Abstract: The aim of this study was to investigate the protective role of Cinnamon Flavonoid Extract (CFE) against histopathological changes in albino rats of Wistar strain treated with Dibutylnitrosamine (DBNA) for 12 weeks. The results indicated that rats treated with DBNA recorded decreasing in the total body and liver weights and increasing in spleen and kidney weights with significant values when compared with the control group all over the experiment period 4, 8 and 12 weeks. Addition of CFE by 150 and 300 mg kg-1 b.wt./day in the presence of nitrosamine induced significant improvements in all organs weights. Also, DBNA treated group had histopathological changes on liver through degeneration hyperemia, inflammatory reaction, kidney through hemorrhages renal casts hyperemia, inflammatory reaction and also urinary bladder through papillary hyperplasia with papillary projection formation in the cell layer of the lining epithelium. The co-treatment of CFE with DBNA leads to prevent some of the previous histopathological changes mainly on liver and urinary bladder and secondary on the kidney. It could be concluded that CFE was effective in protecting against DBNA-induced histopathological changes. These results supported present hypothesis that CFE contains several compounds that are able to prevent or inhibit DBNA toxicity.
INTRODUCTION
Although, efforts in cancer diagnosis and therapy have intensified over the years, many cancers still remain difficult to cure. Several recent studies become more conscious and enthusiastic to improve the process of treatment by searching about new compound, which might be effective toward cancer cells at the same time less harmful for the human health (Newmark et al., 1992).
Nitrosamines are known carcinogenic agents. However, their mechanism of action is not well understood. Generation of reactive oxygen species could be an important cause of toxicity of nitrosamines (Masuda et al., 2000). The presence of Nitrose compounds and their precursors in human environment together with the possibility of their endogenous formation in human body have led to suggestions of their potential involvement in human cancers (Kaplan et al., 1997). The maternal consumption during pregnancy of meat cured with sodium nitrite increases the risk for brain tumors in their offspring. Intake of vitamins C and E inhibit endogenous formation of N-Nitrose compounds and was accepted to be protective (Preston-Martin et al., 1996).
Cinnamon (Cinnamon vernum) is a famous beverage in several countries all over the world including Egypt. It has a long history of medicinal use and is one of the world’s most important spices (Stavric et al., 1992). Extract of cinnamon have also been shown to have antioxidant effects in part through activating antioxidant enzymes in various tissues (Anilakumar et al., 2001). That prevent free radical formation, remove radicals before damage can occur, repair oxidative damage, eliminate damaged molecules, or prevent mutations are important mechanisms in cancer prevention (Gordon, 1996).
Changes in histopathological parameters as the result of sodium nitrate intake have been studied by National Toxicology Program (2001). The mean body weight of females at the highest dose were lower than those of control groups and showed the incidences of squomas cell papilloma and carcinoma (combined) in the forestomach of female mice. The incidence of hyperplasia the glandular epithelium was significantly greater in males at the highest dose than in controls. Szemes and Szamado (1991) found that, rats receiving dibutylnitrosamine were showed histological changes in striated muscle, liver, intact rat urinary bladder suggested that including deep and homogenous necrosis in tract and tumorous tissues. Also, Alam et al. (2005) observed that in mice exposed to hepatocarcinogen N-nitrosodibutylamine an enhanced expression of high molecular weight glycoprotein TAA (Tumor-Associated Antigen) in liver cells and in histological examination DBNA treated animals showed cell distortion and extensive necrosis. Therefore, the present study was aimed to investigate the protective role of Cinnamon Flavonoid Extract (CFE) against histopathological changes in albino rats of Wistar strain treated with dibutylnitrosamine (DBNA) for 12 weeks.
MATERIALS AND METHODS
Materials
The bark of cinnamon (Cinnamomum verum) was obtained from Medical
Plants Institute, Agricultural Research Center, Giza, Egypt. N-dibutylamine
and sodium nitrite recommended by Hashimoto et al. (1976) as nitrosamine
precursors were purchased from Sigma Chemical Co., St. Louis, MO. All chemicals
and solvents used for cinnamon extract preparation were obtained from El-Nasser
Chemical Company, Cairo, Egypt.
Experimental Animals
Ninety female albino rats of Wistar strain weighting 110±10
g per each were obtained from the Laboratory of Animal Colony, Ministry of Health
and Population, Helwan, Egypt. All animals were subjected to acclimatization
for weeks, during which the rats were maintained in Biology Lab, Nutrition and
Food Science Department, Faculty of Home Economics, Minufiya University, Shebin
El-Kom, Egypt. Rats were housed in plastic cages with galvanized iron filter
taps.
Diets
The basal diet consisted of: casein 10%, corn oil 10%, vitamins mixture
1%, salt mixture 4% and starch up to 100%. The salt mixture used was according
to Hegested et al. (1941) and the vitamins mixture was that of Campbell
(1963).
Cinnamon Flavonoid Extract (CFE) Preparation
The Cinnamon Flavonoid Extract (CFE) was prepared according to the procedure
described by Sauvesty et al. (1992). The bark of Cinnamomum verum
was chopped. A known weight of the dried powdered plant tissue was extracted
three times with ethanol, 50%, (1:4) at 40°C for 4 h and the mixture was
centrifuged for 15 min at 3000 rpm at room temperature. The clear supernatants
were combined and the solvent was removed by using rotary evaporator (type 349/2,
Corning Limited Laboratory Division Stone Staffordshire, England) at room temperature.
The final extract contained free and conjugated flavonoids.
Experimental Design
The experiments were conducted in accordance with the provisions of the
guide for care and use of laboratory animals (NIH, 1985). The male albino rats
weighting 100±10 g were kept in the laboratory under constant conditions
of temperature (24±2°C) for at least 1 week before and through the
experimental study, being maintained on a standard diet and water were available
ad libitum. The experiment rats were divided into 6 groups as follow:
| Group 1: | Fed on the basal diet and normal water and left in normal condition for 12 weeks which served as control group |
| Group 2: | Fed on the control diet and drink tap water containing 20 ml L-1 dibutylamine (DBA) and 5 g L-1 sodium nitrite daily for 12 weeks |
| Group 3: | Fed the basal diet supplied with 150 mg kg-1 body weight of powdered Cinnamon Flavonoid Extract (CFE) |
| Group 4: | Fed the basal diet supplied with 300 mg kg-1 body weight of powdered CFE |
| Group 5: | Fed the basal diet supplied with 150 mg kg-1 body weight of CFE and drink tap water containing 20 ml L-1 DBA as well as 5 g L-1 sodium nitrite |
| Group 6: | Fed the basal diet supplied with 300 mg kg-1 CFE and drink tap water containing 20 ml L-1 DBA as well as 5 g L-1 sodium nitrite |
All the diet prepared daily just before the feeding time. The doses of CFE, 150 and 300 mg kg-1, were calculated according to the recommended dose of human. DBN and sodium nitrite were mixed with the drinking water using magnetic stirrer for a period of 20 min. The experiment was terminated at 12 weeks after the dietary supplementation of cinnamon flavonoid extract.
Histopathological Examination
Five rats taken every 4 weeks from the experimental groups were sacrificed
then the target organs such as liver, spleen and kidney were excised and weighted.
Specimens from liver, kidney and urinary bladder were fixed in 15% neutral buffered
formalin (pH 7.0), dehydrated in ethyl alcohol, cleared in xylol and embedded
in paraffin 4-6 mM. Sections were prepared and stained with hematoxyl and eosin
according to Carleton (1976) and Banchraft et al. (1996).
Statistical Analysis
Statistical analysis was performed with the student’s t-test and MINITAB
12 computer program (Minitab Inc., State College, PA).
RESULTS AND DISCUSSION
The Effect of CFE and DBNA on Total Body, Liver, Spleen and Kidney Weights
of Rats
The changes in total body weight (g) of rats during different periods of
feeding diet contains 150 or 300 mg CFE/kg b.wt./day and drinking nitrosamine
precursors (DBA+NO2) (Table 1). In control group
(G1), the total body weight at 12 weeks reached to the highest value 143.40
g. Animal maintained on nitrosamine precursors (G2) showed continuous significant
decreasing during all sampling periods 4, 8 and 12 weeks by the ratio of 17.48,
28.15 and 51.60% (as a percent of control group), respectively. The feeding
on 150 mg CFE/kg b.wt./day induced continuous increasing during all sampling
periods 4, 8 and 12 weeks by the ratio of 3.80, 18.60 and 32.20% (as a percent
of control group), respectively. With the raising of CFE feeding dose, 300 mg
kg-1 b.wt./day, the total body weight was decreased after 4 weeks
by the rate of 3.83%, then started to continuous increasing during the rest
of the sampling periods 8 and 12 weeks by the ratio 5.18 and 21.06% (as percent
of control group), respectively.
| Table 1: | The effect of feeding CFE on total body weight (Mean±SD, g) of rats drinking DBNA |
| *Each value represents the Mean±SD of five rats, **In the same raw, different letter(s) mean significant variations at p<0.05 in comparison to control | |
| Table 2: | The effect of feeding CFE on liver weight (Mean±SD, g) of rats drinking DBNA |
| *Each value represents the Mean±SD of five rats, **In the same raw, different letter(s) mean significant variations at p<0.05 in comparison to control | |
In contrary, feeding with 150 mg CFE/kg b.wt./day in the presence of nitrosamine induced continuous decreasing in total body weight during all sampling periods 4, 8 and 12 weeks by the ratio of 21.17, 20.87 and 20.64% (as a percent of control group), respectively. With the raising of CFE feeding dose in the presence of nitrosamine, 300 mg kg-1 b.wt./day, the rate of decreasing recorded 17.79, 22.27 and 12.97% (as a percent of control group) for the same previously sampling periods, respectively.
The changes in total body weight (g) of rats during different periods of feeding diet contains 150 or 300 mg CFE/kg b.wt./day and drinking nitrosamine precursors (DBA+NO2) (Table 2). In control group (G1), the liver weight at 12 weeks reached to the highest value 4.22 g. Animal maintained on nitrosamine precursors (G2) showed continuous significant decreasing during all sampling periods 4, 8 and 12 weeks by the ratio of 27.34, 35.32 and 27.49% (as a percent of control group), respectively. The feeding on 150 mg CFE/kg b.wt./day not induced any significant changes during all sampling periods 4, 8 and 12 weeks, while the raising of that dose to 300 mg kg-1 b.wt./day, the total body weight was decreased after 4 week by the rate of 20.79%, then started to continuous increasing during the rest of the sampling periods 8 and 12 weeks by the ratio 19.72 and 41.94 % (as percent of control group), respectively. In contrary, feeding with 150 mg CFE/kg b.wt./day in the presence of nitrosamine induced decreasing in liver weight during all sampling periods 4, 8 and 12 weeks by the ratio of 18.22, 12.61 and 8.06% (as a percent of control group), respectively. With the raising of CFE feeding dose in the presence of nitrosamine, 300 mg kg-1 b.wt./day, the rate of decreasing recorded 21.73, 34.40 and 9.00% (as a percent of control group) for the same previously sampling periods, respectively.
The opposite directions were observed for the changes in spleen and kidney
weights. In control group (G1), the spleen weight at 12 weeks reached to the
highest value 0.46 g (Table 3). Animal maintained on nitrosamine
precursors (G2) showed continuous significant increasing during all sampling
periods 4, 8 and 12 weeks by the ratio of 21.95, 25.00 and 30.43% (as a percent
of control group), respectively. The feeding on 150 mg CFE/kg b.wt./day not
induced any significant changes during all sampling periods 4, 8 and 12 weeks,
while the raising of that dose to 300 mg kg-1 b.wt./day, the spleen
weight was increased after 4, 8 and 12 weeks by the rates of 9.76, 4.55 and
2.17%, (as a percent of control group), respectively. In contrary, feeding with
150 mg CFE/kg b.wt./day in the presence of nitrosamine induced increasing in
spleen weight after 4 weeks of feeding by the ratio of 24.39%, while elongation
of the feeding period and/or increasing of the CFE dose leads to decreasing
of the spleen weight by different rates.
| Table 3: | The effect of feeding CFE on spleen weight (Mean±SD, g) of rats drinking DBNA |
| *Each value represents the Mean±SD of five rats, **In the same raw, different letter(s) mean significant variations at p<0.05 in comparison to control | |
| Table 4: | The effect of feeding CFE on kidney weight (Mean±SD, g) of rats drinking DBNA |
| *Each value represents the Mean±SD of five rats, **In the same raw, different letter(s) mean significant variations at p<0.05 in comparison to control | |
For kidney weight, data in Table 4 indicted that the kidney weight of the control group (G1) at 12 weeks reached to the highest value 0.78 g. Animal maintained on nitrosamine precursors (G2) showed continuous significant increasing during all sampling periods 4, 8 and 12 weeks by the ratio of 39.19, 49.38 and 55.13% (as a percent of control group), respectively. The feeding on 150 mg CFE/kg b.wt./day not induced any significant changes during all sampling periods 4, 8 and 12 weeks, while the raising of that dose to 300 mg kg-1 b.wt./day, the spleen weight was increased after 4, 8 and 12 weeks by the rates of 8.11, 18.52 and 38.46%, (as a percent of control group), respectively. In contrary, feeding with 150 mg CFE/kg b.wt./day in the presence of nitrosamine induced increasing in kidney weight after 4 weeks of feeding by the ratio of 33.78% which continuously increased by the elongation of the feeding period and/or increasing of the CFE dose by different rates.
Finally, a comparison between the data of DBN group and DBN drinking +150 mg CFE feeding group or DBN group and DBN drinking+300 mg CFE feeding (Table 1-4) indicated that CFE feeding were significantly effective in protecting against DBNA-induced body, liver, spleen and kidney weight changes.
The Effect CFE and DBNA on Liver, Kidney and Urinary Bladder Histopathology
Liver Histopathology
Liver of rats fed the control diet showed the normal histological structure
of the central veins and surrounding hepatocytes (Fig. 1a).
Rats treated with DBNA for 4 weeks showed severe dilatation and congestion in
the central and portal veins (Fig. 1b, Fig.
1c) and mononuclear leucocytes inflammatory cells infiltration in the portal
area (Fig. 1d). With the elongation of treating period of
DBNA, hyperemic central veins and surrounding sinusoids with degeneration in
the hepatocytes were observed for 8 weeks (Fig. 1e) and hyperemia
in the central veins and sinusoids with diffuse kupffer cells proliferation
for 12 weeks (Fig. 1f). Feeding on diet containing 300 mg
kg-1 b.wt./day CFE for 8 weeks showed hyperemia in the central veins
with degeneration in the hepatocytes (Fig. 1g). Rats fed
diet containing 150 mg kg-1 CFE and administrated DBNA for 4 weeks
showed mononuclear leucocytes inflammatory cells infiltration in the portal
area with diffuse kupffer cells proliferation in between the hepatocytes (Fig.
1h).
| Fig. 1: | The effect of CFE on liver histopathological changes induced by DBNA in rats, (a) normal (control diet), (b, c, d) fed the control diet and administrated DBNA for 4 weeks, (e) fed the control diet and administrated DBNA for 8 weeks, (f) fed the control diet and administrated DBNA for 12 weeks, (g) fed diet containing 300 mg kg¯1 CFE for 8 week, (h) fed diet containing 150 mg kg¯1 CFE and administrated DBNA for 4 weeks and (i) fed diet containing 300 mg kg¯1 CFE and administrated DBNA for 8 weeks (H and E, X40) |
The increasing of CFE dose to 300 mg kg-1 b.wt./day in the same earlier treatment showed dilatated of the central vein and sinusoids with diffuse kupffer cells proliferation in between the hepatocytes (Fig. 1i).
Kidney Histopathology
Kidney of rats fed the control diet showed the normal histological structure
of the glomeruli and renal tubules (Fig. 2a). Rats treated
with DBNA for 4 weeks showed hyperemia in the glomerular tuft of the glomeruli
with periglomerular inflammatory cells infiltration and focal hemorrhages between
the tubules (Fig. 2b), swelling of the lining endothelium
(Fig. 2c), mononuclear leucocytes inflammatory cells infiltration
in between the degenerated renal tubules (Fig. 2d) and focal
haemorrhages the corticomedullary portion (Fig. 2e). Kidney
of rats fed diet containing 150 mg/kg b.wt./day CFE for 8 weeks showed hyperemia
of the glomerular tuft of the glomeruli and intertubular stromal blood vessels
(Fig. 2f) and increasing the previous dose of CFE to 300 mg
kg-1 b.wt./day and elongation of the exposure period induced hyperemia
in the glomerular tufts of the glomeruli (Fig. 2g) and hyperemia
of the intertubular blood vessels (Fig. 2h). The group treated
with DBNA for 8 weeks showed hyperemia in the glomerular tufts of the glomeruli
with focal inflammatory cells infiltration in between the renal tubules in the
cortical portion (Fig. 2i). Rats fed diet containing 150 mg
kg-1 CFE and administrated DBNA for 4 weeks showed degenerative and
necrobiotic changes of the epithelial cells lining the tubules (Fig.
2j). With the elongation of earlier treating period hyperemia of the glomerular
tufts, focal haemorrhages, degeneration in the renal tubules and focal perivascular
oedema with inflammatory cells infiltration were observed for 8 weeks (Fig.
2k) and focal haemorrhages in the medullary portion for 12 weeks (Fig.
2l). On the other side, increasing of the CFE dose to 300 mg kg-1
b.wt./day and administrated DBNA induced degeneration in the epithelial cells
lining the renal tubules for 4 weeks (Fig. 2m), focal haemorrhages
of the medullary portion for 8 weeks (Fig. 2n) and hyperemia
in the glomerular tuft of the glomeruli, perivascular oedema and inflammatory
cells infiltration and degeneration in the renal tubules for 12 weeks (Fig.
2o).
Urinary Bladder Histopathology
Urinary bladder of rats fed the control diet showed the normal histological
structure of the lining mucosal epithelium with underlying lamina propria and
muscularis (Fig. 3a). In rats maintained on DBNA for 8 weeks,
papillary hyperplasia was noticed in the mucosal layer (Fig. 3b).
For rats treated with DBNA and fed diet containing 150 mg kg-1 CFE
for 8 weeks papillary hyperplasia in the lining mucosal layer was observed (Fig.
3c).
Changes in histopathological parameters as the result of nitrosamine subjection
have been studied by many authors. Several universities and academic centers
have paid attention towards the methods could be used successively in reducing
like of these changes. One of the most effective methods commonly tested in
the last decade is the using of origin- plant extract such herbs. Some of these
herbs exhibited significant roles in reducing the adverse effects of nitrosamines
including the histopathological changes. In the present study the effect of
flavonoids containing diet on rats treated with nitrosamines have been investigated.
Such as shown in the earlier data (Table 1-4)
rats treated with DBN recorded decreasing in the total body and liver weights
and increasing in spleen and kidney weights with significant values when compared
with the control group all over the experiment period. All of these results
are partially consistent with the finding of Lee et al. (2003), who stated
that treatment with dimethylnitrosamine (DMN) caused a significant decrease
in body and liver weight. The decreasing weight of these organs could be attributed
to one or more of the following reasons. Nitrosamines produce oxidative stress
due to generation of reactive oxygen species which alter the antioxidant defense
system in the tissues consequently brought hepatic degeneration as evidenced
by the significant decrease in liver weight index (Rockey, 2005; Mittal et
al., 2006).
| Fig. 2: | The effect of CFE on kidney histopathological changes induced by DBNA in rats, (a) normal (control diet), (b-e) fed the control diet and administrated DBNA for 4 weeks, (f) fed diet containing 150 mg kg-1 b.wt./day CFE for 8 weeks, (g) fed diet containing 300 mg kg-1 b.wt./day CFE for 4 weeks, (h) fed diet containing 300 mg kg-1 b.wt./day CFE for 8 weeks, (i) treated with DBNA for 8 weeks, (j) fed diet containing 150 mg kg-1 CFE and administrated DBNA for 4 weeks, (k) fed diet containing 150 mg kg-1 CFE and administrated DBNA for 8 weeks, (l) fed diet containing 150 mg kg-1 CFE and administrated DBNA for 12 weeks , (m) fed diet containing 300 mg kg-1 CFE and administrated DBNA for 4 weeks, (n) fed diet containing 300 mg kg-1 CFE and administrated DBNA for 8 weeks and (o) fed diet containing 300 mg kg-1 CFE and administrated DBNA for 12 weeks (H and E, X40) |
Also, Rajeshkumar et al. (2003) indicated that N-nitrosodiethylamine
(NDEA) induced liver tumors in rats and the liver weights were reduced. Otherwise,
both doses of CFE increased significantly the body weight and non-significantly
the liver, kidney and spleen weights when compared with the control group all
over the experimental periods. These results are partially consistent with the
finding of Okazaki et al. (2002), who noticed that cinnamon component
such isoflavones increased liver weight of rats and Badary et al. (2005),
who found that flavanone produced significant reduction in body weight loss.
| Fig. 3: | The effect of CFE on urinary bladder histopathological changes induced by DBNA in rats, (a) normal (control diet), (b) treated with DBNA and fed control diet for 8 weeks and (c) treated with DBNA and fed diet containing 150 mg kg-1 CFE for 8 weeks (H and E, X40) |
Also, Duarte et al. (2002) found that other cinnamon component i.e., quercetin markedly inhibited the development hypertension. This effect was accompanied by full prevention of most of the effects such as increases kidney weight indices. On the other side, Bruneton (1995) stated that cinnamon bark is used to treat symptoms of digestive disorder, functional asthenias and also for loss of appetite to facilitate weight gain or cinnamon’s constituents eugenol, eugenol acetate and methyl eugenol have been reported to help digestion by enhancing trypsin activity and lipolytic (ability to hydrolyze fats) action (Leung and Foster, 1996). Finally, Yokozawa et al. (2005) stated that the improvement of kidney weight induced by CFE could be attributed to different cinnamon,s constituents in particular polyphenols.
The present study also revealed that nitrosamine alone had a toxic effect on liver through degeneration hyperemia, inflammatory reaction, kidney through haemorrhages renal casts hyperemia, inflammatory reaction and also urinary bladder through papillary hyperplasia with papillary projection formation in the cell layer of the lining epithelium. This finding was confirmed by Zakhary et al. (1994) and Alam et al. (2005), who found that drinking water contain 0.05% DBNA during 3 weeks showed cell distortion, extensive necrosis in the liver and bladder papillomas. Other studies have shown that treatment with DBNA was enhanced the incidences of preneoplastic and neoplastic lesions in the upper digestive tract, liver, pancreas, as well as papillomas of the urinary bladder (Szemes and Szamado, 1991; Shibata et al., 1994) and tumor incidence of other organs, such as the kidney and thyroid (Jang et al., 1991). Recently, Omotosho et al. (2000) have reported that, the N-nitrosation reaction may occur in the human bladder in addition to gastrointestinal tract. This reaction occurs in the presence of bacteria and a precursor secondary amine that associated with toxicity for the liver (as a consequence of the urinary retention) and papillomas of the urinary bladder.
On the other hand, in the present study CFE act as a protector against nitrosamine toxicity mainly on liver and urinary bladder and secondary on the kidney. This finding agreed with that noticed by Wu et al. (2006), who found that administration of flavonoid in the initiation or post initiation phase significantly decreased the incidences of bladder neoplasms and preneoplastic lesions, induced by DBNA. Also, they indicated that all histopathological changes in liver such as fatty infiltration, variation in mitotic figures and focal necrosis, which induced, by DBNA were reversed by the administration of CFE.
In conclusion, CFE was effective in protecting against DBNA-induced histopathological changes. These results supported present hypothesis that CFE contains several free and conjugated compounds that are able to prevent or inhibit DBNA toxicity. Therefore, we recommended the cinnamon to be included in the daily diets and drink but by a moderate amount.
ACKNOWLEDGMENT
This study was financially supported by the Research Box (2005-2007), Minufiya University, Shebin El-Kom, Egypt.