Archives of Toxicology

, Volume 79, Issue 4, pp 208–223

Tryptophan–NAD+ pathway metabolites as putative biomarkers and predictors of peroxisome proliferation

  • Jane Delaney
  • Mark P. Hodson
  • Hansa Thakkar
  • Susan C. Connor
  • Brian C. Sweatman
  • Steve P. Kenny
  • Paul J. McGill
  • Julie C. Holder
  • Kathryn A. Hutton
  • John N. Haselden
  • Catherine J. Waterfield
Molecular Toxicology


The present study was designed to provide further information about the relevance of raised urinary levels of N-methylnicotinamide (NMN), and/or its metabolites N-methyl-4-pyridone-3-carboxamide (4PY) and N-methyl-2-pyridone-3-carboxamide (2PY), to peroxisome proliferation by dosing rats with known peroxisome proliferator-activated receptor α (PPARα) ligands [fenofibrate, diethylhexylphthalate (DEHP) and long-chain fatty acids (LCFA)] and other compounds believed to modulate lipid metabolism via PPARα-independent mechanisms (simvastatin, hydrazine and chlorpromazine). Urinary NMN was correlated with standard markers of peroxisome proliferation and serum lipid parameters with the aim of establishing whether urinary NMN could be used as a biomarker for peroxisome proliferation in the rat. Data from this study were also used to validate a previously constructed multivariate statistical model of peroxisome proliferation (PP) in the rat. The predictive model, based on 1H nuclear magnetic resonance (NMR) spectroscopy of urine, uses spectral patterns of NMN, 4PY and other endogenous metabolites to predict hepatocellular peroxisome count. Each treatment induced pharmacological (serum lipid) effects characteristic of their class, but only fenofibrate, DEHP and simvastatin increased peroxisome number and raised urinary NMN, 2PY and 4PY, with simvastatin having only a transient effect on the latter. These compounds also reduced mRNA expression for aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase, EC, the enzyme believed to be involved in modulating the flux of tryptophan through this pathway, with decreasing order of potency, fenofibrate (−10.39-fold) >DEHP (−3.09-fold) >simvastatin (−1.84-fold). Of the other treatments, only LCFA influenced mRNA expression of ACMSDase (−3.62-fold reduction) and quinolinate phosphoribosyltransferase (QAPRTase, EC (−2.42-fold) without any change in urinary NMN excretion. Although there were no correlations between urinary NMN concentration and serum lipid parameters, NMN did correlate with peroxisome count (r2=0.63) and acyl-CoA oxidase activity (r2=0.61). These correlations were biased by the large response to fenofibrate compared to the other treatments; nevertheless the data do indicate a relationship between the tryptophan–NAD+ pathway and PPARα-dependent pathways, making this metabolite a potentially useful biomarker to detect PP. In order to strengthen the observed link between the metabolites associated with the tryptophan–NAD+ pathway and more accurately predict PP, other urinary metabolites were included in a predictive statistical model. This statistical model was found to predict the observed PP in 26/27 instances using a pre-determined threshold of 2-fold mean control peroxisome count. The model also predicted a time-dependent increase in peroxisome count for the fenofibrate group, which is important when considering the use of such modelling to predict the onset and progression of PP prior to its observation in samples taken at autopsy.


Biomarkers N-Methylnicotinamide N-Methyl-4-pyridone-3-carboxamide N-Methyl-2-pyridone-3-carboxamide Peroxisome proliferation Multivariate data analysis 


  1. Bell FP (1982) Effects of phthalate esters on lipid metabolism in various tissues, cells and organelles in mammals. Environ Health Perspect 45:41–50PubMedGoogle Scholar
  2. Bell FP, Hubert EV (1981) Inhibition of LCAT in plasma from man and experimental animals by chlorpromazine. Lipids 16:814–819Google Scholar
  3. Castagne I, Fourche J, Jensen J, Neuzil E (1987) The reaction of pyridoxal-5′-phosphate with hydrazino compounds: a spectrophotometric study. Biochem Soc Trans, 14:142–143Google Scholar
  4. Cattley RC, Deluca J, Elcombe C, Fenner-Crisp P, Lake BG, Marsman DS, Pastoor TA, Popp JA, Robinson DE, Schwetz B, Tugwood J, Wahli W (1998) Do peroxisome proliferating compounds pose a hepatocarcinogenic hazard to humans? Regul Toxicol Pharmacol 27:47–60CrossRefGoogle Scholar
  5. Chao Y, Chen JS, Hunt VM, Kuron GW, Karkas JD, Liou R, Alberts AW (1991) Lowering of plasma cholesterol levels in animals by lovastatin and simvastatin. Eur J Clin Pharmacol 40 [Suppl 1]:S11–S14Google Scholar
  6. Chiang M-T, Tsai M-L (1995) Effect of fish oil on plasma lipoproteins, liver glucose-6-phosphate dehydrogenase and glucose-6-phosphatase in rats. Int J Vitam Nutr Res 65:276–282PubMedGoogle Scholar
  7. Clark DA, Leeder LG, Foulds EL, Trout DL (1970) Changes in lipids of rat liver after hydrazine injection. Biochem Pharmacol 19:1743–1752CrossRefPubMedGoogle Scholar
  8. Connor SC, Hodson MP, Ringeissen S, Sweatman BC, Waterfield CJ, Haselden JN (2004) Development of a multivariate statistical model to predict peroxisome proliferation in the rat, based on urinary 1H NMR spectral patterns. Biomarkers (in press)Google Scholar
  9. Cornish HH (1968) The role of vitamin B6 in the toxicity of hydrazines. Ann N Y Acad Sci 166:126–145Google Scholar
  10. Dallongeville J, Bauge E, Tailleux A, Peters JM, Gonzalez FJ, Fruchart J-C, Staels B (2001) Peroxisome proliferator-activated receptor alpha is not rate-limiting for the lipoprotein-lowering action of fish oil. J Biolog Chem 276:4634–4639CrossRefGoogle Scholar
  11. De Craemer D, Vamecq J, Roels F, Vallee L, Pauwels M, Van den Branden C (1994) Peroxisomes in liver, heart, and kidney of mice fed a commercial fish oil preparation: original data and review on peroxisomal changes induced by high-fat diets. J Lipid Res 35:1241–1250PubMedGoogle Scholar
  12. Egashira Y, Yamamiya Y, Sanada H (1992) Effects of various dietary fatty acids on amino-carboxymuconate-semialdehyde decarboxylase activity in rat liver. Biosci Biotechnol Biochem 56:2014–2019Google Scholar
  13. Egashira Y, Komine T, Ohta T, Shibata K, Sanada H (1997) Change of tryptophan–niacin metabolism in d-galactosamine-induced liver injury in the rat. J Nutr Sci Vitaminol 43:233–239PubMedGoogle Scholar
  14. Froyland L, Vaagenes H, Asiedu DK, Garras A, Lie O, Berge RK (1996) Chronic administration of eicosapentaenoic acid and docosahexaenoic acid as ethyl esters reduced plasma cholesterol and changed the fatty acid composition in rat blood and organs. Lipids 31:169–178PubMedGoogle Scholar
  15. Fukuoka S-I, Ishiguro K, Yanagihara K, Tanabe A, Egashira Y, Sanada H, Shibata K (2002) Identification and expression of a cDNA encoding human α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD). J Biolog Chem 277:35162–35167CrossRefGoogle Scholar
  16. Fukuwatari T, Suzuki Y, Sugimoto E, Shibata K (2002) Elucidation of the toxic mechanism of the plasticizers, phthalaic acid esters, putative endocrine disrupters: effects of dietary di(2-ethylhexyl)phthalate on the metabolism of tryptophan to niacin in rats. Biosci Biotechnol Biochem 66:705–710CrossRefPubMedGoogle Scholar
  17. Gervois P, Torra IP, Fruchart J-C, Staels B (2000) Regulation of lipid and lipoprotein metabolism by PPAR activators. Clin Chemi Lab Med 38:3–11CrossRefGoogle Scholar
  18. Haghighi, B, Boroumand A, Behmanesh, O (1985) The effects of hydrazine on liver and serum lipids of normal and adrenalectomised rats. Indian J Pharmacol 17:214–218Google Scholar
  19. Halvorsen, B, Rustan AC, Christiansen EN (1995) Effect of long chain mono-unsaturated and n-3 polyunsaturated fatty acids on postprandial blood and liver lipids in rats. Scand J Clin Lab Invest 55:469–475PubMedGoogle Scholar
  20. Hasmall SC, James NH, Macdonald N, Soames AR, Roberts RA (2000) Species differences in response to the plasticizer di-(2-ethylhexyl)phthalate: suppression of apoptosis, induction of DNA synthesis and peroxisome proliferator activated receptor α-mediated gene expression. Arch Toxicol 74:85–91CrossRefPubMedGoogle Scholar
  21. Henninger C, Clouet P, Danh HC, Pascal M, Bezard J (1987) Effects of fenofibrate treatment on fatty acid oxidation in liver mitochondria of obese Zucker rats. Biochem Pharmacol 36:3231–3236CrossRefPubMedGoogle Scholar
  22. Howarth JA, Price SC, Dobrota M, Kentish PA, Hinton RH (2001) Effects on male rats of di-(2-ethylhexyl)phthalate and di-n-hexylphthalate administered alone or in combination. Toxicol Lett 121:35–43CrossRefPubMedGoogle Scholar
  23. Inoue I, Goto S-I, Mizotani K, Awata T, Mastunaga T, Kawai S-I, Nakajima T, Hokari S, Komoda T, Katayama S (2000) Lipophilic HMG-CoA reductase inhibitor has an anti-inflammatory effect. Reduction of mRNA levels for interleukin-1β, interleukin-6, cyclooxygenase-2 and p22phox by regulation of peroxisome proliferator-activated receptor α (PPARα) in primary endothelial cells. Life Sci 67:863–876CrossRefPubMedGoogle Scholar
  24. Jenner AM, Timbrell JA (1994) Effect of acute and repeated exposure to low doses of hydrazine on hepatic microsomal enzymes and biochemical parameters in vivo. Arch Toxicol 68:240–245CrossRefPubMedGoogle Scholar
  25. Lalwani ND, Reddy MK, Qureshi SA, Sirtori CR, Abiko Y, Reddy JK (1983) Evaluation of selected hypolipidaemic agents for the induction of peroxisomal enzymes and peroxisome proliferation in the rat liver. Hum Toxicol 2:27–48PubMedGoogle Scholar
  26. Lazarow PB (1981) Assay of peroxisomal β-oxidation. Methods Enzymol 72:314–319Google Scholar
  27. Loo Y, Shin M, Yamashita Y, Ishigami M, Sasaki M, Sano K, Umezawa C (1995) Effect of feeding clofibrate-containing diet on the hepatic NAD level in rats. J Nutr Sci Vitaminol 41:341–347PubMedGoogle Scholar
  28. Martin G, Duez H, Blanquart C, Berezowski V, Poulain P, Fruchart J-C, Najib-Fruchart J, Glineur C, Staels B (2001) Statin-induced inhibition of the Rho-signalling pathway activates PPAR-α and induces HDL apoA-I. J Clin Invest107:1423–1432Google Scholar
  29. Menahan LA, Williams RH (1971) Interrelationship between fatty acid oxidation and gluconeogenesis from pyruvate. Eur J Biochem 20:488–493PubMedGoogle Scholar
  30. Mitchell FE, Price SC, Hinton RH, Grasso P, Bridges JW (1985) Time and dose response study of the effects on rats of plasticizer di-(2-ethylhexyl)phthalate. Toxicol Appl Pharmacol 81:371–392CrossRefPubMedGoogle Scholar
  31. Mocchiutti NO, Bernal CA (1997) Effects of chronic di-(2-ethylhexyl)phthalate intake on the secretion and removal rate of triglyceride-rich lipoproteins in rats. Food Chem Toxicol 35:1017–1021CrossRefPubMedGoogle Scholar
  32. Nicholson JK, Lindon JC, Holmes E (1999) “Metabonomics”: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica 29:1181–1189CrossRefPubMedGoogle Scholar
  33. Otto DA, Baltzell JK, Wooten JT (1992) Reduction in triacylglycerol levels by fish oil correlates with free fatty acid levels in ad libitum fed rats. Lipids 27:1012–1017Google Scholar
  34. Petit D, Bonnefis MT, Rey C, Infante R (1988) Effects of ciprofibrate and fenofibrate on liver lipids and lipoprotein synthesis in normo- and hyperlipidaemic rats. Atherosclerosis74:214–225Google Scholar
  35. Price SC, Hall DE, Hinton RH (1985) Lipid accumulation in the livers of chlorpromazine-treated rats does not induce peroxisome proliferation. Toxicol Lett 25:11–18CrossRefPubMedGoogle Scholar
  36. Price SC, Hinton RH, Mitchell FE, Hall DE, Grasso P, Blane GF, Bridges JW (1986) Time and dose study on the response of rats to the hypolipidaemic drug fenofibrate. Toxicology 41:169–191CrossRefPubMedGoogle Scholar
  37. Prueksaritanont T, Ma B, Fang X, Subramanian R, Yu J, Lin JH (2001) Beta-oxidation of simvastatin in mouse liver preparations. Drug Metab Dispos 29:1251–1255PubMedGoogle Scholar
  38. Ragazzi E, Costa CV, Caparrotta L, Biasiolo M, Bertazzo A, Allegri G (2002) Enzyme activites along the tryptophan–niacin pathway in alloxan diabetic rabbits. Biochim Biophys Acta 1471:9–17Google Scholar
  39. Reddy JK, Lalwai ND (1983) Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidaemic drugs and industrial plasticizers to humans. Crit Rev Toxicol 12:1–58Google Scholar
  40. Ren B, Thelen AP, Peters JM, Gonzalez FJ, Jump DB (1997) Polyunsaturated fatty acid suppression of hepatic fatty acid synthase and S14 gene expression does not require preoxisome proliferator-activated receptor α. J Biolog Chem 272:26827–26832CrossRefGoogle Scholar
  41. Ringeissen S, Connor SC, Brown HR, Sweatman BC, Hodson MP, Kenny SP, Haworth RI, McGill P, Price MA, Aylott MC, Nunez DJ, Haselden JN, Waterfield CJ (2003) Potential urinary and plasma biomarkers of peroxisome proliferation in the rat: identification of N-methylnicotinamide and N-methyl-4-pyridone-3-carboxamide by 1H nuclear magnetic resonance and high performance liquid chromatography. Biomarkers 8:240–271CrossRefPubMedGoogle Scholar
  42. Roglans N, Sanguino E, Peris C, Alegret M, Vazquez M, Adzet T, Diaz C, Hernandez G, Laguna JC, Sanchez RM (2002a) Atorvastatin treatment induced peroxisome proliferator-activated receptor α expression and decreased plasma nonesterified fatty acids and liver triglyceride in fructose-fed rats. J PharmacolExp Ther 302:232–239Google Scholar
  43. Roglans N, Verd JC, Peris C, Alegret M, Vazquez M, Adzet T, Diaz C, Hernandez G, Laguna JC, Sanchez RM (2002b) High doses of atorvastatin and simvastatin induce key enzymes involved in VLDL production. Lipids 37:445–454PubMedGoogle Scholar
  44. Sakurai T, Miyazawa S, Hashimoto T (1978) Effects of di-(2-ethylhexyl) phthalate administration on carbohydrate and fatty acid metabolism in rat liver. J Biochem 83:312–320Google Scholar
  45. Sasaki J, Funakoshi M, Arakawa K (1985) Lipids and apolipoproteins in patients treated with major tranquilisers. Clin Pharmacol Ther 37:684–687PubMedGoogle Scholar
  46. Scales MDC, Timbrell JA (1982) Studies on hydrazine hepatotoxicity. 1. Pathological findings. J Toxicol Environ Health 10:941–953PubMedGoogle Scholar
  47. Schoonjans K, Peinado-Onsurbe J, Fruchart J-C, Tailleux A, Fievet C, Auwerx J (1999) 3-Hydroxy-3-methylglutaryl CoA reductase inhibitors reduce serum triglyceride levels through modulation of apolipoprotein C-III and lipoprotein lipase. FEBS Lett 452:160–164CrossRefPubMedGoogle Scholar
  48. Senada H (1985) Suppressive effect of dietary unsaturated fatty acids on α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase, a key enzyme of tryptophan–niacin metabolism in rat. J Nutr Sci Vitaminol 31:327–337PubMedGoogle Scholar
  49. Shin M, Asada S, Mizumori N, Sano K, Umezawa C (1998) Effect of thioridazine or chlorpromazine in increased hepatic NAD level in rats fed clofibrate, a hypolipidaemic drug. J Pharm Pharmacol 50:431–436PubMedGoogle Scholar
  50. Shin M, Sano K, Umezawa C (1999) Effects of peroxisome proliferators on the TRP-NAD pathway. In: Heuther G, Kochen W et al. (eds). Tryptophan, serotonin and melatonin: basic aspects and applications. Kluwer Academic/Plenum, New York, Chap 43, pp 333–340Google Scholar
  51. Slaughter MR, O’Brien PJ (2000) Fully-automated spectrophotometric method for measurement of antioxidant activity of catalase. Clin Biochem 7:525–534CrossRefGoogle Scholar
  52. Smit MJ, Temmerman AM, Wolters H, Kuipers F, Beynen AC, Vonk RJ (1991) Dietary fish oil-induced changes in intrahepatic cholesterol transport and bile acid synthesis in rats. J Clin Invest 88:943–951PubMedGoogle Scholar
  53. Staels B, Van Tol A, Fruchart J-C, Auwerx J (1996) Effects of hypolipidaemic drugs on the expression of genes involved in high density lipoprotein metabolism in the rat. Israel J Med Sciences, 32:490–498Google Scholar
  54. Steiner S, Gatlin GL, Lennon JJ, Mcgrath AM, Seonarain MD, Makusky AJ, Aponte AM, Esquer-Blasco R, Anderson NL (2001) Cholesterol biosynthesis regulation and protein changes in rat liver following treatment with fluvastatin. Toxicol Lett 120:369–377CrossRefPubMedGoogle Scholar
  55. Stenson WF, Seetharam B, Talkad V, Pickett W, Dudeja P, Brasitus TA (1989) Effects of dietary fish oil supplementation on membrane fluidity and enzyme activity in rat small intestine. Biochem J 263:41–45PubMedGoogle Scholar
  56. Taskinen MR, Tuusi T (1987) Enzymes involved in triglyceride hydrolysis. Baillieres Clin Endocrinol Metab 1:639–666PubMedGoogle Scholar
  57. Trout DL (1965) Effect of hydrazine on plasma free fatty acid transport. Biochem Pharmacol 14:812–821CrossRefGoogle Scholar
  58. Vamecq J (1987) Chlorpromazine and carnitine-dependency of rat liver peroxisomal β-oxidation of long chain fatty acids. Biochem J 241:783–791PubMedGoogle Scholar
  59. Vamecq J, Roels F, Van den Branden C, Draye J-P (1987) Peroxisomal proliferation in heart and liver of mice receiving chlorpromazine, ethyl 2-(5-(4-chlorophenyl)pentyl)oxiran-2-carboxylic acid or high fat diet: a biochemical and morphometric comparative study. PaediatrRes 22:748–754Google Scholar
  60. Van den Branden C, Vamecq J, Dacremont G, Premereur N, Roels F (1987) Short and long term influence of phenothiazines on liver peroxisomal fatty acid oxidation in rodents. FEBS Lett 222:21–26CrossRefPubMedGoogle Scholar
  61. Van Veldhoven P, Declercq PE, Debeer LJ, Mannaerts GP (1984) Effects of benfluorex and fenofibrate treatment on mitochondrial and peroxisomal marker enzymes in rat liver. Biochem Pharmacol 33:1143–1145CrossRefPubMedGoogle Scholar
  62. Williams ML, Menon GK, Hanley KP (1992) HMG-CoA reductase inhibitors perturb fatty acid metabolism and induce peroxisomes in keratinocytes. J Lipid Res 33:193–208PubMedGoogle Scholar
  63. Willumsen N, Skorve J, Hexeberg S, Rustan AC, Berge RK (1993a) The hypotriglyceridaemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis. Lipids 28:683–690PubMedGoogle Scholar
  64. Willumsen N, Hexeberg S, Skorve J, Lundquist M, Berge RK (1993b) Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats. J Lipid Res 34:12–22Google Scholar
  65. Zipper J (1997) Proliferation of myocardial peroxisomes caused by several agents and conditions. J Mol Cell Cardiol 29:149–161CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Jane Delaney
    • 1
  • Mark P. Hodson
    • 1
  • Hansa Thakkar
    • 1
  • Susan C. Connor
    • 1
  • Brian C. Sweatman
    • 1
  • Steve P. Kenny
    • 1
  • Paul J. McGill
    • 1
  • Julie C. Holder
    • 1
  • Kathryn A. Hutton
    • 2
  • John N. Haselden
    • 1
  • Catherine J. Waterfield
    • 1
    • 3
  1. 1.Safety AssessmentGlaxoSmithKlineWareUK
  2. 2.DMPKGlaxoSmithKlineWareUK
  3. 3.Health AssessmentSyngentaMacclesfieldUK

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