT). While muscle degeneration appeared to attenuate over time, the lipid accumulation did not: need for a biomarker

  6. Lactate Increases Indicative of Lactic Acidosis • Lactate levels were increased predominantly after 2 months of dosing, returning towards normal after 3 months • Correlation with decreases in serum bicarbonate, indicating a lactic acidosis and increased ‘anaerobic’ glycolysis • Consistent with alterations in pyruvate metabolism.

  7. O C H Lactate 3 N O H C H 3 N,N-dimethylglycine Ethylmalonate Lactate Group 4 Week 13 Adipic acid Adipic acid Ethylmalonate Pyruvate Group 1 Week 13 ppm 1D NMR Metabonomics of Dog Urine: Some Distinctly Altered Metabolites

  8. Statistically Significantly Altered Metabolites

  9. 9 weeks 13 weeks 1 week 5 weeks EMA Concentration Profiles by Week

  10. Why Methylsuccinate and Ethylmalonate? • Normally, ethylmalonate and methylsuccinate are not a component of urine in dogs • Increases were evident in methylsuccinate and ethylmalonate after 1 month of dosing, with continued increases after 2 and 3 months • When short-chain acyl-CoA dehydrogenase is inhibited, an alternate biochemical pathway predominates, resulting in the production and excretion of ethylmalonate (EMA) and methylsuccinate (MSA) into the urine.

  11. Biochemical pathway for EMA and related metabolites n > 6 Acyl-CoA oxidation Short chain acyl-Coenzyme A dehydrogenase (ACADS), SCAD 1.3.99.2 Butyryl-CoA PropionylCoA carboxylase 6.4.1.3 hydrolysis EMA S-EthylmalonylCoA AcetylCoA MethylmalonylCoA racemase 5.1.99.1 R-EthylmalonylCoA MethylmalonylCoA mutase 5.4.99.2 MethylsuccinylCoA hydrolysis MSA

  12. Ethylmalonate as Biomarker • Ethylmalonic acid (EMA) was identified as the most significantly treatment-altered metabolite • Increases in EMA correlated to morphologic changes in skeletal muscle due to drug treatment • Increased EMA and related metabolites are indicative of dysregulation in fatty acid oxidation • This provides an association between the increases in EMA and lipid accumulation evident in skeletal muscle • A clinical assay for EMA exists and had been previously established for diagnosis of rare inborn defects.

  13. II. Biomarkers of enzyme induction

  14. DPC-904, a CRF R1 antagonist and multiorgan toxicant • Toxicity Profile • Hepatomegaly and enzyme induction • Rapid decrease in T4 levels • Testicular atrophy • Metabolomics and transcriptomics sample collection • Urine pooled over 24 hr interval • Serum pre dose and at day 4 after start of dosing • Liver, testes, etc. collected for gene expression profiling

  15. 0 1 3 4 2 5 Most significant signals increasing with dose and time cannot be readily assigned Intensity of 4.15 ppm PCA time trajectory at high dose

  16. 1D NMR Selective 1D-TOCSY results for potential biomarker in the mixture treated control

  17. (R,S)? (R,S)? (R,S)? Compound is not found in any standard NMR library or spectral database HMQC spectrum sugests a sugar-alcohol, HMBC shows correlation to a carbonyl group… stereochemistry allows many possible combinations…gluconic, xylonic, galactonic, gulonic, talonic acids

  18. D-Gulono-γ-lactone conversion to D-gulonic acid (NaOH addition) NaOH 1.2 mEq 0.8 mEq 0.6 mEq 0.4 mEq 0.2 mEq 0 mEq Gulonic ac. Lactone

  19. 24 hours 2 hours 1 min Gul. Ac. Lactone Control Spiking gulono-γ-lactone into control urine produces gulonic acid 10 ul of a ~1mM std. gulono-g-lactone solution added to control urine. Incubation at room temperature

  20. Urinary excretion of ascorbic acid in addition to gulonic acid/gulonolactone DPC904 4d 200 mpk Vehicle control 4d Ascorbic acid standard in D2O

  21. Anova results (p-values and fold changes) show ascorbic acid as second most significantly upregulated metabolite

  22. Metabolism of Ascorbic Acid and Hepatic Enzyme Induction • Excretion of ascorbic acid and other metabolites in its biosynthesis pathway correlate to mono-oxygenase function (Brodie 1958, Lake et al 1976, 1977) • Microsomal enzyme induction increases the demand for Vitamin C • Burns et al (1954): barbiturates, DDT increased ascorbate turnover 4-8x • Ascorbic acid is required to support microsomal enzyme induction • Vitamin C-deficient rats show lower P450 levels and decreased induction (Horio et al 1986)

  23. Metabolism of Ascorbic Acid and Hepatic Enzyme Induction • Mamals (except humans, primates, guinea pigs, bats) produce ascorbate in the liver • Excretion of ascorbate upon xenobiotic administration in urine has been observed since the 1940’s (Longnecker et al, Salomon et al, Lake et al, etc.) • Ascorbic acid is synthesized from Glucose via UDP-glucuronic acid (5) • UDP glucose transferase • UDP-glucuronosyl transferase • Beta-glucuronidase • L-gulonolactone oxidase • Gulonolactonase

  24. Recycling of Ascorbate Ascorbate is efficiently recycled from its oxidized form (dehydroascorbate) by two enzymes, glutaredoxin reductase and thioredoxin reductase Linster et al., FEBS Journal 2007, 274: 1-22.

  25. Ascorbic and gulonic acid concentrations increase with time after DPC-904 or Phenobarbital dosing ascorbate gulonate Control urine DPC-904 Day 1 DPC-904 Day 4 PB Day 1 PB Day 4 Gulonic acid std. Ascorbic acid std.

  26. Comparing different cytochrome P450 inducers’ effect on urinary excretion of ascorbic and gulonic acid • Dose daily for 4 days in male SD rats or Wistar for PB group • DPC-904 (904) • Phenobarbital (PB) - known inducer of CAR • Diallyl sulfide (DAS) - known inducer of CAR • Beta-naphtoflavone (BNF) - known inducer of AhR • NMR metabolomics of urine, daily sampling 0-4 days • RT PCR of all genes involved in glucuronate to ascorbate pathway enzymes plus ascorbate recycling • RT PCR of Cyp1a1 and Cyp2b1

  27. Overview of representative spectra Hippurate and other gut flora metabolites Expansion area (next slide) Urea DAS BNF 904 (SD) 904 (Wistar) PB (Wistar) Control

  28. Expansion showing urine spectra at 4 days and ascorbic and gulonic acid standards Gul Asc DAS BNF 904 (SD) 904 (Wistar) PB (Wistar) Control (Wistar) Ascorbate std Gulonate std

  29. Relative quantitation for days 4 and 1: DT08040, DAS and BNF in SD rats All values normalized to a constant internal standard (TSP); n=5 or 6 for each group, mean +/- std; one-tailed t-test * * * p-value for treatment vs control < 0.001

  30. DAS, BNF and controls spectra: Anova showed no significant changes for BNF treatment gulonate ascorbate

  31. Relative quantitation for days 1 and 4: DT06142, PB and 904 in Wistar rats All values normalized to a constant internal standard (TSP); n=5 or 6 for each group, mean +/- std; one-tailed t-test * * * * * * * * p-value for treatment vs control < 0.01

  32. 3-Dehydro-L-gulonate L-Gulonate-3-dehydrogenase (Cryl1) D-Glucuronate L-gulono-1,4-Lactone Glucuronate reductase Gulonolactonase L-Gulonolactone Oxidase UDP-D-Glucuronate UDP glucuronosyltransferase 1A6 3.4 DAS 2.8 904 1.5 PB 1.4 DAS 2.7 904 1.4 PB Glutaredoxin reductase Thioredoxin reductase β-D-Glucuronide Dehydroascorbate RT PCR fold-changes in 5 day treated rats Control = 1 Red - Green - 2.6 DAS 1.3 DAS 2.8 904 1.2 PB 5.0 DAS 5.0 904 2.0 PB L-gulonate 2.5 DAS 7.1 904 DAS 904 PB Ascorbic acid excretion in urine 5.5 DAS 6.0 BNF 4.8 904 3.4 PB

  33. Summary • Ethyl malonic acid and methylsuccinic acid, known markers of hereditary SCAD deficiency, are aproppriate markers of drug-related lipidosis in muscle. • Urinary gulonic acid excretion may be a predictor of hepatic enzyme induction, in particular CAR activated P450. • Metabonomics can be applied in an investigative manner to generate mechanistic hypotheses, by relating the changes in the composition of biofluids to metabolic pathways and, subsequently, to identify small molecule biomarkers. • Integration of metabonomic and transcriptomic data provides a more robust view of the biochemical events.

  34. Acknoledgements AIM (Applied and Investigative Metabonomics) Michael Reily Don Robertson Nathan Siemers Adrienne Tymiak Bruce Car Don Bertolini Pierre Depelteau

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