Journal of Pediatric Gastroenterology & Nutrition:
"Let There Be Bile"-Understanding Hepatic Injury in Cholestasis
Sokol, Ronald J.; Devereaux, Michael; Dahl, Rolf; Gumpricht, Eric
Department of Pediatrics, Pediatric Liver Center and Section of Pediatric Gastroenterology, Hepatology and Nutrition, University of Colorado School of Medicine and The Children's Hospital, Denver, CO
Address correspondence and reprint requests to Ronald J. Sokol, MD, Professor and Vice Chair of Pediatrics, Chief, Section of Pediatric Gastroenterology, Hepatology and Nutrition, Pediatric Liver Center, Box B290; and Department of Pediatrics, The Children's Hospital, 1056 East 19th Avenue, Denver, CO 80218-1088 (e-mail: Sokol.firstname.lastname@example.org).
Supported in part by grants from the National Institutes of Health (RO1-DK38446, UO1-DK062453 and U54-RR019455), The Cystic Fibrosis Foundation, the American Liver Foundation and the Abby Bennett Liver Research Fund.
Cholestatic liver disorders account for a large proportion of chronic liver ailments in adults, children and infants, and are among the leading indications for liver transplantation in all age groups. Recent studies have begun to characterize the cellular and molecular mechanisms of hepatocyte injury caused by the retention of hydrophobic bile acids in cholestasis. Steatocholestasis is the combined presence of hepatic steatosis and cholestasis, common in genetic causes of metabolic liver disease in childhood. Retention of hydrophobic bile acids promotes hepatocellular injury and subsequent portal fibrosis in these conditions. Investigations at the mechanistic level have revealed that activation of hepatocyte death receptors, induction of oxidative stress, mitochondrial perturbations and activation of caspases are intracellular pathways that mediate hepatocyte injury. Several compounds in licorice root have been shown to modulate bile acid-induced apoptosis and necrosis of hepatocytes. Further investigations will be needed to identify novel molecular and cellular targets for which pharmaceuticals might be developed, to reduce liver injury and fibrosis in cholestasis and steatocholestasis.
Cholestatic liver diseases (liver disorders in which there is impaired flow of bile) account for a large proportion of chronic liver ailments in adults, children and infants, and are among the leading indications for liver transplantation in all age groups (1). In adults, primary biliary cirrhosis, primary sclerosing cholangitis and cholestatic forms of chronic autoimmune and viral hepatitis frequently progress to cirrhosis and end-stage liver disease. Despite a growing understanding of the genetic regulation of hepatic and ileal bile acid transporters in the developing neonate, the reasons that the human neonate is particularly prone to cholestatic liver injury are not known. Biliary atresia, idiopathic neonatal hepatitis, paucity of interlobular bile ducts, bile acid synthesis defects and familial cholestatic syndromes, such as progressive familial intrahepatic cholestasis types 1, 2 and 3, are common causes of progressive liver disease in childhood, occurring in 1 in 2500 live births, and together as cholestatic liver diseases comprise the leading indication for liver transplantation in children (2). Although the etiologies of these disorders may differ, the consequence of impaired bile flow in all cholestatic disorders is the retention of bile constituents in the liver, including bile acids.1 In recent years, it has become apparent that another group of disorders causing liver disease in the infant and child are characterized by the presence of concomitant hepatic steatosis and cholestasis. In general, this "steatocholestasis" is caused by genetic metabolic diseases such as cystic fibrosis, hereditary tyrosinemia, galactosemia, citrin deficiency, fatty acid oxidation defects and mitochondrial hepatopathies.
Although recent advances have been made in understanding the mechanisms of hepatocyte injury in a variety of conditions, the development of effective therapies for cholestatic and steatocholestatic disorders has been hampered by a poor understanding of the processes by which cholestasis injures the liver. The most promising agent for treatment of cholestatic disorders, the cytoprotective hydrophilic bile acid, ursodeoxycholic acid, is only of limited benefit in most patients (3), but it underscores the importance of bile acid derivatives in cholestatic liver injury. Unfortunately, all too often, progression to end-stage liver disease is either fatal or requires liver transplantation in these disorders (4). Cholestatic disorders also cause significant morbidity, with signs and symptoms including jaundice, severe pruritus, fatigue, hyperlipidemia and xanthoma formation, all the complications of portal hypertension, and as a result of these ailments, impaired quality of life (5). The scientific basis for the development of new treatments for cholestatic disorders rests on the delineation of new cellular, biochemical and molecular targets for intervention. Gaining an understanding of the intracellular mechanisms causing this liver injury is the major objective of our research program. In this article, we will summarize the current state of knowledge in cholestatic and steatocholestatic liver injury and describe studies of novel licorice compounds that may be potential therapies for cholestasis.
HEPATOCYTE INJURY IN CHOLESTASIS
The hepatocyte, a cell which is injured early in the course of cholestasis and steatocholestasis, is responsible, at least in part, for many of the subsequent inflammatory and fibrinogenic responses of nonparenchymal cells. Hepatocyte swelling and intracellular accumulation of bile pigment are 2 of the earliest histologic findings in experimental 6 and human cholestatic disorders (7). Ultrastructural changes of hepatocyte mitochondria, commonly observed in the cholestatic hepatocyte, include swelling, pleomorphism and abnormal cristae (8) Injured hepatocytes may then secrete molecules (chemokines, cytokines, growth factors and lipid peroxide products, etc.) that amplify the inflammatory response, stimulate fibrogenesis by hepatic stellate cells, or directly injure other nearby cells (9). Retention and accumulation of hydrophobic bile acids within the hepatocyte during cholestasis 10 has long been implicated as a major promotor of liver damage in cholestasis (11-13). This critical role of retained bile acids is underscored by the severe liver disease seen in the congenital defect of hepatocyte bile acid secretion (progressive familial intrahepatic cholestasis (2) caused by deficiency of the ATP-dependent canalicular bile salt export pump (14). Thus, understanding the events that initiate liver injury during cholestasis should focus to a large extent on the hepatocyte and the effects of toxic bile acids on hepatocyte survival and regeneration.
Cholestatic hepatocytes undergo cell injury and death through 2 mechanisms. High concentrations of hydrophobic bile acids (>100 μmol/L) induce cellular oncotic necrosis with typical findings of cell swelling and disruption of intracellular and plasma membranes (15). Oncotic necrosis is the consequence of acute impairment of cellular metabolism leading to ATP depletion, ion dysregulation, mitochondrial and cellular swelling, activation of degradative enzymes, plasma membrane failure and cell lysis releasing intracellular contents (16). Hepatocytes exposed to relatively low concentrations (25-100 μmol/L) of hydrophobic bile acids undergo primarily apoptosis, or programmed cell death, a fundamental, tightly regulated active process in tissue homeostasis, a means of eliminating unwanted, senescent, and damaged cells from tissues in multicellular organisms (17). Dysregulated apoptosis in hepatocytes and bile duct epithelial cells is now recognized as a potential mechanism in the pathogenesis of many liver diseases (17). There is evidence that both mechanisms of cell death occur in human cholestasis, although the contribution of each form is controversial (18). Recent studies demonstrate oncotic necrosis of hepatocytes to be the predominant form of cell death in certain mouse models of extrinsic and intrinsic bile acid toxicity (19). Nevertheless, a large body of literature supports the role of apoptosis in cholestatic injury (17). Most likely both forms of cell death play important roles in clinical cholestasis.
CELL DEATH PATHWAYS IN CHOLESTASIS
Apoptosis of the hepatocyte occurs through activation of specific molecular pathways that are under tight control of a network of proteins and their own endogenous inhibitors. In hydrophobic bile acid toxicity, apoptosis seems to be triggered by the extrinsic pathway, through activation of cell surface death receptors (CD95/Fas and TRAIL), and by the intrinsic pathway, in which intracellular stress induces mitochondrial release of proapoptotic factors (17). Crosstalk between these 2 pathways takes place at several levels, including induction of the mitochondrial permeability transition (MPT) and release of proapoptotic mitochondrial proteins. Both pathways result in activation of intracellular proteases and endonucleases that degrade cellular constituents yielding apoptotic bodies that are taken up by macrophages and hepatic stellate cells. Over the past 5 years, the bile acid-induced hepatocyte apoptosis pathways have received much attention. Hydrophobic, but not hydrophilic, bile acids seem to trigger translocation of the death receptor Fas/CD95 from cytosol to the plasma membrane. This causes ligand-independent oligomerization of Fas/CD95 (and TRAIL R2), which recruits the Fas activated death domain, activating caspase 8, cleaving and translocating Bid (a proapoptotic Bcl-2 protein) to the mitochondria, which results in opening of the MPT pore and release of cytochrome c and other proapoptotic intermembrane space small molecules (17). Cytosolic caspase 9 activation and finally activation of the effector caspase 3 proceed to execute irreversible hepatocyte death. Physiologic concentrations of bile acids have also been shown to directly induce the MPT, generation of reactive oxygen species (ROS) by mitochondria, cytochrome c release from mitochondria and activation of caspase 3 (20,21). How bile acids stimulate Fas/CD95 translocation, and the role of oxidative stress in this process, has been the subject of intense investigation. Reinehr et al. (22) have demonstrated that bile acids induce an almost instantaneous oxidative stress response, which triggers activation of c-Jun-N-terminal kinases (JNK) and activation of the Src kinase, Yes, which is responsible for activating the epidermal growth factor receptor, which associates in a JNK-dependent manner with CD95. The resulting tyrosine phosphorylation of CD95 induces CD95 plasma membrane trafficking, formation of the death-inducing signaling complex (DISC) and induction of apoptosis through activation of caspase 8 and cleavage of Bid. These cascades are summarized in (Figures 1 and 2). Recent evidence from Reinehr et al. (22) suggests that upstream generation of oxidative stress seems to be a key trigger of the apoptosis cascade in bile acid toxicity. Several potential sources of ROS have been proposed, including the mitochondrial respiratory chain (23-25). These authors further suggest that NADPH oxidase may be involved in upstream generation of ROS in CD95-induced hepatocyte apoptosis (26), leading us to propose that NADPH oxidase isoforms or similar oxidases may be activated similarly by bile acids (Fig. 1).
HEPATOCYTE INJURY IN "STEATOCHOLESTATIC" DISORDERS
Genetic metabolic liver disorders are a common cause of chronic liver disease and liver failure in infancy and childhood, accounting for over 30% of all liver transplants performed in children (27). Many metabolic disorders caused by inborn errors of metabolism, such as cystic fibrosis, galactosemia, hereditary tyrosinemia and mitochondrial hepatopathies, Wilson disease and congenital defects of glycosylation, share the histologic and biochemical features of micro- or macrovesicular steatosis in combination with cholestasis (28). We have called this combined liver injury "steatocholestasis" (29) and propose that mechanisms of cell injury may be shared among these disorders regardless of the underlying genetic etiology. Hepatic steatosis had been considered in the past to be a benign component of the pathophysiology of metabolic disorders, perhaps caused by malnutrition (30). It is now believed that steatosis in these disorders is caused, at least in part, by the accumulation of toxic intermediates or the absence of essential cofactors, leading to impaired transport or beta-oxidation of fatty acids in mitochondria or altered mitochondrial respiratory chain activity. In recent years, it has become evident that steatosis plays an important role in the biochemical pathogenesis of other liver disorders such as nonalcoholic steatohepatitis (31,32). Because simple steatosis may be well tolerated by hepatocytes, it is believed that a "second hit," most likely induction of oxidative stress, is necessary to trigger cellular injury in the fat-laden hepatocyte (31). Cholestasis in genetic metabolic disorders results from accumulated toxic metabolites or cytokines that down regulate or interfere with function of bile acid and phospholipid canalicular transport proteins, or that reduce mitochondrial oxidative phosphorylation and impair ATP-dependent bile acid secretion (33). It has been recently demonstrated that steatosis itself may perturb normal bile acid metabolism in the hepatocyte, further aggravating bile secretion (34). We propose that in steatocholestasis, hepatocellular retention of toxic bile acids (35) in fat-laden hepatocytes may provide a "second hit" through stimulation of ROS generation and signaling cascades that trigger hepatocellular injury (36-39). It should be emphasized that the "metabolic syndrome" associated with obesity and nonalcoholic steatohepatitis is not the condition described herein (40), but rather genetic metabolic liver diseases caused by inborn errors of metabolism.
To characterize the mechanisms underlying the hepatocyte response to concurrent bile acid toxicity and steatosis, we have developed a rodent model of steatocholestasis. In this model, freshly isolated rat hepatocytes in suspension from lean and obese Zucker rats were exposed to the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC), in suspension for 4 hours. The results showed that oncotic necrosis was significantly increased and apoptosis was reduced in fat-laden hepatocytes compared with hepatocytes from lean Zucker rats. Necrosis was dependent on both ROS generation and the MPT and was abrogated by treatment of cells with antioxidants and MPT blockers. However, basal and dynamic ATP content and alpha-tocopherol concentrations did not differ between the fat-laden and lean hepatocytes. Furthermore, GCDC stimulated ROS generation, MPT and cytochrome c release to a similar extent in purified mitochondria from both obese and lean rats. Thus, the factors determining the different modes of cell death favored by the fat-laden cells in this model do not seem to include differences in mitochondrial function nor ROS generation by mitochondria. Investigation of other pathways and regulators of apoptosis and necrosis are currently underway in the fat-laden hepatocyte such as death receptor activation, Bcl-2 family proteins, MAP kinases and cell survival signals.
LICORICE COMPOUNDS AND CHOLESTATIC INJURY
Licorice root is an herbal preparation that has been used for many decades to reduce liver injury in a number of clinical disorders. In 1977, Suzuki et al. reported that the principal triterpene component of licorice root, glycyrrhizin (GL), benefited patients with chronic hepatitis C infection (41). Derivatives of licorice root have been used in Asia to treat children with the cholestatic disease, biliary atresia (42), although no controlled clinical trials have been reported. Increasing evidence supports that GL, or its hydrolyzed metabolite 18β-glycyrretinic acid (GA), protects against several models of oxidant-mediated toxicity, including exposure to CCl4, (43), t-butyl hydroperoxide (44) and ischemia-reperfusion injury (45), with GA generally exhibiting greater hepatic protection than GL. Whereas several hypotheses are offered to account for the hepatoprotective effects of GL and GA, the effects of these compounds on molecular and biochemical pathways of cell injury have not been well characterized. We have recently examined the effects of GA and GL on cell pathways of bile acid-induced cytotoxicity in both freshly isolated rat hepatocyte suspensions and purified liver mitochondrial fractions (50). GL and GA had opposing effects toward GCDC-induced cytotoxicity; GA prevented both necrosis and apoptosis, whereas GL enhanced apoptosis. GCDC promoted activation of caspase 10, caspase 3 and PARP; all were inhibited by GA, but not GL. Induction of apoptosis by GCDC was also associated with activation of JNK, which was prevented by GA. Activation of caspase 9 and dissipation of mitochondrial membrane potential were prevented by GA, but not GL. GA reduced GCDC-induced ROS generation to a greater extent than GL. In liver mitochondrial studies, GL and GA were both potent inhibitors of the MPT, ROS generation and cytochrome c release at submicromolar concentrations. GA suppressed activation of JNK, caspase 10 and 3. Thus, GA seems to be a promising agent to reduce hepatocyte injury in cholestasis, although its mechanism of action has not yet been clearly defined.
The chemical structure of GA shares structural features with molecules known to be ligands for nuclear hormone receptors (NHR). NHRs comprise a large family of ligand-activated transcription factors that control gene regulation in many biological processes (47). Several are activated by steroid and sterol molecules, including cholesterol, bile acids, tocopherols, retinoids and bile alcohols (52). Several class II NHRs regulate hepatic specific functions that may modulate hepatic injury in cholestasis. Particularly, CAR, FXR, LXR and PXR are transcription factors for genes that influence synthesis, metabolism, uptake and secretion of bile acids by the hepatocyte, CYP activities, and fatty acid and cholesterol synthesis. Thus, we propose that GA and GL may also function as ligands for one of the families of NRH leading to target gene regulation that is responsible, in part, for the effects of GA and GL on cholestatic (bile acid-induced) hepatic injury. Further exploration of these and related compounds as potential therapies is warranted.
The overall goals of our research program are to define the fundamental mechanisms by which retention of hydrophobic bile acids induce oxidative stress and cell death signaling pathways in cholestasis and steatocholestasis. Through a better understanding of these pathways, new targets for pharmaceutical intervention may be uncovered that may ultimately afford infants and children with cholestasis robust medical treatment to reduce hepatic injury and fibrosis.
The authors thank the post-doctoral fellows and trainees that have participated in the laboratory research studies.
1. Moseley R. Cholestasis. In: Kaplowitz N, ed. Liver and Biliary Diseases
. Williams & Wilkins, 1992:162-81.
2. Arrese M, Ananthananarayanan M, Suchy FJ. Hepatobiliary transport: molecular mechanisms of development and cholestasis. Pediatr Res
3. Lazaridis KN, Gores GJ, Lindor KD. Ursodeoxycholic acid `mechanisms of action and clinical use in hepatobiliary disorders'. J Hepatol
4. Busuttil RW, Farmer DG, Yersiz H, et al. Analysis of long-term outcomes of 3200 liver transplantations over two decades: a single-center experience. Ann Surg
2005;241:905-916. discussion 916-08.
5. Milkiewicz P, Heathcote EJ. Fatigue in chronic cholestasis. Gut
6. Kountouras J, Billing BH, Scheuer PJ. Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol
7. Scheuer PJ. Liver biopsy interpretation. Liver Biopsy Interpretation
. London: Balliere Tindall, 1980:35-59.
8. Phillips MJ, Poucell S, Patterson J, et al. The liver. An Atlas and Text of Ultrastructural Pathology
. New York: Raven Press, 1987:101-58.
9. Maher JJ, Friedman SL. Parenchymal and nonparenchymal cell interactions in the liver. Semin Liver Dis
10. Greim H, Trulzsch D, Czygan P, et al. Mechanism of cholestasis. 6. Bile acids in human livers with or without biliary obstruction. Gastroenterology
11. Attili AF, Angelico M, Cantafora A, et al. Bile acid-induced liver toxicity: relation to the hydrophobic-hydrophilic balance of bile acids. Med Hypotheses
12. Hoffmann AF, Popper H. Ursodeoxycholic acid for primary biliary cirrhosis (letter). In: 2 TLV, ed. The Lancet
. vol. 2;1966:398-9.
13. Javitt NB. Cholestasis in rats induced by taurolithocholate. Nature
14. Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev
15. Spivey JR, Bronk SF, Gores GJ. Glycochenodeoxycholate-induced lethal hepatocellular injury in rat hepatocytes. Role of ATP depletion and cytosolic free calcium. J Clin Invest
16. Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology
17. Guicciardi ME, Gores GJ. Apoptosis: a mechanism of acute and chronic liver injury. Gut
18. Gujral JS, Liu J, Farhood A, et al. Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation. Hepatology
19. Fickert P, Trauner M, Fuchsbichler A, et al. Oncosis represents the main type of cell death in mouse models of cholestasis. J Hepatol
20. Serviddio G, Pereda J, Pallardo FV, et al. Ursodeoxycholic acid protects against secondary biliary cirrhosis in rats by preventing mitochondrial oxidative stress. Hepatology
21. Yerushalmi B, Dahl R, Devereaux MW, et al. Bile acid-induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition. Hepatology
22. Reinehr R, Becker S, Wettstein M, et al. Involvement of the Src family kinase yes in bile salt-induced apoptosis. Gastroenterology
23. Krahenbuhl S, Stucki J, Reichen J. Reduced activity of the electron transport chain in liver mitochondria isolated from rats with secondary biliary cirrhosis. Hepatology
24. Krahenbuhl S, Talos C, Fischer S, et al. Toxicity of bile acids on the electron transport chain of isolated rat liver mitochondria. Hepatology
25. Winklhofer-Roob BM, McKim JM Jr, Devereaux MW, et al. Characterization of site of reactive oxygen. Hepatology
26. Reinehr R, Becker S, Eberle A, et al. Involvement of NADPH oxidase isoforms and SRC family kinases in CD95-dependent hepatocyte apoptosis. J Biol Chem
27. Ryckman FC, Alonso MH, Bucuvalas JC, et al. Liver transplantation in children. In: Suchy FJ, Sokol RJ, Balistreri WF eds. Liver Disease in Children
, 2nd ed. Philadelphia: Lippincott, 2001:949-73.
28. Scheuer PJ. Liver biopsy interpretation. Liver Biopsy Interpretation
. London: Balliere Tindall, 1980:167-88.
29. Kobak GE, Dahl R, Devereaux MW, et al. Increased susceptibility of fat-laden Zucker-rat hepatocytes to bile acid-induced oncotic necrosis: an in vitro model of steatocholestasis. J Lab Clin Med
30. Feranchak AP, Sokol RJ. Cholangiocyte biology and cystic fibrosis liver disease. Semin Liver Dis
31. Pessayre D, Mansouri A, Fromenty B. Nonalcoholic steatosis and steatohepatitis. V. Mitochondrial dysfunction in steatohepatitis. Am J Physiol Gastrointest Liver Physiol
32. Rashid A, Wu TC, Huang CC, et al. Mitochondrial proteins that regulate apoptosis and necrosis are induced in mouse fatty liver. Hepatology
33. Phillips MJ, Suchy FJ. Mechanisms and morphology of cholestasis. In: Suchy FJ, Sokol RJ, Balistreri WF eds. Liver Disease in Children
. Philadelphia: Lippincott, 2001:23-38.
34. Pizarro M, Balasubramaniyan N, Solis N, et al. Bile secretory function in the obese Zucker rat: evidence of cholestasis and altered canalicular transport function. Gut
35. Greim H, Czygan P, Schaffner F, et al. Determination of bile acids in needle biopsies of human liver. Biochem Med
36. Gumpricht E, Devereaux MW, Dahl RH, et al. Glutathione status of isolated rat hepatocytes affects bile acid-induced cellular necrosis but not apoptosis. Toxicol Appl Pharmacol
37. Rodrigues CM, Steer CJ. Mitochondrial membrane perturbations in cholestasis. J Hepatol
38. Sokol RJ, Straka MS, Dahl R, et al. Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria by hydrophobic bile acids. Pediatr Res
39. Sokol RJ, Winklhofer-Roob BM, Devereaux MW, et al. Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic bile acids. Gastroenterology
40. Brunt EM. Nonalcoholic steatohepatitis. Semin Liver Dis
41. Suzuki H, Ohta Y, Takino T, et al. Igaku no Aymi
42. Sokol RJ, Mack C, Narkewicz MR, et al. Pathogenesis and outcome of biliary atresia: current concepts. J Pediatr Gastroenterol Nutr
43. Jeong HG, You HJ, Park SJ, et al. Hepatoprotective effects of 18beta-glycyrrhetinic acid on carbon tetrachloride-induced liver injury: inhibition of cytochrome P450 2E1 expression. Pharmacol Res
44. Kinjo J, Hirakawa T, Tsuchihashi R, et al. Hepatoprotective constituents in plants. 14. B. Effects of soyasapogenol, sophoradiol, and their glucuronides on the cytotoxicity of tert-butyl hydroperoxide to HepG2 cells. Biol Pharm Bull
45. Nagai T, Egashira T, Yamanaka Y, et al. The protective effect of glycyrrhizin against injury of the liver caused by ischemia-reperfusion. Arch Environ Contam Toxicol
46. Gumpricht E, Dahl R, Devereaux MW, et al. Licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid are potent modulators of bile acid-induced cytotoxicity in rat hepatocytes. J Biol Chem
47. Karpen SJ. Nuclear receptor regulation of hepatic function. J Hepatol
48. Sonoda J, Chong LW, Downes M, et al. Pregnane X receptor prevents hepatorenal toxicity from cholesterol metabolites. Proc Natl Acad Sci USA
Hepatocyte apoptosis; Licorice; Genetic metabolic liver disease
© 2006 Lippincott Williams & Wilkins, Inc.
Highlight selected keywords in the article text.
Connect With Us
Visit JPGN.org on your smartphone. Scan this code (QR reader app required) with your phone and be taken directly to the site.