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Chapter 4 Diosmin

Deptt. of Med. Elemn. & Toxicology 81 PhD Thesis

4.1. Introduction

Humans are continuously exposed to a wide array of environmental toxicants including

organic chemicals. 1,1,2-trichloroethylene (TCE) is a widespread organic chemical that is

used in industries as a general purpose solvent and as non-flammable solvent for

degreasing metal parts. It is also an intermediate product in the synthesis of

fluorochemicals. TCE is not found as natural sources, but has been found in measurable

amounts in the food chain and in drinking water. During production, storage, and use

TCE evaporates into the atmosphere and has been identified as a common air

contaminant. In addition, leakage from chemical waste sites has lead to its contamination

of ground water (Lock & Reed, 2006). The maximum permissible level of TCE in

drinking water according to US-Environmental Protection Agency (US-EPA) is 5 ppb

(5µg/l). Recently US-EPA (2011) and International Agency for Research on Cancer has

designated TCE as “carcinogen to human” by all routes of exposure. According to

published reports kidneys and liver to be two most effected organs for human and animal

species after TCE exposure (Bull, 2002; Lash et al., 2000a and Goldstein & Schnellmann,

1996). Further, acute and chronic exposure of TCE results in toxicity in variety of organs

in human and experimental animals (Lock & Reed, 2006; Ruder, 2006).

The bulk of TCE biotransformation occurs in the liver, though metabolic activation of

relatively small quantities of TCE reaching extra-hepatic tissues, such as kidney (Lash et

al., 2000b), testes and lungs (Forkert et al.,2002, 2006), can have toxicologically-

significant impacts in situ. The Toxicity of TCE is dependent on its bio-activation, which

occurs either by glutathione (GSH) conjugation in liver to form dichlorovinyl glutathione

(DCVG) or by cytochrome (P450)-dependent oxidation to form dichlorovinyl cysteine

(DCVC). DCVG is further metabolized in kidney either to cysteine conjugate which is

metabolically activated to a thioacetylating agent by β-lyase or detoxified by N-

acetyltransferase (NAT) and released in urine (Lash et al., 2000a). DCVC can also

undergo sulfoxidation to form a reactive intermediate (Sausen & Elfarra, 1991). Another

metabolite of TCE is formed as a result of FMO mediated sulfoxidation of DCVC.

Chapter 4 Diosmin


DCVC-sulfoxide is reported to be nephrotoxic in rats and also cytotoxic to isolated rat kidney cells (Lash et al., 1994). TCE has shown to have toxic effect on carbohydrate metabolism and brush border enzymes in rat kidney (Khan et al., 2009), it has also shown its damaging effects on skeletal muscles (Vattemi et al., 2005). TCE can induce oxidative stress mediated induction of apoptosis in humans as well as animal model. Chen et al., 2002 showed TCE to induce apoptosis in human lung cancer cells, Similarly Lash et al., 2005 also reported induction of apoptosis in culture of human renal cortical cells.

In recent years considerable interest in chemoprevention has focused our approach on the search for naturally occurring anticancer agents many of which have dietary origin (Bharali et al., 2003). The positive health effects from the consumption of diet rich in fruits and vegetables are mainly due to the presence of antioxidants such as carotenoids, polyphenols and anthocyanins. These compounds have ability to influence gene expression owing to their multifaceted beneficial interactions. Minor dietary non-nutrients such as flavonoids, derived from flavones with various degrees of hydroxylation and glycosidic substitutions, are long accepted as potential cancer chemopreventive agents (Wattenberg, 1985).

Flavonoids are the natural polyphenols ubiquitously present in many plants (Wang and Morris 2007). Epidemiological studies advocate that dietary supplements rich in flavonoids prevent various diseases viz., cancers, diabetes, cardiovascular diseases and neurodegenerative diseases (Clere et al., 2011, Garcia-Lafuente et al., 2009, Fu et al., 2011, Mandel et al., 2008). Diosmin (diosmetin 7-O-rutinoside), a naturally occurring flavone glycoside readily obtained by dehydrogenation of hesperidin, is found abundantly in the pericarp of various citrus (Campanero et al., 2010). Diosmin is biologically very active polyphenol and has been show to possess antioxidant, anti-inflammatory, antidiabetic, anti-atherosclerotic and anti-apoptotic activities (Yasim et al, 2010; Galati et al., 1994; Da silva et al., 1994; Lonchampt et al., 1994; Damon et al., 1987; Ratty & Das, 1988; Dholakiya & Benzeroual, 2011; Manuel et al., 1999). Tanaka et al, (1997a; 1997b and 1997c) reported protective efficacy of Diosmin against N-methyl-N-amylnitrosamine induced esophageal tumrogenesis, azoxymethane-induced rat colon carcinogenesis and 4-nitroquinoline 1-oxide-induced oral carcinogenesis.

Chapter 4 Diosmin


In the light of above inferences from other reports we designed our study to investigate the protective effect of Diosmin against TCE induced renal toxicity and apoptotic response in the normal renal tissue of Wistar rats.

4.2. Results

4.2.1 TCE exposure leads to depletion of renal antioxidants amelioration with Diosmin.

TCE administration caused significant decrease in the activities of glutathione metabolizing enzymes viz. GST (P<0.001), GPx (P<0.001) and GR (P<0.001) when compared with control group (Table 1). Diosmin significantly and dose dependently recovered the activities of GST (D1, P<0.01; D2, P<0.001), GPx (D1, P<0.01; D2, P<0.001) and GR (D1, P<0.01; D2, P<0.001) antioxidant enzymes. No significant change was observed with only Diosmin treated animals as compared to control. GSH data also showed a significant decrease (P<0.001) in animals treated with TCE and treatment with Diosmin significantly recovered GSH levels dose dependently. Further, the exhaustive depletion of GSH dependant enzymes lead to disruption of other oxidative stress signaling cascade enzymes such as decrease in SOD (P<0.001) (Table 3) and CAT (P<0.001) activity (Table 3). Diosmin treatment was effective in increasing the level of these antioxidant enzymes to normal at both the doses.

4.2.2. Effect of Diosmin on renal membrane damage (LPO).

MDA formation in renal PMS was measured to demonstrate the oxidative damage in TCE-induced renal injury of Wistar rats. A significant (P < 0.001) rise in the level of MDA formation was found in the TCE-treated groups in renal tissue when compared with control. We have observed that treatment with Diosmin at both the doses leads to the significant restoration (D1, P < 0.01 and D2, P < 0.001, respectively) of membrane integrity in renal tissue when compared with TCE-treated group (Table 3).

4.2.3. Diosmin treatment inhibits renal damage.

The effect of Diosmin administration on TCE-mediated elevated levels of kidney toxicity

markers (BUN, LDH and creatinine) are shown in Table 2. Rats treated with TCE

showed a significant increase in BUN (P < 0.001), creatinine (P < 0.001), LDH (P<0.001)

Chapter 4 Diosmin


levels when compared with control. Marked inhibition was observed in BUN at D2 (P <

0.001) creatinine level at D2 (P<0.001) and in LDH level at D2 (P<0.01).No significant

difference was found in the only D2 group compared with control (Table 2).

4.2.4. Diosmin decreases number of DNA strand breaks in alkaline unwinding assay

(marker for DNA damage).

In the DNA alkaline unwinding assay (Table 3), a significant decrease in the F-value

(p<0.001) in TCE treated group was noted compared to the control group, whereas the F-

value increased by at D1 (p<0.05) and at D2 (p<0.01) of Diosmin.

4.2.5. Diosmin modulates apoptotic cell death mechanisms

Caspase 3 and 7 are effector caspases and caspase 9 is initiator caspase. The levels of all

these Caspases were measured and steep increase in the activity of all the caspases

(P<0.001) was observed in TCE treated group compared to control. Diosmin treatment

dose dependently and significantly increased the level of all the caspases studied to the

normal (Fig. 4). Genomic instability is basically linked to change in the control of

apoptosis and apoptosis-associated genes, including Bcl-2, Bax, p53 and PARP (poly

ADP-ribose polymerase). The expression of Bax and p53 in renal tissue was examined by

immunohistochemistry. We could not detect any Bax and p53 positive region in the

kidney of normal animals (control), positive staining of these proteins was located in the

cytoplasm of renal cells from TCE treated animals. Higher dose of Diosmin decreased the

Bax (Fig. 2) and p53 (Fig. 3) immunopositivity and as compared to TCE treated group.

There was not much difference in immunereactivity in case of only Diosmin treated

group as compared to control (Data not shown).

4.3. Discussion

TCE is an important volatile solvents widely used in metal degreasing industry and

human beings are exposed either through inhalatory route in the form of TCE vapours or

orally as water contaminant (US-EPA, 2011) .TCE exposure has been associated with a

range of adverse health effects like renal toxicity, hepatotoxicity, neurotoxicity,

immunotoxicity, developmental toxicity, endocrine toxicity, and numerous forms of

Chapter 4 Diosmin


cancers (Lash et al., 2000a, b, 2006; Lash et al., 2001; Lock & Reed, 2006; Chiu et al.,

2006a, b). The present investigation was carried out to understand the effect of Diosmin a

naturally occurring flavone in TCE-induced toxic effects in rat kidneys and to assess its

role in modulation of apoptotic pathway. Flavonoids the secondary plant metabolites are

non nutritive but biologically active polyphenolic compounds (Crozier et al., 2000).

Flavonoids are present in a variety of vegetables, fruits and seeds, many of which have

been found to exhibit a wide range of biological activities including anti-oxidant, anti-

inflammatory, antiallergic and anti-tumor effects (Middleton & Kandaswami, 1992;

Middleton et al., 2000; Nijveldt et al., 2001 and Beecher, 2003). Diosmin a naturally

occurring flavone exhibits anti-inflammatory, antioxidant, and anti mutagenic properties

(Crespo et al., 1999; Del Bano et al., 2004; Le Marchand et al., 2000; Nogata et al.,

2006).

Several investigators have also reported antiproliferative effect of Diosmin in various

animal models and human cancer cell lines (Yang et al., 1997; Kuntz et al., 1999).

Oxidative stress has been suggested to be a possible mechanism in TCE-induced

nephrotoxicity (Cojocel et al., 1989). TCE induced toxicity is mainly dependent on bio-

activation, which occurs by two pathways, glutathione (GSH) conjugation and

cytochrome P450 dependent oxidation (Lash et al., 2000b, 2001). Major biochemical

markers of oxidative stress response include elevation of Lipid peroxidation (LPO) and

decrease of glutathione reduced (GSH) and related redox cycle enzymes like SOD,

Catalase, GR and GPX (Tabrez, & Ahmad, 2011a). Elevated level of LPO indicates TCE

induced oxidative damage to both liver and kidneys (Torasson et al., 1999). An elevated

level of MDA, a lipid peroxidation product, was observed after treatment with TCE

(Toraason et al., 1999; Watanabe & Fukui, 2000). In the present study, TCE-treated rats

showed a significant increase in the level of MDA and Diosmin attenuated its level in

renal tissue (Table 3). Thus, Diosmin exhibited the protective efficacy against TCE-

induced lipid peroxidation in renal tissue. TCE has been shown to inhibit the activity of

antioxidant enzymes (glutathione reductase, glutathione-S-transferase, and catalase) in

renal tissue of rats (Tabrez & Ahmad, 2011a).

Chapter 4 Diosmin


In agreement with previous studies from our lab and others, TCE exposure leads to a

depletion in glutathione content and inhibits the activities of the anti-oxidant enzymes

glutathione reductase, catalase, superoxide dismutase, glutathione peroxidase, and

glutathione S-transferase (Ali & Sultana, 2011; Sharma, et al., 2007). GSH is a low

molecular weight antioxidative tripeptide and has essential role in the detoxification of

chemotherapeutic drugs, toxicants, metabolism of nutrients and regulation of various

pathways to maintain cellular homeostasis. Enzymatic (Catalase, SOD, GR, GPX etc)

and endogenous non-enzymatic antioxidant (GSH) were significantly restored to normal

levels in Diosmin treated groups (Table 1-3). Diosmin treatment preserved the level of all

these antioxidant enzymes as previously reported by Tanrikulu et al., (2011) and few

more (Dholakiya & Benzeroual, 2011; Sezer et al., 2011). The cellular damage exhibits a

good correlation with the enzyme leakage (Rehman & Sultana, 2011). Serum Creatinine,

LDH and BUN are the sensitive markers employed in the diagnosis of TCE induced renal

damage (Cojocel et al., 1989; Khan et al., 2009). The present study entirely agrees with

the above findings that TCE administration leads to elevated levels of serum toxicity

markers of the kidneys (LDH, creatinine and BUN) that is, index of renal dysfunction.

The increase in the activities of these enzymes in the serum and subsequent fall in the

tissue might be due to the leakage of these cytosolic enzymes into the circulatory system

resulting from kidney damage during TCE administration. This is indicative of the onset

of renal-cellular damage due to kidney dysfunction and disturbance in the biosynthesis of

these enzymes, with alteration in the membrane permeability. Diosmin treatment

prevented TCE-induced renal toxicity, as indicated by a drop in serum LDH, creatinine

and BUN activity (Table 2), possibly by maintaining the renal cellular membrane

integrity. This is an indicator of possible nephro-protective efficacy offered by Diosmin

compared with the TCE treated group. Further, TCE induced Oxidative stress can also

cause DNA damage via causing single-strand breaks, DNA-protein cross-links and many

other type of DNA base modification (Clay 2008; Hu et al., 2008; Tabrez & Ahmad,

2011b). The single strand DNA breaks assay detects sites on the DNA where one of the

strands has been nicked: (the more the nicks, more rapidly the DNA unbinds under

alkaline conditions). Our results show that Diosmin as the potent inhibitor of single

strand DNA breaks as it effectively reduced the number of single stranded breaks as

Chapter 4 Diosmin


compared to TCE treated group (Table 3). Ozaki et al., (2011) reported DNA damage to

play an important role in regulation of p53. The tumor suppressor gene, p53, in turn plays

pivotal role in inducing apoptosis (Chumakov, 2007). p53 is a well established marker for

DNA damage and cell death under various conditions. Elevation expression of p53 is

seen with kidney toxicity during drug and chemically induced cellular injury (Kang et al.,

2010; Zhang et al., 2010).

In our study TCE induced the p53 expression in renal tissue in the same manner as in

earlier studies (Chen et al., 2002) and Diosmin down regulated p53 expression in dose

dependent manner (Fig. 2). The Bax gene is an apoptosis-promoting member of the bcl-2

gene family and its up-regulation is one of the key mechanisms in programmed cell death

or apoptosis. Bax protein controls cell death through its participation in disruption of

mitochondria and subsequent cytochrome c release and is also considered to be one of the

primary p53 targets. The activation of this event involves transcription independent and

Bax dependant release of cytochrome C with subsequent permeabilization and disruption

of mitochondrial membrane and activation of caspases (Chumakov, 2007; Menendez et

al., 2010; Wang et al., 2010). Results similar to that of p53 were observed in case of BAX

expression (Fig. 3). Caspases are a family of cysteine proteases implicated in the

biochemical and morphological changes that occur during apoptosis (programmed cell

death) (Radovic et al., 2006).

The release of cytochrome c in conjunction with other mitochondrial proteins causes

further activation of caspase 9. Active caspase 9 triggers activation of caspase 3 and other

downstream events such as PARP cleavage and DNA fragmentation of apoptosis process.

Thus above events conclude that p53 accumulation result in Bax dependant release of

cytochrome C, which further in concert with other cofactors and proteins up-regulate

activation of Caspase 7, caspase 9 and caspase 3. PARP cleavage, DNA fragmentation

and chromatin condensation are other events to complete the apoptotic regime. To assess

the effect of Diosmin on activation of caspase-3, 7 and 9 induced by TCE, caspase-3, 7

and 9 colorimetric assay (ELISA) was carried out. The results generates from our study

showed that all the caspases studied were activated in animals treated with TCE as

compared to control. This activation of caspase was in agreement with previous reports

Chapter 4 Diosmin


which showed activation of caspases on treatment with TCE (Shan et al., 2009; Xu et al.,

2008; Akundi et al., 2004). Diosmin dose dependently decreased level of caspases hence

strengthening our hypothesis that Diosmin plays important role in modulating apoptosis

induced by TCE (Fig. 3-5). In conclusion, Diosmin, a natural occurring flavonoid,

attenuates the nephrotoxicity of TCE in rats. The results provide further insight into the

mechanisms of TCE-induced nephrotoxicity and confirm the antioxidant potential of

Diosmin. Further, Findings of the present study support the role of ROS, Serum enzymes,

apoptotic pathway proteins like caspase-3, caspase-7, caspase-9, Bax and P53 in

pathogenesis of the TCE-induced nephrotoxicity. Diosmin has a potent protective effect

against the nephrotoxicity of this agent, and might be clinically useful. However, there is

a need for further molecular studies in this regard before it can be taken for clinical trials.

Chapter 4 Diosmin


Table.1 Results of pretreatment of diosmin on antioxidant enzymes like GSH, GST, GR

and GPX on TCE induced renal redox imbalance.

Results represent mean ± SE of five animals per group. Results obtained are significantly

different from Control group (***P < 0.001). Results obtained are significantly different from TCE

treated group (#P < 0.05), (##P < 0.01) and (###P<0001). Diosmin; D1= 20mg/kg/b wt; D2 =

40mg/kg/b wt

Treatment regimen per group

GSH (n mol GSH

/g tissue)

GST (n mol CDNB conjugateformed/min/mg protein)

GR (n mol NADPH Oxidized/min/

mg protein)

GPX ( n mol NADPHOxidized/min/

mg protein)

Group I (control) 0.60±0.01 416.7±24.2 404.3±17.9

393.1±46.9

Group II (only TCE) 0.32±0.02*** 168.1±14.9*** 200.2±19.8** 186.8±13.9***

Group III (TCE +DM D1) 0.40±0.01# 217.5±15.7## 271.1±19.2## 267.7±18.3ns

GroupIV (TCE+DM D2) 0.52±0.02### 287.3±14.1## 357.9±18.6### 295.7±18.6#

Group V (only DM D2) 0.65±0.01 417.0±19.5 408.3±17.9 399.1±23.4

Chapter 4 Diosmin


Table.2 Results of pretreatment of diosmin on Serum Markers like BUN, Creatinine and

LDH on TCE induced enhancement.



treated group (#P < 0.05), (##P < 0.01) and (###P<0001).DM= Diosmin; D1= 20mg/kg/b wt; D2 =

40mg/kg/b wt.

Treatment regimen

per group

BUN

(mg / 100 ml)

IU/L

Creatinine

(mg / 100 ml)

IU/L

LDH

(n mol NADH oxidised

/ min/ mg protein)

Group I

(control) 21.87±1.17 1.31±0.19 165.0±16.3

Group II

(only TCE) 57.01±2.93*** 3.63±0.14*** 399.9±36.6***

Group III

(TCE +DM D1) 38.02±3.72# 2.70±0.17# 288.8±13.6#

Group IV

(TCE+DM D2) 31.85±1.23## 1.95±0.08## 212.7±21.6###

Group V

(only DM D2) 20.79±1.68 1.30±0.15 161.9±9.25

Chapter 4 Diosmin


Table.3 Results of pretreatment of diosmin on parameters like MDA, SOD, Catalase and

DNA strand breaks on TCE induced renal toxicity.



treated group (#P < 0.05), (##P < 0.01) and (###P<0001). Diosmin; D1= 20mg/kg/b wt; D2 =

40mg/kg/b wt

Treatment regimen per group

MDA (n mol MDA

formed /g tissue)

SOD ( Units/mg

protein)

Catalase (nmol H2O2

Consumed/min /mg protein)

DNA Strand Breaks

(F-Value)

Group I (control) 2.84±0.35 206.1±11.4 893.7±23.4

0.84±0.05

Group II (only TCE) 5.68±0.51*** 100.3±2.87*** 309.1±32.2***

0.33±0.03***

Group III (TCE +DM D1)

3.68±0.09## 130.6±2.97# 455.4±27.4# 0.46±.07ns

GroupIV (TCE+DM D2)

3.2±0.19### 163.8±6.05### 761.4±39.3### 0.76±.04###

Group V (only DM D2) 2.76±0.18 207.3±6.9 888.5±35.1 0.83±.04

Chapter 4 Diosmin


Figure 1 Mouse kidney histology (400× magnification):

Representative histological abnormalities in the regions including the inner cortical and

outer medullar areas of the kidney, which are the main target sites of TCE nephrotoxicity.

(A) Normal kidney histology from a control rat. (B) After TCE administration, many

cortical convoluted tubules were invaded by necrotic epithelial cells or vacuolated swell

cells, glomeruli were swollen, and the Bowman's capsular space was distorted. (C)

Administration of dose I of DM (20 mg/kg bwt) partially prevented the cytotoxic damage

induced by TCE, as indicated by the slight cellular vacuolization of cortical convoluted

tubules. (D) Dose II of DM (40 mg/kg bwt) almost fully protected the kidney tissue from

destruction induced by TCE, as evident from the normal histology of the inner cortical

region. (40x magnification)

Chapter 4 Diosmin


Figure 2 Immunehistochemistry of p53 (400 X magnifications):

Representative photomicrographs of p53 determined by immunohistochemistry. (A) There is almost no expression of p53 in the kidney sections after 20 days of consecutive TCE administration. (B) TCE administration increased strongly p53 positivity in tubular region (C) There was partial inhibition of p53 expression as evidenced by weak immunostaining in the kidneys treated with lower dose of Diosmin (20 mg/kg bwt). (D) In contrast, there was almost complete suppression of p53 positivity in rats treated with higher dose of Diosmin (40 mg/kg bwt). This was evident from the figure, as the tubular structures did not show any substantial p53. (40x magnification)

TCE = Trichloroethylene; DM = Diosmin; D1=20 mg/kg bwt; D2=40 mg/kg bwt.

Chapter 4 Diosmin


Figure 3 Bax protein immunehistochemical staining of the rat kidney tissues (400 X magnification):

Intense Brown staining in the cytoplasm of renal tissue indicates positive Bax protein

expressions. There is almost no Bax expression in renal sections of control (A). Bax

positive regions could be clearly seen in TCE treated group (B) Diosmin showing

moderate decrease in Bax expression in lower dose (20 mg/kg bwt.) (C), where as higher

dose of Diosmin (40 mg/kg bwt) showed effective decrease in BAX expression as

compared to TCE treated group. (40x magnification)

Chapter 4 Diosmin


Figure 4 Effect of Co-treatment of Diosmin on TCE-induced modulation of

apoptotic proteins Caspase-3, Caspase-7 and Caspase-9

Results represent mean ± SE of six animals per group. TCE administration resulted in

significant increase in the level of Caspase-3, Caspase-7 and Caspase-9 (***p < 0.001).

Co-treatment with DM at both doses significantly modulated the alterations in level of

caspases 3 7 and 9 induced by TCE in rat kidney. DM treatment significantly restored

TCE induced over activation of all the Caspases studied (###p< 0.001), (##p < 0.01) and

(#p < 0.05). Trichloroethylene – TCE, Diosmin – DM; D 1 = 20 mg/kg b.wt. D2 = 40

mg/kg b.wt.

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