Ochratoxin a Induced Early Hepatotoxicity: New 32

OPEN Ochratoxin A induced early

SUBJECT AREAS: hepatotoxicity: new mechanistic insights RISK FACTORS DISEASES from microRNA, mRNA and proteomic

Received profiling studies 20 February 2014 Xiaozhe Qi1*, Xuan Yang1*, Siyuan Chen1, Xiaoyun He1, Harsh Dweep2, Mingzhang Guo1, Accepted Wen-Hsing Cheng3, Wentao Xu1*, Yunbo Luo1, Norbert Gretz2, Qiu Dai1 & Kunlun Huang1 12 May 2014

Published 1Laboratory of food safety and molecular biology, College of Food Science and Nutritional Engineering, China Agricultural 4 June 2014 University, Beijing 100083, P.R. China, 2Medical Faculty Mannheim, Medical Research Center, University of Heidelberg, D- 68167, Mannheim, Germany, 3Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, MS, 39762, USA. Correspondence and requests for materials The mycotoxin ochratoxin A (OTA) is found widely in agricultural commodities. OTA can induce various should be addressed to toxicities. In this study, rats were gavaged with OTA for different weeks. Then, the expression of W.T.X. (xuwentao@ microRNAs, mRNAs and proteins were measured in the rat livers treated with OTA for 13 weeks. Our cau.edu.cn) sequencing data suggests that the medial and the high doses of OTA exert different effects on livers. Five distinctive pathways were induced after OTA treatment as collectively demonstrated at miRNA, mRNA and protein levels. Two (primary bile acid biosynthesis, and metabolism of xenobiotics by cytochrome P450) are * These authors directly associated with liver damage, whereas the remaining pathways (arginine and proline metabolism, contributed equally to cysteine and methionine metabolism, and PPAR signaling pathway) cause metabolic disease. This study reveals OTA-induced early hepatotoxicity for the first time by combining multi-omics methods. The novel this work. metabolic pathways may contribute to the pathogenesis of metabolic diseases later in life.

chratoxin A (OTA) is a low molecular weight mycotoxin produced by certain strains of filamentous fungi of the Aspergillus and Penicillium genera and has been detected in a large variety of agricultural commodities. OTA carries potential health-associated risks and has been classified as a possible human O 1 carcinogen (group 2B) by the International Agency for Research on Cancer . OTA has previously been found to induce various toxic effects including nephrotoxicity, hepatotoxicity, immunotoxicity, genotoxicity, carcinogenicity, teratogenicity, neurotoxicity and mutagenicity. The liver is one of the major target organs of OTA biotransformation. Although, some early hepatotoxicity studies employed relative high concentrations of OTA and found remarkable liver lesion2,3, lower doses of OTA did not provoke significant pathological changes4–6. Most of the previous studies have been extensively focused on OTA-induced kidney damage, less on liver. However, it is still unknown how OTA affects the liver which is the largest detoxification organ of body. Therefore, the identification of early hepatotoxicity can be helpful to understand the mechanism of some diseases that may be developed and progressed by OTA in later stage, and to prevent these diseases in early stage to improve human health. Omics approaches, such as transcriptome and proteome, are promising to detect the pathological changes of liver at a molecular level. Till date, omics technology has been employed only in a few studies to understand the mechanism of OTA hepatotoxicity. Moreover, a high-throughput microarray profiling study on HepG2 liver cell transcriptome demonstrated that multiple hepatic metabolism genes are modulated by OTA exposure7.In addition, proteomic approaches have helped identify key proteins in the livers of pigs treated with OTA8. These omics approaches have widened our view about OTA hepatotoxicity, however the mechanism of OTA actions remains largely unknown. Recently, microRNAs (miRNAs) in animals and plants are shown to play an important role in stress resistance and metabolism. miRNAs are small (,22 nucleotides in length), noncoding and single-stranded RNAs, which negatively regulate gene expression either by translational inhibition or exonucleolytic messenger RNA (mRNA) decay. miRNAs have already been described as crucial regulators of gene expression in a wide range of processes,

SCIENTIFIC REPORTS | 4 : 5163 | DOI: 10.1038/srep05163 1 www.nature.com/scientificreports including induction or maintenance of cell fate in normal, stem and groups. We also found that 17, 11 and 11 miRNAs were confined cancerous cells. With these functions, miRNAs have been extensively to control, medial and high groups respectively. studied as biomarkers for the diagnosis and therapy in diverse range Hierarchical clustering results depicted that miRNA expression of diseases, as well as for their impact on gene regulation. The altogether was similar between control and high-dose groups, but miRNA expression profiling is not only helpful in identifying they were differentially expressed compared to medial-dose group miRNAs capable of regulating a large range of biological processes, (Fig. 1). Our sequencing data indicates that OTA-induced hepato- but also can be systematically utilized to study the gene regulation, toxicity is differentially mediated in medial-dose and high-dose especially when miRNA measurements are combined with mRNA groups. profiling and other genome-scale data. Compared to the control livers, 47 up- and 26 down-regulated Within recent years, the multiple omics profiling technologies are miRNAs were found only in the high-dose group (p , 0.05), while considered powerful to acquire corroborative evidence for hepato- 59 up- and 47 down-regulated miRNAs only in the medial-dose toxicity study. Through a cross-omics method, it is demonstrated group, indicating a differential response to different doses of OTA. that the major metabolic consequences of acute toxicity of aflatoxin In addition, 29 up- and 14 down-regulated miRNAs were noticed in B1, one of the well-known hepatotoxic mycotoxin, are associated both high-dose and medial-dose groups, respectively. Of note, 8 with gluconeogenesis and lipid metabolism pathways9. Moreover, (rno-let-7a-5p, let-7c-5p, let-7e-5p, let-7f-5p, miR-92a-3p, miR- inorganic arsenic methylation in mouse liver may be saturated under 126a-3p, miR-92b-3p, and miR-3596c) and 4 (rno-miR-19a-3p, chronic exposure to the element based on an integrated-omics miR-19b-3p, miR-29b-3p, and miR-532-5p) miRNAs were com- approach10. The toxicity mechanism of another hepatotoxic commonly detected in up- (Fig. 2A) and down-regulated (Fig. 2B) pound EMD 335823 was also elucidated by using modern omics groups, respectively, across the three comparisons of the OTA dose effect. technologies, from which the liver pathologies are linked to deregu- 16 lated PPARa signaling, glucose metabolism, and fatty acid metabol- miRWalk database was employed to collect the putative targets ism11. Furthermore, omics analyses on several hepatotoxicants have for deregulated miRNAs observed in OTA-treated groups by con- enhanced preclinical safety assessment and helped to identify the sidering six different algorithms (miRanda, miRDB, miRWalk, nature of injury. In particular, Wnt/b-catenin signaling has been PITA, TargetScan and RNAhybrid). The targets predicted by at least identified as the biomarker of human hepatocellular carcinoma two different programs were chosen – as many investigators have and gastrointestinal disease12. Altogether, cross-omics methods recently been utilized this ‘‘at least 2 algorithms’’ approach to effectively reduce false positive targets13,17–22. In a next step, further ana- indeed have enhanced the understanding of liver metabolic regu- lysis was carried out on these selected putative target genes. Analyses lation of exogenous chemicals. The exploration of OTA hepatotoxi- of the identified miRNAs by KEGG pathway enrichment demon- city also can be better understood by integration of multiple omic strated 136 and 145 pathways in high-dose and medial-dose groups, platforms (Fig. S1). On the other hand, a whole body model is super- respectively, compared to control group (Table S2A–F). In total, 133 ior to an in vitro model to better reflect the metabolic and physio- overlapping pathways were identified in OTA-treated groups. logical circumstances. Among these, ‘‘pathways in cancer’’, ‘‘MAPK signaling pathway’’, In order to better understand the early hepatotoxic response to and ‘‘metabolic pathways’’ were highly significantly enriched in all OTA exposure in male F344 rats, we herein integrated miRNA, OTA-treated groups. mRNA and proteomic profiling approaches to identify the biological Furthermore, an additional miRNA-target prediction was con- pathways of early OTA-induced hepatotoxicity. ducted with at least three different programs23 and pathway enrichment analysis was carried out. The pathways resulting with ‘‘at least Result two algorithms’’ and ‘‘at least three algorithms’’ were comparable in Health status of rats gavaged with OTA. After the administration of which ‘‘pathways in cancer’’ and ‘‘MAPK signaling pathway’’ were OTA, body mass of the high-dose rats reduced about 7% (p , 0.05) at highly significantly enriched in all OTA-treated groups. 9 and 10 weeks13, but gradually recovered thereafter. The liver OTA differentially affects mRNA expression profiles in the livers weights were comparable between OTA-treated and control rats at . Illumina HiSeqTM2000 was used for identifying the differentially 2, 4 and 13 weeks (Fig. S2). These results are consistent with a expressed mRNAs in livers of OTA-treated and control rats. The previous toxicity study of orally administering OTA for 13 weeks5. differentially expressed genes between two groups were screened LDH of a and c groups, and AST of c group were lower in the high- based on DESeq analysis, and the controlling FDR was set to 5% dose than the control rats, which were consistent with previous level of significance. We identified 28 up-regulated and 17 down- reports14,15. In contrast, ALB activity of c group was slightly higher regulated genes in high-dose compared to control group. Six genes in high-dose group than in control rats13. The liver damage in high- were highly elevated, while three genes were significantly suppressed dose group is more serious than that in medial dose group at 13 in medial-dose group compared to control group. Only one gene was weeks. Hence, we focused on the high-dose group in the subsequent differentially expressed in high-dose compared to medial-dose study. group. These differentially expressed mRNAs (Log2fold change . Analyses of a complete gross necropsy and microscopic anatomic 1, p , 0.05) are shown in Table 1. The KEGG pathway enrichment pathology demonstrated that rat livers treated with OTA for 2, 4 and analysis on these differentially expressed mRNAs resulted in 50 and 13 weeks were comparable to those without OTA treatment. Bile 24 enriched pathways in high-dose and medial-dose groups, duct hyperplasia appeared in rat liver treated with OTA for 26 weeks respectively. Furthermore, there were 20 overlapping pathways, (data not shown). We concluded that OTA did not apparently induce among which ‘‘amino acid metabolism’’, ‘‘xenobiotics biodegrada- pathological damage in the livers of the rats in the 13 weeks, in which tion and metabolism’’, ‘‘energy metabolism’’, and ‘‘environmental point the cross-omics analysis of rat livers was conducted. information processing’’ were enriched.

OTA differentially affects miRNA expression profiles in the livers. Overlapping pathways between deregulated miRNAs and mRNAs. To evaluate miRNA expression profile in livers of rats treated with In order to identify the commonly influenced mechanisms due to OTA, an Illumina Hiseq 2000 platform was utilized. A total of 304, OTA treatments, the significantly enriched pathways of miRNAs and 301 and 304 known mature miRNAs were identified in control, mRNAs analyses were compared. By comparing the mRNA and medial-dose and high-dose groups, respectively. Of these, 272 miRNA results, 21 and 13 commonly enriched pathways were iden- mature miRNAs were found to be expressed in all the three tified in high-dose and medial-dose groups, respectively (Table 2).

SCIENTIFIC REPORTS | 4 : 5163 | DOI: 10.1038/srep05163 2 www.nature.com/scientificreports

Figure 2 | Venn diagrams of differential expression of miRNAs. The differential expression of miRNAs between cH vs cC (HC), cM vs cC (MC) and cH vs cM (HM) group. A. up-modulated miRNAs; B. down- modulated miRNAs.

Seven pathways including amino acid metabolism, lipid metabolism, signaling molecules and interaction, and xenobiotics biodegradation and metabolism were commonly identified in high-dose and medial- dose groups. In the high-dose group, the pathways influenced by OTA were solely associated with circulatory system, digestive system, endocrine system, excretory system, lipid metabolism, and signal transduction. Nevertheless, in the medial-dose group, the effects of OTA were relatively specific, primarily centering on carbohydrate metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and signaling molecules and interaction. These pathway results also suggest that different toxicity mechanisms are provoked by different doses of OTA.

Cross-omics analyses and the identification of five common pathways. Some miRNAs induce mRNAs degradation and/or repress mRNA translation without changing mRNA expressions. A comprehensive regulatory network was constructed by integrating hepatic miRNA, mRNA, and protein profiles in response to OTA treatments. In high-dose group, a proteomic approach was adopted by using two-dimensional gel electrophoresis and applying a threshold cutoff of at least 1.5 range ratio and p , 0.05. A total of 61 proteins were significantly different between the high-dose and control group (Fig. 3). A pathway enrichment analysis on these 61 proteins was performed and compared with those pathways obtained from miRNA and mRNAs profiling studies in the high-dose group. Five pathways encompassing cysteine and methionine metabolism, PPAR signaling, primary bile acid biosynthesis, arginine and proline metabolism, and metabolism of xenobiotics by cytochrome P450 were commonly identified among all the three profiles. Differentially regulated miRNAs among these five different pathways are described in Table 3. The mRNAs and their corresponding miRNAs related to these five most interesting pathways are shown in Table 4. Table 5 depicts the possible interactions among the identified proteins and the deregulated miRNAs in the five most relevant pathways. Interestingly, these miRNAs were found to have a distinct expression pattern in medial-dose and high-dose groups compared to control group. The expression patterns of these miRNAs were found to be dose-related in PPAR signaling, arginine metabolism and proline metabolism pathways. However, in cysteine and methionine metabolism pathway, all of these miRNAs showed highly elevated expressions in medial-dose group compared to high-dose group. In primary bile acid biosynthesis pathway, some but not all of these miRNAs were noticeably dose-related. Although the proteomic ana- Figure 1 | Hierarchical clustering of miRNA expression. miRNA profiles lysis was not conducted for the medial-dose group, analyses of the from three groups of livers were clustered. Treatment groups are in transcriptomic data demonstrated that similar signaling pathways columns, miRNAs in rows. (cysteine and methionine metabolism, arginine and proline meta-

SCIENTIFIC REPORTS | 4 : 5163 | DOI: 10.1038/srep05163 3 www.nature.com/scientificreports

Table 1 | Differentially expressed mRNAs (detected in all cC, cM and cH) in HC, MC and HM comparisons (Log2foldchange .1)

Gene Name Gene ID Log2FoldChange Gene Name Gene ID Log2FoldChange HC group HC group Sds ENSRNOG00000001388 4.8149 Ces1f ENSRNOG00000015317 21.1096 D3ZYL1_RAT ENSRNOG00000017727 3.0524 Ier2 ENSRNOG00000002837 21.1575 Mtmr7 ENSRNOG00000011420 3.043 Pdp2 ENSRNOG00000012343 21.1923 Mbnl3 ENSRNOG00000002487 2.0619 Prkcdbp ENSRNOG00000017914 21.2845 Amdhd1 ENSRNOG00000005266 1.8753 Pcp4l1 ENSRNOG00000003209 21.2962 Acmsd ENSRNOG00000003884 1.8615 Klf10 ENSRNOG00000006118 21.3275 Gls2 ENSRNOG00000031612 1.6723 Hspb1 ENSRNOG00000023546 21.4709 Cyp17a1 ENSRNOG00000020035 1.6398 Chka ENSRNOG00000016791 21.5159 Atp1b1 ENSRNOG00000002934 1.521 Csad ENSRNOG00000011573 21.6345 Ass1 ENSRNOG00000008837 1.4352 Fam129a ENSRNOG00000002403 21.6521 Slc7a2 ENSRNOG00000011016 1.4037 Fgf21 ENSRNOG00000020990 21.801 Ppm1k ENSRNOG00000006893 1.3238 A2m ENSRNOG00000028896 22.0095 D4A608_RAT ENSRNOG00000039494 1.316 LOC100361444 ENSRNOG00000011490 22.0345 Srd5a1 ENSRNOG00000017601 1.2593 Ednra ENSRNOG00000012721 22.2543 Ccdc152 ENSRNOG00000039473 1.248 Ltc4s ENSRNOG00000003244 22.8252 Slc38a4 ENSRNOG00000006653 1.226 Tgm1 ENSRNOG00000020136 22.9146 Cyp2c23 ENSRNOG00000013291 1.2199 Phgdh ENSRNOG00000019328 24.3183 ARLY_RAT ENSRNOG00000000903 1.1981 MC group Il33 ENSRNOG00000016456 1.178 Sds ENSRNOG00000001388 3.7686 Cited2 ENSRNOG00000012193 1.1506 D3ZYL1_RAT ENSRNOG00000017727 3.3628 Cyp8b1 ENSRNOG00000019481 1.1383 RGD1559459 ENSRNOG00000042714 1.4929 Slc16a10 ENSRNOG00000000588 1.1327 Oat ENSRNOG00000016807 1.3765 Tsc22d1 ENSRNOG00000001030 1.1112 D4A608_RAT ENSRNOG00000039494 1.1047 Cps1 ENSRNOG00000013704 1.0899 Ass1 ENSRNOG00000008837 1.0601 LOC310721 ENSRNOG00000015222 1.088 Csad ENSRNOG00000011573 21.0806 F1LXN6_RAT ENSRNOG00000028668 1.0879 Asns ENSRNOG00000007546 22.276 LOC100361238 ENSRNOG00000002345 1.0772 Phgdh ENSRNOG00000019328 24.5691 Car14 ENSRNOG00000023162 1.0277 HM group Hamp ENSRNOG00000021029 2.5253

bolism and metabolism of xenobiotics by cytochrome P450) were group (Fig. 4A). Additionally, qRT-PCR experiments were carried enriched as compared to control group. This suggests that these three out to verify transcriptional results in which the expression patterns pathways are also modulated by medial-dose of OTA. In addition, of Sds and Cps1 were significantly increased in the high-dose group the expression of three genes (Sds, Ass1 and Rgd1559459) were (p , 0.05). Ass, Cyp8b1, and Cyp2c23 were exhibited an upward significantly different between medial-dose and control group. trend (p 5 0.083, 0.051, 0.549, respectively) (Fig. 4B). qRT-PCR These genes are the representative members of cysteine and methio- results of miRNA and mRNA were mostly consistent with the nine metabolism, arginine and proline metabolism, and metabolism sequencing findings, illustrating an agreement with the sequencing of xenobiotics by cytochrome P450 pathways. profiles. Proteins involved in these five most interesting pathways were listed in Table 6. Carbamoyl-phosphate synthase (Cps1) and Three important enzymes related with miRNA processing. The S-adenosylmethionine synthase isoform type-1 (Mat1a) were up- expression of key miRNA processing regulators (Drosha, Dicer1 regulated, while alpha-methylacyl-CoA racemase (Amacr), hydro- and DGCR8) was determined. As shown in Fig. 5, mRNA levels of xymethylglutaryl-CoA synthase (Hmgcs2), glutathione S-transferase DGCR8 and Dicer1 were significantly reduced in high-dose group Y-b subunit (Gstm2), ornithine carbamoyltransferase (Otc) and compared with control group. These results are consistent with pre- cytosol aminopeptidase (Lap3) were down-regulated. Surprisingly, vious reports on liver carcinogenesis and regeneration studies24,25. only carbamoyl-phosphate synthase and its encoding gene both were Also, our proteomic data showed that some of the differentially up-regulated, whereas the remaining six genes were not differentially expressed proteins were closely related with liver regeneration as expressed in the high-dose group based on the transcriptomic data. well (data not shown). These observations suggest a mode of action of miRNAs in which they only inhibit the translational process but not drive mRNA Impact of OTA on the liver. To elucidate the impact of OTA on repression. hepatotoxicity, we further investigated the five most significantly altered pathways (Table 4) induced by OTA. Fig. 6 illustrated the PCR validation of miRNAs and mRNAs in these pathways. The regulatory network of significantly regulated genes, miRNAs and miRNAs and mRNAs involved in these five most relevant pathways proteins by high-dose OTA in the livers. Three miRNAs: rno-miR- were validated by qRT-PCR. The expression of rno-miR-195-5p, 30a-5p, miR-30d-5p, and miR-30e-5p were involved in both cysteine miR-130a-3p, miR-30a-5p, miR-140-5p, and miR-128-3p were and methionine metabolism, as well as in primary bile acid examined. Compared to control livers, the expression of rno-miR- biosynthesis. rno-miR-130a-3p, rno-miR-130b-3p, and Cyp8b1 195-5p was significantly increased, while the expression of rno-miR- were associated with both PPAR signaling pathway and primary 130a-3p was significantly decreased in high-dose group (p , 0.05). bile acid biosynthesis. rno-miR-122-5p was participated in both Despite of being statistically insignificant, rno-miR-30a-5p was primary bile acid biosynthesis and arginine and proline metabo- observed with an up-regulation trend while two other miRNAs: lism, whereas the rno-miR-128-3p was involved in arginine and rno-miR-140-5p and rno-miR-128-3p were identified with a proline metabolism and metabolism of xenobiotics by cytochrome down-regulation trend in the high-dose compared to control P450. Altogether, analyses of these pathways suggest an implication

SCIENTIFIC REPORTS | 4 : 5163 | DOI: 10.1038/srep05163 4 www.nature.com/scientificreports

Table 2 | mRNA and miRNA commonly enriched pathways respectively in high and medium dose groups (p , 0.05)

Pathway Secondary classification Primary classification High dose Arginine and proline metabolism Amino acid metabolism Metabolism Cysteine and methionine metabolism Amino acid metabolism Metabolism Tryptophan metabolism Amino acid metabolism Metabolism Fructose and mannose metabolism Carbohydrate metabolism Metabolism Regulation of actin cytoskeleton Cell motility Cellular Processes Vascular smooth muscle contraction Circulatory system Organismal Systems Gastric acid secretion Digestive system Organismal Systems Salivary secretion Digestive system Organismal Systems PPAR signaling pathway Endocrine system Organismal Systems Aldosterone-regulated sodium reabsorption Excretory system Organismal Systems Metabolic pathways Global and overview maps Metabolism Glycerophospholipid metabolism Lipid metabolism Metabolism Linoleic acid metabolism Lipid metabolism Metabolism Primary bile acid biosynthesis Lipid metabolism Metabolism Steroid hormone biosynthesis Lipid metabolism Metabolism Calcium signaling pathway Signal transduction Environmental Information Processing MAPK signaling pathway Signal transduction Environmental Information Processing Cytokine-cytokine receptor interaction Signaling molecules and interaction Environmental Information Processing Neuroactive ligand-receptor interaction Signaling molecules and interaction Environmental Information Processing VEGF signaling pathway Signaling molecules and interaction Environmental Information Processing Metabolism of xenobiotics by cytochrome P450 Xenobiotics biodegradation and metabolism Metabolism Medium dose Arginine and proline metabolism Amino acid metabolism Metabolism Cysteine and methionine metabolism Amino acid metabolism Metabolism Glycine, serine and threonine metabolism Amino acid metabolism Metabolism Ascorbate and aldarate metabolism Carbohydrate metabolism Metabolism Pentose and glucuronate interconversions Carbohydrate metabolism Metabolism Starch and sucrose metabolism Carbohydrate metabolism Metabolism Metabolic pathways Global and overview maps Metabolism Steroid hormone biosynthesis Lipid metabolism Metabolism Porphyrin and chlorophyll metabolism Metabolism of cofactors and vitamins Metabolism Retinol metabolism Metabolism of cofactors and vitamins Metabolism Cytokine-cytokine receptor interaction Signaling molecules and interaction Environmental Information Processing Neuroactive ligand-receptor interaction Signaling molecules and interaction Environmental Information Processing Metabolism of xenobiotics by cytochrome P450 Xenobiotics biodegradation and metabolism Metabolism of OTA in hepatotoxicity by affecting liver functions or inducing approaches (miRNA, mRNA and proteomic profiles) and identified metabolic diseases. five enriched pathways (primary bile acid biosynthesis, metabolism of xenobiotics by cytochrome P450, PPAR signaling pathway, Discussion cysteine and methionine metabolism, and arginine and proline meta- To the best of our knowledge, this is the first study of its own kind in bolism) that are most likely to be unique and critical in the under- which an integrative approach encompassing miRNA, transcrip- standing of hepatic response to OTA. tomic and proteomic profiling was adopted to explore OTA-induced In the present study, rats were gavaged with OTA doses (0, 70 or early hepatotoxicity. We have employed and integrated three omics 210 mg/kg body weight (bw)), and miRNA, mRNA and protein pro-

Figure 3 | Images of a representative 2D gels from total proteins isolated from rat livers. H, cH group; C, cC group. The numbered spots in H are significantly different between cH and cC groups (range ratio .1.5, p , 0.05).

SCIENTIFIC REPORTS | 4 : 5163 | DOI: 10.1038/srep05163 5 www.nature.com/scientificreports

Table 3 | miRNAs expression changes in five most relevant pathways in cH vs cC (HC) and cM vs cC (MC)

Pathway miRNA MC (fold change) HC (fold change) Cysteine and methionine metabolism rno-miR-103-3p 0.996 1.030 rno-miR-107-3p 0.996 1.030 rno-miR-497-5p 0.750 1.386 rno-miR-15b-5p 1.189 1.136 rno-miR-16-5p 1.305 1.206 rno-miR-195-5p 1.305 1.206 rno-miR-30a-5p 1.099 1.069 rno-miR-30d-5p 1.099 1.069 rno-miR-30e-5p 1.099 1.069 rno-miR-340-5p 1.450 1.103 PPAR signaling pathway rno-let-7d-5p 0.746 1.138 rno-let-7e-5p 1.041 1.377 rno-let-7f-5p 1.041 1.377 rno-miR-130a-3p 0.991 0.800 rno-miR-130b-3p 0.991 0.800 Primary bile acid biosynthesis rno-miR-30a-5p 1.099 1.069 rno-miR-30d-5p 1.099 1.069 rno-miR-30e-5p 1.099 1.069 rno-miR-130a-3p 0.991 0.800 rno-miR-130b-3p 0.991 0.800 rno-miR-148b-3p 0.856 0.946 rno-miR-152-3p 0.856 0.946 rno-miR-190a-5p 1.508 0.394 rno-miR-450a-5p 1.179 0.870 rno-miR-122-5p 0.755 1.155 arginine and proline metabolism rno-miR-128-3p 0.032 1.321 rno-miR-320-3p 0.900 1.126 rno-miR-122-5p 0.755 1.155 rno-miR-449a-5p 0.453 0.280 rno-miR-532-5p 0.886 0.753 rno-miR-140-5p 0.926 0.789 Metabolism of xenobiotics by cytochrome P450 rno-miR-128-3p 0.032 1.321 filing studies were performed in the livers after thirteen weeks of the carbohydrate metabolism pathway has been identified enriched gavage. The rat livers treated with OTA for 2, 4 and 13 weeks were in the medial-dose group29. comparable with controls (without OTA-treatment). However, bile The results of our study provide a comprehensive landscape of duct hyperplasia appeared in rat liver treated with OTA for 26 weeks OTA-induced hepatotoxicity that has never been revealed before. (data not shown). Moreover, lactate dehydrogenase (LDH) and Furthermore, our sequencing data suggest an OTA hepatotoxicity aspartate transaminase (AST) at 13 weeks were lower in the high- based on OTA doses. In particular, the findings resulting from hier- dose compared to control rats, while at 4 weeks, LDH and AST were archical clustering demonstrate that all miRNA profiles in control not significantly different between high-dose group and the control tissue are reminiscent of those in the high-dose group but not similar rats. Both LDH and AST could reflect the liver damage to some in the medial-dose group. Also, the enriched pathways obtained on extent and suggest that the liver damage could be more severe in miRNAs and mRNAs clearly indicate that, although both high- and high-dose group at 13 weeks compared to OTA-treated four-week medial-dose OTA markedly changed the gene expression, their influ- animals. In addition, the differences of the serum biochemical enced effects were quite different. The number of differentially indexes at 13 weeks were higher in high-dose (compared to control) expressed genes by high-dose OTA was marginally higher than those livers than medium-dose (compared to control) livers. Medial-dose observed in medial-dose OTA. These observations suggest that the OTA does not have impact on the serum chemical or histopatholo- high-dose OTA exerts a more extensive effect on the rats, whereas, gical indexes, which is consistent with OTA studies4–6. Nonetheless, the medial-dose OTA acts in a more concentrated manner. the derivatives found in the liver indicates that OTA must be meta- Mechanistically, OTA at the medial dose mainly influences energy bolized in order to act as a carcinogen26. Therefore, we selected 13 metabolism such as carbohydrate metabolism, and basic substance weeks as a critical time point to find out the potential candidates and metabolism (including amino acid metabolism, metabolism of cofac- their signaling cascades which may be closely associated with early tors and vitamins). However, in the high- dose group, the pathways liver damage. No pathological changes were noticed at 13 weeks, but influenced by OTA were solely associated with the different body we wanted to elucidate the early changes in livers caused by OTA systems such as circulatory system, digestive system, endocrine sys- using multi-omics approach which could identify the early metabolic tem, excretory system, lipid metabolism, and signal transduction. and toxicological impacts of OTA and provide an insightful resource Leire et al. reported that the hepatic gene expression changes induced for the prevention and/or attenuation of OTA toxicity in humans. by OTA. The authors also observed that circadian exercise, choles- Some of the OTA pathways resulting from our cross-omics terol biosynthesis and lipid metabolism were influenced in the rat approaches are in line with those reported previously27–29. Briefly, liver which is similar with our high dose group30. In another study in a previous study, three pathways (PPAR signaling pathway, with 300 ml OTA/kg bw, similar dose in our study, carbon metabol- cysteine and methionine metabolism, and arginine and proline meta- ism, amino acid metabolism were influenced in the liver31. This bolism) have been found to be closely associated with metabolic comparative analysis suggests that a high-dose of OTA influences syndrome, especially obesity27. In another study, the digestive system not only the basic metabolism but also endocrine environment, lead- is shown to be impacted by OTA in the high-dose group28. Moreover, ing to the imbalance of whole body, while, a low-dose of OTA enables

Table 4 |miRNAs and their target genes within the five most interested pathways in cH vs cC Cysteine and methionine metabolism mRNA Gene EnsemblID log2 Fold change rno-miR-103- rno-miR-107- rno-miR-15b- rno-miR-16- rno-miR-195- rno-miR-30a- rno-miR-30d- rno-miR-30e- rno-miR-340- rno-miR-497- 3p 3p 5p 5p 5p 5p 5p 5p 5p 5p Sds ENSRNOG00 4.8149 " " """""""" 000001388 PPAR signaling mRNA Gene EnsemblID log2 Fold change rno-let-7d-5p rno-let-7e-5p rno-let-7f-5p rno-miR-130a- rno-miR-130b- 3p 3p Cyp8b1 ENSRNOG00 1.1383 "" "## 000019481 Primary bile acid biosynthesis mRNA Gene EnsemblID log2 Fold change rno-miR-30a- rno-miR-30d- rno-miR-30e- rno-miR-122- rno-miR-130a- rno-miR-130b- rno-miR-148b- rno-miR-152- rno-miR-190a- rno-miR-450a- 5p 5p 5p 5p 3p 3p 3p 3p 5p 5p Cyp8b1 ENSRNOG00 1.1383 " " ""###### 000019481 Arginine and proline metabolism mRNA Gene EnsemblID log2 Fold change rno-miR-128- rno-miR-320- rno-miR-122- rno-miR-449a- rno-miR-532- rno-miR-140- 3p 3p 5p 5p 5p 5p Gls2 ENSRNOG00 1.6723 " " "### 000031612

Ass1 ENSRNOG00 1.4352 - ""-- - www.nature.com/ 000008837 Metabolism of xenobiotics by cytochrome P450 mRNA Gene EnsemblID log2 Fold change rno-miR-128-3p Cyp2c23 ENSRNOG00 1.2199 " 000013291 scientificreports 7 SCIENTIFIC

Table 5 |Proteins and miRNAs in cysteine and methionine metabolism, PPAR signaling pathway, primary bile acid biosynthesis, arginine and proline metabolism and metabolism of xenobiotics by cytochrome P450 pathways. Cps1 gene doesn’t have any 39-UTR known sequence, so its miRNAs could not be detected. 1 means the miRNA targets the protein. 0 means

REPORTS the miRNA does not target the protein.

Protein names

13|DI 10.1038/srep05163 DOI: | 5163 : 4 | Alpha-methylacyl-CoA glutathione S-transferase Hydroxymethylglutaryl- ornithine Alpha-methylacyl-CoA Y-b subunit (EC 2.5.1.18), CoA synthase, cytosol carbamoyltransferase, S-adenosylmethionine MiRNAs Mimatid UpHCmiRs DownHCmiRs racemase partial mitochondrial aminopeptidase mitochondrial precursor synthase isoform type-1 rno-let-7a-5p MIMAT0000774 1 0 0 0 0 0 1 0 rno-let-7b-5p MIMAT0000775 1 0 0 0 0 0 1 0 rno-let-7c-5p MIMAT0000776 1 0 0 0 0 0 1 0 rno-let-7d-5p MIMAT0000562 1 0 0 0 0 0 1 0 rno-let-7e-5p MIMAT0000777 1 0 0 0 1 0 0 0 rno-let-7f-5p MIMAT0000778 1 0 0 0 0 0 1 0 rno-miR-101a-3p MIMAT0000823 0 1 1 0 0 0 1 0 rno-miR-101b-3p MIMAT0000615 0 1 1 0 0 0 1 0 rno-miR-103-3p MIMAT0000824 1 0 1 0 0 1 1 1 rno-miR-107-3p MIMAT0000826 1 0 1 0 0 1 1 1 rno-miR-10a-5p MIMAT0000782 1 0 0 0 1 1 0 0 rno-miR-122-5p MIMAT0000827 1 0 1 0 0 0 1 0 rno-miR-125b-1-3p MIMAT0004730 1 0 1 0 0 0 0 0 rno-miR-125b-5p MIMAT0000830 1 0 1 0 0 1 0 1 rno-miR-128-3p MIMAT0000834 1 0 1 1 1 0 0 0 rno-miR-130a-3p MIMAT0000836 0 1 1 0 0 0 1 0 rno-miR-130b-3p MIMAT0000837 0 1 1 0 0 0 1 0 rno-miR-133a-3p MIMAT0000839 1 0 0 0 1 1 0 0 rno-miR-133b-3p MIMAT0003126 1 0 0 0 1 1 0 0 rno-miR-1-3p MIMAT0003125 1 0 1 0 1 0 1 1 rno-miR-140-5p MIMAT0000573 0 1 1 0 1 0 0 0 rno-miR-141-3p MIMAT0000846 0 1 0 0 1 1 0 1 rno-miR-144-3p MIMAT0000850 0 1 1 0 1 1 0 0 rno-miR-148b-3p MIMAT0000579 0 1 1 1 0 0 0 0 rno-miR-152-3p MIMAT0000854 0 1 1 1 0 0 0 0 rno-miR-15b-5p MIMAT0000784 1 0 1 0 1 0 0 1 www.nature.com/ rno-miR-16-5p MIMAT0000785 1 0 1 0 1 0 0 1 rno-miR-186-5p MIMAT0000863 1 0 0 1 1 1 1 1 rno-miR-190a-5p MIMAT0000865 0 1 1 0 0 1 1 0 rno-miR-191a-5p MIMAT0000866 1 0 0 0 0 1 1 0 rno-miR-192-5p MIMAT0000867 0 1 0 0 1 1 1 1 rno-miR-194-5p MIMAT0000869 1 0 0 0 1 1 0 0 rno-miR-195-5p MIMAT0000870 1 0 1 0 1 0 0 1 rno-miR-19a-3p MIMAT0000789 0 1 1 0 0 1 0 0

rno-miR-19b-3p MIMAT0000788 0 1 1 0 0 1 0 0 scientificreports rno-miR-204-5p MIMAT0000877 1 0 0 0 1 1 0 0 rno-miR-206-3p MIMAT0000879 1 0 1 0 1 0 1 1 rno-miR-20a-3p MIMAT0000603 0 1 0 0 0 1 0 1 rno-miR-211-5p MIMAT0000882 1 0 0 0 1 1 0 0 rno-miR-215 MIMAT0003118 0 1 0 0 1 1 1 1 rno-miR-216a-5p MIMAT0000886 0 1 1 0 0 0 0 0 rno-miR-217-5p MIMAT0000887 0 1 1 0 1 1 0 1 8 SCIENTIFIC

Table 5 |Continued

REPORTS Protein names

Alpha-methylacyl-CoA glutathione S-transferase Hydroxymethylglutaryl- ornithine Alpha-methylacyl-CoA Y-b subunit (EC 2.5.1.18), CoA synthase, cytosol carbamoyltransferase, S-adenosylmethionine 13|DI 10.1038/srep05163 DOI: | 5163 : 4 | MiRNAs Mimatid UpHCmiRs DownHCmiRs racemase partial mitochondrial aminopeptidase mitochondrial precursor synthase isoform type-1 rno-miR-221-3p MIMAT0000890 0 1 1 0 1 1 1 0 rno-miR-223-3p MIMAT0000892 0 1 0 0 0 1 0 1 rno-miR-22-3p MIMAT0000791 1 0 1 0 1 1 0 1 rno-miR-23b-5p MIMAT0017099 1 0 0 0 0 1 0 0 rno-miR-25-3p MIMAT0000795 1 0 1 0 0 1 1 0 rno-miR-26a-5p MIMAT0000796 0 1 1 0 0 1 1 0 rno-miR-27a-3p MIMAT0000799 1 0 0 0 1 0 1 0 rno-miR-27b-3p MIMAT0000798 1 0 0 0 1 0 1 0 rno-miR-29a-3p MIMAT0000802 0 1 0 0 0 1 0 1 rno-miR-29b-3p MIMAT0000801 0 1 0 0 0 1 0 1 rno-miR-29c-3p MIMAT0000803 0 1 0 0 0 1 0 1 rno-miR-30a-5p MIMAT0000808 1 0 1 0 0 1 1 0 rno-miR-30d-5p MIMAT0000807 1 0 1 0 0 1 1 0 rno-miR-30e-5p MIMAT0000805 1 0 1 0 0 1 1 0 rno-miR-320-3p MIMAT0000903 1 0 1 1 0 0 1 1 rno-miR-340-5p MIMAT0004650 1 0 0 0 0 1 1 0 rno-miR-3557-5p MIMAT0017819 1 0 1 1 1 0 0 1 rno-miR-3571 MIMAT0017851 1 0 1 0 0 0 0 0 rno-miR-3587 MIMAT0017883 1 0 0 0 0 0 0 1 rno-miR-3588 MIMAT0017887 1 0 1 0 0 0 0 0 rno-miR-3591 MIMAT0017893 1 0 0 1 0 0 0 0 rno-miR-3596b MIMAT0017871 1 0 0 0 1 0 0 0 rno-miR-3596c MIMAT0017877 1 0 1 0 0 0 0 0 rno-miR-3596d MIMAT0017823 1 0 0 0 0 0 0 1 rno-miR-362-5p MIMAT0012828 0 1 0 0 0 0 0 1 rno-miR-375-3p MIMAT0005307 1 0 1 0 0 1 0 0

rno-miR-378a-3p MIMAT0003379 0 1 1 0 0 1 0 0 www.nature.com/ rno-miR-378a-5p MIMAT0003378 1 0 0 0 0 0 0 1 rno-miR-379-5p MIMAT0003192 1 0 0 0 1 0 0 0 rno-miR-449a-5p MIMAT0001543 0 1 1 0 0 1 1 0 rno-miR-450a-5p MIMAT0001547 0 1 1 0 0 1 0 0 rno-miR-455-3p MIMAT0017308 1 0 0 0 0 0 0 1 rno-miR-455-5p MIMAT0005316 1 0 0 0 0 1 0 0 rno-miR-497-5p MIMAT0003383 1 0 1 0 1 0 0 1 rno-miR-532-5p MIMAT0005322 0 1 0 0 0 1 1 0 rno-miR-542-5p MIMAT0003178 1 0 0 0 1 0 0 0 rno-miR-743b-3p MIMAT0005280 0 1 1 0 0 1 0 1 scientificreports rno-miR-7a-1-3p MIMAT0000607 1 0 0 0 1 0 0 0 rno-miR-7a-5p MIMAT0000606 0 1 0 0 1 1 0 0 rno-miR-7b MIMAT0000780 0 1 0 0 1 1 0 0 rno-miR-92a-3p MIMAT0000816 1 0 1 0 0 1 1 0 rno-miR-92b-3p MIMAT0005340 1 0 1 0 0 1 1 0 rno-miR-96-5p MIMAT0000818 1 0 0 0 0 1 0 0 rno-miR-98-5p MIMAT0000819 1 0 0 0 0 0 1 0 9 www.nature.com/scientificreports

Table 6 | Identification of liver proteins related with cysteine and methionine metabolism, PPAR signaling pathway, primary bile acid biosynthesis, arginine and proline metabolism and metabolism of xenobiotics by cytochrome P450 between cH and cC group using MS/MS analysis.

Spot Protein ID Range ratio Protein name Gene symbol Function

11 gi | 119850777 22.7 Alpha-methylacyl-CoA racemase Amacr Has a role in drug metabolism as well as probably in lipid metabolism. 15 gi | 123330 21.7 Hydroxymethylglutaryl-CoA Hmgcs2 This enzyme condenses acetyl-CoA with acetoacetyl-CoA to synthase, mitochondrial form HMG-CoA, which is the substrate for HMG-CoA reductase 20 gi | 204499 23.0 glutathione S-transferase Gstm2 Conjugation of reduced glutathione to a wide number of Y-b subunit (EC 2.5.1.18), exogenous and endogenous hydrophobic electrophiles. partial The olfactory GST may be crucial for the acuity of the olfactory process. 24 & 50 gi | 8393186 & 3.5 & 2.5 carbamoyl-phosphate synthase Cps1 Involved in the urea cycle of ureotelic animals where the gi | 8393186 enzyme plays an important role in removing excess ammonia from the cell. 29 gi | 77157805 3.0 S-adenosylmethionine synthase Mat1a Catalyzes the formation of S-adenosylmethionine from isoform type-1 methionine and ATP 32 gi | 6981312 26.8 ornithine carbamoyltransferase, Otc Nitrogen metabolism; urea cycle; L-citrulline from L-ornithine mitochondrial precursor and carbamoyl phosphate 40 gi | 58865398 26.6 cytosol aminopeptidase Lap3 Exopeptidase which selectively removes arginine and/or lysine residues from the N-terminus of several peptide substrates.

Spot numbers correspond to those in Fig. 6; Range ratio is the average change in abundance from three independent treatments (cH vs cC); Protein name, matched protein description; Protein ID, accession number from the NCBI database of matched proteins; Function, the funtion of the protein. the imbalance of energy metabolism, but the detoxification of the study. The one of the possible reasons behind this observation could liver still functions. In contrast, a high-dose OTA treatment exhausts be that miRNAs can regulate the gene expression at translational the liver repair system, influencing the overall defense reaction, for level instead of decaying mRNAs35. Another possibility could be that instance, circulatory system and excretory system broken down. the post-transcriptional regulation, such as phosphorylation, ubiqui- Although, our sequencing data suggests that there are differential tination, glycosylation, and acetylation, can make changes on pro- effects between high and medial dose OTA, this could not be con- teins but not on mRNAs36,37. firmed by qRT-PCR. Further studies are needed to confirm our Deregulated PPAR signaling pathway, cysteine and methionine current findings. It should be noted that in the current manuscript, metabolism, and arginine and proline metabolism are known to we only studied the toxicological mechanisms between the high-dose cause metabolic syndrome including obesity, dyslipidemia, hyper- and medial-dose OTA at thirteen weeks. However, various toxic tension, and elevated plasma glucose level. A review of OTA on renal mechanisms could be expected at the different time intervals30. cells has indicated that glomerular capillaries are damaged by hyper- Moreover, several studies have been carried out to elucidate the tension38, which is thought to contribute to OTA-induced disorders. impacts of different doses of other substances. For example, a recent PPARs are closely related to energy status and metabolism. An aber- study demonstrated that a high-dose cyclophosphamide is solely ration of PPARs leads to abnormal expression of genes in metabolic attributed to its direct cytotoxicity in contrast to the response to a pathways, thereby contributing to etiology of metabolic syndrome39. low dose32. In another study, the expression of a few critical genes was In addition, PPARs are the major regulators of lipid and fatty acid altered by medial but not with the high doses of diethylnitrosamine, metabolism. This may explain the metabolic shift of lipid pathways leading to non-linearity effects33. by high dose OTA, as well as changes of protein expression in lipid In the present study, we identified dysregulation in critical path- metabolism based on our proteomic data (data not shown). ways encompassing primary bile acid biosynthesis and metabolism Moreover, coronary artery disease is associated with abnormal of xenobiotics by cytochrome P450 in response to OTA, which pro- methionine metabolism. An elevated level of total homocysteine in vide a pathophysiological explanation to OTA-induced live damage. the blood is a strong risk factor for atherosclerotic vascular disease in Bile acid is a primary component of bile and plays an important role the coronary, cerebral, and peripheral vessels, and for arterial and in the absorption of not only fat and fat-soluble vitamins, but also venous thromboembolism. Altered methionine metabolism also xenobiotics. OTA is absorbed in the upper portion of the gastro- plays a significant role in the protection against oxidative stress, intestinal tract and then transported to the liver. Thereafter, OTA suggesting that OTA could cause oxidative damage. Further future is secreted along with bile acid from the liver to gastrointestinal tract experiments are required to support the notion whether OTA treat- through enterohepatic circulation. An increase in bile acid likely ment promotes cardiovascular disease - as arginine and proline indicates increasing OTA absorption, resulting in the elevation of metabolism signaling was highly significantly enriched in our study. OTA-induced toxicity. Also, aflatoxins-induced acute liver injuries Interestingly, arginine metabolism has previously been shown to play have been shown to be promoted by elevated serum bile acid levels. a major role in cardiovascular physiology and to ameliorate the The metabolism of xenobiotics by cytochrome P450 is another most metabolic syndrome in Zucker diabetic fatty Rats40. Furthermore, relevant pathway for proper liver functioning. Cytochrome P450 OTA has been reported to stimulate ceramide formation which par- system protects liver by metabolizing exogenous substances. We also ticipates in the pathophysiology of cardiovascular diseases27. found a decrease in the expression level of glutathione S-transferase Additionally, glutaminase 2 (Gls2) expression is induced in response (Gstm2). This gene has previously been reported to be significantly to DNA damage or oxidative stress41, suggesting that OTA may also down-regulated during the development of hepatocellular preneo- induces DNA damage or oxidative stress. plastic foci in rats34. In our study, the down-regulation of Gstm2 was Notably, most of the pathways identified in our investigation have only observed at the protein level, but not during mRNA profiling previously been explored in OTA toxicity related studies. In addition,

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Figure 4 | qRT-PCR analyses of miRNAs (A) and mRNAs (B). *: p , 0.05, compared to cC. Values are means 6 SD (n 5 6). the role of biotransformation in OTA toxicity has already been reports found an elevation in their expression43. Furthermore, energy addressed in previous studies. Some of these investigators reported metabolism was deregulated induced by OTA in vitro and in vivo a down-regulation in the expression of the representative genes of studies. Also, many genes (for example, Glutathione S-transferase) transporters and xenobiotic metabolism30,42, while the remaining involved in energy and xenobiotic metabolism of HepG2 liver cell exposed to OTA were down-regulated7. Our results also showed a reduction in this gene. Male Tsc2 EKER rats gavaged with OTA also changed their energy provision43. In addition, liver lipid metobolism was down-regulated of male F344 rats after OTA treatments in several studies30,31. In our result, the protein (such as Hmgcs2) was down-regulated in PPAR pathway, which is the major regulator of lipid and fatty acid metabolism. Also, the amino acids metabolism (such as alanine and aspartame metabolism, tryptophane metabolism) was affected in rats’ liver30. We compared the miRNA pathways with at least two and three algorithms, and found that the lost pathways with at least three algorithms mainly concentrated on amino acid metabolism, carbohydrate metabolism, glycan biosynthesis, lipid metabolism, and metabolism of cofactors and vitamins. In previous studies on OTA, amino acid metabolism, carbon metabolism pathways were influenced in the liver31. In addition, genes related with glycan biosynthesis in HepG2 cells are changed in response to OTA treatment7. Figure 5 | qRT-PCR analyses of Drosha, DGCR8 and Dicer1 mRNA Also, the lipid metabolism was influenced in the rat liver induced by levels. Drosha, DGCR8 and Dicer1 mRNA levels in livers of the rats in cC, OTA30. Moreover, vitamins are important cofactors of many cM and cH groups were shown. Expression levels were normalized using enzymes and thereby influence metabolic activities, playing a part those of b-actin. Values are mean 6 SD (n 5 6). in the effect of mycotoxins44. We therefore think that pathways

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Figure 6 | A comprehensive picture of miRNAs, mRNAs and proteins. miRNAs, mRNAs and proteins participated in the five pathways, describing the impact of OTA on hepatic pathophysiology in the rats. Red and green color represents up and down-regulation, respectively. resulting from at least two algorithms are more comprehensive. complementary to their mRNA targets and they inhibit protein syn- Among the five interested pathways (primary bile acid biosynthesis, thesis through an unknown mechanism that preserves the stability of and metabolism of xenobiotics by cytochrome P450, arginine and the mRNA target. Apart from the repression, several groups have proline metabolism, cysteine and methionine metabolism, and recently reported that miRNAs can also activate rather than repress PPAR signaling pathways) in the current study, metabolism of xeno- their mRNA targets under certain conditions, which is in contrast to biotics by cytochrome P450, and arginine and proline metabolism the known ‘‘dogma’’ of miRNA regulation as was known previously. were not identified with at least three algorithms. Cytochrome P450s It is probably the reason that rno-miR-195-5p induction by OTA are the critical biotransformation enzymes involved in OTA tox- treatment along with its mRNA target (Sds) induction as well (Fig. 4). icity45. These pathways are lost either due to this highly stringent Therefore, it is ideal and necessary to incorporate results from sys- criterion (at least 3 algorithms) to reduce the number of false positive tematic analyses of miRNA profiling, transcriptomics and proteo- targets or could be that these pathways may be slightly modulated by mics for making a comprehensive and robust conclusion. miRNAs, but previous studies suggest their potential relationship The combination of omics technologies allows an improved with OTA. Pathways resulting with at least two algorithms contain detailed analysis and identification of potential mechanisms to ad- more information; hence, the method using at least 2 algorithms is vance the overall understanding of OTA hepatotoxicity. Our cross- reasonable. omics results demonstrate that different doses of OTA exert a diverse The proteomic and transcriptomic expression results of carba- pathophysiological impact and elucidate OTA-induced five most moyl-phosphate synthase out of the five most interesting pathways relevant pathways (cysteine and methionine metabolism, PPAR sig- were comparable; however, unfortunately, the correlation between naling, primary bile acid biosynthesis, arginine and proline metabol- mRNA and protein results was relatively low. Recently, the relation- ism, and metabolism of xenobiotics by cytochrome P450) are ship between miRNA, mRNA and proteins has been explored by induced by OTA. Nonetheless, future investigations are required to many researchers. In principle, a perfect or near-perfect Watson- study the mechanisms of OTA hepatotoxicity by further exploring Crick complementarity between the miRNA and its target mRNA these five pathways. results in mRNA cleavage and decay, whereas, an imperfect complementarity directs the translational repression. Such imperfect inter- Methods actions of miRNAs regulate the gene expression at translational level Ethics statement. Every possible effort was made to minimize animal suffering. All instead of decaying mRNAs. Most animal miRNAs are imprecisely procedures were approved by the the Ethics Committee of China Agricultural

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University (permission number: 120020). All experiments were performed in Dithiothreitol (DTT), 1% (v/v) pH 4–7 IPG buffer (GE Healthcare, NJ, USA) and accordance with relevant guidelines and regulations. 0.5% (v/v) protease inhibitor cocktail, sonicated intermittently for 90 s and then centrifuged at 15000 g for 20 min at 4uC. The supernatants were mixed (six rats per Animals. Specific pathogen-free male F344 rats were obtained at the age of six weeks group) and carried out in triplicate. Protein concentrations were determined by the and were kept in accordance with institutional guidelines [The Supervision and Coomassie blue method46. For the first dimension, samples containing 550 mg total Testing Center for GMOs food safety, Ministry of Agriculture (Beijing, China) with proteins were mixed in 250 mL of rehydration buffer containing 7 M urea, 2 M the license number SYXK (Beijing) 2010–0036]. Rats were housed three per cage in a thiourea, 2% (w/v) CHAPS, 65 mM DTT, 2% (v/v) pH 4–7 IPG buffer (GE temperature controlled room (22 6 2uC) with a relative humidity of 40 , 70% and a Healthcare, NJ, USA), and 0.002% bromophenol blue. Isoelectric focusing (IEF) was 12 h light-dark cycle. Feed and sterilized water were consumed ad libitum. Rats (six performed in IPG strips (pH 4–7, 13 cm, GE Healthcare) on a Multiphor IIIsystem per group) were randomly assigned to control group (C), medial-dose group (M), (GE Healthcare, NJ, USA) using the following program: 500 V for 500 Vh, 1000 V high-dose group (H), gavaged with OTA at 0, 70 or 210 mg/kg bw, for 2, 4, 13 and 26 for 800 Vh, 8000 V for 11500 Vh, 8000 V for 7500 Vh, 500 V for 10 h. The second weeks (designated as a, b, c and d) respectively. OTA was dissolved in corn oil (Aladin, dimension electrophoresis was performed in 12.5% SDS polyacrylamide gels at 2 W/ Shanghai, China). Body weights were recorded weekly. After the gavage, rats were gel for 45 min, followed by separation at 4 W/gel until the dye front reached the anesthetized using chloral hydrate (6%, 5 ml/kg bw, ip). Blood samples were collected bottom of the gel. The proteins were visualized with Coomassie Brilliant Blue R-250 from the inner canthus before decapitation for determination of blood parameters after a 1 h protein fixation in 50% ethanol, 10% acetic acid and 40% water. Gels were including alanine (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), destained with a solution of 30% ethanol, 8% acetic acid and 62% water for 2 h. The albumin (ALB), creatinine (CREA), glucose (GLU), blood urea nitrogen (BUN), resulting digital images were analyzed and quantified using the Image-Master 2D 7.0 lactate dehydrogenase (LDH), high-density lipoprotein (HDL) and low-density software (GE Healthcare, NJ, USA). Three independent experiments were conducted. lipoprotein (LDL) using a Hitachi 7020 automatic biochemical analyzer (Hitachi, One-way ANOVA (p , 0.05) was used for selecting the different spots between the Tokyo, Japan)13. two groups (cH vs cC). For mass spectrometry (MS), spot picking of interest was carried out with pre- Preparation of livers. The liver was weighed, and a complete liver necropsy was parative gels and subjected to in-gel trypsin digestion according to Sun et al.47 with performed. The livers were fixed in 4% buffered formaldehyde for at least 24 h before minor modifications. Matrix-assisted laser desorption/ionization time-of-flight histological processing, embedded in paraffin, sectioned to 4–6 mm thick, and then (MALDI-TOF) and tandem TOF/TOF MS analyses were carried out on a 4800 stained with hematoxylin–eosin (H&E). Histopathological examination of tissue Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA) according to Sun sections was conducted by a pathologist at the Experimental Animal Research Center, et al.48. GPS ExplorerTMsoftware, version 3.6 (Applied Biosystems, Foster City, CA, China Agricultural University. Part of livers were frozen immediately in liquid USA) was used for creating and searching files with the MASCOT (Matrix Science, nitrogen and kept at 280uC for further studies. http://www.matrixscience.com/) for peptide and protein identification. The highest protein score (top rank) was singled out from the multiprotein family. Precursor error miRNA profiling and statistical analysis. Small RNA-containing total RNA was tolerance was set to 60.2 Da, and MS/MS fragment error tolerance was set to extracted from the thirteen-week livers using mirVanaTM miRNA isolation kit 60.3 Da. All of the identified proteins had a protein score .66 with an expected p- (Ambion, Austin, TX, USA). Six samples from one treatment were pooled together to value less than 0.05. generate one group. RNA quality was assessed and verified by using a BioAnalyzer 2100 (Agilent Technology, Santa Clara, USA). For each sample, small RNA enriched Reverse transcription and quantitative real-time PCR (qRT-PCR) analysis of by PEG8000 precipitation from 10 mg total RNA was used for library constructions miRNAs and mRNA. Isolated miRNAs and mRNAs were reverse transcribed by following the Illumina’ protocol. The small RNAs ligated with 59 and 39 adaptors were using miRcute microRNA first-strand cDNAsynthesis kit (Tiangen, Beijing, China) reverse transcribed and amplified. The libraries were quantified by ECO (Illumina, and TIANscript RT Kit (Tiangen, Beijing, China), respectively. Quantitative real-time San Diego, USA) and sequenced by using the Solexa’s proprietary sequencing-by- PCR (qRT-PCR) analyses on the miRNAs and the mRNAs were performed by using synthesis method. The resulting images were analyzed to generate raw digital-quality the RealMasterMix (SYBR green I) (Tiangen, Beijing, China) in a Bio-Rad CFX96 data. After masking adaptor sequences and removal of contaminated reads, clean Real-time PCR System (Bio-Rad, Hercules, CA, USA). The thermal cycling program reads were selected for further analysis. These clean reads were aligned with the Rfam was set as follows: 95uC for 5 min, followed by 40 cycles of 95uC for 30 s, 60uCfor database (ftp://selab.janelia.org/pub/Rfam) to match with known rRNA, snRNA, 30 s, and 72uC for 30 s. Following the amplification, melting curve analysis was snoRNA and tRNA sequences. After non-coding RNAs were removed, the remaining conducted by heating from 65uCto95uC with increments of 0.5uCfor5sto clean reads were further compared with the pre-miRNAs database in miRBase. The determine the specificity of the PCR reactions. The internal controls for miRNA49 and matched reads were used to identify mature miRNAs, and their reads were counted. mRNA50 normalization were 5 s RNA and b-actin. The primer sequences were shown Chi-square test was applied to calculate the difference between groups with 5% level in Table S1. Relative gene expression was evaluated using the 22DDCT method. Six of significance. The numbers of miRNA reads were normalized by tags per million biological replicates were used, and each were performed in triplicates. (TPM) values (TPM 5 (miRNA total reads/total clean reads) 3 106). Different genes were set by the threshold of false discovery rate (FDR) , 0.05. Hierarchical clustering based on average linkage was performed using J-express. Kyoto Encyclopedia of Statistics. The false discovery rate (FDR) corrected p-value of 0.05 was considered as Genes and Genomes (KEGG) analysis on differentially expressed miRNAs was statistically significant. KEGG analysis on differentially expressed miRNAs was performed using R script by implementing Fishers’ exact test with the Benjamini and performed using a customized ‘‘R script’’ by implementing Fishers’ exact test with the Hochberg (BH) as multiple testing methods with 5% level of significance. Benjamini and Hochberg (BH) as multiple testing method with 5% level of significance. For RT-PCR experiments, results were statistically evaluated by using mRNA sequencing and statistical Analysis. Total RNA in the livers of rats gavaged ANOVA with Duncan test and were given as mean 6 standard deviation (SD). A p- for thirteen weeks were extracted by using mirVanaTM miRNA isolation kit (Ambion, value of ,0.05 was considered statistically significant. Austin, TX, USA). Two rats were randomly choosen as biological replicates in one treatment. RNA quality was assessed by using a BioAnalyzer 2100 (Agilent Technology, Santa Clara, USA). Enriched mRNAs were used for library constructions 1. International Agency for Research on Cancer (IARC). Some naturally occurring following Illumina’s protocols. The mRNAs were reversely transcribed, end repaired, substances: food items and constituents, heterocyclic aromatic amines and and then the adaptor addition PCR products were enriched. The library was assessed mycotoxins. IARC Monogr Eval Carcinog Risks Hum. 56, 489–521 (1993). using a BioAnalyzer 2100 (Agilent Technology, Santa Clara, USA) and the 2. Aydin, G., Ozcelik, N., Cicek, E. & Soyoz, M. Histopathologic changes in liver and concentration reached 2 nM. mRNA was sequenced on the Illumina HiSeqTM2000 renal tissues induced by Ochratoxin A and melatonin in rats. Hum Exp Toxicol. (Illumina, San Diego,USA). Clean reads were acquired after masking adaptor 22, 383–391 (2003). sequences and removing reads of low quality. Gene expression level was estimated by 3. Ferrante, M. C. et al. Expression of COX-2 and hsp72 in peritoneal macrophages using Reads Per Kilo bases per Million reads (RPKM) method. Different genes were after an acute ochratoxin A treatment in mice. Life Sci. 79, 1242–1247 (2006). detected based on DESeq, controlling FDR , 0.05. KEGG analysis on differentially 4. Palabiyik, S. S. et al. ochratoxin A causes oxidative stress and cell death in rat liver. expressed genes was performed by using KOBAS software. World mycotoxin J. 5, 377–384 (2012). 5. Rached, E. et al. Ochratoxin A: 13-week oral toxicity and cell proliferation in male miRNA target predictions and enrichment analysis. An ‘‘in-silico’’ screening of F344/n rats. Toxicol Sci. 97, 288–298 (2007). miRNA binding site prediction was carried out within deregulated genes by adopting 6. Kamp, H. G. et al. Ochratoxin A induces oxidative DNA damage in liver and the miRWalk database15, including miRanda, miRDB, miRWalk, PITA, TargetScan kidney after oral dosing to rats. Mol Nutr Food Res. 49, 1160–1167 (2005). and RNAhybrid. The targets predicted by at least two different programs were chosen 7. Hundhausen, C. et al. Ochratoxin a lowers mRNA levels of genes encoding for key for further analysis to effectively reduce false positive targets13,17–22. An in-silico proteins of liver cell metabolism. Cancer Genomics Proteomics 5, 319–332 (2008). analysis was used for identifying possible interactions between miRNAs and mRNAs. 8. Roncada, P. et al. Swine ochratoxicosis: proteomic investigation of epatic The predicted target genes were subjected to KEGG enrichment analysis with bioindicators. Vet Res Commun. 28, 371–375 (2004). Benjamini and Hochberg (BH) as multiple testing method (background correction) 9. Lu, X. et al. Integrated analysis of transcriptomics and metabonomics profiles in with 5% level of significance. aflatoxin B1-induced hepatotoxicity in rat. Food Chem Toxicol. 55, 444–455 (2013). Relative quantitative protein profiling and mass spectrometry. The livers of rats 10. Garcia-Sevillano, M. A., Garcia-Barrera, T., Navarro, F. & Gomez-Ariza, J. L. gavaged for thirteen weeks at th cC and cH were homogenized and re-suspended in a Analysis of the biological response of mouse liver (Mus musculus) exposed to AsO lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (w/v) DL- based on integrated -omics approaches. Metallomics 5, 1644–1655 (2012).

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Possible involvement of oxidative stress in fenofibrate-induced unless indicated otherwise in the image credit; if the image is not included under hepatocarcinogenesis in rats. Arch Toxicol. 82, 641–654 (2008). the Creative Commons license, users will need to obtain permission from the license 35. Gunaratne, P. H., Creighton, C. J., Watson, M. & Tennakoon, J. B. Large-scale holder in order to reproduce the image. To view a copy of this license, visit integration of MicroRNA and gene expression data for identification of enriched http://creativecommons.org/licenses/by/3.0/

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