Comfrey was one of the most popular herbal teas in the world, including the United States. Although its popularity has declined due to the understanding of its dangers, it is still available commercially in several forms. The regular use of comfrey is a potential health risk owing to the presence of PAs. Comfrey contains as many as nine PAs, including acetyl intermedine, acetyl lycopsamine, echimidine, intermedine, lasiocarpine, lycopsamine, symlandine, symphytine, and symviridine [13
]. The PA content of comfrey is less than 1% and depends on the plant part [15
]. Higher PA concentrations occur in the roots of comfrey than in the leaves, and commercially available comfrey tablets containing high levels of PAs are likely to be derived from comfrey roots [16
]. PAs are the leading plant toxins associated with disease in humans and animals.
Several cases of VOD/SOS associated with comfrey ingestion have been reported in humans [6
], as well as in experimental animals [21
]. Deleve and her colleagues [22
] have developed a reproducible animal model of hepatic VOD, in which the rats are gavaged with a single dose of PA, monocrotaline. The model exhibits the characteristic clinical and histological features of hepatic VOD, including the earliest manifestations (progressive injury to the sinusoidal endothelial cells and central vein endothelium), early VOD (centrilobular coagulative necrosis and severe sinusoidal injury, hemorrhage, and central vein endothelial damage), and late VOD (fibrotic occulusion of the central veins) [22
]. Comfrey and the specific alkaloids in comfrey (e.g., symphytine and lasiocarpine) induce hepatoadenoma and hemangioendothelial sarcomas in rats [9
]. The mechanisms by which toxicity and carcinogenicity are produced are still not fully understood. Gene expression profiling offers a powerful approach for identifying differentially expressed genes and identifying mechanisms.
Gene expression was markedly affected (Figure ) in the livers of rats exposed to 8% comfrey root, a dose that resulted in significant decreases in body weight (Figure ) and increases in liver MF (Figure ). Out of 26,857 genes evaluated, the expression of 4,132 (15%) and 9,937 (37%) genes were altered more than 2-fold and 1.5-fold, respectively. At P
-values of 0.01 and 0.05, 7,518 (28% of expressed genes) and 1,0341 (39%) genes, respectively, displayed a significant effect after comfrey-treatment compared to control group. In this study, differential gene expression was considered significant for genes showing at least a 2-fold up- or down-change, and a P
< 0.01. In total, 2,726 genes (10%) satisfied the requirements and about half of them were down-regulated and half up-regulated in response to comfrey exposure (Figure ). Such a large number of significantly altered genes may partly reflect the therapeutic effects of comfrey exerted through plant components other than PAs [15
]. In the present study, we concentrated on the analysis of genes involved in metabolism, injury of endothelial cells, and liver injury and abnormalities.
Liver is the major organ for biotransformation of xenobiotics and drugs. PAs are metabolically activated to toxic, alkylating pyrroles by mixed-function oxidases. The cytochrome P450 (Cyp) superfamily contains 57 genes and plays a critical role in the phase I metabolism of a variety of xenobiotics including drugs, carcinogens, steroids and eicosanoids [25
]. In the present study, comfrey exposure resulted in changes in the expression of 19 Cyp genes (Table ). Among phase I, II, and III drug metabolizing genes, Cyp2c12, Cyp7a1, Cyp26, Gsta3, and Abcc3 were increased 6-21-fold. In contrast, Cyp2c, Cyp39a1, Gstm3, Abcc8, and others were reduced in the comfrey-treated livers. It is known that many herbal/dietary constituents form reactive intermediates capable of irreversibly inhibiting various Cyps (reviewed in [25
]). The resultant metabolites lead to Cyp inactivation by chemical modification of the heme, the apoprotein, or both, as a result of covalent binding of modified heme to the apoprotein. Phase II consists of conjugating enzymes, such as glutathione S
-transferases (GSTs), UDP-glucuronosyltransferases (UGTs), and sulfatases. GSTs also participate in oxidative stress release pathways. The altered expression of phase I and phase II enzymes along with altered drug transport proteins (phase III) could contribute to the increased susceptibility of rats to carcinogenic chemicals, such as comfrey.
Sinusoidal endothelial cells are more susceptible than hepatocytes to PAs that cause VOD/SOS [22
]. Functional annotation extracted from Ingenuity Pathway Analysis revealed that many of the transcriptional responses were associated with the apoptosis, cell death, adhesion, and cell movement of endothelial cells (Table ). The genes in these pathways were highly expressed in comfrey-treated livers; these genes included the endothelial cell markers plasminogen activator inhibitor type 1 (PAI-1, also known as Serpine1 and Serpine2) and tissue plasminogen activator (Plat), and the cytokine tumor necrosis factors (Tnfrsf6, Tnfrsf12a, and Tnfsf10), and transforming growth factors (Tgfb1 and Tgfb2). Induction of a number of genes involved in the injury of endothelial cells was also detected, including endothelin 1 (Edn1), urokinase plasminogen activator receptor (Plaur), collagen type IV alpha 2 (Col4a2), matrix metalloproteinase 2 (Mmp2), mitogen-activated protein kinase 9 (Mapk9), and secreted phosphoprotein 1 (Spp1). It has been reported that Edn1 is a mediator of hepatic sinusoidal constriction, and increased activity of matrix metalloproteinases is responsible for changes of sinusoidal endothelial cells [11
]. Elevated plasma PAI-1 levels are useful in distinguishing VOD/SOS [26
]. Endothelial injury is the initiating event in the cascade of events leading to the hepatic changes and clinical manifestation of VOD/SOS [27
]. Our results offer a more comprehensive overview of the molecular responses to comfrey exposure by expression of multiple genes in liver endothelial cells.
PAs in comfrey can reach the hepatocytes via the sinusoidal blood, and their toxic metabolites lead to immediate damage to the hepatocytes [4
]. Genes involved in liver cell death and growth were also induced or repressed in response to comfrey treatment (Table ), including tumor necrosis factors, transforming growth factor β, chemokine receptor CCR2 gene (Ccr2), heme oxygenase 1 (Hmox1), immediate early response 3 (Ier3), cyclin-dependent kinase inhibitor (Cdkn1a), inhibin beta A (Inhba), and tissue inhibitor of metalloproteinase 1 (Timp1). It is well known that Hmox1 induction is a protective mechanism against the oxidative stress associated with liver injury [28
], and that elevated Hgf could protect hepatocytes from injury or promote hepatocellular regeneration [29
]. Since Inhba, so-called activin A, is a negative regulator of hepatocyte cell growth [30
], the decreased expression of Inhba observed after comfrey treatment suggests the induction of hepatic growth.
Necrosis of hepatocytes and mesenchymal cells follows comfrey-induced liver cell injury, and functional cells are replaced by fibrotic tissues [27
]. In the present study, we observed 16 genes involved in the function of liver fibrosis by Ingenuity Pathway Analysis (Table ). Liver fibrosis is characterized by cell proliferation and the accumulation of extracellular matrix components and is mediated by cytokines and growth factors, of which TGF-β1 appears to be a key mediator [32
]. The up-regulation of cytokines Hgf and Tnfrsf6 and down-regulation of Igf1 and Lif play an important role in the pathogenesis of liver injury and fibrosis. Decreased serum Igf-1 levels provide a useful index of hepatocellular dysfunction and impaired nutritional status, and increased Hgf appears to limit liver fibrosis [33
]. Agt and Agtr1a, cytokines with vasoactive properties, also regulate liver fibrogenesis. Chemokines have a much wider biological role including angiogenesis, carcinogenesis, and cell cycle control [34
]. Chemokines in the liver (Cxcl4 and Cxcl12) may modulate the progression of liver fibrosis through their actions on hepatic stellate cells.
PA-induced DNA damage in the liver (endothelial cells and hepatocytes), if not repaired prior to DNA synthesis, might produce replication errors and mutations, which eventually could result in the development of neoplasmas in the treated animals. We determined MFs in the liver cII
gene of Big Blue transgenic rats. After feeding with 8% comfrey root for 12 weeks, we observed a 4-fold higher MF in the liver cII
gene compared to the controls (Figure ). The induction of mutation was similar to that reported previously for rats fed with 2% comfrey root [10
]. These observations suggest that the rats could not tolerate the feeding of roots in concentrations over 2%, in terms of mutation induction. Furthermore, the overall pattern of mutations induced by 8% comfrey in liver was similar to that in the livers of rats fed 2% comfrey root (Table ), whereas both the 2% and 8% comfrey-induced mutation spectra were significantly different from liver controls. In contrast to the G:C → A:T transition that was the predominant mutation in the controls, the major type of mutation in the 8% comfrey-fed rats was G:C → T:A transversion (41%), a mutation that was also induced by riddelliine, a representative genotoxic PA that is tumorigenic for rat liver [35
]. In addition, 13% of mutations from the 8% comfrey-fed rats were tandem base substitution, which has been suggested as a mutational signature for the genetic damage of PAs [36
]. G:C → T:A transversion may cause the initiation of tumors in the liver of rats fed with comfrey, because it has been reported that more than half of riddelliine-induced liver hemangiosarcomas have a G → T mutation at K-ras
codon 12 [37
mutation also has been detected at an early stage of riddelliine exposure [38
]. Mutations are thought to be involved in carcinogenesis because the transition from a normal somatic cell to a cancer cell is due to mutations in protooncogenes, tumor suppressor genes and/or genes that function in the maintenance of genomic stability [39
]. Comfrey-induced neoplasms in the rat were mostly found in the liver. Hepatocellular adenomas were induced in all experimental groups that received diets containing 1–8% comfrey root or 8–33% comfrey leaves. In addition, a few rats bearing hepatocellular adenomas simultaneously had hemangioendothelial sarcoma of the liver [9
]. The first hepatocellular carcinoma (HCC) appearance was 7 months after initiating the 8% comfrey diet.
Ingenuity Pathway Analysis found 8 genes (2 down- and 6 up -regulated) involved in liver cancer development that were altered due to comfrey treatment (Table 5). In mammals, the nucleotide excision repair process is the most important repair pathway for elimination of DNA damage caused by exogenous agents, including UV light, DNA-reactive carcinogens, and some endogenously generated oxidative lesions [41
]. Xeroderma pigmentosum group C (Xpc) is implicated in the early steps of this repair pathway. A significantly higher incidence of chemically induced liver and lung tumors is observed in Xpc null mice [42
]. Gap junction membrane channel protein beta 1 (Gjb1), also called connexin 32 (Cx32), is the main gap junction protein in hepatocytes and plays an important role in the regulation of signal transfer and growth control in the liver. It has been reported that Cx32 expression decreases gradually as liver disease progresses to cirrhosis and HCC [43
], and a low expression of Cx32 mRNAs in HCC tissues is also predictive of the postoperative recurrence of HCCs [44
]. Bcl-2 is characterized as an antiapoptotic/oncogenic protein and also functions as an antioxidant. Increased Bcl-2 expression in cirrhotic patients correlates with the development of HCC [45
]. Bcl-2 is also expressed in HCC tissues and the increasing Bcl-2 expression associated with HCC progression suggests that the Bcl-2 protein takes part in the formation of HCC [46
]. Hgf, identified originally as the most potent mitogen for hepatocytes, is now known to be a cytokine with numerous functions in a wide variety of cells [47
]. It is up-regulated in inflammatory liver diseases and stimulates DNA synthesis preferentially in initiated hepatocytes, presumably resulting in tumour promotion [48
]. N-myc downstream-regulated gene 1 (Ndr1, or Ndrg1) plays a role in growth arrest and cell differentiation, is induced by several stress conditions, and is overexpressed in many cancers [49
]. Expression of Ndrg1 was significantly up-regulated in HCC tissues compared to that of noncancerous and normal liver tissues [50
]. Cyclin E (Ccne1), a regulatory subunit of cyclin-dependent kinase 2, is an important regulator for entry into the S
phase of the mammalian cell cycle. Overexpression of Ccne1 has been observed in many tumors including primary HCCs [51
]; overexpression results in chromosome instability and thus may contribute to tumorigenesis [52
]. Growth arrest and DNA damage 45 alpha (Gadd45a) is a nuclear protein involved in the maintenance of genomic stability, DNA repair, and the suppression of cell growth [53
]. Gadd45a protein levels are higher in liver cirrhotic and neoplastic tissues [54
]. Tissue inhibitor of metalloproteinase 1 (Timp1) is a contributory factor to fibrosis of a variety of organs including the liver. Timp1 and other extracellular matrix remodeling genes are implicated in the transition from mild to moderate fibrosis in patients with chronic hepatitis C [55