The effect of Viagra (sildenafil citrate) on liver injury caused by chronic ethanol intragastric feeding in rats
Introduction
To enhance the effects of nitric oxide (NO) in improving liver blood flow, rats were fed Viagra (sildenafil citrate) with ethanol and a liquid diet intragastrically for 1 month. To measure the improvement in liver, blood flow pO2 and perfusion units were measured on the surface of the liver in rats where the blood alcohol levels (BAL) and urinary ethanol levels (UAL) were high during the UAL cycle.
Viagra selectively inhibits cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) resulting in enhanced smooth muscle relaxation initiated by nitric oxide (NO). Endothelial NO derived from NO synthase enters smooth muscle cells and activates guanylate cyclase, resulting in an increase in cGMP levels. This is responsible for the vasodilator effects of NO (Moncada and Higgs, 1993). In the case of the liver, liver sinusoidal endothelial cells are responsible for nitric oxide modulation of resistance to blood flow in the sinusoids (Shah et al., 1997). The half-life of cGMP is short, since it is rapidly degraded by PDE5 (Gibson, 2001). In the present study, the PDE5 inhibitor sildenafil (Viagra) was fed with ethanol to improve liver blood flow and prevent the liver hypoxia that develops during recovery from a brief ischemia episode induced by clamping the portal blood flow for 2 min.
There is evidence that an acute high does of ethanol causes an increase in NO levels (Baraona et al., 2002) as a response to adenosine release cause by ethanol (Orrego et al., 1988). Nitric oxide modulates hepatic vascular tone in normal rat liver (Mittal et al., 1994). A similar increase in NO occurs in binge drinking in man (Oekonomaki et al., 2004). In rats fed with ethanol intragastrically, nitrites increase in the liver to the same extent at high and low blood alcohol levels indicating that NO production is chronically increased in the liver regardless of the blood alcohol level (Bardag-Gorce et al., 2002). The question addressed in the present study is: will enhancement of NO effects change the circulation dynamics in the liver to alter the alcohol liver damage in chronic ethanol-fed rats? Nanji et al. (2001) showed that l-arginine supplement to enhance NO synthesis reversed the pathologic changes induced by intragastric ethanol feeding.
The intragastric ethanol feeding rat model used here is characterized by a cyclic change in blood alcohol levels (BAL) and urinary alcohol levels over a span of 7–10 days (Tsukamoto et al., 1985). Liver hypoxia occurs only when the BAL and UAL are very high during the cycle (Arteeel et al., 1997, French, 2004, Li et al., 2004a). Arteeel et al. (1997) likened this model to ischemic–reperfusion injury because liver hypoxia occurs when the BAL is high and reoxygenation occurs when the BAL is low during the cycle. Sinusoidal endothelial-derived NO has a protective effect during cold ischemia (Oishi et al., 2001). NO prevents hypoxia/reoxygenation injury in mouse liver (Taniai et al., 2004). NO protects rat hepatocytes against reperfusion injury by preserving the mitochondrial permeability transition (Kim et al., 2004). The role of NO as a cytoprotective agent during I/R injury, however, is complicated by the cytotoxic effects NO (Shah and Kamath, 2003). Thus, it is difficult to predict how NO enhancement will effect liver hypoxia and alcohol-induced liver damage. The results of this study indicate that NO enhancement did not effect the UAL cycle but improved the post-ischemic recovery of liver pO2. Paradoxically, liver injury was worse when Viagra was fed with ethanol.
The feeding experiment was performed using the intragastric tube-feeding model in which ethanol was fed at a constant rate for 1 month. In this model, the BAL cycles up and down over a 7- to 10-day period as previously described (Tsukamoto et al., 1985). At the end of the experiment, pO2 was monitor on the surface of the liver to measure ischemia–reperfusion injury after 2 min clamping of the porta hepatis.
Eight Wistar male rats, weighing about 275 g, were fed with ethanol at 13 g/kg/day for 6 weeks. Four of the rats were fed with Viagra (10 mg/kg/day) in the ethanol diet. Eight pair-fed control rats were fed a dextrose solution isocaloric to the ethanol. Four of the pair-fed control rats had Viagra (10 mg/kg/day) added to the diet and were pair-fed with ethanol + Viagra-fed rats. The diet provided 271 kcal/kg/day in all four groups. The diet used included a salt and vitamin mix (Dyets, Bethlehem, PA) as described by the American Institute of Nutrition Manual Mix and Vitamin Mix for Optimum Growth of Rats (French et al., 1997). The source of protein was lactalbumin. The diet was supplemented with 500 mg of choline and 1 g of methionine per liter of diet. The dietary calories were derived as follows: 33% from fat, 25% from protein, 6.7% from dextrose, and 34% from ethanol.
Rats fed with ethanol were monitored for blood ethanol levels (BAL) at the termination of the experiment. Urinary alcohol levels (UAL) were determined daily on 24 h urines collected under toluene to prevent evaporation. The UAL was measured daily to identify the peak and trough UALs in order to document the UAL cycle. UALs were measured using the QUED Saliva Alcohol Text Kit A150 (STC Technologies, Bethlehem, PA). Blood levels of serum alanine aminotransferase were measured by using a clinical analyzer (A kinetic rate method on Synch Ronex Systems; Beckman Instruments) at the beginning and the end of the experimental period. Body weights were measured weekly and at termination. Liver weights were measured at termination and the liver weight/body weight ratio calculated. The procedures were approved by the Research and Education Institute Animal Care Committee in accordance with guidelines for animal care as described by the National Academy of Sciences (1996).
Liver histology was examined after fixation of the liver in Zinc formalin and stained with hematoxylin and eosin. The liver pathology score was determined blinded to the experimental treatments of the rats using a previously reported scoring method (Bardag-Gorce et al., 2002). PCNA-positive hepatocytic nuclei were quantitated morphometrically using Nikon metamorph software. The liver sections were stained with a mouse monoclonal antibody to rat PCNA (clone PC10) (Signet Pathology Systems Inc., Dedham, MA).
This experiment was designed to test whether Viagra affected pO2 and perfusion unit levels before, during or after clamping of the portal hepatis. pO2 (mm Hg) was measured using an O2 probe on the surface of the liver during laparotomy under isofluorane anesthesia. The pO2 probe was calibrated to room air (159–160 mm Hg) using an automatic internal barometer for transcutaneous oxygen pO2 (tcp O2) before every use. The pO2 probe module used was PE 50 ys. (Perimed, North Royalton, OH). Perfusion arbitrary units (PU) were simultaneously measured as an indication of blood flow using a laser Doppler perfusion instrument (Laser Doppler System PR407-1 master probe). The laser Doppler module used was PF5010. (Perimed, North Royalton, OH). The Doppler probe was calibrated from zero PU and 250 PU using a motility standard which simulates random movement times velocity of concentrated moving blood cells. The data were analyzed using Data Acquisition Software PSW 325 (L) with the assistance of Robert Kostelny (North Royalton, OH).
The pO2 and perfusion units (PU) were recorded simultaneously using combined laser Doppler and transcutaneous oxygen probes. The perfusion unit reflects the movement of blood, which includes Brownian movement and movement caused by respiration and the excursion of the diaphragm, causing micromotion of the blood in the liver. When the diaphragm contracts downward, the pressure in the thorax becomes negative which increases hepatic venous outflow. When the diaphragm relaxes, the intrathoracic pressure increases and hepatic venous flow decreases. This is why the flow oscillates in synchrony with respiration. Because of this mechanism, when the portal blood flow is clamped, the baseline measurements still record movement of blood in synchrony with respiration.
Recording of pO2 and the laser Doppler PU levels were taken over a 4-min baseline. Then, the hepatic portal vessels at the porta hepatis were clamped for 2 min. The clamp was then released and the recovery of pO2 and blood flow were continuously monitored for 4 min more. The rats were then terminated by exsanguination. Data for each point were calculated as the Mean ± SEM in the 4 rats/group, during the preclamp, clamp, and post-clamp recovery.
P values were determined by t test or one-way ANOVA, and all pair-wise multiple comparison procedures such as Bonferroni, or the Student–Newman–Keuls method (Sigma-Stat Software, San Francisco, CA).
Section snippets
Results
The rats in all four groups gained the same amounts of weight (Fig. 1). The weight of the liver was increased in both ethanol-fed and ethanol-plus Viagra-fed groups compared to their dextrose pair-fed controls (P < 0.001) (Fig. 2). The liver weights were not different when the ethanol-fed and the ethanol-plus Viagra-fed groups were compared. Likewise, the liver weight/body weight ratio was increased in the ethanol-fed and ethanol-plus Viagra-fed groups when compared to their controls (P <
Discussion
There is evidence that the UAL cycle causes ischemic/reperfusion (I/R) injury in the liver due hypoxia at the peaks of the cycle and reoxygenation at the troughs of the cycle (Arteeel et al., 1997, Bardag-Gorce et al., 2002, French, 2004). In I/R injury of the liver, post-ischemic oxidant stress increases the expression of inducible nitric oxide synthase (iNOS) among other factors (Jaeschke, 2003).
NO is potent vasodilator which acts intracellularly by activation of guanylate cyclase which
Acknowledgments
The authors would like to thank Adriana Flores for typing the manuscript and Fawzia Gorce-Bardag, PhD for digitizing the figures. Supported by NIH/NIAAA grants 8116 and P50-011999 Alcohol Center Grant on Alcoholic Liver and Pancreatic Diseases Morphology Core.
References (35)
- et al.
Mediation by nitric oxide of the stimulatory effects of ethanol on blood flow
Life Sci.
(2002) - et al.
The importance of cycling of blood alcohol levels in the pathogenesis of experimental alcoholic liver disease in rats
Gastroenterology
(2002) - et al.
Control of mitochondrial respiration by NO: effects of low oxygen and respiratory state
J. Biol. Chem.
(2003) The role of hypoxia in the pathogenesis of alcoholic liver disease
Hepatol. Res.
(2004)Phosphodiesterase 5 inhibitors and nitrergic transmission—From Za prinast to sildenafil
Eur. J. Pharmacol.
(2001)- et al.
Ethanol-induced disturbance of hepatic microcirculation and hepatic hypoxia
Biochem. Pharmacol.
(1991) - et al.
Liver hypoxia and lack of recovery after reperfusion at high blood alcohol levels in the intragastric feeding model of alcohol liver disease
Exp. Mol. Pathol.
(2004) - et al.
Catecholamines are involved in the mechanism of the urinary alcohol level cycle in rats fed ethanol intragastrically at a constant rate
Life Sci.
(2004) - et al.
The effect of nitric oxide production by sinusoidal endothelial on preservation injury during cold ischemia
Hepatol. Res.
(2001) - et al.
Accumulation of HIF-1α under the influence of nitric oxide
Blood
(2001)
Nitric oxide in liver transplantation: pathobiology and clinical implications
Liver Transplant
Chronic alcohol consumption increases the sensitivity of rat liver mitochondrial respiration to inhibition by nitric oxide
Hepatology
Expression of type I plasminogen activator inhibitor and nitric oxide generation in ischemic preconditioning of the rat liver
Hepatol. Res.
The paradox of nitric oxide in cirrhosis and portal hypertension: too much or too little
Hepatology
Role of nitric oxide in the regulation of HIF-1α expression during hypoxia
Am. J. Physiol.: Cell Physiol.
Chronic ethanol treatment causes hypoxia in rat liver in vivo
Hepatology
Role of superoxide as a signaling molecule
News Physiol. Sci.
Cited by (19)
Protective effect of sildenafil on liver injury induced by intestinal ischemia/reperfusion
2013, Journal of Pediatric SurgeryCitation Excerpt :According to Ji et al. [8], sildenafil citrate did not induce significant changes in hepatic function and regenerative activity after partial hepatectomy in alcohol-fed rats. However, Li et al. [30] used PCNA to quantify the mitotic index in an experimental study designed to investigate the effects of sildenafil on liver injury caused by chronic ethanol feeding. They found an increase in the numbers of liver cells in the S phase in rats fed ethanol plus sildenafil.
Vardenafil Reduces Testicular Damage Following Ischemia/Reperfusion Injury in Rats
2009, Kaohsiung Journal of Medical SciencesEffects of graded doses of sildenafil citrate on liver microanatomy in male albino rats
2023, National Journal of Clinical AnatomyStructure and Function Changes of the Organ of Corti After Use of Sildenafil Citrate (VIAGRA®), in Adult Male Guinea Pigs
2022, Egyptian Journal of Ear, Nose, Throat and Allied SciencesSildenafil beyond erectile dysfunction and pulmonary arterial hypertension: Thinking about new indications
2021, Fundamental and Clinical PharmacologyEffect of sildenafil citrate on the liver structure and function in obstructive jaundice: An experimental study
2018, Turkish Journal of Surgery