Effect of Clerodendron inerme on Erythrocyte Membrane Integrity During 7,12-dimethylbenz(a)anthracene Induced Skin Carcinogenesis in Swiss Albino Mice

MATERIALS AND METHODS

Chemicals
The carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA) was purchased from Sigma Aldrich Chemical Pvt Ltd., Bangalore, India. All other required chemicals used were of analytical grade.

Animals
Male Swiss Albino mice 4-6 weeks old, weighing 15-20 g were purchased from National Institute of Nutrition, Hyderabad, India and maintained in the Central Animal House, Rajah Muthaiah Medical College and Hospital, Annamalai University. The animals were housed in groups of four or five in polypropylene cages and provided standard pellet diet and water ad libitum and maintained under controlled conditions of temperature and humidity, with a 12 h light/dark cycle as per the principles and guidelines of the ethical committee for animal care of Annamalai University in accordance with Indian National Law on animal care and use.

Plant Material
Clerodendron inerme (L.) Gaertn was collected in and around Chidambaram and Cuddalore, Tamil Nadu, India. Dr. R. Panneer Selvam, Botanist, Department of Botany, Annamalai University verified the identity of the plant and a voucher specimen was also deposited in the Department of Botany, Annamalai University.

Ethanolic Extract of Clerodendron inerme Leaves
The ethanolic extract of Clerodendron inerme leaves was prepared according to the method of Hossain et al. (1992). Five hundred gram of fresh leaves of Clerodendron inerme leaves were dried, powdered and socked in 1500 mL of 95% ethanol over night. After filtration, the residue obtained was again resuspended in equal volume of 95% ethanol for 48 h and filtered again. The above two filtrates were mixed and the solvents was evaporated in a rotovapour at 40-50 °C, under reduced pressure. A 14% semisolid light greenish yellow material obtained was stored at 0-4 °C until used. A known volume of the residual extract is suspended in distilled water and was orally administered to the animals during the experimental period.

Experimental Design
The experimental design was approved (Proposal No. 315; dated 15-02-2006) by the Annamalai University animal ethical committee (Reg. No: 160/1999/CPCSEA), Annamalai University, Annamalainagar. The present study was conducted during 2005-2006 in the Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, Tamilnadu, India.

A total number of 40 male Swiss albino mice were divided into four groups of 10 each. Skin carcinogenesis was developed in Swiss albino mice according to the method of Azuine and Bhide (1992). Depilatory cream was applied to remove hair from the back of each mouse and the mice were left untreated for two days. Mice having no hair growth after two days were selected for the present study.

The depilated back of group I mice was painted with acetone alone (0.1 mL^-1 mouse) twice weekly for 8 weeks (vehicle treated control). The depilated back of group II and III mice were painted with DMBA (25 μg in 0.1 mL acetone/mouse) twice weekly for 8 weeks. Group II mice received no other treatments. Group III mice were orally administered with ethanolic extract of Clerodendron inerme leaves (300 mg kg^-1 b.wt. in 1 mL distilled water, three times per week) starting 1 week before the exposure to the carcinogen and continued throughout the experimental period. Group IV animals received CILEE alone (300 mg kg^-1 b.wt. in 1 mL distilled water) three times per week throughout the experimental period. At the end of experimental period all the animals were sacrificed by cervical dislocation.

CILEE Dose Fixation
Previous studies from our laboratory have identified the pharmacological dose (300 mg kg^-1 b.wt.) of CILEE for experimental cancer studies (Manoharan et al., 2006; Renju et al., 2007).

Toxicity Studies
The toxicity studies for the administrated dose of CILEE were evaluated by histological studies and estimating the activities of serum aspartate transaminase (AST) and alanine transaminase (ALT) (Mohun and Cook, 1957) and γ-glutamyl transferase (GGT) (Fiala et al., 1972). Measurement of these enzymes activities help to assess the extent of tissue toxicity and damage.

Histopathological Studies
For histopathological examination, skin tissues were fixed in 10% formalin and embedded with paraffin, 2-3 μm sections were cut in a rotary microtome and stained with hematoxylin and eosin.

Biochemical Analysis
After plasma separation, the erythrocyte membrane was prepared by the method of Dodge et al. (1968) modified by Quist (1980). The precipitate obtained after treating the plasma with 95% ethanol was used for the estimation of protein bound hexose and hexosamine. Similarly, the precipitate obtained after treating the erythrocyte membranes with 1% phosphotungstic acid followed by 5% TCA was used for the estimation of protein found hexose and hexosamine. The protein bound hexose, hexosamine, total sialic acid and fucose in plasma and erythrocyte membrane were estimated by the methods of Niebes (1972), Wagner (1979), Warren (1959) and Dische and Shettles (1948), respectively. Plasma lipid bound sialic acid level was determined by the method of Katopodis and Stock (1980). The levels of thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) in erythrocyte membrane were measured by the methods of Donnan (1950) and Beutler and Kelley (1963), respectively. Osmotic fragility was determined by the method of Parpart et al. (1946) and mean corpuscular fragility was calculated by recording the saline concentration, which would have resulted in 50% hemolysis.

Statistical Analysis
The data are expressed as mean ± SD. Statistical comparisons were performed by One way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT) and Student’s t-test. The results were considered significant if the p-values were less than 0.05.

RESULTS

The activities of serum AST, ALT and GGT in control animals (60.4 ± 4.8, 27.5 ± 1.9 and 1.47 ± 0.08, respectively) and in control animals treated with CILEE alone (61.8 ± 5.1, 28.1 ± 2.1 and 1.53 ± 0.09, respectively) showed no significant difference between them.

The levels of total hemoglobin and red blood cell count were significantly decreased (p<0.001) where as the mean corpuscular fragility was increased in tumor bearing animals as compared to control animals. Oral administration of CILEE at a dose of 300 mg kg^-1 b.wt. to DMBA painted animals restored the status of above said parameters (p<0.001). Mice treated with CILEE alone showed no significant differences in the levels of total hemoglobin, red blood cell count and mean corpuscular fragility as compared to control animals (Table 1).

A significant increase in TBARS (p<0.001) and decrease in GSH (p<0.001) were observed in tumor bearing animals as compared to control animals. Oral administration of CILEE at a dose of 300 mg kg^-1 b.wt. to DMBA painted animals restored the status of TBARS and reduced glutathione (p<0.001). Mice treated with CILEE alone showed no significant differences in TBARS and GSH levels as compared to control animals (Table 2).

Table 1:	Levels of total hemoglobin, red blood cell count and mean corpuscular fragility in control and experimental animals in each group (n = 10)
Values are expressed as mean ± SD, (n = 10). Values not sharing a common superscript significantly different at p<0.05 (DMRT); *: Significantly different from control animals p< 0.001 (Student’s t-test); #: Significantly different from control animals p< 0.05 (Student’s t-test); # : Significantly different from DMBA alone painted animals p<0.001 (Student’s t-test); CILEE-Clerodendron inerme ethanolic leaf extract; Δ: Concentration of NaCl solution (g%) at 50% hemolysis

Table 2:	Levels of TBARS and reduced glutathione in erythrocyte membranes of control and experimental animals in each group (n = 10)
Values are expressed as mean ± SD, (n = 10). Values not sharing a common superscript significantly differ at p<0.05 (DMRT); *: Significantly different from control animals p<0.001 (Student’s t-test); #: Significantly different from control animals p<0.05 (Student’s t-test); #: Significantly different from DMBA alone painted animals p<0.001(Student’s t-test); CILEE-Clerodendron inerme ethanolic leaf extract

Table 3:	Protein bound hexose, hexosamine, total sialic acid, lipid bound sialic acid and fucose in plasma of control and experimental animals in each group
Values are expressed as mean ± SD, (n = 10), Values not sharing a common superscript significantly differ at p<0.05 (DMRT); *: Significantly different from control animals p<0.001 (Student’s t-test); # -significantly different from control animals p<0.05 (Student’s t-test); #: Significantly different from DMBA alone painted animals p<0.001(Student’s t-test); CILEE-Clerodendron inerme ethanolic leaf extract

Table 4:	Protein bound hexose, hexosamine and total sialic acid levels in erythrocyte membranes of control and experimental animals in each group (n = 10)
Values are expressed as mean ± SD, (n = 10). Values not sharing a common superscript significantly differ at p<0.05 (DMRT); *: Significantly different from control animals p<0.001 (Student’s t-test); #: Significantly different from control animals p<0.05 (Student’s t-test); #: Significantly different from DMBA alone painted animals p<0.001(Student’s t-test); CILEE-Clerodendron inerme ethanolic leaf extract

The levels of these glycoconjugates were significantly increased (p<0.001) in plasma of tumor bearing animals as compared to control animals. Oral administration of CILEE at a dose of 300 mg kg^-1 b.wt. to DMBA painted animals restored the status of glycoconjugates (p<0.001). Mice treated with CILEE alone showed no significant difference in glycoconjugates levels as compared to control animals (Table 3).

The levels of these glycoconjugates were significantly decreased (p<0.001) in erythrocyte membranes of tumor bearing animals as compared to control animals. Oral administration of CILEE at a dose of 300 mg kg^-1 b.wt. to DMBA painted animals restored the status of glycoconjugates (p<0.001). Mice treated with CILEE alone showed no significant differences in glycoconjugates levels as compared to control animals (Table 4).

Fig. 1:	Osmotic fragility curve of control and experimental animals in each group. The degree of haemolysis was calculated by comparing with 0.1% NaCl solution which represented 100% lysis

Fig. 2:	Histopathological features of control and experimental animals in each group

The fragility curve of tumor bearing animals was shifted to the right for the control animals. The mean corpuscular fragility was also significantly higher in tumor bearing animals as compared to control. Treatment of tumor bearing animals with CILEE at a dose of 300 mg kg^-1 b.wt. shifted the curve to the left of cancer animals. Mean Corpuscular Fragility (MCF) values did not differ significantly in animals treated with CILEE alone as compared to control animals (Fig. 1 ).

The skin tissues from DMBA treated mice (Group II) revealed severe keratosis, hyperplasia, dysplasia and well differentiated squamous cell carcinoma. A mild to moderate preneoplastic lesions (hyperplasia (+) keratosis (++) and dysplasia (+) were noticed in group III animals (DMBA+CILEE). We did not observe any histological changes in the skin tissues of mice treated with CILEE alone (Fig. 2).

DISCUSSION

The simplicity, availability and ease of isolation make erythrocyte membrane as an excellent model for membrane studies. The goal of the present study was to investigate the protective effect of Clerodendron inerme on erythrocyte membrane integrity in DMBA induced skin carcinogenesis. The membrane integrity was assessed by measuring the status of RBC membrane glycoconjugates and red blood cell osmotic fragility in experimental animals.

Control animals treated with acetone alone (vehicle) showed well defined skin and the presence of subcutaneous tissues. DMBA alone treated animals showed 100% tumor incidence and the tumor was histopathologically diagnosed as well differentiated squamous cell carcinoma. The tumor cells have pleiomorphic, hyperchromatic nuclei with epithelial pearl formation. Oral administration of CILEE completely prevented the formation of well differentiated squamous cell carcinoma. We have however observed precancerous lesions such as hyperplasia and dysplasia. Present results thus indicate that CILEE has suppressing effect on cell proliferation.

Oral administration of CILEE alone at a dose of 300 mg kg^-1 b.wt. did not produce any histological changes in liver, kidney and skin of control animals. We have also assessed the toxic effect of CILEE by estimating the activities of AST, ALT and GGT in serum. No significant difference in the activities of AST, ALT and GGT in control animals treated with CILEE alone. Our results thus suggest that the administered dose has no toxic effect to the host tissues.

Erythrocytes of tumor bearing mice were more fragile than those from control mice. Osmotic fragility has been found to be altered in various pathological conditions including cancers (Abou-Seif et al., 2000; Kolanjiappan et al., 2002). Increased osmotic fragility in cancer animals can be correlated to elevated lipid peroxidation in erythrocytes. Lipid peroxidation has been implicated in the alterations of membrane structure and functions. Increased lipid peroxidation has been reported to cause an increase in osmotic fragility and decrease in cell fluidity (Manoharan et al., 1996; Kolanjiappan et al., 2002).

Several studies have reported that reduced glutathione plays a central role in cellular defense against reactive oxygen species (McLellan and Wolf, 1999). Decline in red blood cell reduced glutathione in cancer mice is partially responsible for the increased osmotic fragility of erythrocytes. The tripeptide glutathione plays an important role in protecting the erythrocytes from free radical-induced lipid peroxidation. Hence, we feel that the decrease in red blood cell glutathione due to increased lipid peroxidation may in turn leads to changes in red blood cell osmotic fragility. Oral administration of CILEE protected the changes in red blood cell fragility in DMBA alone painted animals. This is probably due to antilipidperoxidative and antioxidant potential of CILEE. In the present study, we also observed that CILEE significantly restored the levels of total Hemoglobin and red blood count in DMBA painted animals. This suggests that CILEE protected the alterations in red blood cells during skin carcinogenesis.

In the present study, an increase in plasma glycoconjugates and decrease in erythrocyte membrane glycoconjugates levels were noticed in tumor bearing animals as compared to control animals. Analysis of glycoconjugates, the vital components of cell surface, has been the field of intensive investigation due to the fact that they play a crucial role during neoplastic transformation (Dabelsteen, 1996; Suresh et al., 2007). Altered glycosylation of glycoconjugates is one of the important molecular changes that accompany malignant transformation. Dabelsteen (1996) have reported that epithelial cell surface carbohydrates were lost during experimental carcinogenesis. The decrease in erythrocyte membrane glycoproteins can therefore be related to increased membrane degradation during malignancy.

Increased concentration of serum glycoproteins have been reported in human and experimental carcinogenesis (Manoharan et al., 2004; Suresh et al., 2007). Macbeth and Bekesi (1964) have reported that the presence of neoplastic cells in the body stimulate the synthesis of glycoproteins in the liver, which subsequently enter into circulation. Increased levels of plasma glycoproteins in tumor bearing mice are either due to spontaneous release of glycoproteins from tumor cells or due to increased hepatic synthesis with subsequent shedding into plasma.

Sialic acids, group of acylated neuraminic acid, impart a net negative charge to cell membrane and has important role in cell-cell and cell-matrix interactions. Sialic acid concentration on the surface of malignant cell can be used to correlate directly with its ability to metastasize (Sebzda et al., 2006). Elevated levels of total sialic acid and lipid bound sialic acid were reported in several cancers (Manoharan et al., 2004; Suresh et al., 2007). Elevated plasma sialic acid concentration is probably either due to shedding or secretion of sialic acid from tumor cell surfaces or due to membrane degradation (Macbeth and Bekesi, 1964; Manoharan et al., 2004). The observed increase in plasma lipid bound sialic acid is probably due to shedding of aberrant sialic acid rich glycolipids into circulation.

A positive association between serum fucose level and cancer progression has been reported (Manoharan et al., 2004). The observed increase in plasma total sialic acid and fucose in tumor bearing mice could be either due to increased turnover of malignant cells or shedding from tumor tissues and membranes into circulation. In the present study, oral administration of Clerodendron inerme ethanolic leaf extract restored the status of plasma and erythrocyte membrane glycoconjugates in DMBA painted mice. Our results thus suggest that Clerodendron inerme protected the levels of plasma and erythrocyte membrane glycoconjugates by maintaining the structural integrity and stability of the erythrocyte membrane during DMBA induced skin carcinogenesis.

Increased membrane lipid peroxidation, insufficient antioxidant potential and loss of membrane glycoconjugates caused structural and functional abnormalities in the erythrocytes of skin tumor bearing mice. Oral administration of CILEE restored these abnormalities during DMBA induced skin carcinogenesis. The protective effect of CILEE on membrane integrity is probably due to the presence of one or more bioactive constituents such as flavonoids and isoflavones and their synergistic interactions. The present study thus demonstrates the protective effect of CILEE on red blood cell membrane integrity during DMBA induced skin carcinogenesis.