Oxidative Stress in the Liver of Diabetic Rats Treated with a Combination of Sildenafil Citrate and a Free Radical Scavenger
Erectile Dysfunction (ED) is a common problem within
diabetic patients. The liver is one of the most affected vital organs
by diabetic consequences. Oxidative stress is the most known intermediary
pathway initiating liver diseases among diabetics. The present study was
designed to investigate the protective effect of alpha toccopherol (α-TP)
against possible oxidative stress, that may be elicited by administration
of Sildenafil Citrate (SC) and to assess whether SC may negatively affect
the liver in an experimental diabetic model. SC was given to groups of
normo-glycemic and diabetic rats, either alone or in combination with
α-TP, by oral route for two weeks. Hepatic tissue content of malondialdehyde-a
thiobarbituric acid reactive oxygen species (TBARS) and reduced glutathione
(GSH) were determined as biomarkers for oxidative stress in liver tissue.
TBARS was significantly up-regulated in diabetic than normo-glycemic rats.
SC significantly down-regulated TBARS content, an effect which was synergized
by α-TP co-administration. SC treatment depleted GSH in both normo-glycemic
and hyperglycemic rats, this effect was completely reversed by α-TP
co-administration. α-TP could not correct the effect of diabetes
on liver GSH and TBARS contents and it couldn`t restore theses parameters in diabetic to non-diabetic values.
In conclusion, our study explored the usefulness of α-TP co-administration
in protecting the liver against GSH depletion, induced by SC administration.
We also elucidated that SC down-regulated TBARS in liver tissue, an effect
which was potentiated by α-TP co-administration. We recommend the
use of α-TP as an adjuvant therapy to SC, specially for diabetic
patients who are considered to be the most extensive users of the drug.
Oxidative stress is believed to mediate the development of diabetes-associated
vasculopathy, endothelial dysfunction and neuropathy within erectile tissue.
It was proposed that adequate levels of a free radical scavenger, as vitamin
E, improves Erectile Dysfunction (ED), with a synergistic potential to
phosphodi-esterase type 5 (PDE-5) inhibitors (De Young et al.,
In addition, diabetes is often associated with hypogonadism, both conditions
represent major risk factors for ED. Testosterone normalization in diabetic
models maintains neural nitric oxide, PDE-5 and reinstates sensitivity
to relaxant stimuli and responsiveness to Sildenafil Citrate (SC) (Zhang
et al., 2006).
However, SC enhanced liver injury caused by ethanol (Li et al.,
2005). It is metabolized by cytochrome p-450, 3A4 and 2 C and any inhibitor
of these enzymes, may result in delayed metabolism and require dose adjustment
(McCullough, 2002). Diabetes represents a major risk factor for ED as
reported in people with long term Insulin Dependent Diabetes Mellitus
(IDDM) (Klein et al., 1996). This is why special attention should
be paid for patients with ED-risk factors as hypertension, diabetes and
coronary heart diseases (Kalsi and Kell, 2004). In experimental models
of diabetic ED, it was found that increased levels of free radicals in
diabetes can divert nitric oxide away from erectogenic pathway, through
its conversion to peroxynitrite (Khan et al., 2001). Pharmacokinetic
studies of SC demonstrated similarities between the rat and human in metabolite
formation in vivo (Walker et al., 1999). This drug was widely
used for male ED, showing a selective PDE 5 inhibitory potential, preventing
cycloguanosine monophosphate (cGMP) degradation. These actions enhance
the effect of nitric oxide at the target tissue (Leung and Yip, 1999).
In addition, high levels of malondialdehyde-MDA-(lipoperoxide product)
and low levels of nitric oxide in peripheral blood of diabetic men, having
ED correlates strongly with the severity of ED (El-Latif et al.,
2006). Malondialdehyde is always up-regulated in liver cells in response
to hepatocellular injury (El Sisi et al., 1993). Biomarkers of
oxidative stress such as MDA and reduced glutathione (GSH) have been considered
as specific indicators to oxidative status (Mayne, 2003). Data provided
evidence that nitric oxide deficiency, possibly due to the membrane lipid
peroxidation and defective glutathione levels, may contribute to the development
of diabetic ED and thus is involved in the pathogenesis of ED in diabetic
patients (Alper et al., 2003).
The present study was designed to
investigate the possible effect of SC on liver tissue oxidative status
in diabetic rats, trying α-TP as a free radical scavenger in both
normo-glycemic and hyperglycemic subjects.
MATERIALS AND METHODS
Animals and Experimental Design
Sixty four male Wistar rats weighing 100-120 g of ages 6-8 weeks were
purchased from the animal house of the college of medicine, Assuit University,
Egypt, around December 2004. The animals were kept in polyethylene cages
of 60x40x30 cm dimensions at temperature
range of 15-20°C, in fairly humid room at 12 h light/12
h dark adjusted cycles. Rats were fed standard rat chow and allowed to
drink normal tap water ad libitum and left for 10 days to acclimatize
before dosing started. They were classified into 8 equal groups and assigned
from 1 to 8.
Group 1 was left as control and did not given any medication. Group 2
was given SC (formal chemical name (IUPAC) : 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]
pyrimidin-5-yl) phenylsulfonyl]-4-methylpiperazine, Pfizer, USA), as single
dose of 3 mg kg-1 body weight by oral gavage
(Baratti and Boccia, 1999). Group 3 was given α-TP (Farco, Egypt),
dissolved in olive oil, as 300 mg kg-1 daily and orally (Fu
and Liu, 1992). Group 4 was given both SC, then α-TP, 30 minutes
apart, in the same doses on a daily basis. The first four groups were
considered as normo-glycemic. The animals of group 5 were given 150 mg
kg-1 alloxan, intra-peritoneally, blood glucose was measured
daily, by blood puncture, the dose could be repeated till blood glucose
level reached about 270 mg dL-1. This level was considered
to be the diabetic level (Kisel et al., 2001). This group served
as diabetic control. Groups 6-8 ` animals were diabetized by the same
way, then group 6 animals were given SC doses as group 2. Group 7 animals
were given α-TP alone, those of group 8 were given SC plus α-TP
as group 4. Dosing for both normo-glycemic and diabetic animals continued
for two consecutive weeks. Groups 5-8 were considered as diabetic subjects.
All animals were killed in the next morning of the last dose. Fasting
blood samples were withdrawn, centrifuged, sera were divided into aliquots,
livers were excised, blotted in filter papers, frozen in liquid nitrogen
and all samples were kept frozen at -80°lC right analysis.
Tissue content of GSH was colorimetrically determined, utilizing a
reaction with 5, 5-dithiobis (2-nitrobenzoic acid, Illman`s reagent) (Moron
et al., 1979). Hepatic tissue lipid peroxidation product was estimated
by thiobarbituric acid reaction (Ohkawa et al., 1979).
Results were expressed as mean±SEM. The inter group variations
was measured by one way analysis of variance (ANOVA). Statistical significance
was considered at p<0.05. Analysis was by one way ANOVA and differences
were calculated using Duncan`s new multiple range test (Duncan`s, 1955).
RESULTS AND DISCUSSION
Thiobarbituric acid reactive oxygen species, TBARS (a lipid peroxidation
product) was significantly reduced by SC and more significantly by α-TP
when given alone or as an adjuvant to SC (p<0.01). GSH was significantly
down-regulated by SC, but administration of α-TP alone significantly
up-regulated GSH, which was non-significantly up-regulated by the combination
of SC with α-TP (Table 1). Effect of diabetes on
liver contents of both TBARS and GSH is shown in Table 2.
Malondialdehyde is a known by-product of lipid peroxidation usually considered
as a biomarker for oxidative stress. Peroxidation and reduced antioxidant
reserve play an important role in the pathogenesis of diabetic vascular
complications (Piconi et al., 2003). In our study, TBARS is significantly
up-regulated in diabetic, compared to control normo-glycemic animals (Table
2). This observation is in agreement with that reported by Griesmacher
et al. (1995). In addition, El-Latif et al.
||Effect of Sildenafil citrate (SC, 3 mg kg-1,
body weight) combination with α-TP (300 mg kg-1, body
weight) on liver lipid peroxide and glutathione contents in normo-glycemic
and diabetic rats after two weeks of daily oral administration
|Values are expressed in mean±SE (n = 8), *: Significantly
different from control at p<0.01
||Role of diabetes on TBARS and GSH liver contents in
rats treated by SC (3 mg kg-1, body weight) and α-TP
(300 mg kg-1, body weight) combination after two weeks
of daily oral administration
|Values are expressed in mean±SE (n = 8), D =
Diabetic, N = Normo-glycemic, **: Significantly different from normo-glycemic
(2006) registered that peripheral and cavernous TBARS levels were higher
in diabetic, compared to normo-glycemic men. TBARS up-regulation was strongly
correlated to ED in diabetic patients (Sozmen et al., 1999). SC
significantly down-regulated hepatic TBARS in normo-glycemic rats (compared
to normo-glycemic control) and similarly in diabetic in comparison to
control diabetic rats. This is might be due to maintaining nitric oxide
production within a physiologic level acting as internal antioxidant (Tooby
et al., 2004). It is also clear that diabetes played a significant
role in TBARS regulation in SC users, which is mostly due to diabetes-induced
vasculopathy, taking into account that SC mainly acts on vascular bed.
On the contrary of that, a combination of SC and alcohol was reported
to up-regulate TBARS in rat testicular tissues after the same period of
administration. This is mostly due to increased tissue oxygenation and
vascular congestion by both SC and alcohol (Sivasankaran et al.,
2007). However, tissue response may differ in regard to SC vascular activity.
In our findings, both SC and α-TP could nearly restore TBARS to normal
non-diabetic control, specially when given together. α-TP administration
for both normo- and hyperglycemic rats down-regulated TBARS, whether given
alone, or as an adjuvant to SC. Literatures correlating SC to hepatic
TBARS content seem to be very sparse. This is why our observation may
be reported for the first time. SC treatment exhibited a prominent antioxidant
activity on hepatocellular level, comparable to α-TP and its combination
to α-TP showed an additional synergistic effect as an antioxidant,
taking into account that α-TP was reported to be hepatocellular antioxidant
(Yakaryilmaz et al., 2007).
GSH content was significantly lower in diabetic than normo-glycemic subjects.
In both cases, SC significantly depleted hepatic GSH, which was corrected
by α-TP co-administration to control, non-treated levels, but it
was up-regulated by α-TP when given alone. Our finding is comparable
to that recently noticed by Sivasankaran et al. (2007), who found
that SC and alcohol combination significantly depleted testicular GSH
in treated rats. α-TP was reported to correct glucose-induced vascular
dysfunction in diabetics (Kinlay et al., 1999). In addition, our
results agree with that reported by De Young et al. (2003), who
registered that α-TP combined with SC is better than either of each,
when given solely in animal model of diabetes. Additionally, reactive
oxygen species seems to be double-edged sword, serving as a key signal
molecule in physiological processes, but also have a role in pathological
pathways, which was greatly corrected by α-TP supplementation (Agarwal
et al., 2005). However, diabetes played a significant role in GSH
down-regulation than normal rats. Existence of diabetes prohibited both
SC (solely) or combined with α-TP from restoring GSH level to normal
non-diabetic control, although α-TP co-administration significantly
alleviated SC effect on GSH level (Table 2).
In conclusion, our study revealed that diabetes plays a key role in the
effect of SC on oxidative stress and anti-oxidant therapy fails to combat
this problem. SC can attenuate lipoperoxidation in hepatic tissue, which
was significantly synergized by α-TP co-administration, in both diabetic
and normo-glycemic rats. Meanwhile, it significantly depleted liver GSH,
an effect could be combated by α-TP supplementation. It is suggested
that, GSH depletion is mostly consumed in other alternative pathways than
lipid peroxidation. By instance, SC administration by hepatic diseased
and diabetic patients can be encouraged when used simultaneously with
α-TP, although SC can`t be considered as an adjuvant treatment for
diabetic-induced GSH depletion, but greatly corrected reflected lipoperoxidation
Agarwal, A., S. Gupta and R.K. Sharma, 2005. Role of oxidative stress in female reproduction. Reprod. Biol. Endocrinol., 3: 28-47.
Baratti, C.M. and M.M. Boccia, 1999. Effects of sildenafil on long-term retention of inhibitory avoidance response in mice. Behav. Pharmacol., 10: 731-737.
De Young, L., D. Yu, D. Freeman and G.B. Brock, 2003. Effects of PDE5 inhibition combined with free oxygen scavenger therapy on erectile function in a diabetic animal model. Int. J. Impot. Res., 15: 347-354.
De Young, L., D. Yu, R.M. Bateman and G.B. Brock, 2004. Oxidative stress and antioxidant therapy: Their impact in diabetes-associated erectile dysfunction. J. Androl., 25: 830-836.
Direct Link |
Duncan, D.B., 1955. Multiple range and multiple F test. Biometrics, 11: 1-42.
Direct Link |
El Sisi, A.E., D.L. Earnest and I.G. Sipes, 1993. Vitamin A potentiation of carbon tetrachloride hepatotoxicity: Role of liver macrophages and active oxygen species. Toxicol. Applied Pharmacol., 119: 295-301.
CrossRef | PubMed |
El-Latif, M.A., A.A. Makhlouf, Y.M. Mostafa, T.E. Gouda and C.S. Niederberger et al., 2006. Diagnostic value of nitric oxide, lipoprotein (a) and malodialdehyde levels in the peripheral venous and cavernous blood of diabetics with erectile dysfunction. Int. J. Impotence Res., 18: 544-549.
Fu, T. and G. Liu, 1992. Protective effects of dimethyl-4, 4-dimthoxy-5,6,5-, 6-dimethylenedio-xybiphenyl-2,2-dicarboxylate on damages of isolated rat hepatocytes induced by carbon tetrachloride and D-galactosamine. Biomed. Environ. Sci., 5: 185-194.
Griesmacher, A., M. Kindhauser, S.E. Andert, W. Schreiner and C. Toma et al., 1995. Enhanced serum levels of thiobarbituric-acid-reactive substances in diabetes mellitus. Am. J. Med., 98: 469-475.
CrossRef | PubMed |
Kalsi, J.S. and P.D. Kell, 2004. Update on oral treatment for male erectile dysfunction. J. Europ Acad Derm Vinereol. (JEADV), 18: 267-274.
Khan, M.A., C.S. Thompson, J.Y. Jeremy, F.H. Mumtaz and P. Mikhailidis and R.J. Morgan, 2001. The effect of superoxide dismutase on nitric oxide-mediated and electrical field-stimulated diabetic rabbit cavernosal smooth muscle relaxation. BJU Int., 87: 98-103.
Kinlay, S., J.C. Fang, H. Hikita, I. Ho and D.M. Delagrange et al., 1999. Plasma α-toccopherol and coronary endothelium-dependent vascular function. Circulation, 100: 219-221.
Direct Link |
Kisel, M.A., L.N. Kulik, I.S. Tsybovsky, A.P. Vlasov and M.S. Vorob'yov et al., 2001. Liposomes with phosphatidyl ethanol as a carrier for oral delivery of insulin: Studies in the rat. Int. J. Pharm., 216: 105-114.
Klein, R., B.E. Klein, K.E. Lee, S.E. Moss and K.J. Cruickshanks, 1996. Prevalence of self-reported erectile dysfunction in people with long-term IDDM. Diabetes Care, 19: 135-141.
CrossRef | Direct Link |
Leung, L.S. and A.W.C. Yip, 1999. Sildenafil (Viagra) and erectile dysfunction. Am. Coll. Surg, 4: 99-102.
Li, J., P. Fu, M. Deleon, B.A. French and S.W. French, 2005. The effect of viagra (sildenafil citrate) on liver injury caused by chronic ethanol intragastric feeding in rats. Exp. Mol. Pathol., 78: 101-108.
Mayne, S.T., 2003. Antioxidant nutrients and chronic disease: Use of biomarkers of exposure and oxidative stress status in epidemiologic research. J. Nutr., 133: 933S-940S.
Direct Link |
McCullough, A.R., 2002. Four-year review of sildenafil citrate. Rev. Androl., 4: 26-38.
Direct Link |
Moron, M.S., J.W. Depierre and B. Mannervik, 1979. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica Biophysica Acta, 582: 67-78.
Ohkawa, H., N. Ohishi and K. Yagi, 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem., 95: 351-358.
Piconi, L., L. Quagliaro and A. Ceriello, 2003. Oxidative stress in diabetes. Clin. Chem. Lab. Med., 41: 1144-1149.
Sivasankaran, T.G., R. Udayakumar, K. Panjamurthy and V. Albert Singh, 2007. Impact of sildenafil citrate (Viagra) and ethanol interaction on antioxidant defense system in the adult male albino rats. Int. J. Pharmacol., 3: 55-60.
Sozmen, B., Y. Delen, F.K. Girgin and E.Y. Sozmen, 1999. Catalase and paraoxonase in hypertensive type 2 diabetes mellitus: Correlation with glycemic control. Clin. Biochem., 32: 423-427.
Tooby, J.T., N. Smith, J. Kenneth and K.J. Broadley, 2004. Effect of phosphodiesterase-5 inhibitor, sildenafil (viagra), in animal models of airways disease. Am. J. Resp. Crit. Care Med., 169: 227-234.
Direct Link |
Tuncayengin, A., H. Biri, M. Onaran, I. Sen and O. Tuncayengin et al., 2003. Cavernosal tissue nitrite, nitrate, malondialdehyde and glutathione levels in diabetic and non-diabetic erectile dysfunction. Int. J. Androl., 26: 250-254.
Walker, D.K., M.J. Ackland, G.C. James, G.J. Muirhead and D.J. Rance et al., 1999. Pharmacokinetics and metabolism of sildenafil in mouse, rat, rabbit, dog and man. Xenobiotica, 29: 297-310.
Direct Link |
Yakaryilmaz, F., S. Guliter, B. Savas, O. Erdem and R. Ersoy et al., 2007. Effects of vitamin E treatment on peroxisome proliferator-activated receptor-α expression and insulin resistance in patients with non-alcoholic steatohepatitis: Results of a pilot study. Int. Med. J., 37: 229-235.
Zhang, X.H., S. Fillipi, A. Morelli, L. Vignozzi and M. Luconi et al., 2006. Testosterone restores diabetes-induced erectile dysfunction and sildenafil responsiveness in two distinct animal models of chemical diabetes. J. Sex Med., 3: 253-266.