The 5-HT1A Agonist Buspirone Decreases Liver Oxidative Stress and Exerts Protective Effect Against CCl4– Toxicity

We aimed to study the effect of buspirone, an anxiolytic drug and 5-HT1A agonist on liver injury induced by carbon tetrachloride (CCl4) in rats. Rats were orally treated with CCl4 (2.8 mL/kg in olive oil) along with buspirone at 10, 20 or 30 mg/kg once daily starting with CCl4 and for one week thereafter. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as well as alkaline phosphatase (ALP) activities were determined in the serum. Markers of oxidative stress: lipid peroxidation (malondialdehyde; MDA), reduced glutathione (GSH), nitric oxide (nitrite/nitrate) levels were measured in the liver. Moreover, paraoxonase 1 activity was determined in the liver and serum. The administration of CCl4 led to significant increases in serum ALT, AST, and ALP activities. Results showed that there were significantly increased hepatic MDA, nitrite and decreased GSH levels. PON1 activity decreased both in the liver and serum, respectively. The immunohistochemical investigations using anti-caspase-3 antibody revealed that CCl4 caused apoptosis to many hepatocytes. DNA studies showed that CCl4 caused hypoploidy in hepatocytes. Rats treated with 20-30 mg/kg buspirone showed significant decrease in serum ALT and AST by 19.5-34.3% and 24.2-31.4%, respectively. Serum ALP decreased by 21.7% after 30 mg/kg buspirone. In the liver, the higher dose of the drug resulted in decreased MDA (by 15.8%), decreased nitric oxide (17.4%) and increased GSH (by 20.1%). Significantly increased serum PON1 activity by 43.9-53.5% was observed after treatment with 20-30 mg/kg buspirone. On histopathologic examination of liver sections, there was mild protective effect for the drug at 30 mg/kg. Sections stained with anticaspase3 confirmed the results obtained from histopathological examination. Moreover, buspirone given at 30 mg/kg resulted in an increase in % of cells containing normal values of DNA. These results indicate that buspirone decreases liver oxidative stress and exerts protective effect against CCl4toxicity. The study thus indicates more beneficial effects of buspirone as an anxiolytic drug and that the drug could be used safely in patients with liver disease. JOURNAL OF EXPERIMENTAL AND CLINICAL TOXICOLOGY ISSN NO: Coming soon RESEARCH DOI : coming soon Corresponding author: Omar M.E.Abdel-Salam, Department of Toxicology and Narcotics, National Research Centre, Dokki 112311, Cairo, Egypt, omasalam@hotmail.com


Introduction
Buspirone is a partial 5-HT 1A receptor agonist that is widely used in treating anxiety disorders and depression [1].5-HT 1A receptors are presynaptically located as somatodendritic receptors on the 5-HT neurons in the midbrain raphe nuclei and also postsynaptically in limbic and cortical regions.
Stimulation of 5-HT 1A receptors decreases the firing of 5 -HT neurons and 5-HT terminal release.This results in inhibition of serotonergic neurotransmission [2].The drug in addition has a weak affinity for 5-HT 2 receptors and act as an antagonist at dopamine D-2, D-3 and D-4 receptors [3][4][5].In animal models of pain, buspirone exerted analgesic action increasing the threshold to thermal, electrical, chemogenic, and visceral pain.The drug inhibits gastric acid secretion and exerts gastric mucosal protective and anti-inflammatory effects [6].
Serotonin has wide distribution in the brain and gut.In brain, serotonin plays an important role in mood, behavior, aggression, and sexual function [7].
Serotonin is decreased in depression and thus drugs which increase brain serotonergic activity eg., the serotonin reuptake inhibitors (SSRIs) are the most common agents used nowadays in treating depressive disorders [8] including depression that occurs in the course of liver disease and/or results from antiviral therapy [9].There is also evidence involving the brain serotonergic system in the development of depression in patients with chronic liver disease [10] and in hepatic encephalopathy [11] and it is likely that changes in brain serotonin could modulate liver injury.
In the body, serotonin exists mainly in the gut, being produced by the enterochromaffin cells, whilst a small amount is present in plasma stored in platelets [12].Studies suggested an important role for serotonin derived from platelets in liver regeneration [13,14] and also in causing hepatic injury [15,16].5-HT 2A receptors are upregulated in activated hepatic stellate cells, the principal cells mediating liver fibrosis and 5 -HT 2A antagonists inhibit activation of HSCs [17].
There is also a decrease in

Animals
The study was conducted using Sprague-Dawley rats of both sexes (130-140 g of body weight).
Rats were fed with standard laboratory chow and water ad libitum.

Serum liver enzymes
At the end of the study, retro-orbital vein plexus blood samples were obtained under light ether anaesthesia.The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in serum were then measured colorimetric ally using commercially available kits (BioMérieux, France).

Liver lipid peroxidation
The measurement of malondialdehyde (MDA) was used to determine the extent of lipid peroxidation in the liver tissue.The method used is that of Ruiz-Larrea et al. [25].In this assay, thiobarbituric acid reactive substances (TBARS) react with thiobarbituric acid to produce TBA -MDA adduct with a red color that can be determined using spectrophotometer at 532 nm.

Liver reduced glutathione
The method used is that of Ellman

Studies
The liver sections of each rat were fixed in

DNA Ploidy Studies
For DNA analysis, we used Feulgen stained sections countered stained with Light green.Analysis was carried out using Leica Quin 500 image cytometry (Pathology Department, NRC, Cairo).For each section (100-120) cells were randomly measured [29].

Statistical Analysis
Data

Serum Liver Enzymes
Results are presented in Table 1 2).

Serum and Liver Paraoxonase-1 Activity
In rats treated with CCl 4 , the activity of PON1 in the liver and serum was significantly depressed by 38.7% and 60.6%, respectively (Table 3).Treatment with buspirone had no significant effect on liver tissue PON1 activity.
However, significantly increased serum PON1 activity by 43.9% and 53.5% was observed after treatment with 20 and 30 mg/kg buspirone, respectively (Table 3).

Histopathological Results
Sections from CCl 4 only-treated rats stained with Hx & E revealed severe damage of hepatocytes and liver tissue in the form of marked vacuolar degeneration and/or acidification of many cells, congestion of blood sinusoids and cellular infiltration (Fig. 1A).Buspirone given at 10 mg/kg showed no protection against CCl 4 -induced liver damage as vacuolar degeneration of hepatocytes, cellular infiltration and nuclear changes were still observed (Fig. 1B) whereas a low ameliorating effect on the degree of liver damage was observed at buspirone dose of 20 mg/kg

Caspase-3 immunostaining
The immunohistochemical investigations using anti-caspase-3 antibody revealed that CCl 4 caused apoptosis to many hepatocytes (Fig. 2, A).Buspirone had a very weak protecting effect against the damaging effect of CCl 4 at doses of 10 and 20 mg/kg as many positively stained hepatocytes were still observed (Fig. 2B & C).The highest dose of buspirone (30 mg) showed slight reduction of positively stained cells denoting mild protecting effect against CCl 4 induced apoptosis (Fig. 2D).

DNA ploidy results
In        Glutathione is essential for hepatoceullar integrity.This    In the peripheral tissues, synthesis of serotonin is carried out by the enterochromaffin cells of the gut.
Serotonin is then released into circulation and most of this circulating serotonin is actively taken up, sequestrated within vesicles in platelets and released upon their stimulation [7,45].This platelet-derived serotonin has been implicated in both liver protection [46,47] and regeneration [14,15] but also in the development of hepatic injury [16,17].Thus, in the aged

hepatic 5 -
HT 1A receptor function during hepatocyte regeneration and neoplasia while stimulation of 5-HT 1A receptor inhibited hepatocyte DNA synthesis [19].The above data point to the important role of serotonin in modulating the integrity of the liver.The administration of the serotonin reuptake inhibitors or serotonergic antagonists eg., trazodone and nefazodone protected against the CCl 4 -induced hepatic toxicity in rodents [20-22].The mechanism by which these drugs decrease the toxin-induced liver damage is not clear, but increased central serotongeric activity [20], prevention of the metabolic derangement induced by CCl 4 [22], reduced platelet serotonin or decreased liver nitric oxide [21] have been suggested.In this study, our aim was to investigate the possible modulatory effect of buspirone on the development of hepatic injury caused by CCl 4 .The latter is a widely used industrial solvent which is known to cause heptotoxicity in humans and rodents.The acute administration of CCl 4 causes fatty degeneration, and hepatocellular death with the mechanism largely involving free radical-mediated oxidative damage to cellular biomolecules[23,24].
10% neutral-buffered formal saline for 72 hours at least and processed routinely for the microscopic examination.Serial sections (5 μm thick) were cut and stained with haematoxylin and eosin (Hx & E) and examined by light microscopy.Caspase-3 immunohistochemical staining was performed with the use of streptavidin-biotin.In brief, deparaffinized sections (4 μm thick) were incubated for 30 min with fresh 0.3% hydrogen peroxide in methanol at room temperature, followed by incubation with anti caspase-3 antibodies (1: 100 dilution).The specimens were counter stained with H & E. In negative controls, normal mouse serum was substituted for anti caspase-3 antibodies.All sections were investigated by the light microscope.Adobe Photoshop version 8.0 was used for capturing and processing images.
the present study, the Qwine 500 image analyzer was used to evaluate the DNA content of examined cells.The image analysis system automatically expresses the DNA content of each individual cell measured then gives the percentage of each cell out of the total number of cells examined.Also, it classifies the cells into four groups; diploid (2C), proliferating cells (3C), tetraploid (4C) and aneuploid cells (>5C).The proliferating cells are further subclassified into; (<10%) low proliferation index, (10-20%) medium proliferation index and (>20%) high proliferation index [29].Normal distribution of DNA content in the liver cells of the control group (G. 1) showed that 13.2% of the examined cells contained DNA (<1.5C),75.47% contained diploid DNA value (2C), 11.32% contained (3C) DNA value (medium proliferation index) and 0.0% of the examined cells at (4C) area (Fig. 3 & table 4).

Fig. 1 :
Fig. 1: Hx & E stained liver tissue from (A) control rat receiving CCl 4 showing severe degenerative changes in liver tissue in the form of marked vacuolar degeneration of many hepatocytes (black arrow), acidified hepatocytes (green arrowhead), congestion of blood sinusoids (green arrow) and cellular infiltration either diffuse in the center of the lobule or in blood sinusoids (black arrowhead).The lower right part of the figure shows focal aggregation of cellular infiltrates.(B) CCl 4 and 10 mg/ kg buspirone showing no protective effect against the damaging effect of CCl 4 as acidified cells (arrow), vacuolar degeneration of most hepatocytes and cellular infiltration are still observed.(C) CCl 4 and 20 mg/kg buspirone showing minimal protection against the damaging effect of CCl 4 as some cells appear with normal nuclei (arrow) and others are with pyknotic nuclei (arrowhead), although many cells show vacuolar degeneration, cellular infiltration in the center of the lobule is still present and architecture of liver tissue is markedly deformed.(D) CCl 4 and 30 mg/kg buspirone showing mild protection of the drug against the damaging effect of CCl 4 .Cellular infiltration is localized at focal areas beside central vein (arrowhead) with restriction of vacuolar degeneration to the area around central vein (arrow).

Fig. 2 :
Fig. 2: A photomicrograph of a section of liver tissue stained with anti-caspase-3 antibody with streptavidin-biotin from (A) control rat receiving CCl 4 showing many hepatocytes with positive immune-reaction to the stain (arrow).(B) CCl 4 and 10 mg/kg buspirone showing a result close to that of the previous group.(C) CCl 4 and 20 mg/kg buspirone showing minimal reduction of the positively stained cells.(D) CCl 4 and 30 mg/kg buspirone showing mild reduction of the positively stained cells

CCl 4 a c t i v i t y o c c u r s w h e n e v e r t h e r e i s a s t a
and 30 mg/kg of buspirone (G. 5) showed that 24.77% of examined cells contained (< 1.5 C), 46.78% of examined cells contained (2 C) value of DNA.Cells contained (3C) value were 20.18%, while 6.42% of examined cells contained (4C) value of DNA (Fig.4D & table 4).From the above results it was clear that CCl 4 caused hypoploidy in the examined cells as percentage of cells containing DNA value less than the normal was increased markedly, while percentage of cells containing the normal value of DNA was greatly reduced.Nearly the same results were obtained after treatment with the lowest dose of buspirone, whereas buspirone given at 20 mg/kg to CCl 4 -treated rats caused hyperploidy as the percentage of cells containing DNA value higher than normal was increased at the expense of cells containing normal value of DNA.The highest dose of the drug showed mild amelioration of DNA values in hepatic cells as the percentage of cells containing normal values of DNA was increased, although the percentage of cells containing less or more DNA values was still higher than normal.DiscussionT h e r e s u l t s o f t h e p r e s e n t s t u d y i n d i c a t e t h a t t r e a t m e n t w i t h b u s p i r o n e w a s a s s o c i a t e d w i t h a d e c r e a s e i n C C l 4 -i n d u c e d h e p a t o t o x i c i t y .B u s p i r o n e t r e a t m e n t a t 2 0 -30 m g / k g r e s u l t e d i n a s i g n i f i c a n t d e c r e a s e i n t h e a c t i v i t i e s o f t h e h e p a t o ce l l u l a r e n z y m e s A L T a n d A S T i n s e r u m .I t a l s o r e d u c e d t h e l e a k a g e o f A L P i n t o t h e p l a s m a .T h e r e l e a s e o f t h e s e i n t r a c e l l u l a r e n z y m e s i n t o t h e c i r c u l a t i o n i s a n i m p o r t a n t m a r k e r f o r d e t e c t i n g h e p a t o c y t e i n j u r y [ 3 0 ] .T h i s e n z y m e i s p r e s e n t a t t h e h e p a t o c y t e ' s c a n a l i c u l a r m e m b r a n e a n d i n c r e a s e d e n z y m e g n a t i o n o f b i l e f l o w [ 3 1 ] .I t i s t h e r e f o r e c l e a r t h a t t h e d e c r e a s e i n t h e a c t i v i t y o f t h e s e e n z y m e s i s a f u n c t i o n o f a d e c r e a s e d e x t e n t o f l i v e r d a m a g e a n d t h i s n o t i o n w a s s u p p o r t e d b y h i s t o p a t h o l o g i c a l e x a m i n a t i o n o f t h e l i v e r a n d b y t h e D N A s t u d i e s .T h e a d m i n i s t r a t i o u o l a r d e g e n e r a t i o n a n d / o r a c i d i f i c a t i o n o f m a n y c e l l s .S t u d y o f t h e D N A c o n t e n t o f h e p a t i c t i s s u e i n d i c a t e d t h a t C C l 4 c a u s a t o c y t e s ( h y p o p l o i d y ) .B u s p i r o n e d e c r e a s e d h e p a t i c v a c u o l a r d e g e n e r a t i o n , t h e e x p r e s s i o n o f t h e a p o p t o t i c m a r k e r c a s p a s e -3 a n d t h e C C l 4 -i n d u c e d c h a n g e s i n D N A v a l u e s i n l i v e r c e l l s .T h e a b o v e f i n d i n g s c o l l e c t i v e l y i n d i c a t e a b e n e f i c i a l e f f e c t f o r t h e d r u g u p o n CCl 4 h e p a t o t o x i c i t y .The drug exerted an antioxidant action against the liver oxidative stress caused by CCl 4 .The latter is a well known hepatic toxin in humans and in experimental animals.This is largely due to its metabolic activation by cytochrome (CYP)2E1, CYP2B1 or CYP2B2, and the formation of the trichloromethyl radical, CCl 3 , capable of causing lipid peroxidation, protein and DNA damage [32].In this study, the administration of CCl 4 caused a marked increase in liver content of the lipid peroxidation product malondialdehyde.This occurred along with depletion of the antioxidant reduced glutathione.In the cell, glutathione (L-g-glutamyl-L-cysteinyl-glycine) is the most abundant thiol in the cystosol, present mainly in its reduced form.Glutathione is a direct free radical scavenger and is also a co-factor for glutathione peroxidase and glutathione reductase enzymes [33].

Fig. 3 :
Fig. 3: (A) A chart showing the distribution of DNA content in normal hepatic cells.Notice that most of cells contain the normal content of DNA (2C).(B) Shows abnormal mitosis in Feulgen-stained sections of liver tissue.

Fig. 4 .
Fig. 4. (A) A chart of DNA content in liver cells of a rat treated with CCl 4 shows deviation to the left (<2C).(B) A chart of DNA content in liver cells of a rat treated with CCl 4 and 10 mg/kg buspirone shows the same result as the previous (C) A chart of DNA content in liver cells of a rat treated with CCl 4 and 20mg/ kg buspirone shows deviation to the right (> 2C).(D) A chart of DNA content in liver cells of a rat treated with CCl 4 and 30 mg/kg buspirone shows increase in percentage of cells containing (2C value or less) and decrease in percentage of cells containing (>2C).
is because cellular glutathione depletion resulted in hepatic steatosis, inflammation and cell death in mice[34].On the other hand, the glutathione donor N-acetylcysteine was able to correct the biochemical and the pathological changes in the liver of glutathione deficient mice[35].In this study, the increase in liver lipid peroxidation and the depletion of the liver tissue content of reduced glutathione was significantly decreased by the administration of 30 mg/kg of buspirone, possibly due to lowered of oxidative stress by the drug.Our results also indicated markedly increased hepatic nitric oxide content following CCl 4 administration.In the liver, nitric oxide that is constitutively formed by the action of endothelial isoform of nitric oxide synthase (eNOS) maintains hepatic microcirculation and the integrity of endothelium.In contrast, the increased generation of nitric oxide by the inducible form of NOS (iNOS) due to the action of inflammatory cytokines contributes to hepatocyte apoptosis, liver tissue damage and fibrosis under such conditions as ischaemicreperfusion injury and also after exposure to CCl 4 [36,37].Moreover nitric oxide synthase inhibitors were shown to prevent hepatic necrosis and to decrease the expression of tumour necrosis factor alpha (TNF-α) and cycloozygenase-2 in liver tissue of CCl 4 treated rats [38].In this study, the administration of buspirone at 30 mg/ kg was associated with a significant decrease in liver nitric oxide in CCl 4 intoxicated rats.The finding in the present study of the decrease in lipid peroxidation and nitric oxide as well as the increase in reduced glutathione after treatment with CCl 4 and buspirone might therefore indicate improved cell-redox state by the drug and/or a lower degree of tissue damage due to other mechanism of buspirone.The present study also demonstrated markedly reduced paraoxonase 1 (PON1) activity in the liver tissue and serum from CCl 4 -treated rats.This enzyme is synthesized in the liver and is found in plasma in association with high-density lipoproteins to prevent their oxidation.Paraoxonase 1 is also endued with xenobiotic metabolizing and antioxidant activities [39].Paraoxonase-1 exerts an antioxidant and antiinflammatory actions in the liver and is considered a biomarker of liver diseases [40-42].Mice deficient in paraoxonase 1 fed high fat and cholesterol diet exhibited increased extent of fatty degeneration as well as increased lipid peroxides and oxidative stress markers relative to their wild-type counterparts on the same diet [43].It is conceivable that the serum level of PON1 depends on the ability of the liver to synthesize the enzyme [44].The decrease in PON1 activity observed in the current study might thus reflects decreased synthesis by the intoxicated liver cells and the recovery in PON1 could be the result of reducing oxidative stress and improving the condition of hepatocytes by buspirone treatment.

rat stimulation of 5 -
hydroxytryptamine receptor 2 resulted in improving sinusoidal perfusion and in restoring the deficit in liver regeneration via vascular endothelial growth factor [46].In mice, absence of peripheral serotonin caused increased acetaminopheninduced liver damage [47].Moreover, mice lacking tryptophan hydroxylase 1 which is the rate-limiting enzyme for synthesizing serotonin in periphery showed blunted liver regeneration [48].Serotonin administration increases hepatic glycogen synthesis and concentration [49].This would provide a substrate for glycolysis and cellular ATP synthesis.On the other hand, in a murine model of non-cytopathic lymphocytic choriomeningitis viral infection, platelets recruited to the liver, and their activation resulted in reduced sinusoidal microcirculation, and delayed virus elimination while increasing liver damage.Fluoxetine, a SSRI resulted in a reduction of hepatocyte damage [15].Platelet serotonin increases neutrophil recruitment to the sites of inflammation and this could be decreased by fluoxetine [16].Studies have shown that the SSRIs inhibitors fluoxetine, sertraline, citalopram, fluvoxamine or the serotonergic antagonists trazodone and nefazodone were able to protect against the hepatotoxic effect of CCl 4 [20-22].These agents inhibit the serotonin transporter, thereby inhibiting the uptake of serotonin into platelets and impairing platelet aggregation [50].

Freely
Available Online www.openaccesspub.org| JECT CC-license DOI : coming soon Vol-1 Issue 1 Pg.no.-24The same mechanism could possibly be also involved in their hepatic protective effects described earlier by increasing the plasma serotonin and therefore serotonin availability for liver cells.Buspirone, however, does not affect the reuptake of serotonin into platelets and in healthy subjects causes an increase in plasma levels of free serotonin without affecting platelet serotonin or platelet aggregation [51].5-HT 1A agonist buspirone reduced experimental liver injury induced by CCl 4 in the rat.Buspirone displayed antioxidant action, reduced apoptosis and improved the CCl 4 -induced changes in DNA values in hepatic cells.These data suggest that the drug is safe in patients with liver disease.

Table 1 .
The effect of buspirone on serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activities in CCl 4 -treated rats Results are mean ± S.E.Six rats were used per each group.The percent change from the CCl 4 -control group is also shown in parenthesis.Data were analyzed by one way ANOVA and means of different groups were compared by Duncan's multiple range test.P < 0.05 was considered statistically significant.*: P<0.05 vs vehicle-treated group.+: P<0.05 vs the CCl 4 control group.

Table 2 .
The effect of buspirone on liver tissue levels of malondialdehyde (MDA), nitric oxide (NO), and reduced glutathione (GSH) in CCl 4 -treated rats Results are mean ± S.E.Six rats were used per each group.The percent change from the CCl 4 -control group is also shown in parenthesis.Data were analyzed by one way ANOVA and means of different groups were compared by Duncan's multiple range test.P < 0.05 was considered statistically significant.*: P<0.05 vs vehicle-treated group.+: P<0.05 vs the CCl 4 control group.

Table 3 .
The effect of buspirone on liver tissue and serum paraoxonase 1 activity (PON1) in CCl 4 -treated rats Results are mean ± S.E.Six rats were used per each group.The percent change from the CCl 4 -control group is also shown in parenthesis.Data were analyzed by one way ANOVA and means of different groups were compared by Duncan's multiple range test.P < 0.05 was considered statistically significant.*:P<0.05vsvehicle-treated group.+:P<0.05vs the CCl 4 control group.Examination of cells from G. 2 (control +ve group) treated with CCl 4 showed that the cells contained contained (4C) DNA value (Fig. 4 C & table4).Examination of cells from the group treated with

Table 4 .
The effect of buspirone on DNA content in CCl 4 -treated rats