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Anticlastogenic effects of ascorbic acid against the genotoxic damage induced by norethynodrel.

Introduction

Synthetic progestins are widely used as oral contraceptives in addition to their use in the treatment of various menstrual disorders, cancers and in hormone replacement therapy. For contraception they are used either alone or in combination with estrogens [9]. Norethynodrel is a synthetic progestin used either as single entity drug or in combination with estrogen, such as ethinylestradiol or mestranol in oral contraceptives [9]. Norethynodrel alone and in combination with mestranol was tested for its carcinogenicity in mice, rats hamsters and monkeys orally and by subcutaneous implantation[7]. Increased incidences of pituitary, mammary, vaginal and cervical tumors were found in female mice and of pituitary tumors in male mice both with and without mestranol [7]. Norethynodrel, increases tumors and decreased the latency period for tumor development in castrated male mice [15]. It fails to induced aneuploidy in cultured human cells, unscheduled DNA synthesis in cultured rat hepatocytes and was also negative in Ames test [8]. In our earlier study norethynodrel was found to be genotoxic at 60 mg/ml in cultured human lymphocytes in the presence of metabolic activation [17]. The prolonged use of synthetic progestins/oral contraceptives has been reported to developed various types of cancers in humans [9]. In this context the natural antioxidants/ plant products may provide protection against the genotoxic damage of synthetic steroids, thereby decreasing the possibility of carcinogenesis in the frequent use s of synthetic steroids or contraceptives [19,23,21,18,20].

The present work deals with the study of the effect of ascorbic acid against the genotoxic damage induced by norethynodrel using chromosomal aberrations and sister chromatid exchanges as parameters.

Materials and methods

Chemicals

Norethynodrel (CAS No.: 68-23-5) (17a)-17- Hydroxy-19-norpregn-5(10)en-20-yn-3-one (Sigma); R PMI 1640, Fetal calf serum, Phytohaemagglutinin-M, Antibiotic-antimycotic mixture (Gibco); Dimethylsulphoxide, Colchicine, Giemsa Stain (Merck); Hoechst 33258 (Sigma); 5-Bromo-2-deoxyuridine, Ascorbic acid (SRL, India).

Lymphocyte culture

Duplicate peripheral blood lymphocyte culture were performed according to Carballo et al. [2]. Briefly, 0.5 ml of heparinized blood samples were obtained from two healthy females donors and were placed in a sterile culture bottle containing 7 ml of RPMI 1640 medium, supplemented with 1.5 ml of fetal calf serum, 0.1 ml of phytohaemagglutinin and 0.1 ml of antibiotic-antimycotic mixture and were placed in an incubator at 37[degrees]C for 24 hr.

Preparation of S9 mix

Liver microsomal fraction (S9 mix) was prepared from Swiss albino healthy rats (Wistar strain) each weighin about 200 g. The rats were given 0.1% of Phenobarbital (1 mg/ml) in drinking water for 1 week for the induction of liver enzymatic activities. The S9 mix was freshly prepared as per standard procedures of Maron and Ames [12]. The animals were he sacrificed and livers were obtained immediately. The liver slices were the carefully homogenized at 4[degrees]C in 25 mM phosphate buffer (pH 7.2), containing 27 mM KCl and 21 mM Mg[Cl.sub.2]. The homogenate was centrifuged at 4[degrees]C for 10 min at 9000 rpm. The S9 fraction was enhanced by addition of 5 mM of NADP and 10 mM of glucose-6-phosphate just before use. An amount of 0.5 ml of S9 mix was given along with each of the treatment. After 2 hr the S9 mix and drugs were removed by washing. Negative and positive controls were also given, 0.5 ml of S9 mix after 24 hr of the initiation of culture.

Chromosomal aberration analysis

After 24 hr, the treatment of norethynodrel (60 mg/ml) was given separately and along with 20, 60 and 80 mM of ascorbic acid. The cells were cultured for another 48 hr at 37[degrees]C in an incubator. An amount of 0.2 ml of colchicine (02. mg/ml) was added to the culture flask, 1 hr prior to harvesting. Cells were then centrifuged at 1000 rpm for 10 min. The supernatant was removed and 5 ml of prewarmed (37[degrees]C) 0.075 Molar KCl (hypotonic solution) was added. Cells were resuspended and incubated at 37[degrees]C for 15 min. The supernatant was removed by centrifugation and subsequently 5 ml of chilled fixative (methanol: glacial acid; 3:1) was added. The fixative was removed by centrifugation and the procedure was repeated twice. Slides were prepared and stained in 3% Giemsa solution in phosphate buffer (pH 6.8) for 15 min. Three hundred metaphases were examined for the occurrence of different types of abnormalities. Criteria to classify different types of aberrations were in accordance with the recommendation of EHC 46 for Environmental Monitoring of Human Population [10].

Sister chromatid exchange analysis

For sister chromatid exchange analysis, bromodeoxyuridine (BrdU, 10 mg/ml) was added at the beginning of the culture. After 24 hr, separate treatment of norethynodrel (60 mg/ml) and with 20, 40 and 80 mM of ascorbic acid were given. The cells were cultured for another 48 hr at 37[degrees]C in an incubator. Mitotic arrest was attempted 1 hr prior to harvesting by adding, 0.2 ml of colchicine (0.2 mg/ml). Hypotonic treatment and fixation were done in the same way, as described for chromosomal aberrations analysis. The slides were processed according to Perry and Wolff[14]. The sister chromatid exchange induction was analysed from 50 plates of second division mitosis per dose.

Statistical analysis

Student's t-test was used for the analysis of chromosomal aberrations and sister chromatid exchanges. Regression analysis was performed using Statistica Soft Inc.

Results and discussions

Results

The results of the present study reveal dose dependent significant decrease in the number of abnormal metaphases when norethynodrel (16 mg/ml) was treated with ascorbic acid (20, 40 and 80 mM) (Table 1). The chromatid exchanges induced by norethynodrel were completely eliminated by even the lowest dose of ascorbic acid (20 mM) (Table 1). A significant decrease in the sister chromatid exchanges per cell was observed when norethynodrel (60 mg/ml) was treated with the different dosages of ascorbic acid i.e. 20, 40 and 80 mM (Table 2). Regression analysis was also performed to determine the dose effect of ascorbic acid on 60 mg/ml of norethynodrel for number of abnormal metaphases and sister chromatid exchanges. A decrease in the slope of linear regression lines was observed as the dose of the ascorbic acid was increase, in each of the treatment. For abnormal metaphases, the treatment of 60 mg/ml of norethynodrel (F=8.33; P<0.08), with the increase in the dosages of ascorbic acid result in the decrease in slope of the linear regression line (Fig. 1). For sister chromatid exchange analysis, the treatment of 60 mg/ml of norethynodrel (F=3.15; P<0.03), with the increase in the dosages of ascorbic acid, the decrease in slope of linear regression line was observed (Fig. 2).

Discussion

The results in the present study clearly demonstrate that ascorbic acid is potent in reducing the genotoxic damage of norethynodrel. The metabolic activation of norethynodrel, may results in the generation of reactive species that are responsible for the genotoxic damage. Similar observations on the DNA damaging properties of synthetic steroids as is evident from chromosomal damage, induction of sister chromatid exchanges [24,16,3,18] have also been reported earlier. Cytochrome P450 in liver S9 mix plays an important role in activation of promutagens to mutagens [12].

Metabolic activation of estrogens such as estradiol and ethinylestradiol give rise to various forms of quinones that are responsible for the genotoxic damage in various tests systems [4,11,5,18,1,6]. Vitamins act as an antioxidant and free radical scavengers, thereby acting as an anticarcinogenic, anticlastogenic and antimutagenic agents. Chromosomal aberrations are the changes in chromosome structure resulting from the break or an exchange of chromosomal material. Most of the chromosomal aberrations are lethal, but there are many corresponding aberrations that are viable and can cause genetic effects either somatic or inherited. High frequency of chromosomal aberrations is the indication of the possibility of the carcinogenesis [25]. Sister chromatid exchanges is a more sensitive indicator of genotoxic effects [27].

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

There is a correlation between the carcinogenicity and SCE inducing ability of a large number of chemicals [25]. Our earlier studies with ascorbic acid and other synthetic progestins have also been shown to possess antigenotoxic and anticlastogenic potential in both in vivo and in vitro studies [23,19,20,22,21]. Ascorbic acid is an antioxidant and plays a role in scavenging of free radicals [26]. The protective effect observed in the present study, i.e. significant reduction in the frequency of cell with chromosomal damage and sister chromatid exchanges may be due to the antioxidant property of ascorbic acid. In a very recent "Multi Centre Study" on oral contraceptives and liver cancer the project team came to the conclusion that oral contraceptives may enhance the risk of liver carcinomas [13]. The treatment of ascorbic acid reduced the frequency of chromosomal aberrations and sister chromatid exchanges in the test system, thereby indicating the possibility of reducing the changes of carcinogenesis during the steroid therapy in patients.

Conclusion

It is concluded that ascorbic acid it potent enough to reduced the genotoxic damage of norethynodrel in the presence of metabolic activation and may reduce the possible risk of carcinogenesis during the steroid therapy in patients.

Acknowledgments

Thanks are due to the CSIR, New Delhi for awarding grant No. 9/112(355)/2003-EMR to the author (YHS) and to the Chairman, Department of Zoology, Aligarh Muslim University, Aligarh, 202002 (U.P) for laboratory facilities.

References

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[9.] IARC, 1999. International Agency for Research on Cancer: Monograph on the evaluation of carcinogenic risk to humans. Hormonal contraception and post-menopausal hormone therapy. Lyon France, 72: 49-338.

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[13.] Martelli, A., F. Mattioli, M. Angiola, R. Reimann and G. Brambilla, 2003. Species, sex and inter individual differences in DNA repair induced by nine sex steroids in primary cultures of rat and human hepatocytes. Mutation Research, 561: 69-78.

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[15.] Rudali, G., 1975. Induction of tumours in mice with synthetic sex hormones. Gann Monograph, 17: 243-252.

[16.] Siddique, Y.H. and M. Afzal, 2004. Evaluation of genotoxic potential of synthetic progestin ethynodioldiacetate in human lymphocytes in vitro. Current Sciene, 86: 1161-1165.

[17.] Siddique, Y.H. and M. Afzal, 2005a. Evaluation of genotoxic potential of norethynodrel in human lymphocytes in vitro. Journal of Environmental Biology, 26: 387-392.

[18.] Siddique, Y.H. and M. Afzal, 2005b. Protective role of allicin and L-ascorbic acid against the genotoxic damage indued by chlormadinone acetate in cultured human lymphocytes. Indian Journal of Experimental Biology, 43: 769-772.

[19.] Siddique, Y.H., G. Ara, T. Beg and M. Afzal, 2006b. Effect of vitamin C on cyproterone acetate induced genotoxic damage in mice. Research Journal of Biological Sciences, 1: 69-73.

[20.] Siddique, Y.H., G. Ara, T. Beg and M. Afzal, 2007. Additive action of vitamin C and E against norgestrel induced genotoxicity. Biomedical Research, 18: 155-160.

[21.] Siddique, Y.H., T. Beg and M. Afzal, 2005a. Antigenotoxic effects of ascorbic acid against megestrol acetate induced genotoxicity in mice. Human and Experimental, Toxicology, 24: 121-127.

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[23.] Siddique, Y.H., T. Beg and M. Afzal, 2006a. Protective effect of nordihydroguaiaretic acid (NDGA) against norgestrel induced genotoxic damage. Toxicology in vitro, 20: 227-233.

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Corresponding Author

Yasir Hasan Siddique, Human Genetics and Toxicology Laboratory, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh (U.P.) 202002, India.

Email: yasir_hasansiddique@rediffmail.com

Yasir Hasan Siddique, Tanveer Beg, Mohammad Afzal

Human Genetics and Toxicology Laboratory, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh (U.P.) 202002, India.

Yasir Hasan Siddique, Tanveer Beg, Mohammad Afzal,: Anticlastogenic Effects of Ascorbic Acid Against the Genotoxic Damage Induced by Norethynodrel, Am.-Eurasian J. Sustain. Agric., 1(1): 2732, 2007
Table 1: Effect of ascorbic acid on chromosomal aberrations
induced by norethynodrel.

 Abnormal metaphases without gaps
Treatment Cells
 scored Number % [+ or -] S.E.

Norethynodrel
60 mg/ml 300 30 10 [+ or -] 1.73 (a)
Norethynodrel
(mg/ml) + AA (mM)
60 + 20 300 15 5.0 [+ or -] 1.25 (b)
60 + 40 300 10 3.33 [+ or -] 1.03 (b)
60 + 80 300 7 2.33 [+ or -] 2.87 (b)
AA (mM)
20 300 2 0.67 [+ or -] 0.47
40 300 3 1.00 [+ or -] 0.57
80 300 3 1.00 [+ or -] 0.57
Untreated 300 2 0.67 [+ or -] 0.47
Negative control 300 3 1.00 [+ or -] 0.57
(DM SO, 5 ml/ml)
Positive control 300 140 46.7 [+ or -] 2.88
CP (0.16 mg/ml)

 Chromosomal aberrations
Treatment
 Gaps CTB CSB CTF DIC

Norethynodrel
60 mg/ml 24 31 11 2 --
Norethynodrel
(mg/ml) + AA (mM)
60 + 20 10 18 6 -- --
60 + 40 6 11 3 -- --
60 + 80 4 8 2 -- --
AA (mM)
20 1 2 -- -- --
40 1 2 1 -- --
80 2 2 1 -- --
Untreated 1 2 -- -- --
Negative control 1 2 1 -- --
(DM SO, 5 ml/ml)
Positive control 56 58 38 6 4
CP (0.16 mg/ml)

AA: Ascorbic acid; CTB: Chromatid break; CSB: Chromosome break;
DIC: Dicentric; CTE: Chromatid exchange; CP: Cyclophosphamide;
DMSO: Dimethylsulphoxide

(a) Significant with respect to untreated (P<0.01).

(b) Significant with respect to Norethynodrel (P<0.05)

Table 2: Effect of ascorbic acid on sister chromatid exchanges
induced by norethynodrel.

Treatments SCEs/Cell (Mean [+ or -] SE) Range

Norethynodrel (mg/ml)
60 8.64 [+ or -] 0.52 (a) 2-9
Norethynodrel (mg/ml)
 + AA (mM)
60 + 20 3.32 [+ or -] 0.38 (b) 2-5
60 + 40 3.02 [+ or -] 0.36 (b) 1-5
60 + 80 2.94 [+ or -] 0.34 (b) 1-5
AA (mM)
20 2.02 [+ or -] 0.22 0-5
40 2.26 [+ or -] 0.28 0-5
80 2.84 [+ or -] 0.32 1-5
Untreated 1.68 [+ or -] 0.18 0-5
Negative control
 (DMSO, 5 ml/ml) 1.74 [+ or -] 0.20 0-5
Positive control CP
 (0.16 mg/ml) 14.42 [+ or -] 0.64 (a) 2-15

AA: Ascorbic acid; DMSO: Dimethylsulphoxide; CP:
Cyclophosphamide.

(a) Significant with respect to untreated (P<0.01).

(b) Significant with respect to Norethynodrel (P<0.05).
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Title Annotation:Original Article
Author:Siddique, Yasir Hasan; Beg, Tanveer; Afzal, Mohammad
Publication:Advances in Environmental Biology
Article Type:Report
Date:Sep 1, 2007
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