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Chapter 6 Chrysin

Deptt. of Med. Elemn. & Toxicology 110 PhD Thesis

6.1. Introduction

Renal cell carcinoma (RCC) represents the most common and lethal cancer in urinary system (Liou et al., 2004). The average survival, following metastatic RCC, is about 4 months, and only 10% of patients survive for one year. Despite the increase in the understanding of molecular mechanisms and the subsequent development of many novel chemotherapeutic agents over the past decade, RCC still remains an incurable and lethal disease (Bullock et al., 2010). RCC development has been linked with numerous risk factors including environmental exposure to various toxicants (Jemal et al., 2010). Nitrilotriacetate (NTA) is an environmental toxicant frequently used in detergent making industry as an alternative for polyphosphates, it enters into water bodies in the form of wash offs of soap and detergents from hospitals, household and industries. Mizuno et al. (2006) reported that repeated i.p. administration of Ferric nitrilotriacetate (Fe-NTA) induced acute and sub-acute renal proximal tubular damage and subsequent development of RCC in rats and mice at high incidence (60–92%). It is also reported that Fe-NTA administration can specifically causes allelic loss of the p16 tumor suppressor gene in renal tubular cells (Hiroyasu et al., 2002). Fe-NTA is a known pro-oxidant and carcinogen causing renal and hepatic damage. Irwing and Miles (1966) reported that NTA has capability to form complex with Fe2+ and Cu2+ and iron overload itself is associated with renal carcinogenesis (Huang, 2003). Renal toxicity of Fe-NTA is assumed to be caused by the increase of free serum iron concentration, following its reduction at the luminal side of the proximal tubule, generating reactive oxygen species (ROS), which ultimately causes lipid peroxidation and subsequent oxidative damage (Hamazaki et al., 1989). Accumulation of tubular injury and oxidative damage ultimately leads to a high incidence of RCC (Toyokuni, 1998). Experimental studies from our lab and others have demonstrated that Fe-NTA can induce renal toxicity and renal tumor formation by inducing oxidative stress, DNA damage, cellular proliferation and inflammation (Rehman and Sultana, 2011; Ahmad et al., 2011; Iqbal et al., 2003). ROS have key role in initiation as well as tumor promotion as they can alter various signaling pathways involved in cellular proliferation and inflammation by modulation of redox sensitive nuclear transcription factor kappa B (NFkB), Proliferating cell nuclear antigen (PCNA), Cycloxygenase-2 (COX-2) and several other enzymes involved in cellular signaling.

Chapter 6 Chrysin


Renal tumorigenesis could be intercepted through modulating its pathophysiological

events of initiation and/or promotion which forms the basic axis of various promising

anticancer therapeutics. Clear and precise knowledge of these core events is pre-requisite

for the successful anticancer drug designing. Recently, newer methods involving

administration of natural and dietary agents of flavonoids, terpenoids, polyphenols,

indoles and organosulphides origin are employed to impede, preclude and restore the

various molecular pathological happenings characteristic of cancer. Ethnobotanicals are

currently in vogue for the targeted and differential chemopreventive strategy against

oncogenic phenotype. One of the major and highly valued advantage associated with

these agents are minimum or low unwanted effects which are generally associated with

classical and contemporary modern synthetic anticancer medicines. Another non-

ignorable feature involves the therapeutic action specifically against transformed cells

and destroying the healthy and normal cellular population to the minimum. Thus, taking

in to account the ever increasing statistics of the cancer incidences, and also still devoid

of any valuable contemporary medicinal tool, the new discipline of cancer

chemoprevention through natural agents should be encouraged and explored for its

beneficial effects on human health.

Epidemiological and chemopreventive studies in both animals and humans have shown

that usual consumption of fruits, vegetables, and tea is associated with decreased risk of

cancer (Surh, 2003).They provide essential nutrients and many diet-derived phenolics

particularly flavonoids which have been demonstrated to exert potential anticarcinogenic

activities (Middelton et al., 2000). Flavonoids are natural polyphenolic phytochemicals

that are ubiquitous in plants and present in the normal human diet, are safe and associated

with extremely low toxicity which makes them first rated candidate for chemoprevention

(Wang and Morris, 2007).

Flavonoids are effective in decreasing the risk of various disease like cancer (Ross and

Kasum, 2002; Clere et al., 2011; Pierini et al., 2008) and also reported to be effective in

case of diabetes (Fu et al., 2011) Cardiovascular (Garcia-Lafuente et al., 2009) and

neurodegenerative disease (Mandel et al., 2008). These suggested protective effects of

flavonoids, together with their potent antioxidative and free radical scavenging activities

Chapter 6 Chrysin


observed in in-vivo studies have increased the public’s interest in the use of flavonoids

for their potential health benefits. Chrysin (5, 7-dihydroxyflavone) is a natural flavonoid

found in many plant extracts, honey, and propolis (Barbaric et al., 2011, Pichichero et al.,

2010). Chrysin exhibits many biological activities and pharmacological effects, including

antioxidant, anti-inflammatory, anti-aging and anticancer (Lukacinova et al 2008;

Goncalves et al., 2011; Cardenas et al., 2006; Wang et al., 2004; Miyamoto et al.,

2006).Chrysin has also been reported to improve bowel diseases (Eun Kyung Shin et al

2010). Khan et al (2010) recently reported chrysin exerts hepato-protective effect and

significantly inhibits nodule formation. We have also reported recently that chrysin is

effective in preventing ethanol induced organ toxicity and protects against colon and

jejunum toxicity (Tahir and sultana 2011; Khan et al., 2011 a, b).

There are no previously published reports regarding chemopreventive effect of chrysin on

renal carcinogenesis. Therefore, the present study was planned to investigate the efficacy

of chrysin against two-stage renal carcinogenesis in animal model and to probe into the

mechanism(s) that might be implicated in its anticancer activity. The effect of chrysin

was investigated on key aspects of carcinogenesis with a major focus on inflammation

and Tumor promotion.

6.2. Results

6.2.1. Effect of chrysin on Fe-NTA-Induced Oxidative Stress, related Damage, and

cellular proliferation

Result of chrysin pretreatment on Fe-NTA-induced depletion in the levels of GSH and

activities of antioxidant and phase II enzymes is shown in Table 1&2. Single

intraperitoneal administration of Fe-NTA significantly depleted renal GSH content

(P<0.001), and activities of GR, CAT, GPX and SOD as compared to the saline treated

controls. Pretreatment of rats with chrysin resulted in a significant recovery in GSH

levels and activities of almost all the investigated enzymes (Table 1). There was a

concomitant and significant decrease in the activity of Phase-II metabolizing enzymes

viz. GST and QR (p < 0.001) in the Fe-NTA treated group. But this decrease in their

activity was attenuated substantially by the prophylactic treatment of both the dose of

Chapter 6 Chrysin


chrysin (p < 0.001) (Table 2). Fe-NTA-induced increase in iron-ascorbate mediated lipid

peroxidation of renal tissue is shown in Table 2. Treatment of Fe-NTA significantly

enhanced the vulnerability of renal microsomal membrane for LPO. However, chrysin

(20 and 40 mg/kg BW) pretreatment significantly (p<0.001) suppressed this enhancement

(Table 3). As shown in Table 3, Fe-NTA administration resulted in a significant increase

in serum blood urea nitrogen (BUN) (p<0.001) and creatinine levels (P<0.001) as

compared to control animals. Pretreatment with chrysin (20 and 40 mg/kg) one hour

before Fe-NTA administration markedly improved renal dysfunction (indicated by

significant reduction in serum creatinine and BUN).similar pattern of results was

observed in case of other serum toxicity parameters viz. LDH and �-GGT.

Apart from inducing oxidative stress, Fe-NTA is also known to cause tumor promotion

by inducing cellular proliferation. Fe-NTA exposure significantly (P<0.001) increased

renal ODC which is a hallmark of tumor promotion and is greatly induced during

tumorigenesis. Intraperitoneal application of Fe-NTA significantly elevated ODC activity

However, chrysin pretreatment in both the doses (20 & 40 mg/kg BW) down regulated

Fe-NTA-induced ODC activity (Figure 1).

Further, quantification of PCNA, well known cell proliferation markers by

immunohistochemistry has been used to characterize the proliferation of cells in many

fields, such as in tumor studies. The semi-quantitative expression of PCNA protein in all

the groups of long term renal tumorigenesis study is given in Figure 3. According to

Figure the number of PCNA positive cells increased substantially in group II (DEN + Fe-

NTA) (p < 0.001) indicating the proliferative potential of Fe-NTA. Higher dose of

chrysin suppressed the proliferation of tubular epithelium cells significantly (p < 0.001)

as revealed in Figure.

6.2.2. Inhibitory effect of chrysin on DEN initiated Fe-NTA promoted renal

Carcinogenesis

Renal cancer in rats was initiated with single intraperitoneal injection of DEN (200mg/kg BW).Twice weekly treatment of 9mg/kg BW of Fe-NTA for 16 weeks was used as Promotion agent. Table 4 represents the Data generated as a result of Bioassay

Chapter 6 Chrysin


conducted to establish chemopreventive effect of chrysin in case of chemically induced renal tumors. Saline alone treated groups did not show any tumors, DEN only group showed only 5.88% tumors. Animals treated with Fe-NTA and initiated with DEN increased the incidence of Renal Cell Tumors (RCTs) by 81.8% and the animals in the group treated with Fe-NTA alone led to the development of RCTs in 20% of the animals studied. Chrysin significantly lowered the percentage of tumor bearing animals (tumor incidence) at the termination of experiment. The tumor incidence was decreased in the group of animals pretreated with Chrysin at lower dose of 20 mg/kg BW to 50% whereas in the group treated with higher dose of chrysin 40 mg/kg BW, the tumor incidence was decreased to 26.6%.

The representative pictures of histopathological examination in the renal tissue are shown in Figure 3.The tissue sections from kidney of rats treated with DEN and Fe-NTA, either with or without pre-treatment of chrysin, were examined for the degree of infiltration of leukocytes, tumour cells and hyperchromatism. Sections from kidneys of control rats demonstrated intact tubular architecture with normal convoluted tubules and glomeruli within the cortex. The tissue sections from DEN initiated and Fe-NTA promoted group showed enormous focal collection of leukocytic infiltratory cells and adenocarcinomas with hyperchromatism and enlargement of nuclei in the tubular epithelium. Lower dose of chrysin +DEN+ Fe-NTA group showed mild inflammatory invasion with lesser tubular congestion and glomerular damage while as chrysin at higher dose was quite effective in restoration of almost normal histo-architecture of renal sections which was comparable with untreated control.

6.2.3. Effect of chrysin on DEN induced and Fe-NTA- promoted elevation in levels of proinflammatory cytokines in serum.

Proinflammatory cytokines like IL-6, TNF-α, and PGE2 whose secretion is known to be enhanced by Fe-NTA, play an important role in tumorigenesis (Okazaki et al 1999; Kaur et al 2009). Significant levels of TNF-α, IL-6 and PGE2 could be detected in serum of rats exposed to tumor promotion with Fe-NTA for 16 wk (Table 5).chrysin pretreatment at both the doses significantly restores the increased level of all the three proinflammatory cytokines studied to normal.

Chapter 6 Chrysin


6.2.4. Effect of chrysin on Fe-NTA induced Nuclear Localization of NF-κB p65in

kidney of wistar rats.

Genes necessary for the induction of COX-2, iNOS and other inflammatory cytokines are

transcribed by redox sensitive transcription factor NF-kB (Braynes et al., 1999; May and

Ghosh, 1998). NF-kB has been shown to have role in organ toxicities such as liver,

kidney, pancreas (Orfila et al., 2005; Sun and Andersson, 2002; Tugcu et al., 2006) .To

evaluate the effect of chrysin on Fe-NTA induced NF-kB activation in renal tissue, we

investigated the levels of nuclear translocation of NF-kB by IHC. Exposure to Fe-NTA

led to a significant elevation in NF-kBp65 indicating Fe-NTA to cause activation of NF-

kB (Figure 4). However, chrysin pretreatment I hr prior to Fe-NTA exposure caused a

marked attenuation in elevated levels of NF-kBp65.

6.2.5. Effect of chrysin on COX-2 and iNOS Expression

The effect of chrysin was investigated on iNOS and COX-2 expression in Fe-NTA-

administered rats as well as in DEN+ Fe-NTA-induced renal tumors. IHC revealed Fe-

NTA to significantly up-regulated the expression of iNOS and COX-2 in rat kidney

(Figure 5&6). Both, iNOS and COX-2 were hardly detected in kidney of control group.

Pretreatment with chrysin significantly attenuated Fe-NTA-induced expression of iNOS

and COX-2 (Figure 5&6). In accordance to these results, immunehistochemical analysis

of samples showed intense staining of iNOS and COX-2 in DEN + Fe-NTA-induced

renal tumors. In kidney sections from rats that received Chrysin application plus DEN +

Fe-NTA treatment, a significant reduction in staining of both, COX-2 and iNOS was

observed. In control kidney tissue only mild staining of COX-2 and iNOS was observed.

6.3. Discussion

The incidence of RCC is increasing annually owing to lack of early warning signs and

resistance to various kinds of therapies (Kaul et al., 2011). Limited effectiveness of

modern medicinal therapies for treatment of renal cancer has shifted our focus on

development of alternative strategies. The most valuable option to prevent or delay

carcinogenesis is offered by the use safe plant based compounds, hence there is rise in

Chapter 6 Chrysin


exploration of safe and effective phytochemicals for the management of renal cancer.

Chemoprevention has the potential to be a major component of cancer control. Several

herbs, vegetables fruits and plants with diversified pharmacological properties have been

shown to be rich sources of micro chemicals with the potential to prevent human cancer.

Reports from our laboratory and others indicates that various food ingredients may also

play an essential role in renal cancer prevention (Rehman and Sultana, 2011; Jahangir

and Sultana, 2007).Chrysin is a naturally occurring flavonoid present in number of fruits

and vegetables and has displayed numerous chemopreventive properties against

chemically induced toxicities (Khan et al 2011 a, b in press; Tahir and Sultana, 2011).

Chrysin has also shown to be effective against various tumors like hepatocellular

carcinomas (Khan et al., 2011; Yang et al., 2010), thyroid tumors (Phan et al., 2011) in

animal models and in different cancer cell lines like lung cancer cell line (Brechbuhl,

2011), prostate cancer cell line (Samarghandian, 2011) and human colorectal cell line

(Galijatovic et al., 2001).

Recent Reports have already indicated association between inflammation and oxidative

stress (Bicker and Athar 2006). We have also reported that oxidative stress and

inflammation plays an important role in pathogenesis of nephrotoxicity caused by Fe-

NTA (Rehman and Sultana, 2011; Jahangir and Sultana, 2007; Kaur et al., 2009).

Further, Fe-NTA exposure elevates the levels of redox active iron, which is known to

induce the formation of ROS that can readily attack the cellular molecules leading to lipid

peroxidation, oxidative damage and increase of serum toxicity markers. These events

lead to modulation in the reduced thiol pool and enzymatic and antioxidants (Gpx, GR

catalase, SOD etc), which have important role in protection of renal tissue by quenching

free radicals. In agreement with previous studies we found Fe-NTA to downregulated

both concentration of antioxidants and activities of all antioxidant enzymes (Kaur et al.,

2007; Iqbal et al., 2003). Chrysin supplementation augmented the level of GSH,

glutathione redox cycle enzymes and also phase-II metabolizing enzymes like GST and

QR in renal tissue as also previously reported from our lab (Khan R et al 2011; Tahir S et

al 2011). Lipid peroxidation is an outcome of oxidative stress and incredible elevation in

the level of malondialdehyde (MDA), a lipid peroxidation product, was observed after

Chapter 6 Chrysin


treatment with Fe-NTA (Iqbal et al., 2003). In the present study, Fe-NTA treated rats

showed a remarkable increase in the level of MDA and chrysin significantly attenuated

its level in renal tissue. Thus, chrysin exhibited the protective efficacy against Fe-NTA-

induced lipid peroxidation in renal tissue. Studies from our laboratories have also shown

that chrysin decreases the level of MDA in kidney, liver and colon after treatment with

ethyl alcohol and cisplatin respectively (Tahir and Sultana 2011; Khan et al 2011a, b).

Moreover, lipid peroxidation and the associated membrane damage are implicated in the

pathophysiology of a number of diseases including renal disorders. Renal dysfunction is

followed by the elevated levels of serum enzymes indicating cellular leakage and loss of

functional integrity of renal membrane. It correlates with our results, which showed

increased activities of LDH, BUN and creatinine in the serum of Fe-NTA-treated rats.

Rats treated with chrysin had LDH, BUN and creatinine significantly lower than those

receiving only Fe-NTA. These results suggested that chrysin may protect against Fe-NTA

induced renal toxicity.

Renal sections of rats treated for 16 weeks with Fe-NTA, on histopathological

examination revealed more widespread tubular necrosis, massive inflammatory response,

loss of cellular differentiation, dilated tubules and numerous renal cell tumors.

Histological evaluation showed that chrysin administration suppressed the inflammatory

responses in the renal tissue by decreasing the intense infiltration. It also reduced the

severity of tubular degeneration and loss of cellular differentiation induced by Fe-NTA

treatment. Chrysin treatment was thus instrumental in amelioration of renal toxicity and

tumorigenesis.

Our study mainly focuses on unraveling the mechanisms of anti-inflammatory and anti-

promoting activity of chrysin in Fe-NTA model of renal cancer. Present data suggest that

chrysin markedly inhibits Fe-NTA induced tumor promotion in DEN initiated rat kidney.

Fe-NTA application for 16 week produced renal tumors in agreement with previous

reports (Iqbal et al., 2007; Jahangir et al., 2006). Chrysin drastically ameliorated PCNA-

positive cells in the renal tissue. Hall et al., (1990) reported PCNA, a 36 kDa co-factor of

DNA polymerase-δ, is one of the downstream effectors of the activation of

Chapter 6 Chrysin


MAPK/ERK1/2 signaling and is very useful molecular biomarker of

hyperproliferationation. Ornithine decarboxylase (ODC) is another important and widely

used marker to study tumor promotion. ODC is a rate limiting enzyme in the biosynthesis

of polyamines, spermine, spermidine and putrecines. Auvenin, (1992) reported

transformed cell lines to have elevated levels of ODC activity and elevation of ODC

activity is closely related to tumor promotion and carcinogenesis (Pegg et al., 1995).

Chrysin treatment significantly attenuated ODC activity in rat kidneys exposed to Fe-

NTA suggesting it to have a potent anti-hyperproliferative activity. A decrease observed

in PCNA positive cells and activity of ODC following chrysin pretreatment indicated

suppression induced by chrysin in cellular proliferation and hence tumor promotion.

The activation of NFκB linked regulatory pathways generally underlies inflammatory

processes, and an increase in the nuclear translocation of NFκB has been demonstrated in

all cancers including RCC (Morais et al., 2011). The transcription factor NFκB helps to

regulate the expression of several genes activated during inflammation and is implicated

in several other aspects of oncogenic process such as cellular proliferation, preclusion of

apoptosis, conferring the tumor cells a metastatic and angiogenic ability etc (Brown et al.,

2008). NFκB is induced by various cell stress associated stimuli including growth factors,

vasoactive agents, cytokines, and oxidative stress (Yamamoto and Gaynor, 2001; Li and

Verma, 2002; Karin and Greten, 2005). NFκB in turn controls the regulation of genes

encoding proteins involved in immune and inflammatory responses (i.e., cytokines,

chemokines, growth factors, immune receptors, cellular ligands, and adhesion

molecules). Thus, inhibition of NFκB is nowadays documented as a valuable approach to

control the carcinogenic development. Since NFκB is a redox sensitive transcription

factor it is activated by oxidants generated by Fe-NTA. In agreement with previous

published reports Fe-NTA exposure was found to activate NFκB in renal tissues (Kaur et

al., 2009). Chrysin was found to potently inhibit NFκB activation. It noticeably decreased

the levels of phosphorylated form of inhibitor of kappa B (IκB) and also suspended the

nuclear translocation of NFκB-p65. These observations are consistent to reports in

literature where chrysin has been shown to significantly inhibit NFκB activation (Li et

al., 2010)

Chapter 6 Chrysin


The proteins iNOS and COX-2 are also under the transcriptional regulation of NFκB

(Surh et al., 2001) and are reported to be associated with renal inflammation and tumor

promotion (Kaur et al., 2009), thus Fe-NTA associated increase in their expression may

also be mediated through NFκB activation. Further, Surh et al (2001) reported

overexpression of COX-2 to mediate both inflammation and tumor promotion. Our data

showed chrysin to effectively inhibit Fe-NTA mediated overexpression of COX-2 which

was in agreement with report of woo kJ et al., (2001). Khan et al., (2009) reported

chrysin to downregulate COX-2 expression in NDEN (N-nitrosodiethylamine) induced

hepatocellular carcinoma and have suggest COX-2 inhibition by chrysin to play a pivotal

role in its antitumor activity. Thus, inhibition of Fe-NTA induced COX-2 expression by

chrysin may also plausibly be implicated in protection against Fe-NTA induced renal

carcinogenesis. Pathological and chronic inflammatory reactions can be triggered by

nitric oxide production in renal tissue by the inducible nitric oxide synthase (iNOS)

enzyme (Cattell, 2002; Kashem et al., 1996). Moreover, selective iNOS inhibitor can

suppress renal toxicity, suggesting that iNOS signaling could serve as an important target

for prevention and treatment of renal cancer, suggesting that iNOS signaling could serve

as an important target for prevention and treatment of renal diseases. Recent evidences

suggest that iNOS plays a crucial role in development and progression of renal cancer

(Fukumura et al., 2006). In our study Fe-NTA caused significant induction of iNOS in

renal tissues in accordance with previously published paper of Wu et al., (2001) where in

Fe-NTA was demonstrated to induce NO generation in cultured proximal tubule cells.

Additionally, we have shown that chrysin is able to decrease the elevated expression of

iNOS in the renal cancer tissue.

Fe-NTA exposure induced the expression of proinflammatory cytokines IL-6, TNF-α,

and PGE2 which are under direct transcriptional regulation of NFκB. These cytokines

have important role in inflammation, vascular permeability as well as proliferation. Thus,

inhibition in their secretion by chrysin seems to play an important role in its protective

effect against renal tumorigenesis Our results are in agreement with the findings of Ha et

al., (2010) who have demonstrated chrysin to inhibit the secretion of these cytokines in

LPS stimulated proinflammatory response in microglia cells. Thus, Inhibition of

Chapter 6 Chrysin


proinflammatory cytokines by chrysin treatment may be now accepted as yet another

valuable strategy to control the carcinogenic development.

A number of studies have established that oxidative stress and inflammation contributes

to initiation, promotion and progression of renal carcinogenesis. In this study we

presented data demonstrating that chrysin inhibits DEN initiated and Fe-NTA promoted

renal carcinogenesis in animal model. Chrysin treatment resulted in marked decline in

renal hyperplasia, renal ODC activity and protein expression of PCNA, iNOS, COX-2

and secretion of proinflammatory cytokines, all of which are traditional markers of

inflammation and tumor promotion. In addition, our data also revealed that chrysin

treatment maintained antioxidant armory and also suppresses activation of redox active

transcription factor NFκB. These results supported by published literature reports suggest

chrysin to be a potential candidate for prevention of renal carcinogenesis, since it

restrains several biomarkers of tumor promotion in rat model of renal carcinogenesis.

Chapter 6 Chrysin


Table.1 Results of pretreatment of chrysin on antioxidant enzymes like GSH, GR, SOD,

GPX and Catalase on Fe- NTA induced renal redox imbalance.

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

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

from Fe-NTA treated group (#P < 0.05), (##P < 0.01) and (###P<0.001).Chy= Chrysin;

D1= 20mg/kg/b wt; D2 = 40mg/kg/b wt.

Treatment regimen

per group

GSH (n mol GSH

/g tissue)

GR (nmolNADPH Oxidized/min/

mg protein)

GPX (n mol NADPH Oxidized/mi /mg

protein)

Catalase (nmol H2O2

Consumed/min /mg protein)

SOD (IU/ mg

protein

Group I (control)

0.63±0.02

291.7±16.0 258.2±13.9 308.5±51.9 171.4±13.4

Group II (Fe-NTA

only) 0.40±0.02* 187.1±15.2*** 120.8±14.0** 86.80±10.0*** 119.2±14.7

Group III ( Fe-NTA+

Chy D1) 0.51±0.02# 221.4±15.1# 188.2.0±8.7# 217.9±18.8### 150.6±14.5

Group IV (Fe-NTA+ Chy D2)

0.56±0.03# 284.4±14.9## 211.2±11.9## 247.1±28.1### 157.0±13.4

Group V (only ChyD2) 0.65±0.01 292.0±15.1 276.2±16.0 324.4±65.3 171.4±14.2

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Table.2 Results of pretreatment of chrysin on parameters like GST, QR and MDA

formation in Fe- NTA induced renal Toxicity



from Fe-NTA treated group (#P < 0.05), (##P < 0.01) and (###P<0.001). Chy= Chrysin;


Treatment regimen

per group

GST

(n mol CDNB

Conjugate formed

/min/mg protein)

QR

(nmol NADPH oxidized

/min/mg protein)

MDA

(nmoles of MDA

formed/g tissue)

Group I

(control) 269.2±22.4 232.6±16.5 2.27±0.04

Group II

(Fe-NTA only) 531.7±46.8*** 134.8±9.06*** 5.28±0.28***

Group III

( Fe-NTA+ Chy D1) 347.0±24.6# 201.5±17.4# 3.13±0.13###

Group IV

(Fe-NTA+Chy D2) 314.1±41.6## 218.6±19.2## 3.93±0.19###

Group V

(only Chy D2) 251.6±36.5 236.6±6.60 2.15±0.15

Chapter 6 Chrysin


Table.3 Results of pretreatment of Chrysin on Toxicity Markers like BUN, Creatinine,

LDH and GGT on Fe-NTA induced enhancement.

Treatment

regimen

per group

BUN

(mg / 100 ml)

IU/L

Creatinine

(mg / 100 ml)

IU/L

LDH

(n mol NADH

oxidized / min/ mg

protein)

γ-GGT

(nmoles p

nitroaniline

formed/min/mg

protein)

Group I

(control) 20.04±1.0 1.58±0.07 225.4±25.2 269.2 ± 22.4

Group II

(Fe-NTA

only)

52.07±3.2*** 3.85±0.11*** 453.9±44.49*** 531.7± 46.8***

Group III

(Fe-NTA+ Chy

D1)

38.17±2.9## 3.20±0.19# 298.4±24.28# 347.0 ± 24.6#

Group IV

(Fe-NTA+

Chy D2)

28.26±2.8### 1.96±0.13### 301.5±31.34# 314.1 ± 41.6###

Group

(only Chy D2) 18.99±1.1

1.51±0.08

228.5±27.30

271.6 ± 36.5



from Fe-NTA treated group (#P < 0.05), (##P < 0.01) and (###P<0.001).Chy= Chrysin;


Chapter 6 Chrysin


Table.4 Summary of tumor data of the effects of chrysin treatment on DEN initiated and

Fe- NTA- induced renal cell tumors.

Treatment

regimen

per group

Number of

animals

treated

Number of animals

studied

Histopathologically

Number of

Animals

With cell

tumors

Incidence of

renal cell

tumors (%)

Group I 20 19 0 0

Group II 20 11 9 81.81

Group III 20 14 7 50.00

Group IV 20 15 4 26.66

Group V 20 16 0 0

Group VI 20 18 3 22.22

Group I (control): Normal Saline; Group II (toxicant):DEN + Fe-NTA; group III :DEN +

Fe-NTA + Chy (20 mg/kg b wt); group IV :DEN + Fe-NTA + Chy (40 mg/kg b wt);

Group V :DEN only and Group; VI :Fe-NTA only. Chy – chrysin; Fe-NTA – ferric

nitrilotriacetate. Toxicant group showed highest percentage of tumor incidences which

was abrogated by the administration of chrysin in groups III and IV. Whereas, group V

(DEN only) and group VI (Fe-NTA only) did not develop significant no of tumors.

Chapter 6 Chrysin


Table.5 Effect of Chrysin on elevation in serum level of cytokines (TNF- α, PGE2 and

IL-6). DEN initiated rats were promoted twice weekly application Fe-NTA for 16 weeks

with chrysin treatment.

Treatment regimen

per group

TNF-α

(TNF-α

pg/ml)

PG E2

(PGE2 pg/ml)

IL

(IL pg/ml)

Group I (control) 333.4±41.9 49.20±4.44 827.8±39.6

Group II (Fe-NTA

only) 815.4±27.4*** 155.6±10.3*** 2295 ± 73.2***

Group III (Fe-NTA+

Chy D1) 474.3±54.4# 102.6±9.66# 1966± 99.0#

Group IV (Fe-NTA+

Chy D2) 28.26±2.8### 86.8±15.8### 1495 ± 88.8###

Group (only Chy D2) 327.6±44.1 46.6±4.23 829.2 ± 40.8


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

different from Fe-NTA treated group (#P < 0.05), (##P < 0.01) and (###P<0.001).Chy=

Chrysin; D1= 20mg/kg/b wt; D2 = 40mg/kg/b wt.

Chapter 6 Chrysin


Figure 1: Effect of treatment of chrysin against Fe-NTA promoted Ornithine

decarboxylase (ODC level) in kidney of wistar rats.

Data were expressed as mean ± SEM (n=6) and measured ODC activity was measured as

pmol 14CO2 released/min/mg protein) ODC levels were significantly increased

(***p<0.001) in Fe-NTA treated group as compared to control group. Chrysin

significantly attenuated the level of ODC in Fe-NTA + lower dose of chrysin treated

group (###p<0.05) and Fe-NTA + higher dose of chrysin treated group (###p<0.001) as

compared to only Fe-NTA treated group.

Chapter 6 Chrysin


Figure 2: Representative photomicrographs of PCNA by immunohistochemistry.

(A) No expression of PCNA was observed in case of control rats (B) DEN + Fe-NTA

administration increased the number of PCNA positive cells in cortical and tubular region

of renal sections of animals represented by red arrows in the figure (C) DEN + Fe-NTA +

chrysin (20mg/kg BW) treated animals showed slightly lesser number of PCNA positive

cells as compared to group B as is evident from the figure. (D) DEN + Fe-NTA + chrysin

(40mg/kg BW) treated animals showed significantly lesser number of PCNA positive

cells (E) Only chrysin treatment did not show any change in PCNA reactivity as

compared to control (40x magnification).

Chapter 6 Chrysin


Figure 3: Effect of Chrysin treatment on renal histological alterations caused by

DEN and Fe-NTA application

(A) Kidneys showed normal architecture with no signs of infiltration and tubular or

glomerular damage. (B) DEN + Fe-NTA treated kidney showing areas of massive

inflammatory cell invasion, hyperchromatism, glomerular and tubular congestion. (C)

DEN + Fe-NTA + chrysin (20 mg/kg BW) treated rats showed mild inflammation and

cell invasion as compared to group B (D) DEN + Fe-NTA + chrysin (40 mg/kg BW)

treated animals showed almost normal renal histology (E) Shows the kidneys of the

animals treated with the higher dose of Chrysin only with no significant change as

compared with control (40x magnification).

Chapter 6 Chrysin


Figure 4: Representative photomicrographs of NFkB by immunohistochemistry.

(A) There is almost no expression of NFkB in the renal sections of control group. (B)

DEN + Fe-NTA administration increased strongly NFkB expression in renal sections. (C)

There was partial inhibition of NFkB expression as evidenced by weak immunostaining

in the rat kidneys treated with lower dose of chrysin (20 mg/kg BW). (D) In contrast,

there was almost complete suppression of NFkB in rats treated with higher dose of

chrysin (40 mg/kg BW) this was evident from the figure, as the tubular structures within

the inner cortical regions do not show any substantial immunostaining. The picture is

taken at 40X magnification (40x magnification).

Chapter 6 Chrysin


Figure 5: Representation of photomicrographs of i-NOS reactivity in renal tissue

(A) i-NOS staining of control kidneys showing almost negligible expression of i-NOS

(B) i-NOS staining in DEN initiated and Fe-NTA promoted group shows marked

expression. (C) Chrysin pre-treatment at lower dose (20mg/kg BW) showed partial

inhibition of i-NOS in renal sections treated with DEN and Fe-NTA. (D) DEN + Fe-

NTA+ chrysin (40mg/kg) treatment showed there was almost complete suppression of i-

NOS with higher dose of chrysin (E) Shows the kidneys of the animals treated with the

higher dose of Chrysin only with no significant change as compared with control (40x

magnification).

Chapter 6 Chrysin


Figure 6: Photomicrographs of immunehistochemical detection of COX-2 in renal

tissue

(A) COX-2 staining of control kidneys, There was no expression of COX-2 (B) COX-2

staining in DEN-Fe-NTA induced renal tumors was very intense (C) DEN-initiated,

chrysin pretreated (20mg/kg BW) and Fe-NTA promoted kidney sections show partial

inhibition of COX-2 (D) DEN + Fe-NTA+ chrysin (40mg/kg) treatment showed there

was almost complete suppression of COX-2 with higher dose of chrysin (E) shows renal

section of the groups treated with higher dose of chrysin only (40mg/kg BW) with no

significant change as compared to control (40x magnification).

chapter 6 chrysin - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/9250/11/11_chapter...

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