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Homocysteine alterations in experimental cholestasis and its subsequent cirrhosis
Life Sciences 76 (2005) 2497 – 2512 www.elsevier.com/locate/lifescie
Homocysteine alterations in experimental cholestasis and its subsequent cirrhosis Mohammad R. Ebrahimkhania, Hamed Sadeghipoura, Mehdi Dehghania, Samira Kiania, Seyedmehdi Payabvasha, Kiarash Riazia, Hooman Honara, Parvin Pasalarb, Naser Mirazic, Massoud Amanloud, Hassan Farsamd, Ahmad R. Dehpoura,* a
Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran c Department of Biology, Bu-Ali Sina University, Hamadan, Iran d Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
b
Received 14 June 2004; accepted 28 December 2004
Abstract Homocysteine (Hcy), an intermediate in methionine metabolism, has been proposed to be involved in hepatic fibrogenesis. Impaired liver function can alter Hcy metabolism. The aim of the present study was to determine plasma Hcy alterations in acute obstructive cholestasis and the subsequent biliary cirrhosis. Cholestasis was induced by bile duct ligation and sham-operated and unoperated rats were used as controls. The animals were studied on the days 7th, 14th, 21st and 28th after the operation. Plasma Hcy, cysteine, methionine, nitric oxide (NO) and liver S-adenosyl-methionine (SAM), S-adenosyl-homocysteine (SAH), SAM to SAH ratio and glutathione were measured. Chronic L-NAME treatment was also included in the study. Plasma Hcy concentrations were transiently elevated by the day 14th after bile duct ligation (P b 0.01) and subsequently returned to control levels. Similar relative fluctuations in plasma Hcy were observed in BDL rats after intraperitoneal methionine overload. Plasma methionine, cysteine and nitrite and nitrate were significantly increased after bile duct ligation. SAM to SAH ratio was diminished by the 1st week of cholestasis and remained significantly decreased throughout the study. These events were accompanied by a decrease in GSH to GSSG ratio in the liver. Chronic L-NAME treatment improved SAM to SAH ratio and prevented the elevation of plasma Hcy and methionine (P b 0.05) while couldn’t influence the other parameters. In conclusion, this study demonstrates alterations in plasma Hcy and liver SAM and SAH contents in precirrhotic
* Corresponding author. Tel.: +98 21 611 2802; fax: +98 21 640 2569. E-mail address: [email protected] (A.R. Dehpour). 0024-3205/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.lfs.2004.12.009
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stages and in secondary biliary cirrhosis, for the first time. In addition, we observed that plasma Hcy concentrations in BDL rats follow a distinct pattern of alteration from what has been previously reported in other models of cirrhosis. NO overproduction may contribute to plasma Hcy elevation and liver SAM depletion after cholestasis. D 2005 Published by Elsevier Inc. Keywords: Cholestasis; Cirrhosis; Homocysteine; Methionine; Nitric oxide; S-adenosyl-methionine
Introduction Homocysteine (Hcy), an essential intermediate of normal mammalian methionine metabolism, can promote oxidative stress and endothelial dysfunction (Loscalzo,1996). Very recent reports suggest that Hcy interferes with the structure and function of various proteins by forming Hcy-N-protein (N-homocysteinylation)(Jakubowski, 2004). Hcy directly induces the expression of procollagen type I and tissue inhibitor of metalloproteinases-1 genes in hepatocytes and stellate cells in vitro (Torres et al., 1999). These suggest that Hcy may be an effective inducer of liver fibrogenesis (Torres et al., 1999; Garcia-Tevijano et al., 2001). Cholestasis is accompanied by endothelial dysfunction (Namiranian et al., 2001), oxidative stress (Ljubuncic et al., 2000) and progressive fibrogenesis (Parola et al., 1996), whose bases have not been well defined. Knowledge of initial signals that upset the maintenance of proper liver function or trigger necrosis and fibrogenesis will be extremely valuable in understanding the causes of liver cirrhosis and in working towards its treatment. Thus measurement of Hcy in this clinical setting has a potential clinical relevance (Carmel and Jacobsen, 2001). S-adenosyl-methionine (SAM), an important molecule in normal cell function and survival, is central to many biological processes from detoxification to gene regulation (Mato et al., 2002). It is a ubiquitous methyl donor to a wide spectrum of compounds such as DNA, RNA, proteins and phospholipids (Mato et al., 1997; Aleynik and Lieber, 2000). SAM is synthesized from methionine and is an intermediate in methionine-Hcy cycle (Fig. 1). Liver, where 85% of the whole body transmethylation capacity resides (Finkelstein, 1990), is central in Hcy metabolism (Garcia-Tevijano et al., 2001; Stead et al., 2000). Therefore, impaired liver function can influence Hcy metabolism and its plasma level (Garcia-Tevijano et al., 2001; Stead et al., 2000). Chronic treatment of experimental animals with ethanol or CCl4 was associated with hyperhomocysteinemia (Varela-Moreiras et al., 1995; Stickel et al., 2000) while the hepatoprotective effect of SAM on experimental cirrhosis was accompanied by a decrease in plasma Hcy concentration (Varela-Moreiras et al., 1995). Elevated plasma Hcy level in liver cirrhosis has been also shown by some human studies (Torres et al., 1999; Ferre et al., 2002; Bosy-Westphal et al., 2001). However the majority of patients in those studies, suffered from either alcoholic or viral liver cirrhosis and there were not enough data regarding cholestatic liver disease. Distinct patterns of plasma amino acid alterations have been elucidated in two models of cirrhosis: obstructive liver cirrhosis and CCL4-induced cirrhosis, which suggest different metabolic abnormalities in response to diverse liver injuries (Rosen et al., 1997; Weisdorf et al., 1990). It has been demonstrated that nitric oxide (NO) inactivates rat hepatic methionine adenosyl transferase (MAT) by S-nitrosation, in vivo (Ruiz et al., 1998; Perez-Mato et al., 1999). It can also inactivate
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Methionine
MAT
S-adenosyl-methionine a
DMG MS
MTs
BHMT betaine
a-CH3 S-adenosylhomocysteine Ado
SAHH Homocysteine Ado
CBS Cystathionine
Cysteine
Glutathione
Fig. 1. Pathways of Methionine and Homocysteine metabolism. S-adenosyl-methionine (SAM) is synthesized from methionine and ATP by methionine adenosyltransferase (MAT). SAM is converted to S-adenosyl-homocysteine (SAH) after donation of its methyl group, a reaction catalyzed by numerous methyltransferases(MTs). Subsequently SAH is converted into adenosine and Hcy by SAH-hydrolase(SAHH). This is a reversible reaction, however under normal conditions the catabolism of SAH is favored by the rapid removal of the reaction products adenosine and Hcy. Depending on the cellular needs for methionine, Hcy may undergo remethylation to methionine or enter transsulfuration pathway where it serves as a precursor for cysteine and glutathione. BHMT indicates Betaine-Homocysteine methyltransferase; CBS, cystathionine h-synthase; DMG, dimethylglycine; MS, methionine synthase.
methionine synthase (MS), the enzyme that participates in Hcy remethylation reaction (Danishpajooh et al., 2001; Nicolaou et al., 1996). To our knowledge, there are not any data regarding plasma Hcy and liver SAM and SAH levels in cholestatic liver disease and subsequent biliary cirrhosis. It is also not clear whether NO overproduction can influence the methionine cycle in this model. The present study was designed to investigate the potential alterations of plasma Hcy, and liver SAM and SAH levels in cholestasis and its subsequent cirrhosis in which increased systemic production of NO occurs (Marley et al., 1999; Nahavandi et al., 2001; Mani et al., 2002). We also tried to disclose some probable roles for NO overproduction in this regard.
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Material and methods Reagents All materials were purchased from Sigma. Animals and Experimental procedures Animals Adult male Sprague-Dawley rats (230–245 g, Pasteur Institute of Iran, Tehran, Iran) were used throughout this study. The animals were housed in a temperature-controlled room (23 F 1 8C), on a 12-h regular light /dark cycle with free access to standard rodent chow and water. They were handled in accordance with the criteria outlined in the "Guide for the Care and Use of Laboratory animals" (NIH US publication no. 85–23 revised 1985). All experiments were performed in line with the ethical considerations, recommended by the Pasteur Institute of Iran. Study design and operations Age and weight matched animals were studied in three groups: bile duct ligated (BDL), sham operated (SO) and unoperated control rats. Laparatomy was performed under general anesthesia (Ketamine HCl 50mg/kg and Xylazine HCl 10 mg/kg i.p.). The bile duct was exposed and double ligated, then cut through between the ligations. Sham operation consisted of laparatomy, bile duct identification and manipulation without ligation. After 7,14,21 and 28 days, animals were anesthetized with ether and blood collections were performed after cardiac puncture. There were 10–12 rats per group at the time of sampling. Sample collections took place at a same particular time in the morning. For methionine loading experiments, methionine was administered in different doses (40 mg/kg– 100mg/kg i.p.) and blood was collected 2 hours later. The appropriate dosage was selected previously in a pilot study. To investigate the effect of NO overproduction on Hcy metabolism, N(N)-nitro L-arginine methyl ester (L-NAME)(3 mg/kg/day, i.p.) or normal saline (1 ml/kg/day, i.p.) was administered to the SO and BDL animals for 2 weeks (Sadeghipour et al., 2003). The experiment was performed either in the first 2 weeks (day 0 to 14) or in the second 2 weeks of the study (day 14 to 28). Analytical techniques Total plasma Hcy, cysteine and methionine Centrifugation, plasma separation and freezing at –70 8C were done immediately after sampling. Measurements of total plasma Hcy and cysteine concentrations were carried out by High-performance liquid chromatography (HPLC) with fluorescence detection, as previously described (Bosy-Westphal et al., 2001; Hyland and Bottiglieri, 1992). Methionine was measured after deproteinization with sulfosalicylic acid, by HPLC-UV detection (Jayatilleke and Shaw, 1993). Hepatic SAM and SAH Liver specimens were snap frozen in liquid nitrogen for subsequent measurements of SAM and SAH. At the time of measurements, liver tissues were homogenized in 4 volumes of 0.4 M HClO4 then
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centrifuged at 10,000 g for 20 min. SAM and SAH levels in liver homogenates were determined by isocratic HPLC analysis with ultraviolet detection as described (She et al., 1994). The method provides rapid resolution of both compounds in a single run by direct injection of perchloric acid extracts so that sampling procedures and analytical errors can be reduced. Total protein was measured using the Lowry protein assay (Lowry et al., 1951). Bovine serum albumin was used as a standard. All measurements are reported in nanomoles per milligram of protein to adjust for possible changes in milligrams of protein per gram of liver. Hepatic glutathione Glutathione levels (both reduced and oxidized forms) were measured by fluorometric assay (Hissin and Hilf, 1976) .In Brief, ~250 mg of liver tissue was homogenized on ice in 3.75 ml of Phosphate-EDTA (P-E) buffer. The homogenate was centrifuged at 10,000 g for 30 min at 4 8C. 0.5 ml of the homogenate was diluted further in 4.5 ml of P-E (1:10). 0.1 ml of the resulting mixture was added to 1.8 ml of P-E buffer (pH 8.0) followed by 100 Al of the O-phthalaldehyde (OPT). Analysis was performed fluorometrically at 420 nm, with excitation at 350 nm. GSSG was measured by the same procedure after the incubation of tissue homogenates with N-ethylmaleimide (NEM) for 30 minutes in room temperature, to block any free thiol residues. Instead of P-E buffer, 0.1 N NaOH was employed for GSSG assay. Plasma nitrate and nitrite Plasma nitrate and nitrite levels were measured as indicators of NO production. The measurements were done according to method by Miranda et al (Miranda et al., 2001). Samples were deproteinized by centrifugation through a 30-kDa molecular weight filter (Centricon Millipore) at 14,000 rpm, for 1.5–3 h at 4 8C. After loading the plate with samples (100 Al), addition of saturated solution of VCl3 (100 Al) to each well was rapidly followed by addition of the Griess reagents (50 Al each). Sulfanilamide and Naphthylethylenediamine dihydrochloride were applied for preparation of Griess reagents. The plate was incubated at 37 8C for 30 minutes and then absorbance at 540 nm was measured using standard plate reader. Fresh standard solutions of nitrate were included in each experiment. Hepatic and renal function Total plasma bilirubin, alkaline phosphatase (ALP), alanineaminotransferase (ALT) and creatinine were determined with commercially available kits (Zistshimi. Iran). BDL rats that failed to show characteristics of cholestasis were excluded. Histology Liver cirrhosis was histologically confirmed after 28 days of bile duct ligation. Those, who were not confirmed histologically cirrhotic, were not included in our study. Statistical analysis All data are expressed as mean F SEM. Statistical evaluation of data was performed using analysis of variances (ANOVA), followed by Tukey post hoc test. P-values less than 0.05 were considered statistically significant.
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Results Characterization of BDL and SO rats One day after laparotomy BDL rats showed manifestations of cholestasis (jaundice, dark urine). Plasma bilirubin and liver enzyme activities increased in BDL animals consistent with biliary obstruction (P N 0.01) and remained significantly elevated all over the study. During the 1st week of the study, daily food intake of BDL animals was less than SO group (15.1 F 0.3 g/day/rat vs 21.0 F 0.3 g/day/rat, mean daily food intake of BDL and SO groups respectively; P b 0.01). Concurrently, body weight of BDL animals decreased during the 1st week of cholestasis (252 F 1 g vs 267 F 1 g, mean weight after one week, BDL and SO groups respectively; P b 0.01). In the 2nd, 3rd and 4th weeks of the study food intake and weight gain were not significantly different among different groups under study (P = NS). Basal and post methionine load plasma Hcy A gradual rise was observed in Hcy values after bile duct ligation. Plasma Hcy concentration reached its maximum level on day 14th after bile duct ligation and then by day 28th, decreased to values that were not significantly different from controls (Table 1, Fig. 2). Methionine load resulted in elevation of plasma Hcy levels both in control and BDL animals (Fig. 2). The elevation in plasma Hcy after methionine load was more prominent in BDL rats than in controls; however, this effect was observed only in the first 2 weeks after bile duct ligation (Fig. 2). There were not significant differences between unoperated controls and SO animals. Plasma methionine and cysteine Plasma levels of the sulfur-containing amino acids, cysteine and methionine, increased in cirrhotic animals compared to the SO group (Table 1). By the 3rd week after operation, this difference became significant (Table 1). There was not any significant difference between unoperated controls and SO groups. Hepatic SAM and SAH levels Table 2, shows that significantly reduced SAM level was detected by the 3rd and 4th weeks after bile duct ligation. Liver SAH content increased transiently after bile duct ligation; it was higher than SO animals during the 1st and 2nd weeks and returned to control values in the 3rd and 4th weeks of the Table 1 Plasma concentrations of Methionine, Cysteine and NO2 + NO3 in control and bile duct ligated (BDL) rats Group
Methionine
Cysteine
NO2 + NO3
Control BDL(1st wks) BDL(2nd wks) BDL(3rd wks) BDL(4th wks)
41.1 56.2 55.1 69.0 70.3
12.9 17.8 22.7 27.9 24.7
27.1 52.1 48.4 52.9 50.1
F F F F F
4.5 7.8 5.9 5.7* 6.0*
F F F F F
1.9 3.0 2.3 3.5* 2.8*
NOTE. Results are expressed in AM as means F SEM for 10–12 animals per group. * P b 0.01 versus control group. ** P b 0.001 versus control group.
F F F F F
2.5 4.7** 2.4** 4.5** 4.5**
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Fig. 2. Basal and post methionine load plasma Homocysteine (Hcy) concentrations in plasma of control and 7, 14, 21, 28 day bile duct ligated (BDL) rats. Plasma Homocysteine was assessed in basal state and also after methionine administrations. Methionine was administered in different doses (40 mg/kg-100 mg/kg i.p.) and blood was collected 2 hours later. Values represent Mean + SEM (n = 10–12 per group). aP b 0.01 in comparison with control group(Basal state). bP b 0.01 in comparison with control group(after 40 mg/kg methionine load). cP b 0.01 in comparison with control group( after 100 mg/ kg methionine load).
study (Table 2). These fluctuations were in accordance with the above mentioned changes in plasma Hcy levels. The SAM to SAH ratio (methylation ratio) fell by 45% by the 1st week of cholestasis and remained significantly decreased, thereafter (Fig. 3). There was not any significant difference among unoperated control and sham groups. Hepatic glutathione Cholestasis was associated with decreased GSH to GSSG ratio (GSH/GSSG) from the 1st week of bile duct ligation (P b 0.01)(Fig. 3b). The exact values of GSH and GSSG of the liver in different time Table 2 Hepatic concentrations of SAM, SAH, GSH and GSSG in cholestatic and control rats Group
SAM
Control BDL(1st wks) BDL(2nd wks) BDL(3rd wks) BDL(4th wks)
0.50 0.46 0.41 0.30 0.25
F F F F F
SAH 0.03 0.02 0.03 0.02** 0.01**
0.13 0.22 0.25 0.18 0.18
GSH F F F F F
0.01 0.01** 0.02** 0.01 0.01
30.3 36.4 27.8 20.7 19.3
GSSG F F F F F
0.9 1.8* 1.3 1.7** 1.5**
NOTE. Results are expressed in nmol/mg protein as means F SEM for 10–12 animals per group. Abbreviations: SAM, S-adenosyl-methionine; SAH, S-adenosyl-homocysteine. * P b 0.05 versus control group. ** P b 0.01 versus control group.
3.1 F 0.1 10.0 F 0.5** 7.1 F 0.9** 8.0 F 0.7** 6.8 F 0.6**
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Fig. 3. (a, b). Liver S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH) ratio and GSH to GSSG ratio in 7, 14, 21, 28 day bile duct ligated (BDL) rats and their sham operated (SO) ones. Both ratios differed significantly from SO groups 7, 14, 21 and 28 days after operation. Values represent Mean F SEM (n = 10–12 per group). *P b 0.01 in comparison with the SO group.
points of study have been shown in Table 2. There was a transient increase in GSH level in the 1st week of cholestasis (P b 0.05). But the early increase of GSH was followed by a continuous decrease over later times of study.
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Plasma nitrate and nitrite and creatinine clearance Cholestatic animals had significantly higher plasma nitrite and nitrate concentrations compared with SO ones, through out different times of study, indicative of increased NO synthesis in cholestasis (Table 2). This is in agreement with our previous data in BDL rats (Nahavandi et al 2001; Mani et al., 2002).
Fig. 4. (a, b). Basal plasma level of Homocysteine (Hcy) and liver S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH) ratio in 14 day bile duct ligated (BDL) and sham operated (SO) rats given saline or L-NAME. Plasma Hcy increased after 14 days of bile duct obstruction while SAM to SAH ratio decreased in this period. Chronic treatment with L-NAME (day 0 to 14) prevented these alterations in BDL rats. Data are shown as Mean + SEM (n = 10–12). aP b 0.01 in comparison with SHAM groups. bP b 0.05 in comparison with BDL/L-NAME animals.
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Creatinine clearance fell by 33% by the 1st week of cholestasis (1.03 F 0.04 ml/min/100g vs 0.70 F 0.03 ml/min/100 g, SO and BDL respectively; P b 0.001). However, there were no significant changes in plasma NO levels and creatinine clearance among subjects in different stages of cholestasis (P = NS). Furthermore, statistical analysis failed to detect any correlation between plasma creatinine levels and Hcy values.
Fig. 5. (a, b). Plasma methionine and cysteine levels in 28 day bile duct ligated (BDL) and sham operated (SO) rats given saline or L-NAME. Both methionine and cysteine levels increased after 28 days of bile duct obstruction. Chronic treatment with LNAME (day 14 to 28) prevented the increase of plasma methionine level in BDL rats while it did not influence plasma cysteine level significantly. Data are shown as Mean + SEM (n = 10–12). aP b 0.01 in comparison with SHAM groups. bP b 0.05 in comparison with BDL/L-NAME animals.
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Plasma and liver measurements following L-NAME treatment L-NAME treatment during the first 2 weeks of study L-NAME treatment significantly decreased NO production, as assessed by plasma NO2 + NO3 concentration in BDL or SO groups (P b 0.05). Increase in plasma Hcy did not occur in 14 day BDL animals, which were chronically treated by L-NAME through the study (Fig. 4). L-NAME treatment improved liver SAM level in BDL animals (0.35 F 0.03 nmol/mg protein in BDL/Saline and 0.54 F 0.03 in BDL/L-NAME groups, P b 0.05) while it couldn’t influence the SAH values, significantly (0.30 F 0.02 nmol/mg protein in BDL/Saline and 0.24 F 0.04 in BDL/L-NAME groups, P = NS). Consequently, hepatic SAM to SAH ratio also increased in the BDL/L-NAME group (P b 0.05)(Fig. 4). Chronic L-NAME didn’t have any significant effect on plasma Hcy level and SAM to SAH ratio in SO animals (P = NS)(Fig. 4). There were not any significant alterations in plasma cysteine, methionine, and liver glutathione by our L-NAME intervention in 14-day BDLs or corresponding SO ones (P = NS). In addition, despite a significant increase of plasma ALT activity after bile duct ligation, L-NAME treatment did not influence its value in either BDL or SO groups (36 F 8 U/L vs 43 F 7 U/L, Sham/Saline and Sham/L-NAME groups respectively; P = NS)(101 F 21 U/L vs 120 F 18 U/L, BDL/Saline and BDL/L-NAME groups respectively; P = NS). Plasma ALP activity followed a pattern similar to alterations of ALT activity. Creatinine clearance was not influenced by the treatment in the studied groups (1.03 F 0.04 ml/min/ 100g vs 0.99 F 0.06 ml/min/100 g, SO and SO/L-NAME respectively; P = NS)(0.70 F 0.03 ml/min/100 g vs 0.68 F 0.051 ml/min/100 g, BDL and BDL/L-NAME respectively; P = NS). L-NAME treatment during the second 2 weeks of study L-NAME treatment in BDL and SO groups, during the last 2 weeks of study, didn’t effect plasma Hcy and cysteine levels (P = NS), while in BDL group it decreased plasma methionine concentration (Fig. 5), improved hepatic SAM level and consequently, increased liver SAM to SAH ratio (Table 3). Liver SAH level was not different between BDL/L-NAME and BDL/Saline groups (0.19 F 0.01 nmol/mg protein in 28 day BDL/Saline and 0.16 F 0.02 in BDL/L-NAME groups, P = NS). Liver GSH or GSSG was not influenced by L-NAME treatment in studied groups (Table 3). The other studied parameters were not altered either by L-NAME treatment in this time period, quite similar to what was observed during the first 2 weeks of L-NAME treatment.
Table 3 Hepatic level of SAM, SAM to SAH ratio, GSH and GSSG in 28 day bile duct ligated (BDL) and sham operated (SO) rats given saline or L-NAME from day 14 to 28 Group
SAM
Sham/Saline Sham/L-NAME BDL/Saline BDL/L-NAME
0.48 0.50 0.24 0.40
F F F F
0.04 0.03 0.01a,b 0.02
SAM/SAH
GSH
4.01 3.61 1.31 2.44
30.1 27.0 17.6 16.3
F F F F
0.40 0.25 0.10a,b 0.13a
GSSG F F F F
0.9 0.8 1.1a 1.1a
NOTE. Results are expressed in nmol/mg protein as means F SEM for 10–12 animals per group. Abbreviations: SAM, S-adenosyl-methionine; SAH, S-adenosyl-homocysteine. a P b 0.05 versus Sham groups. b P b 0.05 versus BDL/L-NAME group.
3.4 3.0 6.5 6.9
F F F F
0.1 0.1 0.2a 0.9a
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Discussion The present results show that basal and post methionine-load Hcy concentrations were transiently elevated by day 14th after bile duct ligation and later returned to control levels. The increased plasma Hcy level 14 days after bile duct ligation, was prevented by chronic L-NAME treatment. Transient increase of SAH and continuous decrease of SAM values were also detected in the study. SAM to SAH ratio dropped by day 7 and increased following L-NAME treatment for 14 days. Plasma cysteine and methionine levels rose 21 days after cholestasis. Chronic L-NAME prevented the increase in plasma methionine level, while didn’t influence plasma cysteine concentration in 28 day BDL rats. We have also shown increased plasma level of NO2 + NO3 , in different time points after cholestasis. Plasma basal and post methionine load concentrations of Hcy, observed in this study, follow a different pattern from what has been reported previously in other models of cirrhosis. For instance, Mato and his group demonstrated increased Hcy concentration in 9-week CCl4 treated rats (Varela-Moreiras et al., 1995). What we detected was a transient rise in plasma Hcy concentration 14 days after bile duct ligation that returned to the normal level in cirrhotic rats. Although precise interpretation of these results needs detailed biochemical characterization, we should consider different pathogenic processes and metabolic abnormalities that lead to the onset of cirrhosis, such as plasma amino acid profiles involved in BDL and CCl4 experimental models (Weisdorf et al., 1990; Bauer et al., 1990). In CCl4induced cirrhosis, contrary to BDL, bile flow is not interrupted (Sokal et al., 1996; Krahenbuhl and Reichen, 1988), and the metabolic zonation pattern appears the reverse of what is discovered in biliary cirrhosis (Sokal et al., 1992). The dissimilarities between these two models may thus explain the variations noticed in the results. Following bile duct ligation, there are intense lipid peroxidation and oxidative injury in the liver. These were addressed in our study by decreased hepatic GSH to GSSG ratio as a reliable indicator of redox state and oxidative stress. Gamma-lutamylcysteine synthetase (GCS) activity is down regulated during cholestasis (Neuschwander-Tetri et al., 1996). This can lead to the accumulation of the precursor of the enzyme and reduction of the final product, which are cysteine and GSH, respectively. Our findings in the study were in accordance with the mentioned concept. However, the transient increase, observed in the GSH level in our study (Table 2), is attributed to the inhibition of glutathione efflux in cholestasis (Ookhtens et al., 1988; Pastor et al., 2000). Secondary biliary cirrhosis is also associated with increased formations of S-nitrosothiols and nitrotyrosine, indicatives of increased nitrosative stress in this model (Ottesen et al., 2001). Increased plasma Hcy level along with observed NO overproduction, favors S-Nitroso-Hcy formation, which exerts deleterious effects by peroxynitrite formation and by incorporation into proteins, resulting in protein damage (Jakubowski, 2004). A 71% increase in MAT activity has been demonstrated following 17 days of L-NAME treatment in BDL rats (Tunon et al., 2001). Supporting this data, we demonstrated that chronic inhibition of NO synthesis, increased liver SAM level and its methylation capacity in BDL rats. Decreased Hepatic SAM level along with methionine accumulation in the plasma can be the consequence of liver MAT inactivation by NO via S-nitrosation (Ruiz et al., 1998). Previous studies were suggesting that SAM serves as a metabolic switch and its reduced content can lead to cystathionine h-synthase (CBS) inactivation and increased Hcy methylation (Fig. 1) (Finkelstein et al., 1975; Finkelstein and Martin, 1984). Recent data propose that the redox state in the cells can be a significant regulatory factor in the distribution of Hcy between the methionine cycle and transsulfuration pathway. Increased oxidative
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stress results in MS inactivation and favors transsulfuration by CBS activation (Aleynik and Lieber, 2000). Remethylation of Hcy can be influenced by other factors, as well. NO can inactivate MS, which participates in Hcy remethylation (Danishpajooh et al., 2001; Nicolaou et al., 1996). This NO induced inactivation is related to its binding capacity to cobalt moiety of vitamin B12 (Danishpajooh et al., 2001; Brouwer et al., 1996). A recent report by Forestier et al demonstrated that the expression and activity of betaine homocysteine methyltransferase (BHMT) decrease in biliary cirrhosis (Forestier et al., 2003) and these further prevent the remethylation process. So, according to the mentioned evidences, there is a complex interaction between different factors, which can lead to decreased Hcy utilization. Furthermore, The transient rise in plasma Hcy along with the increased liver SAH level, 14 days after bile duct ligation, can be correlated to the inflammatory repair process of the liver, which accelerates specific methylation reactions, generates SAH and releases Hcy (Dudman, 1999). This event, together with above mentioned limitations in the usage of produced Hcy, lead to Hcy accumulation in the hepatocytes and as a result, in plasma, in the first 2 weeks. Persistent NO overproduction during four weeks can influence MAT activity, negatively; so, there would be a restriction in SAM production following cholestasis. Meanwhile, GSH is a crucial regulator of MAT activity in vivo (Corrales et al., 1999), which decreased considerably during the second 2 weeks of our study. This can further inhibit MAT activity during that period. Supporting this, we found increased methionine level at the same time. This event in addition to the continuous consumption of SAM in the first 2 weeks of cholestasis, results in the SAM depletion later on. Diminution of the only precursor of the pathway, thus leads to decreased formation of SAH and Hcy and the remaining Hcy gradually enters the metabolic pathways. So plasma Hcy level eventually falls during the last 2 weeks of study. But we should not neglect the possible activation of compensatory roots for Hcy usage in this period, too. Data obtained from L-NAME administration in our study, enhance the possible contribution of NO to Hcy regulation. Increased SAM level following chronic L-NAME treatment during the first 14 days in BDL rats can activate transsulfuration pathway via improving the level of CBS activity, resulting in attenuation of Hcy level. Decreased creatinine clearance in BDL rats limits the availability of Hcy to be metabolized by kidneys. In this study, chronic L-NAME administration failed to influence creatinine clearance in BDL animals though prevented Hcy rise after 14 days of bile duct ligation. As a result, the increase in plasma Hcy due to the decrease in creatinine clearance is less probable. Cholestatic subjects are prone to Anorexia (Padillo et al., 2001) and this may affect the amount of methionine intake, subsequently. But the difference in the amount of food intake between BDL and SO groups was significant just in the 1st week of cholestasis, which disappeared afterward, in our study. Meanwhile, we detected hypermethioninemia in our BDL animals, which suggests methionine metabolism defect possibly due to liver MAT inactivation. So decrease in Hcy values as a consequence of decreased methionine intake is unlikely. Depletion of SAM, an important intracellular signal, can have many adverse effects on essential hepatic functions like sensitivity to injury (Mato et al., 2002). Our findings of impaired hepatic SAM synthesis in an experimental model of biliary obstruction provide a rationale for the beneficial role of SAM supplementation in preserving liver function which has been mentioned previously (Pastor et al., 1996; Gonzalez-Correa et al., 1997). One of the important mechanisms through which SAM exerts its hepatoprotective effects, is likely by the repletion of the glutathione pool. We observed that chronic nitric oxide synthase (NOS)
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inhibition, increased liver SAM level and reduced plasma methionine and Hcy concentrations, whereas it failed to modulate liver GSH or GSSG content and plasma liver enzyme activity. This proposes impairments in the pathway of glutathione production from SAM in BDL model, which can not be overcome by NOS inhibition. Furthermore, we have found out that the observed effects of L-NAME are not simply due to the attenuation of underlying liver damage and can be attributed to its effects on methionine cycle. In the present study, we investigated the time dependent effect of bile duct ligation on Hcy metabolism. We suggest that plasma Hcy follows a different pattern of alteration in BDL model in comparison to the other models of cirrhosis. Moreover, NO overproduction have an important role in regulation of plasma Hcy concentration and liver SAM content in this model.
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Sadeghipour, H., Dehghani, M., Dehpour, A.R., 2003. Role of opioid and nitric oxide systems in the nonadrenergic noncholinergic-mediated relaxation of corpus cavernosum in bile duct-ligated rats. European Journal of Pharmacology 460, 201 – 207. She, Q.B., Nagao, I., Hayakawa, T., Tsuge, H., 1994. A simple HPLC method for the determination of S-adenosylmethionine and S-adenosylhomocysteine in rat tissues: the effect of vitamin B6 deficiency on these concentrations in rat liver. Biochemistry Biophysics Research Communication 205 (3), 1748 – 1754. Sokal, E.M., Baudoux, M.C., Collette, E., Hausleithner, V., Lambotte, L., Buts, J.P., 1996. Branched chain amino acids improve body composition and nitrogen balance in a rat model of extra hepatic biliary atresia. Pediatric Research 40 (1), 66 – 71. Sokal, E.M., Mostin, J., Buts, J.P., 1992. Liver metabolic zonation in rat biliary cirrhosis: distribution is reverse of that in toxic cirrhosis. Hepatology 15 (5), 904 – 908. Stead, L.M., Brosnan, M.E., Brosnan, J.T., 2000. Characterization of homocysteine metabolism in the rat liver. Biochemical Journal 350, 685 – 692. Stickel, F., Choi, S.W., Kim, Y.I., Bagley, P.J., Seitz, H.K., Russell, R.M., Selhub, J., Mason, J.B., 2000. Effect of chronic alcohol consumption on total plasma homocysteine level in rats. Alcoholism-Clinical and Experimental Research 24 (3), 259 – 264. Torres, L., Garcia-Trevijano, E.R., Rodriguez, J.A., Carretero, M.V., Bustos, M., Fernandez, E., Eguinoa, E., Mato, J.M., Avila, M.A., 1999. Induction of TIMP-1 expression in rat hepatic stellate cells and hepatocytes: a new role for homocysteine in liver fibrosis. Biochimica et Biophysica Acta 1455 (1), 12 – 22. Tunon, M.J., Criado, M., Vazquez-Gil, M.J., Mesonero, M.J., Esteller, A., Gonzalez-Gallego, J., 2001. Reduction of fibrosis by inhibition of nitric oxide synthase is not associated to prevention of oxidative stress in rats with bile duct ligation. Journal of Hepatology 34 (S1), 53. Varela-Moreiras, G., Alonso-Aperte, E., Rubio, M., Gasso, M., Deulofeu, R., Alvarez, L., Caballeria, J., Rodes, J., Mato, J.M., 1995. Carbon tetrachloride-induced hepatic injury is associated with global DNA hypomethylation and homocysteinemia: effect of S-adenosylmethionine treatment. Hepatology 22, 1310 – 1315. Weisdorf, S.A., Freese, D.K., Radmer, W.J., Dehner, L.P., Cerra, F.B., 1990. Plasma amino acids in long-term models for obstructive versus toxic liver injury in developing rats. Journal of Pediatric Gastroenterology and Nutrition 10 (3), 371 – 379.
Homocysteine alterations in experimental cholestasis and its subsequent cirrhosis Mohammad R. Ebrahimkhania, Hamed Sadeghipoura, Mehdi Dehghania, Samira Kiania, Seyedmehdi Payabvasha, Kiarash Riazia, Hooman Honara, Parvin Pasalarb, Naser Mirazic, Massoud Amanloud, Hassan Farsamd, Ahmad R. Dehpoura,* a
Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran c Department of Biology, Bu-Ali Sina University, Hamadan, Iran d Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
b
Received 14 June 2004; accepted 28 December 2004
Abstract Homocysteine (Hcy), an intermediate in methionine metabolism, has been proposed to be involved in hepatic fibrogenesis. Impaired liver function can alter Hcy metabolism. The aim of the present study was to determine plasma Hcy alterations in acute obstructive cholestasis and the subsequent biliary cirrhosis. Cholestasis was induced by bile duct ligation and sham-operated and unoperated rats were used as controls. The animals were studied on the days 7th, 14th, 21st and 28th after the operation. Plasma Hcy, cysteine, methionine, nitric oxide (NO) and liver S-adenosyl-methionine (SAM), S-adenosyl-homocysteine (SAH), SAM to SAH ratio and glutathione were measured. Chronic L-NAME treatment was also included in the study. Plasma Hcy concentrations were transiently elevated by the day 14th after bile duct ligation (P b 0.01) and subsequently returned to control levels. Similar relative fluctuations in plasma Hcy were observed in BDL rats after intraperitoneal methionine overload. Plasma methionine, cysteine and nitrite and nitrate were significantly increased after bile duct ligation. SAM to SAH ratio was diminished by the 1st week of cholestasis and remained significantly decreased throughout the study. These events were accompanied by a decrease in GSH to GSSG ratio in the liver. Chronic L-NAME treatment improved SAM to SAH ratio and prevented the elevation of plasma Hcy and methionine (P b 0.05) while couldn’t influence the other parameters. In conclusion, this study demonstrates alterations in plasma Hcy and liver SAM and SAH contents in precirrhotic
* Corresponding author. Tel.: +98 21 611 2802; fax: +98 21 640 2569. E-mail address: [email protected] (A.R. Dehpour). 0024-3205/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.lfs.2004.12.009
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stages and in secondary biliary cirrhosis, for the first time. In addition, we observed that plasma Hcy concentrations in BDL rats follow a distinct pattern of alteration from what has been previously reported in other models of cirrhosis. NO overproduction may contribute to plasma Hcy elevation and liver SAM depletion after cholestasis. D 2005 Published by Elsevier Inc. Keywords: Cholestasis; Cirrhosis; Homocysteine; Methionine; Nitric oxide; S-adenosyl-methionine
Introduction Homocysteine (Hcy), an essential intermediate of normal mammalian methionine metabolism, can promote oxidative stress and endothelial dysfunction (Loscalzo,1996). Very recent reports suggest that Hcy interferes with the structure and function of various proteins by forming Hcy-N-protein (N-homocysteinylation)(Jakubowski, 2004). Hcy directly induces the expression of procollagen type I and tissue inhibitor of metalloproteinases-1 genes in hepatocytes and stellate cells in vitro (Torres et al., 1999). These suggest that Hcy may be an effective inducer of liver fibrogenesis (Torres et al., 1999; Garcia-Tevijano et al., 2001). Cholestasis is accompanied by endothelial dysfunction (Namiranian et al., 2001), oxidative stress (Ljubuncic et al., 2000) and progressive fibrogenesis (Parola et al., 1996), whose bases have not been well defined. Knowledge of initial signals that upset the maintenance of proper liver function or trigger necrosis and fibrogenesis will be extremely valuable in understanding the causes of liver cirrhosis and in working towards its treatment. Thus measurement of Hcy in this clinical setting has a potential clinical relevance (Carmel and Jacobsen, 2001). S-adenosyl-methionine (SAM), an important molecule in normal cell function and survival, is central to many biological processes from detoxification to gene regulation (Mato et al., 2002). It is a ubiquitous methyl donor to a wide spectrum of compounds such as DNA, RNA, proteins and phospholipids (Mato et al., 1997; Aleynik and Lieber, 2000). SAM is synthesized from methionine and is an intermediate in methionine-Hcy cycle (Fig. 1). Liver, where 85% of the whole body transmethylation capacity resides (Finkelstein, 1990), is central in Hcy metabolism (Garcia-Tevijano et al., 2001; Stead et al., 2000). Therefore, impaired liver function can influence Hcy metabolism and its plasma level (Garcia-Tevijano et al., 2001; Stead et al., 2000). Chronic treatment of experimental animals with ethanol or CCl4 was associated with hyperhomocysteinemia (Varela-Moreiras et al., 1995; Stickel et al., 2000) while the hepatoprotective effect of SAM on experimental cirrhosis was accompanied by a decrease in plasma Hcy concentration (Varela-Moreiras et al., 1995). Elevated plasma Hcy level in liver cirrhosis has been also shown by some human studies (Torres et al., 1999; Ferre et al., 2002; Bosy-Westphal et al., 2001). However the majority of patients in those studies, suffered from either alcoholic or viral liver cirrhosis and there were not enough data regarding cholestatic liver disease. Distinct patterns of plasma amino acid alterations have been elucidated in two models of cirrhosis: obstructive liver cirrhosis and CCL4-induced cirrhosis, which suggest different metabolic abnormalities in response to diverse liver injuries (Rosen et al., 1997; Weisdorf et al., 1990). It has been demonstrated that nitric oxide (NO) inactivates rat hepatic methionine adenosyl transferase (MAT) by S-nitrosation, in vivo (Ruiz et al., 1998; Perez-Mato et al., 1999). It can also inactivate
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Methionine
MAT
S-adenosyl-methionine a
DMG MS
MTs
BHMT betaine
a-CH3 S-adenosylhomocysteine Ado
SAHH Homocysteine Ado
CBS Cystathionine
Cysteine
Glutathione
Fig. 1. Pathways of Methionine and Homocysteine metabolism. S-adenosyl-methionine (SAM) is synthesized from methionine and ATP by methionine adenosyltransferase (MAT). SAM is converted to S-adenosyl-homocysteine (SAH) after donation of its methyl group, a reaction catalyzed by numerous methyltransferases(MTs). Subsequently SAH is converted into adenosine and Hcy by SAH-hydrolase(SAHH). This is a reversible reaction, however under normal conditions the catabolism of SAH is favored by the rapid removal of the reaction products adenosine and Hcy. Depending on the cellular needs for methionine, Hcy may undergo remethylation to methionine or enter transsulfuration pathway where it serves as a precursor for cysteine and glutathione. BHMT indicates Betaine-Homocysteine methyltransferase; CBS, cystathionine h-synthase; DMG, dimethylglycine; MS, methionine synthase.
methionine synthase (MS), the enzyme that participates in Hcy remethylation reaction (Danishpajooh et al., 2001; Nicolaou et al., 1996). To our knowledge, there are not any data regarding plasma Hcy and liver SAM and SAH levels in cholestatic liver disease and subsequent biliary cirrhosis. It is also not clear whether NO overproduction can influence the methionine cycle in this model. The present study was designed to investigate the potential alterations of plasma Hcy, and liver SAM and SAH levels in cholestasis and its subsequent cirrhosis in which increased systemic production of NO occurs (Marley et al., 1999; Nahavandi et al., 2001; Mani et al., 2002). We also tried to disclose some probable roles for NO overproduction in this regard.
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Material and methods Reagents All materials were purchased from Sigma. Animals and Experimental procedures Animals Adult male Sprague-Dawley rats (230–245 g, Pasteur Institute of Iran, Tehran, Iran) were used throughout this study. The animals were housed in a temperature-controlled room (23 F 1 8C), on a 12-h regular light /dark cycle with free access to standard rodent chow and water. They were handled in accordance with the criteria outlined in the "Guide for the Care and Use of Laboratory animals" (NIH US publication no. 85–23 revised 1985). All experiments were performed in line with the ethical considerations, recommended by the Pasteur Institute of Iran. Study design and operations Age and weight matched animals were studied in three groups: bile duct ligated (BDL), sham operated (SO) and unoperated control rats. Laparatomy was performed under general anesthesia (Ketamine HCl 50mg/kg and Xylazine HCl 10 mg/kg i.p.). The bile duct was exposed and double ligated, then cut through between the ligations. Sham operation consisted of laparatomy, bile duct identification and manipulation without ligation. After 7,14,21 and 28 days, animals were anesthetized with ether and blood collections were performed after cardiac puncture. There were 10–12 rats per group at the time of sampling. Sample collections took place at a same particular time in the morning. For methionine loading experiments, methionine was administered in different doses (40 mg/kg– 100mg/kg i.p.) and blood was collected 2 hours later. The appropriate dosage was selected previously in a pilot study. To investigate the effect of NO overproduction on Hcy metabolism, N(N)-nitro L-arginine methyl ester (L-NAME)(3 mg/kg/day, i.p.) or normal saline (1 ml/kg/day, i.p.) was administered to the SO and BDL animals for 2 weeks (Sadeghipour et al., 2003). The experiment was performed either in the first 2 weeks (day 0 to 14) or in the second 2 weeks of the study (day 14 to 28). Analytical techniques Total plasma Hcy, cysteine and methionine Centrifugation, plasma separation and freezing at –70 8C were done immediately after sampling. Measurements of total plasma Hcy and cysteine concentrations were carried out by High-performance liquid chromatography (HPLC) with fluorescence detection, as previously described (Bosy-Westphal et al., 2001; Hyland and Bottiglieri, 1992). Methionine was measured after deproteinization with sulfosalicylic acid, by HPLC-UV detection (Jayatilleke and Shaw, 1993). Hepatic SAM and SAH Liver specimens were snap frozen in liquid nitrogen for subsequent measurements of SAM and SAH. At the time of measurements, liver tissues were homogenized in 4 volumes of 0.4 M HClO4 then
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centrifuged at 10,000 g for 20 min. SAM and SAH levels in liver homogenates were determined by isocratic HPLC analysis with ultraviolet detection as described (She et al., 1994). The method provides rapid resolution of both compounds in a single run by direct injection of perchloric acid extracts so that sampling procedures and analytical errors can be reduced. Total protein was measured using the Lowry protein assay (Lowry et al., 1951). Bovine serum albumin was used as a standard. All measurements are reported in nanomoles per milligram of protein to adjust for possible changes in milligrams of protein per gram of liver. Hepatic glutathione Glutathione levels (both reduced and oxidized forms) were measured by fluorometric assay (Hissin and Hilf, 1976) .In Brief, ~250 mg of liver tissue was homogenized on ice in 3.75 ml of Phosphate-EDTA (P-E) buffer. The homogenate was centrifuged at 10,000 g for 30 min at 4 8C. 0.5 ml of the homogenate was diluted further in 4.5 ml of P-E (1:10). 0.1 ml of the resulting mixture was added to 1.8 ml of P-E buffer (pH 8.0) followed by 100 Al of the O-phthalaldehyde (OPT). Analysis was performed fluorometrically at 420 nm, with excitation at 350 nm. GSSG was measured by the same procedure after the incubation of tissue homogenates with N-ethylmaleimide (NEM) for 30 minutes in room temperature, to block any free thiol residues. Instead of P-E buffer, 0.1 N NaOH was employed for GSSG assay. Plasma nitrate and nitrite Plasma nitrate and nitrite levels were measured as indicators of NO production. The measurements were done according to method by Miranda et al (Miranda et al., 2001). Samples were deproteinized by centrifugation through a 30-kDa molecular weight filter (Centricon Millipore) at 14,000 rpm, for 1.5–3 h at 4 8C. After loading the plate with samples (100 Al), addition of saturated solution of VCl3 (100 Al) to each well was rapidly followed by addition of the Griess reagents (50 Al each). Sulfanilamide and Naphthylethylenediamine dihydrochloride were applied for preparation of Griess reagents. The plate was incubated at 37 8C for 30 minutes and then absorbance at 540 nm was measured using standard plate reader. Fresh standard solutions of nitrate were included in each experiment. Hepatic and renal function Total plasma bilirubin, alkaline phosphatase (ALP), alanineaminotransferase (ALT) and creatinine were determined with commercially available kits (Zistshimi. Iran). BDL rats that failed to show characteristics of cholestasis were excluded. Histology Liver cirrhosis was histologically confirmed after 28 days of bile duct ligation. Those, who were not confirmed histologically cirrhotic, were not included in our study. Statistical analysis All data are expressed as mean F SEM. Statistical evaluation of data was performed using analysis of variances (ANOVA), followed by Tukey post hoc test. P-values less than 0.05 were considered statistically significant.
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Results Characterization of BDL and SO rats One day after laparotomy BDL rats showed manifestations of cholestasis (jaundice, dark urine). Plasma bilirubin and liver enzyme activities increased in BDL animals consistent with biliary obstruction (P N 0.01) and remained significantly elevated all over the study. During the 1st week of the study, daily food intake of BDL animals was less than SO group (15.1 F 0.3 g/day/rat vs 21.0 F 0.3 g/day/rat, mean daily food intake of BDL and SO groups respectively; P b 0.01). Concurrently, body weight of BDL animals decreased during the 1st week of cholestasis (252 F 1 g vs 267 F 1 g, mean weight after one week, BDL and SO groups respectively; P b 0.01). In the 2nd, 3rd and 4th weeks of the study food intake and weight gain were not significantly different among different groups under study (P = NS). Basal and post methionine load plasma Hcy A gradual rise was observed in Hcy values after bile duct ligation. Plasma Hcy concentration reached its maximum level on day 14th after bile duct ligation and then by day 28th, decreased to values that were not significantly different from controls (Table 1, Fig. 2). Methionine load resulted in elevation of plasma Hcy levels both in control and BDL animals (Fig. 2). The elevation in plasma Hcy after methionine load was more prominent in BDL rats than in controls; however, this effect was observed only in the first 2 weeks after bile duct ligation (Fig. 2). There were not significant differences between unoperated controls and SO animals. Plasma methionine and cysteine Plasma levels of the sulfur-containing amino acids, cysteine and methionine, increased in cirrhotic animals compared to the SO group (Table 1). By the 3rd week after operation, this difference became significant (Table 1). There was not any significant difference between unoperated controls and SO groups. Hepatic SAM and SAH levels Table 2, shows that significantly reduced SAM level was detected by the 3rd and 4th weeks after bile duct ligation. Liver SAH content increased transiently after bile duct ligation; it was higher than SO animals during the 1st and 2nd weeks and returned to control values in the 3rd and 4th weeks of the Table 1 Plasma concentrations of Methionine, Cysteine and NO2 + NO3 in control and bile duct ligated (BDL) rats Group
Methionine
Cysteine
NO2 + NO3
Control BDL(1st wks) BDL(2nd wks) BDL(3rd wks) BDL(4th wks)
41.1 56.2 55.1 69.0 70.3
12.9 17.8 22.7 27.9 24.7
27.1 52.1 48.4 52.9 50.1
F F F F F
4.5 7.8 5.9 5.7* 6.0*
F F F F F
1.9 3.0 2.3 3.5* 2.8*
NOTE. Results are expressed in AM as means F SEM for 10–12 animals per group. * P b 0.01 versus control group. ** P b 0.001 versus control group.
F F F F F
2.5 4.7** 2.4** 4.5** 4.5**
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Fig. 2. Basal and post methionine load plasma Homocysteine (Hcy) concentrations in plasma of control and 7, 14, 21, 28 day bile duct ligated (BDL) rats. Plasma Homocysteine was assessed in basal state and also after methionine administrations. Methionine was administered in different doses (40 mg/kg-100 mg/kg i.p.) and blood was collected 2 hours later. Values represent Mean + SEM (n = 10–12 per group). aP b 0.01 in comparison with control group(Basal state). bP b 0.01 in comparison with control group(after 40 mg/kg methionine load). cP b 0.01 in comparison with control group( after 100 mg/ kg methionine load).
study (Table 2). These fluctuations were in accordance with the above mentioned changes in plasma Hcy levels. The SAM to SAH ratio (methylation ratio) fell by 45% by the 1st week of cholestasis and remained significantly decreased, thereafter (Fig. 3). There was not any significant difference among unoperated control and sham groups. Hepatic glutathione Cholestasis was associated with decreased GSH to GSSG ratio (GSH/GSSG) from the 1st week of bile duct ligation (P b 0.01)(Fig. 3b). The exact values of GSH and GSSG of the liver in different time Table 2 Hepatic concentrations of SAM, SAH, GSH and GSSG in cholestatic and control rats Group
SAM
Control BDL(1st wks) BDL(2nd wks) BDL(3rd wks) BDL(4th wks)
0.50 0.46 0.41 0.30 0.25
F F F F F
SAH 0.03 0.02 0.03 0.02** 0.01**
0.13 0.22 0.25 0.18 0.18
GSH F F F F F
0.01 0.01** 0.02** 0.01 0.01
30.3 36.4 27.8 20.7 19.3
GSSG F F F F F
0.9 1.8* 1.3 1.7** 1.5**
NOTE. Results are expressed in nmol/mg protein as means F SEM for 10–12 animals per group. Abbreviations: SAM, S-adenosyl-methionine; SAH, S-adenosyl-homocysteine. * P b 0.05 versus control group. ** P b 0.01 versus control group.
3.1 F 0.1 10.0 F 0.5** 7.1 F 0.9** 8.0 F 0.7** 6.8 F 0.6**
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Fig. 3. (a, b). Liver S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH) ratio and GSH to GSSG ratio in 7, 14, 21, 28 day bile duct ligated (BDL) rats and their sham operated (SO) ones. Both ratios differed significantly from SO groups 7, 14, 21 and 28 days after operation. Values represent Mean F SEM (n = 10–12 per group). *P b 0.01 in comparison with the SO group.
points of study have been shown in Table 2. There was a transient increase in GSH level in the 1st week of cholestasis (P b 0.05). But the early increase of GSH was followed by a continuous decrease over later times of study.
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Plasma nitrate and nitrite and creatinine clearance Cholestatic animals had significantly higher plasma nitrite and nitrate concentrations compared with SO ones, through out different times of study, indicative of increased NO synthesis in cholestasis (Table 2). This is in agreement with our previous data in BDL rats (Nahavandi et al 2001; Mani et al., 2002).
Fig. 4. (a, b). Basal plasma level of Homocysteine (Hcy) and liver S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH) ratio in 14 day bile duct ligated (BDL) and sham operated (SO) rats given saline or L-NAME. Plasma Hcy increased after 14 days of bile duct obstruction while SAM to SAH ratio decreased in this period. Chronic treatment with L-NAME (day 0 to 14) prevented these alterations in BDL rats. Data are shown as Mean + SEM (n = 10–12). aP b 0.01 in comparison with SHAM groups. bP b 0.05 in comparison with BDL/L-NAME animals.
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Creatinine clearance fell by 33% by the 1st week of cholestasis (1.03 F 0.04 ml/min/100g vs 0.70 F 0.03 ml/min/100 g, SO and BDL respectively; P b 0.001). However, there were no significant changes in plasma NO levels and creatinine clearance among subjects in different stages of cholestasis (P = NS). Furthermore, statistical analysis failed to detect any correlation between plasma creatinine levels and Hcy values.
Fig. 5. (a, b). Plasma methionine and cysteine levels in 28 day bile duct ligated (BDL) and sham operated (SO) rats given saline or L-NAME. Both methionine and cysteine levels increased after 28 days of bile duct obstruction. Chronic treatment with LNAME (day 14 to 28) prevented the increase of plasma methionine level in BDL rats while it did not influence plasma cysteine level significantly. Data are shown as Mean + SEM (n = 10–12). aP b 0.01 in comparison with SHAM groups. bP b 0.05 in comparison with BDL/L-NAME animals.
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Plasma and liver measurements following L-NAME treatment L-NAME treatment during the first 2 weeks of study L-NAME treatment significantly decreased NO production, as assessed by plasma NO2 + NO3 concentration in BDL or SO groups (P b 0.05). Increase in plasma Hcy did not occur in 14 day BDL animals, which were chronically treated by L-NAME through the study (Fig. 4). L-NAME treatment improved liver SAM level in BDL animals (0.35 F 0.03 nmol/mg protein in BDL/Saline and 0.54 F 0.03 in BDL/L-NAME groups, P b 0.05) while it couldn’t influence the SAH values, significantly (0.30 F 0.02 nmol/mg protein in BDL/Saline and 0.24 F 0.04 in BDL/L-NAME groups, P = NS). Consequently, hepatic SAM to SAH ratio also increased in the BDL/L-NAME group (P b 0.05)(Fig. 4). Chronic L-NAME didn’t have any significant effect on plasma Hcy level and SAM to SAH ratio in SO animals (P = NS)(Fig. 4). There were not any significant alterations in plasma cysteine, methionine, and liver glutathione by our L-NAME intervention in 14-day BDLs or corresponding SO ones (P = NS). In addition, despite a significant increase of plasma ALT activity after bile duct ligation, L-NAME treatment did not influence its value in either BDL or SO groups (36 F 8 U/L vs 43 F 7 U/L, Sham/Saline and Sham/L-NAME groups respectively; P = NS)(101 F 21 U/L vs 120 F 18 U/L, BDL/Saline and BDL/L-NAME groups respectively; P = NS). Plasma ALP activity followed a pattern similar to alterations of ALT activity. Creatinine clearance was not influenced by the treatment in the studied groups (1.03 F 0.04 ml/min/ 100g vs 0.99 F 0.06 ml/min/100 g, SO and SO/L-NAME respectively; P = NS)(0.70 F 0.03 ml/min/100 g vs 0.68 F 0.051 ml/min/100 g, BDL and BDL/L-NAME respectively; P = NS). L-NAME treatment during the second 2 weeks of study L-NAME treatment in BDL and SO groups, during the last 2 weeks of study, didn’t effect plasma Hcy and cysteine levels (P = NS), while in BDL group it decreased plasma methionine concentration (Fig. 5), improved hepatic SAM level and consequently, increased liver SAM to SAH ratio (Table 3). Liver SAH level was not different between BDL/L-NAME and BDL/Saline groups (0.19 F 0.01 nmol/mg protein in 28 day BDL/Saline and 0.16 F 0.02 in BDL/L-NAME groups, P = NS). Liver GSH or GSSG was not influenced by L-NAME treatment in studied groups (Table 3). The other studied parameters were not altered either by L-NAME treatment in this time period, quite similar to what was observed during the first 2 weeks of L-NAME treatment.
Table 3 Hepatic level of SAM, SAM to SAH ratio, GSH and GSSG in 28 day bile duct ligated (BDL) and sham operated (SO) rats given saline or L-NAME from day 14 to 28 Group
SAM
Sham/Saline Sham/L-NAME BDL/Saline BDL/L-NAME
0.48 0.50 0.24 0.40
F F F F
0.04 0.03 0.01a,b 0.02
SAM/SAH
GSH
4.01 3.61 1.31 2.44
30.1 27.0 17.6 16.3
F F F F
0.40 0.25 0.10a,b 0.13a
GSSG F F F F
0.9 0.8 1.1a 1.1a
NOTE. Results are expressed in nmol/mg protein as means F SEM for 10–12 animals per group. Abbreviations: SAM, S-adenosyl-methionine; SAH, S-adenosyl-homocysteine. a P b 0.05 versus Sham groups. b P b 0.05 versus BDL/L-NAME group.
3.4 3.0 6.5 6.9
F F F F
0.1 0.1 0.2a 0.9a
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Discussion The present results show that basal and post methionine-load Hcy concentrations were transiently elevated by day 14th after bile duct ligation and later returned to control levels. The increased plasma Hcy level 14 days after bile duct ligation, was prevented by chronic L-NAME treatment. Transient increase of SAH and continuous decrease of SAM values were also detected in the study. SAM to SAH ratio dropped by day 7 and increased following L-NAME treatment for 14 days. Plasma cysteine and methionine levels rose 21 days after cholestasis. Chronic L-NAME prevented the increase in plasma methionine level, while didn’t influence plasma cysteine concentration in 28 day BDL rats. We have also shown increased plasma level of NO2 + NO3 , in different time points after cholestasis. Plasma basal and post methionine load concentrations of Hcy, observed in this study, follow a different pattern from what has been reported previously in other models of cirrhosis. For instance, Mato and his group demonstrated increased Hcy concentration in 9-week CCl4 treated rats (Varela-Moreiras et al., 1995). What we detected was a transient rise in plasma Hcy concentration 14 days after bile duct ligation that returned to the normal level in cirrhotic rats. Although precise interpretation of these results needs detailed biochemical characterization, we should consider different pathogenic processes and metabolic abnormalities that lead to the onset of cirrhosis, such as plasma amino acid profiles involved in BDL and CCl4 experimental models (Weisdorf et al., 1990; Bauer et al., 1990). In CCl4induced cirrhosis, contrary to BDL, bile flow is not interrupted (Sokal et al., 1996; Krahenbuhl and Reichen, 1988), and the metabolic zonation pattern appears the reverse of what is discovered in biliary cirrhosis (Sokal et al., 1992). The dissimilarities between these two models may thus explain the variations noticed in the results. Following bile duct ligation, there are intense lipid peroxidation and oxidative injury in the liver. These were addressed in our study by decreased hepatic GSH to GSSG ratio as a reliable indicator of redox state and oxidative stress. Gamma-lutamylcysteine synthetase (GCS) activity is down regulated during cholestasis (Neuschwander-Tetri et al., 1996). This can lead to the accumulation of the precursor of the enzyme and reduction of the final product, which are cysteine and GSH, respectively. Our findings in the study were in accordance with the mentioned concept. However, the transient increase, observed in the GSH level in our study (Table 2), is attributed to the inhibition of glutathione efflux in cholestasis (Ookhtens et al., 1988; Pastor et al., 2000). Secondary biliary cirrhosis is also associated with increased formations of S-nitrosothiols and nitrotyrosine, indicatives of increased nitrosative stress in this model (Ottesen et al., 2001). Increased plasma Hcy level along with observed NO overproduction, favors S-Nitroso-Hcy formation, which exerts deleterious effects by peroxynitrite formation and by incorporation into proteins, resulting in protein damage (Jakubowski, 2004). A 71% increase in MAT activity has been demonstrated following 17 days of L-NAME treatment in BDL rats (Tunon et al., 2001). Supporting this data, we demonstrated that chronic inhibition of NO synthesis, increased liver SAM level and its methylation capacity in BDL rats. Decreased Hepatic SAM level along with methionine accumulation in the plasma can be the consequence of liver MAT inactivation by NO via S-nitrosation (Ruiz et al., 1998). Previous studies were suggesting that SAM serves as a metabolic switch and its reduced content can lead to cystathionine h-synthase (CBS) inactivation and increased Hcy methylation (Fig. 1) (Finkelstein et al., 1975; Finkelstein and Martin, 1984). Recent data propose that the redox state in the cells can be a significant regulatory factor in the distribution of Hcy between the methionine cycle and transsulfuration pathway. Increased oxidative
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stress results in MS inactivation and favors transsulfuration by CBS activation (Aleynik and Lieber, 2000). Remethylation of Hcy can be influenced by other factors, as well. NO can inactivate MS, which participates in Hcy remethylation (Danishpajooh et al., 2001; Nicolaou et al., 1996). This NO induced inactivation is related to its binding capacity to cobalt moiety of vitamin B12 (Danishpajooh et al., 2001; Brouwer et al., 1996). A recent report by Forestier et al demonstrated that the expression and activity of betaine homocysteine methyltransferase (BHMT) decrease in biliary cirrhosis (Forestier et al., 2003) and these further prevent the remethylation process. So, according to the mentioned evidences, there is a complex interaction between different factors, which can lead to decreased Hcy utilization. Furthermore, The transient rise in plasma Hcy along with the increased liver SAH level, 14 days after bile duct ligation, can be correlated to the inflammatory repair process of the liver, which accelerates specific methylation reactions, generates SAH and releases Hcy (Dudman, 1999). This event, together with above mentioned limitations in the usage of produced Hcy, lead to Hcy accumulation in the hepatocytes and as a result, in plasma, in the first 2 weeks. Persistent NO overproduction during four weeks can influence MAT activity, negatively; so, there would be a restriction in SAM production following cholestasis. Meanwhile, GSH is a crucial regulator of MAT activity in vivo (Corrales et al., 1999), which decreased considerably during the second 2 weeks of our study. This can further inhibit MAT activity during that period. Supporting this, we found increased methionine level at the same time. This event in addition to the continuous consumption of SAM in the first 2 weeks of cholestasis, results in the SAM depletion later on. Diminution of the only precursor of the pathway, thus leads to decreased formation of SAH and Hcy and the remaining Hcy gradually enters the metabolic pathways. So plasma Hcy level eventually falls during the last 2 weeks of study. But we should not neglect the possible activation of compensatory roots for Hcy usage in this period, too. Data obtained from L-NAME administration in our study, enhance the possible contribution of NO to Hcy regulation. Increased SAM level following chronic L-NAME treatment during the first 14 days in BDL rats can activate transsulfuration pathway via improving the level of CBS activity, resulting in attenuation of Hcy level. Decreased creatinine clearance in BDL rats limits the availability of Hcy to be metabolized by kidneys. In this study, chronic L-NAME administration failed to influence creatinine clearance in BDL animals though prevented Hcy rise after 14 days of bile duct ligation. As a result, the increase in plasma Hcy due to the decrease in creatinine clearance is less probable. Cholestatic subjects are prone to Anorexia (Padillo et al., 2001) and this may affect the amount of methionine intake, subsequently. But the difference in the amount of food intake between BDL and SO groups was significant just in the 1st week of cholestasis, which disappeared afterward, in our study. Meanwhile, we detected hypermethioninemia in our BDL animals, which suggests methionine metabolism defect possibly due to liver MAT inactivation. So decrease in Hcy values as a consequence of decreased methionine intake is unlikely. Depletion of SAM, an important intracellular signal, can have many adverse effects on essential hepatic functions like sensitivity to injury (Mato et al., 2002). Our findings of impaired hepatic SAM synthesis in an experimental model of biliary obstruction provide a rationale for the beneficial role of SAM supplementation in preserving liver function which has been mentioned previously (Pastor et al., 1996; Gonzalez-Correa et al., 1997). One of the important mechanisms through which SAM exerts its hepatoprotective effects, is likely by the repletion of the glutathione pool. We observed that chronic nitric oxide synthase (NOS)
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inhibition, increased liver SAM level and reduced plasma methionine and Hcy concentrations, whereas it failed to modulate liver GSH or GSSG content and plasma liver enzyme activity. This proposes impairments in the pathway of glutathione production from SAM in BDL model, which can not be overcome by NOS inhibition. Furthermore, we have found out that the observed effects of L-NAME are not simply due to the attenuation of underlying liver damage and can be attributed to its effects on methionine cycle. In the present study, we investigated the time dependent effect of bile duct ligation on Hcy metabolism. We suggest that plasma Hcy follows a different pattern of alteration in BDL model in comparison to the other models of cirrhosis. Moreover, NO overproduction have an important role in regulation of plasma Hcy concentration and liver SAM content in this model.
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