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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
Deptt. of Med. Elemn. & Toxicology 111 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 112 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 113 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 114 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 115 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 116 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 117 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 118 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 119 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 120 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 121 PhD Thesis
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
Chapter 6 Chrysin
Deptt. of Med. Elemn. & Toxicology 122 PhD Thesis
Table.2 Results of pretreatment of chrysin on parameters like GST, QR and MDA
formation in Fe- NTA induced renal Toxicity
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
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
Deptt. of Med. Elemn. & Toxicology 123 PhD Thesis
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
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.
Chapter 6 Chrysin
Deptt. of Med. Elemn. & Toxicology 124 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 125 PhD Thesis
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
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.
Chapter 6 Chrysin
Deptt. of Med. Elemn. & Toxicology 126 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 127 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 128 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 129 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 130 PhD Thesis
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
Deptt. of Med. Elemn. & Toxicology 131 PhD Thesis
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).