Systems Responses of Rats to Aflatoxin B1 Exposure Revealed with Metabonomic Changes in Multiple Biological Matrices Limin Zhang, † Yangfang Ye, †,‡ Yanpeng An, †,§ Yuan Tian, †,§ Yulan Wang,* ,† and Huiru Tang* ,† State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, P. R. China, Graduate University of the Chinese Academy of Sciences, Beijing 100049, P. R. China, and Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University, Ningbo 315211, P. R. China Received July 30, 2010 Exposure to aflatoxins causes liver fibrosis and hepatocellular carcinoma posing a significant health risk for human populations and livestock. To understand the mammalian systems responses to aflatoxin-B1 (AFB1) exposure, we analyzed the AFB1-induced metabonomic changes in multiple biological matrices (plasma, urine, and liver) of rats using 1 H NMR spectroscopy together with clinical biochemistry and histopathologic assessments. We found that AFB1 exposure caused significant elevation of glucose, amino acids, and choline metabolites (choline, phosphocholine, and glycero- phosphocholine) in plasma but reduction of plasma lipids. AFB1 also induced elevation of liver lipids, amino acids (tyrosine, histidine, phenylalanine, leucine, isoleucine, and valine), choline, and nucleic acid metabolites (inosine, adenosine, and uridine) together with reduction of hepatic glycogen and glucose. AFB1 further caused decreases in urinary TCA cycle intermediates (2-oxoglutarate and citrate) and elevation of gut microbiota cometabolites (phenylacetylglycine and hippurate). These indicated that AFB1 exposure caused hepatic steatosis accompanied with widespread metabolic changes including lipid and cell membrane metabolisms, protein biosynthesis, glycolysis, TCA cycle, and gut microbiota functions. This implied that AFB1 exposure probably caused oxidative-stress-mediated impairments of mitochondria functions. These findings provide an overview of biochemical consequences of AFB1 exposure and comprehensive insights into the metabolic aspects of AFB1-induced hepatotoxicity in rats. Keywords: hepatotoxicology • metabonomics • aflatoxin B1 • nuclear magnetic resonance (NMR) Introduction Aflatoxins are produced by fungi, such as Aspergillus flavus and Aspergillus parasiticus, 1 and commonly found in food and feedstuff 2 containing cereal grains, oilseeds, and peanuts in humid environments. The exposure to aflatoxins can lead to liver injuries, liver fibrosis, and hepatocellular carcinoma (HCC) and hence poses a significant health risk for human popula- tions and livestock. 3,4 There are many natural aflatoxins such as aflatoxin B1, B2, G1, and G2, among which aflatoxin B1 (AFB1) is the most frequently occurring and highly toxic one. 5,6 Traditional biochemical studies have shown that the hepa- totoxicity of AFB1 is related to its interactions with biomol- ecules such as nucleic acid and proteins. 7-9 AFB1 is initially oxidized by the microsomal mono-oxygenases in the liver cytochrome P450 system, 10,11 yielding highly reactive AFB1- exo-8,9-epoxide as the ultimate carcinogen. AFB1-exo-8,9- epoxide is a short-lived electrophile and can readily form an 8,9-dihydro-8-(N 7 -guanyl)-9-hydroxy-AFB1 (AFB1-N 7 -Gua) ad- duct by binding to the guanine residues of DNA. 7 AFB1-exo- 8,9-epoxide can also undergo rapid nonenzymatic hydrolysis to form AFB1-dihydrodiol, 12 which in turn undergoes slow base-catalyzed ring-opening to form a dialdehyde phenolate ion. Such an ion can interact with protein amine groups to form AFB1-protein adducts, such as aflatoxin-albumin. 13 The formation of these DNA and protein adducts causes serious DNA, RNA, and proteins dysfunctions. 14-16 Consequently, AFB1 exposure causes alterations in metabolic processes, such as glycogenolysis/glycolysis 17 and phospho- lipidation, 18 and changes in amino acid transportation. 14 AFB1 exposure can also indirectly result in damages to the structural integrity of cell membranes and ultimately lead to cell death. 19 Therefore, AFB1 exposure may cause changes in metabolisms related to the cell membrane although no such reports have been published so far. With the traditional biochemical method focusing on a single metabolic pathway at a time, 17 however, it remains to be systematically investigated whether AFB1 exposure causes other metabolic alterations especially me- * To whom correspondence should be addressed. H.T.: e-mail, Huiru.tang@ wipm.ac.cn; tel, +86-27-87198430; fax, +86-27-87199291. Y.W.: e-mail, Yulan.wang@wipm.ac.cn; tel, +86-27-87197143; fax, +86-27-87199291. † State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics. ‡ Ningbo University. § Graduate School of the Chinese Academy of Sciences. 614 Journal of Proteome Research 2011, 10, 614–623 10.1021/pr100792q  2011 American Chemical Society Published on Web 11/17/2010