- Open Access
Comparison of gene expression profiles altered by comfrey and riddelliine in rat liver
© Guo et al; licensee BioMed Central Ltd. 2007
- Published: 01 November 2007
Comfrey (Symphytum officinale) is a perennial plant and has been consumed by humans as a vegetable, a tea and an herbal medicine for more than 2000 years. It, however, is hepatotoxic and carcinogenic in experimental animals and hepatotoxic in humans. Pyrrolizidine alkaloids (PAs) exist in many plants and many of them cause liver toxicity and/or cancer in humans and experimental animals. In our previous study, we found that the mutagenicity of comfrey was associated with the PAs contained in the plant. Therefore, we suggest that carcinogenicity of comfrey result from those PAs. To confirm our hypothesis, we compared the expression of genes and processes of biological functions that were altered by comfrey (mixture of the plant with PAs) and riddelliine (a prototype of carcinogenic PA) in rat liver for carcinogenesis in this study.
Groups of 6 Big Blue Fisher 344 rats were treated with riddelliine at 1 mg/kg body weight by gavage five times a week for 12 weeks or fed a diet containing 8% comfrey root for 12 weeks. Animals were sacrificed one day after the last treatment and the livers were isolated for gene expression analysis. The gene expressions were investigated using Applied Biosystems Rat Whole Genome Survey Microarrays and the biological functions were analyzed with Ingenuity Analysis Pathway software. Although there were large differences between the significant genes and between the biological processes that were altered by comfrey and riddelliine, there were a number of common genes and function processes that were related to carcinogenesis. There was a strong correlation between the two treatments for fold-change alterations in expression of drug metabolizing and cancer-related genes.
Our results suggest that the carcinogenesis-related gene expression patterns resulting from the treatments of comfrey and riddelliine are very similar, and PAs contained in comfrey are the main active components responsible for carcinogenicity of the plant.
- Ingenuity Pathway Analysis
- Pyrrolizidine Alkaloid
- Gene Expression Alteration
- Main Active Component
- Gene Encode Heat Shock Protein
Comfrey (Symphytum officinale) is consumed by humans as a vegetable and a tea. It has been used as an herbal medicine for more than 2000 years to treat broken bones, tendon damage, ulcerations in the gastrointestinal tract, lung congestion, and joint inflammation, and to promote wound healing . It, however, has been reported that comfrey is hepatotoxic in livestock and humans, and carcinogenic in experimental animals. Comfrey induced hepatic veno-occlusive lesion (VOD) in humans [2–4] and hepatocellular adenomas and hemangioendothelial sarcomas in rats . Therefore, the regular use of comfrey is a potential health risk for development of liver cancers. In 2001, the US Food and Drug Administration requested voluntary compliance for the removal of products containing comfrey .
It is still not clear about the mechanism of tumor induction by comfrey because comfrey is a mixture of many different substances and the active components responsible for the carcinogenesis have not been identified. It has been suggested that the induction of hepatic tumors has been associated with the pyrrolizidine alkaloids (PAs) that are present in comfrey [7–9] since PAs are genotoxic and carcinogenic in liver [10, 11]. Recently, we demonstrated that the PAs in the comfrey plant appear to be responsible for mutation induction in rat liver . Mutations are involved in the etiology of cancer . Research results in molecular cancer genetics have identified inherited and somatic cell mutations associated with cancer in oncogenes, tumor suppressor genes, DNA repair genes and other related genes [14–17]. Therefore, we hypothesize that PAs contained in comfrey are the main active components resulting in tumors in liver.
Riddelliine is one of the tumorigenic PAs and has been studied as a prototype of PA. The toxicity and carcinogenicity of riddelliine have been studied by the National Toxicological Program (NTP) [18–20], and the mechanism of riddelliine-induced tumorigenicity in experimental animals has been studied at the National Center for Toxicological Research (NCTR) [21–27]. Results showed that (1) riddelliine was metabolized to the major metabolites 6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (DHP) and riddelliine N-oxide; (2) DHP-derived DNA adducts were formed both in vivo and in vitro; (3) G:C → T:A transversions were the major type of mutation induced in the liver of riddelliine-treated rats; and (4) riddelliine caused liver tumors in male mice and both sexes of rats, mononuclear cell leukemia in rats, and lung neoplasms in female mice.
DNA microarray, a key advanced technology, has developed rapidly because of its ability to examine the expression levels of thousands of genes simultaneously. It is used increasingly for identifying biomarkers, elucidating patterns of gene expression, and understanding the mechanism of disease and toxicity. Toxicogenomics applies high through-put genomics tools to the study of toxicology and gene expression microarrays have been used extensively . The interpretation of gene expression data, however, can be complicated by the gene expression alterations caused by environmental factors such as diet  and time of day [30, 31]. That is, it can be difficult to discern toxin-specific gene expression changes from those due to environmental effects. This can be complicated further when the test article is a complex food such as comfrey.
To examine the possible PA-induced affects on gene expression caused by comfrey, the gene expression profiles in the livers of comfrey-treated rats were compared to the gene expression profiles from rats treated with the purified PA riddelliine. The correlation of the gene expression and biological functions related to carcinogenesis between the two treatments were explored.
Previously, we investigated the mutagenicity of comfrey and riddelliine in rats [26, 32] under conditions which have been shown to result in liver tumors [5, 20]. In the original mutation studies, 8% comfrey treatment induced about a 4-fold higher mutant frequency (MF) over the control group , while 1 mg/kg riddelliine exposure resulted in about a 3-fold increase in MF compared to controls . In these studies, the rats were exposed to the compounds for 12 weeks and then sacrificed. No tumors were visible at this time in our study, although Hirono et al. reported that liver tumors developed in the rats as early as 7 weeks after initiation of feeding comfrey . The mutation spectra for comfrey- and riddelliine-treated rats were significantly different from the controls, but there was no significant difference between the spectra for comfrey and riddelliine. G:C → T:A transversion (35–42%) was the major type of mutation in both comfrey and riddelliine treated rats, with 8–17% tandem base substitutions . These mutational data from comfrey-treated rats suggests that PAs in the plant are responsible for mutation induction and tumor initiation in rat liver.
To further investigate the effects of comfrey and riddelliine on gene expression changes in liver, microarray analysis was performed on liver samples from these high-dose groups. The details of each experiment and gene expression analysis were reported previously [32, 33]. In the present study, gene expression alterations caused by exposure to comfrey were compared to those caused by exposure to the representative PA riddelliine.
Comparison of gene expression profiles induced by comfrey and riddelliine
Common drug metabolizing genes induced by comfrey and riddelliine
Genes involved in drug metabolism altered by comfrey and riddelliine treatments in liver
Locus link ID
Phase I metabolism
carboxylesterase 2 (intestine, liver)
cytochrome P450, subfamily IIC
cytochrome P450, family 2, subfamily c
cytochrome P450, family 2, subfamily e
flavin containing monooxygenase 5
Phase II metabolism
glutathione S-transferase, alpha 3
NAD(P)H dehydrogenase, quinone 1
sulfotransferase family, cytosolic,1C, member1
Phase III metabolism
ATP-binding cassette, sub-family B
ATP-binding cassette, sub-family B, member 9
ATP-binding cassette, sub-family C, member 3
ATP-binding cassette, sub-family C, member 8
ATPase type 13A5
solute carrier family 13, member 5
solute carrier family 16, member 4
solute carrier family 22, member 6
solute carrier family 22, member 8
solute carrier family 25, member 21
solute carrier family 25, member 30
There are postulated to be three phase 1 pathways for the metabolism of PA; (1) Oxidation via cytochrome P450 (Cyp), (2) N-oxidation via flavin-containing monooxygenases (Fmo), and (3) hydrolysis via carboxylesterases. Hepatic cytochrome P450, Cyp3a and Cyp2b isoforms, were the major metabolizing enzymes involved in PA metabolism in humans . One gene of the Cyp2b family (Cyp2b15) and three genes of the Cyp3a family (Cyp3a2, 3a9 and 3a18) were contained on the microarray. Although riddelliine elevated the level of Cyp3a9 RNA, no significant changes in the expression of these genes was induced by both riddelliine and comfrey exposure in our study. However, a significant induction of Cyp2c12 (Cyp2c40) was found with the expression increased by 8.7- and 42-fold by the treatments with comfrey and riddelliine, respectively (Table 1). This suggests that Cyp2c may play an important role in metabolizing comfrey and riddelliine in addition to Cyp3a and 2b.
Flavin-containing monooxygenases were reported to be involved in the biotransformation of PAs to the N-oxide metabolites , and N-oxidation appears to be one of the crucial pathways in determining the toxicity of PAs. However, it is not well known which particular family member contributes to this metabolism and toxicity. Among five flavin-containing monooxygenase genes (Fmo1-5) contained on microarray, Fmo5 was induced about 4-fold by both comfrey and riddelliine treatments. Hydrolysis of PAs is considered to be a major detoxification pathway in phase I. As shown in Table 1, carboxylesterase 2 (Ces2) was induced by the treatments of comfrey and riddelliine in our study. Our results not only confirm the findings reported in the literature but also provide additional detail that Fmo5 may be the form of flavin-containing monooxygenases family involved in N-oxidation and carboxylesterase 2 may play a role in hydrolysis of these chemicals.
Phase II and phase III enzymes usually function as detoxication. Phase II enzymes involve in conjugation of the polar functional groups of phase I metabolites and phase III enzymes are transporters for these metabolites. In general, exposure to some xenobiotics can trigger cellular "stress" response leading to an increase in the gene expression of phases II and III, which ultimately enhances the elimination and clearance of these xenobiotics. Treatment with comfrey and riddelliine has been shown to alter the expression of a number of phases II and III genes along with phase I genes (Table 1). The expression of the glutathione S-transferase, alpha 3 (Gsta3) gene increased 22- and 13-fold over the control with comfrey and riddelliine treatment, respectively. Glutathione S-transferases are major phase II detoxification enzymes found mainly in the cytosol. It has been reported that PAs can be conjugated with glutathione by these enzymes, reduce glutathione concentrations in liver, and increase the activity of these conjugation enzymes after PAs treatment [35–37]. This result is consistent with those previously studied. In the phase III genes, ATP-binding cassette, sub-family B (Abcb1) was induced by comfrey and riddelliine 97- and 11-fold over the control, respectively. No publications have been found about the relationship between PA treatment and induction of ATP-binding cassette. Future research is encouraged for the role of ATP-binding cassette in PAs' metabolism and carcinogenesis.
Common biological processes and genes associated with carcinogenesis
The major relevant functions altered by both comfrey and riddelliine treatments in liver
Number of processes
Small molecule biochemistry
Cell-to-cell signaling and interaction
Genes involved in carcinogenesis altered by comfrey and riddelliine treatments in liver
Locus link ID
aldo-keto reductase family 1, member C3
androgen receptor (dihydrotestosterone receptor)
Rho GDP dissociation inhibitor (GDI) gamma
chromosome 5 open reading frame 13
Calpastatin (includes EG:831)
CD36 molecule (thrombospondin receptor)
cyclin-dependent kinase inhibitor 1C (p57, Kip2)
CHK1 checkpoint homolog (S. pombe)
cAMP responsive element modulator
docking protein 1, 62kDa (downstream of tyrosine kinase 1)
endothelial cell growth factor 1 (platelet-derived)
early growth response 1
fatty acid binding protein 2
Fas (TNF receptor superfamily, member 6)
fasciculation and elongation protein zeta 1 (zygin I)
FYN oncogene related to SRC, FGR, YES
growth differentiation factor 15
hepatocyte growth factor (hepapoietin A; scatter factor)
HRAS-like suppressor 3
heat shock 70kDa protein 1A
inhibitor of DNA binding 1,
insulin-like growth factor binding protein 2, 36kDa
integrin, alpha 4 (antigen CD49D)
laminin, alpha 5
LIM domain kinase 2
mannoside acetylglucosaminyltransferase 5
metallothionein 1A (functional)
natural cytotoxicity triggering receptor 1
NAD(P)H dehydrogenase, quinone 1
peroxisome proliferative activated receptor, gamma
PTK6 protein tyrosine kinase 6
serpin peptidase inhibitor, clade A, member 5
ST6 beta-galactosamide alpha-2,6-sialyltranferase 1
ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1
TIMP metallopeptidase inhibitor 3
tumor necrosis factor (TNF superfamily, member 2)
tumor necrosis factor (ligand) superfamily, member 10
tubulin, gamma 1
Most of genes involved in apoptosis (Cdkn1c, Egr1, Fas, Gdf15, Hgf, Hrasls3, Hspa1a, Lama5, Smox, Tnfsf10) were up-regulated and may be explained by the removal of cells damaged by PAs. For example, Egr1 (early growth response 1), whose expression increased 12- and 2-fold over the control with comfrey and riddelliine treatments, respectively, has the ability to function in numerous capacities, including differentiation, growth, growth inhibition, and apoptosis depending on the cell type and the stimulus. In response to stress, Egr1 displays a remarkable functional similarity to p53 and p73  and may be involved in protection of the cell against the toxicity of these compounds. We also observed that comfrey and riddelliine significantly up-regulated Hspa1a, the gene encoding heat shock protein (HSP) 70 family members. High levels of inducible HSP70s prevent stress-induced apoptosis and block caspase activity, mitochondrial damage, and nuclear fragmentation . These genes may all be involved in repair and protection of the cell against the damage caused by the PAs.
Among the common cancer-related genes, the expressions of two genes (Fez1 and Mgat5) were altered in opposite directions (down-regulated by comfrey and up-regulated by riddelliine). Mgat5 encodes N-acetylglucosaminyltransferase V (GlcNAc-TV), the Golgi enzyme required in the N-glycan processing pathway that modifies glycoproteins, including the cytokine receptors. Mgat5 gene expression is up-regulated by Ras pathway activation . In Mgat5-/- mice, tumor latency is longer and metastasis is reduced compared with Mgat5+/+ mice . FEZ1 (Fasciculation and elongation protein zeta-1) is a tumor suppressor gene that maps to chromosome 8p22, a chromosomal region frequently deleted in many human malignancies. The FEZ1 gene is expressed almost ubiquitously in normal tissues, and is prone to inactivation in human cancer . In addition, introduction of FEZ1 into Fez1-negative cancer cells results in suppression of tumorigenicity and reduced cell growth with accumulation of cells at late S-G2/M stage of the cell cycle . These two genes appear to be important in the carcinogenesis process and the differences in their expression in the two treatment groups may be an indication that the livers in the two groups are in different stages of the carcinogenic process. Alternatively, this may reflect the contribution of other components in comfrey.
Previously we found that the mutagenicity of comfrey was associated with the PAs contained in the plant. Therefore, we suggested that the toxicity and carcinogenicity of comfrey resulted from these PAs. In this study, we analyzed gene expression profiles in the liver of rats treated with riddelliine, a widely studied genotoxic PA and a proven rodent mutagen and carcinogen, and comfrey, an herbal tea that contains as many as nine PAs. We used the systematic approaches of HCA, PCA, functional analysis, and biological process analysis. Although there were large differences between the genes whose expression was significantly altered by these two treatments, there are common genes and function processes which may be indicative of effects due to PAs. In support of this is the finding that there were strong correlations between the gene expression fold-change alterations caused by the two treatments, in particular, drug metabolizing genes and cancer-related genes. These results suggest that comfrey and riddelliine may share common mechanism(s) of carcinogenesis, and further support our hypothesis that PAs are the main active components for carcinogenesis of comfrey.
Treatments with comfrey and riddelliine
The details on the description of the in vivo portion of this study has been described previously [26, 32]. Briefly, Big Blue Fisher 344 transgenic rats were fed with a diet containing 8% comfrey root for 12 weeks or gavaged with 1 mg/kg riddelliine 5 times a week for 12 weeks. One day after the last treatment, 6 rats from each vehicle control, comfrey-treated, and riddelliine-treated groups were sacrificed, and the livers were isolated and stored at -80°C for mutagenesis and microarray analysis. Big Blue transgenic rats were obtained from Taconic Laboratories (Germantown, NY) through purchase from Stratagene (La Jolla, CA). All animal procedures followed the recommendations of the NCTR Institutional Animal Care and Use Committee for the handling, maintenance, treatment, and sacrifice of the rats.
The detailed description of the microarray processing of this study has been reported previously [32, 33]. Briefly, total RNA was isolated from liver tissues of 6 control, 6 comfrey-fed, and 6 riddelliine-treated rats using an RNeasy system (Qiagen, Chatsworth, CA). All RNA targets were labeled using the Applied Biosystems RT-IVT Labeling Kit Version 2.0. Digoxigenin labeled cRNA targets were hybridized to Applied Biosystems Rat Whole Genome Survey Microarrays using the Applied Biosystems Chemiluminescent Detection Kit. Images were auto-gridded and the chemiluminescent signals were quantified, corrected for background, and finally, spot- and spatially-normalized using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software version 1.1.
Microarray data analysis
Raw microarray intensity data from the Applied Biosystems' Rat Genome Survey Microarray, which is a one channel microarray with chemiluminescence detection and contains 26,857 probes, were normalized with quantile normalization which is recommended by the manufacturer. The normalized data were then input to ArrayTrack, a software system developed by the NCTR for the management, analysis, visualization and interpretation of microarray data . The identification of differentially expressed genes based on fold-change and t-tests cutoffs, and HCA and PCA were conducted within ArrayTrack. Ingenuity Pathway Analysis (Mountain View, CA) was used for network and function analysis.
The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.
This article has been published as part of BMC Bioinformatics Volume 8 Supplement 7, 2007: Proceedings of the Fourth Annual MCBIOS Conference. Computational Frontiers in Biomedicine. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/8?issue=S7.
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