In vitro cytogenetic effects of Andrographis paniculata (kalmegh) on arsenic.
An agent containing arsenic.
Of, relating to, or containing arsenic.
1. pertaining to arsenic.
2. a compound containing arsenic. compounds are environmental toxins with multiple effects in animal and human populations. The US Environmental Protection Agency has placed arsenic at the top of its Superfund contamination list (Hei and Filipic, 2004). Epidemiological data have shown that chronic levels of exposure to arsenite are associated with the cancer of skin, lungs bladder, and kidney (Mo et al., 2006). Accumulated evidence has shown that arsenite-induced cytogenetic cytogenetic /cy·to·ge·net·ic/ (-je-net´ik)
1. pertaining to chromosomes.
2. pertaining to cytogenetics.
pertaining to or originating from the origin and development of the cell. alterations are associated with its carcinogenicity. Arsenical compounds are potent clastogens in many cell types inducing chromosomal aberrations, sister chromatid chromatid (krō`mətəd): see chromosome; crossing over. exchanges (SCEs), cell cycle arrest, mitosis disturbance, aneuploidy aneuploidy /an·eu·ploi·dy/ (an?u-ploi´de) any deviation from an exact multiple of the haploid number of chromosomes, whether fewer or more.
n. , cytoskeleton cytoskeleton
System of microscopic filaments or fibres, present in the cytoplasm of eukaryotic cells (see eukaryote), that organizes other cell components, maintains cell shape, and is responsible for cell locomotion and for movement of the organelles within it. disruption, apoptosis, and chromosomal loss in both human and rodent cells in culture. Such disparities has also been noted in peripheral lymphocytes of human populations chronically exposed to arsenic in endemic areas (Basu et al., 2004; Yih et al., 2002). Although precise mechanism of action of arsenic remains unknown, it has been postulated that arsenic may act as a carcinogen though inhibition of DNA repair mechanisms leading indirectly to increased mutations (Andrew et al., 2003). However, induction of oxyradicals and various kinds of oxidative DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. damages has been considered to be an underlying basis of genotoxic genotoxic /ge·no·tox·ic/ (je´no-tok?sik) damaging to DNA: pertaining to agents known to damage DNA, thereby causing mutations, which can result in cancer.
adj. effects caused by arsenic (Kessel et al., 2002; Liu et al., 2001).
The leaves and aerial parts of Andrographis paniculata (Burm. F.) Nees (Family: Acanthaceae) have been used in traditional systems of medicine for the treatment of hepatitis, bronchitis, colitis, cough, fever, mouth ulcers, sores, tuberculosis, bacillary dysentery, venomous snake bites, common cold, urinary tract infections, and acute diarrhea (Panossian et al., 2002). Over the last decade, Andrographis has been incorporated into the practice of contemporary western herbal medicines. A decoction DECOCTION, med. jurisp. The operation of boiling certain ingredients in a fluid, for the purpose of extracting the parts soluble at that temperature. Decoction also means the product of this operation.
2. of this plant is a blood purifier and is prominent in atleast 26 ayurvedic formulas, whereas in traditional Chinese medicine Traditional Chinese Medicine Definition
Traditional Chinese medicine (TCM) is an ancient and still very vital holistic system of health and healing, based on the notion of harmony and balance, and employing the ideas of moderation and prevention. , Andrographis is an important 'cold property' herb. It is used to rid the body of heat, fevers and to dispel toxins from the body. Andrographis is sometimes called 'Indian Echinacea' and is credited with the stopping of Indian flu epidemic of 1919. The active (main) ingredients of A. paniculata are diterpene di·ter·pene
Any of a class of terpenes containing 20 carbon atoms and 4 branched methyl groups.
highly irritant plant diterpenoid esters, e.g. daphnane, tigliane, ingemane. lactones, the most prominent being andrographolide ([C.sub.20][H.sub.30][O.sub.5]) with molecular weight 350 having the structure as shown in Fig. 1 (Saxena et al., 1998).
Materials and methods
Chemical reagents: Arsenic trioxide ([As.sub.2][O.sub.3]) and ethyl methanesulphonate (EMS) were obtained from HiMedia Laboratories Ltd., Mumbai, India. A. paniculata (30% andrographolide) was procured from Natural Remedies, Banglaore, India. Phytoheamagglutinin (PHA-P), Hoechst 33258, Bromodeoxyuridine (BrdU) and colchicine colchicine (kŏl`chəsēn'), alkaloid extracted from plants of the genus Colchicum and especially from the corms of the autumn crocus, Colchicum autumnale (see meadow saffron). were obtained from Sigma Chemicals, USA.
Cell cultures: Peripheral blood lymphocyte cultures were carried out according to the standard protocol (Hungerford, 1965) with slight modifications. Blood samples from healthy donors were collected by vein puncture in heparinized vacutainers. Lymphocyte cultures were set up by adding 0.5 ml whole blood to 7 ml of RPMI-1640 medium supplemented with 7% heat-inactivated fetal calf serum. Lymphocytes were stimulated by 1% PHA PHA
phytohemagglutinin, a plant lectin. and incubated for 72 h at 37 [degrees]C. Two cultures per individual were established. A final concentration of 80 [micro]l BrdU/7ml culture media at 0 h was added. At 69 h, 30 [micro]l colchicine (1 mg/5 ml) was added to the cultures to arrest cell division at metaphase metaphase /meta·phase/ (met´ah-faz) the second stage of cell division (mitosis or meiosis), in which the chromosomes, each consisting of two chromatids, are arranged in the equatorial plane of the spindle prior to separation. stage. The cultures were harvested after 72 h of incubation by first centrifuging at 1200r.p.m. for 10 min and treated with hypotonic hypotonic /hy·po·ton·ic/ (-ton´ik)
1. denoting decreased tone or tension.
2. denoting a solution having less osmotic pressure than one with which it is compared. solution (20 min in 0.075 M potassium chloride (KC1) at 37 [degrees]C). Cells were fixed in 3:1 (v/v) methanol:acetic acid solution acetic acid solution Lugol's solution, see there . The fixation step was repeated twice and the resulting cells were resuspended in a small volume of fixative fixative /fix·a·tive/ (fik´sit-iv) an agent used in preserving a histological or pathological specimen so as to maintain the normal structure of its constituent elements.
adj. and dropped onto clean slides.
[FIGURE 1 OMITTED]
Arsenic treatment: Stock solution (0.5 mg/ml or 25.2 x [10.sup.2] [micro]M) of [As.sub.2][O.sub.3] was prepared in double distilled water Double distilled water (abbreviated "ddH2O" or "Bidest. water") is prepared by double distillation of water. It is used, among other things, when single distillation does not lead to sufficiently pure water for some applications in biochemistry. and sterilized by autoclaving. Working concentrations were prepared by diluting the stock with double distilled water. Following three doses of arsenic were selected:
Low Dose [As.sub.2][O.sub.3] (LD): 0.5 [micro]g (3.6 x [10.sup.-4][micro]M/7ml culture), Medium Dose [As.sub.2][O.sub.3] (MD): 1.0 [micro]g (1.4 x [10.sup.-3] [micro]M/7 ml culture). High Dose [As.sub.2][O.sub.3] (HD): 2.0 [micro]g (0.72 x [10.sup.-3] [micro]M/7 ml culture).
Arsenic was added at 48th h after setting the culture (so as to give 24 h exposure). These doses were selected based on a study by Nair et al. (2004) followed by further standardization in our laboratory.
Kalmegh treatment: 4.2 mg of kalmegh was dissolved in 1 ml of double distilled water and sterilized by passing through a 0.22 [micro]m syringe filter. Working concentrations were prepared by diluting stock with double distilled water. Final concentration of 0.01 [micro]g/7ml culture media kalmegh was added at 48 h alone and in combination with HD arsenic. The dose was based on a study by Panossian et al. (2002) followed by further standardization in our laboratory.
EMS treatment: Lymphocytes treated with EMS at the dose of 240 [micro]g/ml (Dean and Danford, 1984) were given 24 h exposure period so as to assess SCEs and other genotoxic endpoints.
Experimental groups: Based on the treatments mentioned above, following groups were established:
Group I: Untreated (Control)
Group II: EMS (Positive Control)
Group III: Low dose [As.sub.2][O.sub.3] (LD)
Group IV: Medium dose [As.sub.2][O.sub.3] (MD)
Group V: High dose [As.sub.2][O.sub.3] (HD)
Group VI: Kalmegh alone
Group VII: High dose [As.sub.2][O.sub.3] + Kalmegh
Fluorescence-plus-Giemsa staining for SCE: The slides were stained with fluorescent dye Hoechst 33258 (stock: 1 mg/1 ml Sorenson's buffer, pH 6.8; this solution was diluted 10 times to prepare working solution) for 45 min in the dark. Slides were then kept in 2X saline sodium citrate (SSC solution--1.77g NaCl and 0.88 g sodium citrate in 100 ml distilled water) and then exposed in UV for 15 min. After exposure, slides were washed in distilled water and immediately stained in 2% Giemsa stain prepared in Sorenson's buffer for 10 min and then scored.
Analysis of cell cycle proliferative index/replicative index (CCPI/RI): Metaphases in their second in vitro division were selected for scoring on basis of the spreading of chromosomes and differentiation of chromatids. Hundred metaphases from each culture were analyzed for SCE. In these preparations, cells dividing for the first ([M.sub.1]), second ([M.sub.2]) and third ([M.sub.3]) divisions in the culture containing BrdU were designated by the differential staining pattern of sister chromatids. [M.sub.1] cells contained chromosomes with both sister chromatids stained uniformly dark. [M.sub.2] cells contained only differentially stained chromatids with one chromatid darkly stained and its sister chromatid light stained, whereas [M.sub.3] cells had (a) both the sister chromatids stained lightly, or (b) chromosomes with a dark and a faint chromatid (similar to [M.sub.2]), or (c) chromosomes with a portion that has sister chromatid differentiation and the remaining portion that has both lightly stained chromatids. Total 100 metaphases were analyzed for each individual classifying them as first ([M.sub.1]), second ([M.sub.2]) or third ([M.sub.3]) generation cells.
The CCPI for each individual was calculated according to the following formula:
CCPI/RI = (1 x %[M.sub.1]) + (2 x %[M.sub.2]) + (3 x %[M.sub.3])/Total plates (100).
SCE/plate and SCE/chromosome:
SCE/plate = total SCE scored/total [M.sub.2] metaphase plates observed
SCE/chromosome = SCE/plate/total no. of chromosomes in metaphase plates (= 46)
Average Generation Time (AGT AGT antiglobulin test. ) and Population Doubling Time (PDT):
The cell cycle time AGT is studied as the ratio of BrdU time (h) and replicative index (CCPI/RI)
AGT (h) = 72h (BrdU time)/[CCPI/RI].
PDT is the time in which the cells divide; i.e. in one cell cycle of 24h
PDT (h) = 24 h/[CCPI/RI].
Statistical analysis: Data was analyzed using Student's It test.
The results of the present study are shown as Tables 1 and 2.
SCE/cell and SCE/chromosome: A highly significant (p<0.001) increase was observed in the mean frequency of SCE/cell in arsenic treated cultures (group III-IV) compared to control (group I). In the cultures where kalmegh was supplemented along with arsenic (group VII), mean frequency of SCE/cell decreased (p<0.01) compared to that of high dose arsenic (group V).
Mean SCE/chromosome increased non-significantly with the increase in the dose of [As.sub.2][O.sub.3] (p<0.001) compared to control. However, compared to high dose [As.sub.2][O.sub.3] (group V), average SCE/chromosome declined non-significantly in cells co-cultured with kalmegh (Table 1).
CCPI/RI: The average CCPI decreased in a dose-dependent manner in arsenic treated cultures (groups III-V; non-significant in low and medium doses, p<0.05 in high dose). Co-culture of kalmegh with high dose arsenic (group VII) showed that mean value of CCPI recuperated (non-significantly) compared to high dose arsenic treated cultures (group V) (Table 1).
%[M.sub.1], [M.sub.2] and [M.sub.3]: Arsenic treated cultures (groups III-V) observed a significant (p<0.001) increase in %[M.sub.1] and a decrease in %[M.sub.2] plates as compared to control cultures. However, arsenic + kalmegh group (group VII) exhibited decrease (p<0.01) in %[M.sub.1] and an increase in %[M.sub.2] plates when compared to high dose arsenic group. [M.sub.3] plates were present only in the control cultures (Table 1).
AGT and PDT: Both PDT and AGT showed a remarkable (p<0.001) increase in the average frequencies with the increase in the dose of arsenic. However, cells co-cultured with arsenic and kalmegh (group VII) showed considerable revival (p<0.05) in mean values for AGT and PDT (non-significant) compared to high dose arsenic treated cultures (Table 2).
Positive control group, which was treated with EMS showed a significant (p<0.001) increase in the average incidence of SCE/cell, SCE/chromosome together with AGT and PDT when compared to control.
Arsenic is a recognized human multisite carcinogen, presently affecting millions of people worldwide as an environmental contaminant. The aim of this study was to evaluate the exposure of a variety of doses of [As.sub.2][O.sub.3] upon human peripheral blood lymphocytes Peripheral Blood Lymphocytes (PBL): These are the mature lymphocytes (small white immune cells) that are found circulating in the blood, as opposed to organs, such as the lymph nodes, spleen, thymus, liver or bone marrow. These cells consist of T cells, NK cells and B cells. and the likely favorable consequence of kalmegh upon arsenic-induced genotoxieity. The role of a known mutagen mutagen: see mutation.
Any agent capable of altering a cell's genetic makeup by changing the structure of the hereditary material, DNA. Many forms of electromagnetic radiation (e.g. EMS was also evaluated, which is considered mandatory while assessing compounds of unknown genetic activity.
SCEs are a consequence of the interchange of replicating DNA between chromatids at apparently homologous loci. They are thought to be a result of DNA breakage and reunion. The increased SCE/cell and SCE/chromosome after different arsenic treatments in the present study are in agreement with the other reports in human peripheral lymphocytes (HPLs) as well as in Syrian Hamster embryo cells (SHE) (Gebel, 2001; Rudel et al., 1996). The results of earlier work done in our laboratory (Nair et al., 2004) are also in agreement with that of present study.
Decreased CCP (Certified Computer Professional) The award for successful completion of a comprehensive examination on computers offered by the ICCP. See ICCP and certification.
1. (language) CCP - Concurrent Constraint Programming.
2. 1 and alterations in the %[M.sub.1], [M.sub.2], and [M.sub.3] in the arsenic treated cultures was noted, which is anticipatable, since arsenic caused a delay in the cell cycle. A number of reports on the cell cycle arrest and mitosis disturbance (Yih et al., 2002; Huang and Lee, 1998) and decreased CCPI/RI (Nair et al., 2004; Gonsebatt et al., 1992) induced by arsenic treatment have been reported previously.
Cell division tracking provides a unique opportunity for the analysis of cell growth kinetics. These include the average time between successive divisions (AGT) and time required to increase the total cell number to twofold (PDT). The amplified values for AGT and PDT obtained in the present study are in accordance with a pilot study on individuals chronically exposed to arsenic in Mexico, where AGT was longer in highly exposed group (Ostrosky-Wegman et al., 1991). Many investigators (Chatterjee and Giri, 1998; Pacchierotti et al., 2002) have reported increased AGT in vivo in mouse/human affecting delay in cell cycle progression with different test chemicals.
Though its precise mechanism of action remains unknown, arsenic does not directly damage DNA, but may act as a carcinogen through inhibition of DNA-repair mechanisms leading indirectly to increased mutations. A number of mechanisms are postulated for arsenic-induced genotoxicity Genotoxic substances are a type of carcinogen, specifically those capable of causing genetic mutation and of contributing to the development of tumors. This includes both certain chemical compounds and certain types of radiation. , the foremost of them being induction of free radicals and oxidative stress (Hei and Filipic, 2004; Ho et al., 2000). Such reports are further supported by the fact that distinguished antioxidants like ascorbic acid, N-acetyl cysteine cysteine (sĭs`tēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of mammalian protein. , melatonin, glutathione, catalase catalase /cat·a·lase/ (kat´ah-las) a hemoprotein enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen, protecting cells. , squalene squalene (skwäˑ·lēn),
n a popular traditional Asian remedy derived from the liver oil of sharks. , etc. reduce arsenite-induced chromosomal aberrations, SCEs and micro-nuclei, and protect cells from arsenite insults (Nair et al., 2004; Rao and Tiwari, 2006; Ho et al., 2000). Therefore, this study was undertaken to evaluate the role of A. paniculata extract, having extensive antioxidant properties (Singh et al., 2001; Wang et al., 1997) upon [As.sub.2][O.sub.3]-induced genotoxicity.
Andrographolide, the active principle of the herb A. paniculata is implicated for its pharmacological activity. Though kalmegh has been used traditionally and its beneficial characters remain variegated, nothing has been known about its antigenotoxic potential. This plant extract also has been accounted for subordinating the oxidative stress in various animal models besides preventing oxygen radical and [H.sub.2][O.sub.2] production in human neutrophils, thus suggesting that ROS ROS,
n.pr See reactive oxygen species. can be modulated by it (Zhang and Tan, 2000: Shen et al., 2002). Pharmacological studies have demonstrated that andrographolide supports conceptual hydrogen transfer, which would be thermodynamically favoured for its allylic al·lyl
The univalent, unsaturated organic radical C3H5.
[Latin allium, garlic + -yl (so called because it was first obtained from garlic). hydrogen on carbon C-11. Such hydrogen transfer results in andrographolyl radical, curtailing lipid peroxide formation. It is thus proposed that besides inhibiting lipid peroxidation by chain breaking, these could also scavenge other free radicals, mainly superoxides (Kam-dem and Ho, 2002), thus exerting antioxidant potential on arsenic genotoxicity. Though exact mechanism of action of Andrographis extract is not known, however, based on the above reports it is plausible that scavenging of free radicals and hydrogen transfer by andrographolide might play an important role in providing protection against arsenic-induced oxidative and genotoxic damage as evident from this study.
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Chatterjee, A., Giri, S.D., 1998. Modulation of mitomycin mitomycin /mi·to·my·cin/ (mi?to-mi´sin)
1. any of a group of antitumor antibiotics (e.g., mitomycin A, B, C) produced by Streptomyces caespitosus.
2. mitomycin C; used as a palliative antineoplastic. C-induced sister chromatid exchanges and cell cycle delay by buthionine sulfoximine and reduced glutathione in mouse bone marrow cells in vivo. Mutat. Res. 413, 227-234.
Dean, B.J., Danford, N., 1984. Assays for the detection of chemically induced chromosome damage in cultured mammalian cells. In: Venitt, S., Parry, J. (Eds.), Mutagenicity mutagenicity /mu·ta·ge·nic·i·ty/ (-je-nis´it-e) the property of being able to induce genetic mutation.
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1. in ciliate protozoa, the smaller of two types of nucleus in each cell, which functions in sexual reproduction; cf. macronucleus.
2. a small nucleus. in human fibroblasts. Mutat. Res. 452, 41-50.
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pertaining to mitosis.
degree to which a cell population is proliferating; used as an index of tumor aggression. division and perturbs spindle dynamics in HeLa S3 cells. Carcinogenesis 19, 889-896.
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1. hypertrophy of the stratum corneum of the skin, or any disease so characterized.
2. hypertrophy of the cornea. in inner Mongolia. Environ. Health Perspect. 114, 835-841.
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G. Avani*, M.V. Rao
Department of Zoology, University School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380 009, Gujarat, India
Received 28 December 2006; accepted 20 March 2007
Abbreviations: [As.sub.2][O.sub.3], arsenic trioxide; AGT, average generation time; BrdU, bromodeoxyuridine; CCPI/RI, cell cycle proliferative index/replicative index; EMS, ethyl methanesulphonate; HD, high dose [As.sub.2][O.sub.3]; HPL HPL - Language used in HP9825A/S/T "Desktop Calculators", 1978(?) and ported to the early Series 200 family (9826 and 9836, 68000). Fairly simple and standard, but with extensive I/O support for data acquisition and control (BCD, Serial, 16 bit custom and IEEE 488 interfaces), , human peripheral lymphocytes; KCI, potassium chloride; LD, low dose [As.sub.2][O.sub.3]; MD, medium dose [As.sub.2][O.sub.3]; PDT, population doubling time; PHA, phytoheamagglutinin; SCE, sister chromatid exchanges
E-mail address: firstname.lastname@example.org (G. Avani).
Table 1. SCE and CCPI in arsenic- and kalmegh-exposed cultures Parameters Group I Control Group II EMS SCE/cell 3.61 [+ or -] 0.27 7.42 [+ or -] 0.31* SCE/chromosome 0.08 [+ or -] 0.04 0.18 [+ or -] 0.09 (NS) CCPI/RI 1.34 [+ or -] 0.10 1.31 [+ or -] 0.07 (NS) %[M.sub.1] 62.2 [+ or -] 0.10 80.2 [+ or -] 0.51* %[M.sub.2] 36 [+ or -] 0.93 25.18 [+ or -] 0.57* %[M.sub.3] 2.20 [+ or -] 0.44 0.0 [+ or -] 0.0 Group III Group IV Parameters LD [As.sub.2][O.sub.3] MD [As.sub.2][O.sub.3] SCE/cell 6.47 [+ or -] 0.26* 9.81 [+ or -] 0.51* SCE/chromosome 0.14 [+ or -] 0.04 (NS) 0.21 [+ or -] 0.08 (NS) CCPI/RI 1.23 [+ or -] 0.05 (NS) 1.17 [+ or -] 0.07 (NS) %[M.sub.1] 76.8 [+ or -] 0.48* 86 [+ or -] 0.72* %[M.sub.2] 23.20 [+ or -] 0.48* 17 [+ or -] 0.72* %[M.sub.3] 0.0 [+ or -] 0.0 0.0 [+ or -] 0.0 Group V Group VI Parameters HD [As.sub.2][O.sub.3] Kalmegh alone SCE/cell 15.00 [+ or -] 0.49* 4.63 [+ or -] 0.35 SCE/chromosome 0.33 [+ or -] 0.07* 0.10 [+ or -] 0.05 CCPI/RI 1.11 [+ or -] 0.06* 1.28 [+ or -] 0.08 %[M.sub.1] 89.2 [+ or -] 0.59* 70.40 [+ or -] 0.97 %[M.sub.2] 10.80 [+ or -] 0.59* 29 [+ or -] 0.88 %[M.sub.3] 0.0 [+ or -] 0.0 0.0 [+ or -] 0.0 Group VII Parameters HD [As.sub.2][O.sub.3] + Kalmegh SCE/cell 11.39 [+ or -] 0.67 (#) SCE/chromosome 0.25 [+ or -] 0.10 (&NS) CCPI/RI 1.13 [+ or -] 0.07 (&NS) %[M.sub.1] 86.8 [+ or -] 0.75* %[M.sub.2] 13.20 [+ or -] 0.75 (#) %[M.sub.3] 0.0 [+ or -] 0.0 Values are Mean[+ or -]S.E. (#) p<0.01. &NS = Not Significant; when compared with group V. *p<0.001, NS = Not Significant; when compared with group I. Table 2. Population Doubling Time (PDT) and Average Generation Time (AGT) in arsenic- and Kalmegh-exposed cultures Group III LD [As.sub.2] Parameters Group I Control Group II EMS [O.sub.3] PDT 17.91 [+ or -] 20.51 [+ or -] 19.48 [+ or -] 0.35 0.21* 0.19* AGT 53.72 [+ or -] 57.45 [+ or -] 58.45 [+ or -] 0.61 0.28* 0.33* Group IV Group V Parameters MD [As.sub.2][O.sub.3] HD [As.sub.2][O.sub.3] PDT 20.52 [+ or -] 0.30* 21.67 [+ or -] 0.26* AGT 61.57 [+ or -] 0.53* 65.00 [+ or -] 0.45* Group VI Group VII HD Parameters Kalmegh alone [As.sub.2][O.sub.3] + Kalmegh PDT 18.70 [+ or -] 0.30 21.21 [+ or -] 0.32 (NS) AGT 53.40 [+ or -] 0.75 62.85 [+ or -] 0.51 (#) Values are Mean [+ or -] S.E. (#) p<0.05, NS = Not Significant: when compared with group V. *p<0.001 when compared with group I.