Chromosomal damage induced by androgenic anabolic steroids, stanozolol and trenbolone


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Naturally occurring anabolic steroids are synthesized in the testis testis (t***277;s`t***301;s) or testicle (t***277;s`t***301;k***601;l), one of a pair of glands that produce the male reproductive cells, or sperm. , ovary and adrenal gland from cholesterol via pregnenolone. Anabolic steroids bind to specific receptors present especially in reproductive tissue, muscle and fat [16]. Stanozolol is a synthetic androgenic anabolic steroid, similar to the naturally occurring androgen testosterone. It is used in the treatment of anemia and hereditary angioedema, which causes episodes of swelling of the face, extremities, genitals, bowel wall and throat [7]. Stanozolol may decrease the frequency of these attacks. It is one of the anabolic steroids commonly used by athletes and bodybuilders, although there is no clear evidence that anabolic steroids enhance overall athletic performance [5].

Like stanozolol, trenbolone is also a synthetic steroid, usually used by veterinarians on livestock as a growth promotant in animal husbandry [19]. To increase its effective half-life, trenbolone is not used in an unrefined form, but is rather administered as trenbolone acetate or trenbolone enanthate. Trenbolone is then produced as a metabolite by the reaction of these compounds with androgen receptor. No trenbolone compounds have been approved by the Food and Drug Administration, USA, for human use, due to a lack of clinical applications and considerable negative side effects. However, bodybuilders use the drug illegally to increase body mass and strength more effectively than by weight training alone.

The extensive use of these two steroids, legally and illegally, by humans and their ability to act as 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. agents, as suggested by earlier studies using mammalian systems, was a good enough reason for us to investigate their genotoxic potential using cultured human lymphocytes.

Materials and methods

Chemicals and reagents

Stanozolol (Sigma); Trenbolone (Sigma); RPMI1640 (Gibco); Fetal calf serum (Gibco); 0.1 ml Antibiotic-antimycotic mixture (Gibco); Phytohaemagglutinin-M (Gibco); Colchicine colchicine (k***335;l`ch***601;s***275;n'), alkaloid extracted from plants of the genus Colchicum and especially from the corms of the autumn crocus, Colchicum autumnale (see meadow saffron). (Microlab); Hoechst 33258 stain (Fluka); 3% Giemsa solution in phosphate buffer, pH 6.8 (Merck); 5-Bromo-2-deoxyuridine, BrdU (Sigma); Dimethylsulphoxide, DMSO DMSO dimethyl sulfoxide.

Dimethyl sulfoxide; a colorless hygroscopic liquid obtained from lignin, used as a penetrant to convey medications into the tissues.

n. (Merck); Mitomycin C (Merck); Cyclophosphamide cyclophosphamide /cy·clo·phos·pha·mide/ (-fos´fah-mid) a cytotoxic alkylating agent of the nitrogen mustard group; used as an antineoplastic, as an immunosuppressant to prevent transplant rejection, and to treat some diseases , CP (Sigma). For S9 mix preparation, Swiss albino healthy rats (Wistar strain) and 0.1% Phenobarbital were used.

Lymphocyte culture and chromosomal aberration analysis

Peripheral blood cultures were done in duplicate [3]. Heparinized blood (0.5 ml) samples were taken from healthy female donors, and were placed in sterile tubes containing 7 ml of RPMI1640, supplemented with 1.5 ml of fetal calf serum and 0.1 ml of phytohaemagglutinin Phytohaemagglutinin (PHA, or phytohemagglutinin) is a lectin found in plants, especially beans. It is found in the highest concentrations in uncooked red kidney beans (Phaseolus vulgaris), and it is also found in lower quantities in many types of green bean. , and incubated at 37 C for 24 hours Adv. 1. for 24 hours - without stopping; "she worked around the clock"
around the clock, round the clock . Stanozolol and trenbolone, both o dissolved in dimethyl sulphoxide (DMSO), were added separately to different cultures at the doses of 1, 10, 20, 40 and 60 [micro]M. DMSO (5 ml/ml) served as negative control. For metabolic activation experiments, liver S9 fraction was freshly prepared as per standard procedure [14]. 0.5 ml of S9 mix was added along with each treatment after 24 hours of the commencement of culture. Negative control was also given 0.5 ml of S9 mix.

Two hours before harvesting, 0.2 ml of colchicine (0.2 mg/ml) was added to the culture tubes. Cells were centrifuged at 800-1000 rpm for 10 min. The supernatant was removed and 5 ml of prewarmed (37[degrees]C) 0.075 M KCl 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 was added. Cells were resuspended and incubated at 37[degrees]C for 15 min. The supernatant was removed by centrifugation and 5 ml 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. (methanol: glacial acetic acid glacial acetic acid
Acetic acid that is at least 99.8 percent pure. , 3:1) was added. The fixative was removed by centrifugation and the procedure was repeated twice. The slides were stained in Giemsa solution in phosphate buffer for 15 min. At least 300 metaphases were analyzed for different types of chromosome breakage frequencies. Classification of aberrations was done according to the guidelines of the International Programme on Chemical Safety The International Programme on Chemical Safety (IPCS) is a collaboration between three United Nations bodies—the World Health Organization, the International Labour Organization and the United Nations Environment Programme. , WHO, Geneva Geneva, canton and city, Switzerland
Geneva (j***601;n***275;`v***601;), Fr. Genève, canton (1990 pop. 373,019), 109 sq mi (282 sq km), SW Switzerland, surrounding the southwest tip of the Lake of Geneva. , for the study of genetic defects in human population.

Sister chromatid exchange Sister chromatid exchange is the exchange of genetic material between two identical sister chromatids.

It was firstly discovered by using giemsa staining method on one chromatid belonging to the sister chromatid complex before anaphase in mitosis. analysis

For SCE analysis, BrdU (10 mg/ml) was added at the beginning of the culture. After 24 hours, same treatments were given as for CA analysis. Two hours before harvesting, 0.2 ml of colchicine (0.2 mg/ml) was added, followed by hypotonic treatment and fixation and processing of slides [18] was done as for CA analysis. Slides were stained for 20 min in 0.05% (w/v) Hoechst 33258 solution, rinsed with tap water and placed under a UV lamp for 90 min covered with Sorensen's buffer (pH 6.8) and stained with Giemsa solution in phosphate buffer for 15 min. The sister chromatid exchange average was taken from an analysis of 50 metaphases.

Statistical analysis

Student's t-test was used for calculating the statistical significance in CAs and SCEs. The level of significance was tested using standard statistical tables [23].

Results and discussions


The number of abnormal cells with CAs was insignificantly low at treatment doses of 1, 10, 20 mM, respectively, for both steroids being tested for 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. , in the presence as well as absence of metabolic activation i.e. S9 mix (Tables 1 and 2). A dramatic increase in genotoxic damage was noticed when the dosage was increased to 40 and 60 mM. At this dosage level, the abnormal cells more than doubled, although the dose-dependent increase in CAs was small from 40 to 60 mM.

SCE frequency in human lymphocytes treated separately with stanozolol and trenbolone (Tables 3 and 4), again with and without S9 mix, followed a pattern similar to the one observed for CAs. At 1, 10 and 20 mM, the SCE frequency remained quite low and showed little increase. But as the dosage increased to 40 mM and then 60 mM, a more than three fold increase in SCEs per cell was observed. Again, the dose-dependent rise in SCE frequency was small going from 40 to 60 mM.


The use of stanozolol in sports is illegal and banned by the International Association of Athletics Federations. It has a large oral bioavailability due to 17 a C alpha-alkylation which allows the hormone to survive first pass liver metabolism. High doses of stanozolol could exert a proliferative effect on liver cells [2]. Acute overdosage can produce nausea and gastrointestinal upset. Chronic use of stanozolol can cause menstrual irregularities and virilization virilization /vir·il·iza·tion/ (vir?i-li-za´shun) masculinization; usually used for that occurring in a female or prepubertal male.

n. in women and impotence, premature cardiovascular disease and prostatic hypertrophy in men. Precocious prostatic cancer has been described after long term anabolic steroid abuse [20]. Cases where hepatic cancers have been associated with anabolic steroid abuse have been reported [17]. Also, androgen ingestion by a pregnant mother can cause virilization of a female fetus [4].

Trenbolone compounds have a binding affinity for the androgen receptor thrice as high as that of testosterone. Once metabolized, the drugs have the effect of increasing nitrogen uptake by muscles, leading to an increase in the rate of protein synthesis. They also have the secondary effects of stimulating appetite, reducing the amount of fat being deposited in the body, and decreasing the rate of catabolism. Some short term side effects include insomnia, high blood pressure and increased aggression and libido. However, women suffer virilization effects even at small doses [29].

The only legitimate therapeutic indications for such anabolic steroids are in the replacement of male sex steroids in men who have androgen deficiency, e.g. due to loss of both testes, in the treatment of certain rare forms of aplastic anemia which are or may be responsive to anabolic anabolic

pertaining to or arising from anabolism.


anabolic steroid
steroids with a tissue-building effect. Testosterone is an example of a natural anabolic steroid with the, sometimes undesirable, effect of causing masculinization. androgens, and in certain countries to counteract catabolic states, e.g. after major trauma.

This experiment implies that the synthetic anabolic steroids, stanozolol and trenbolone, have the potential to cause genotoxic damage in human lymphocytes in vitro at higher dosage both in the presence and absence of S9 mix. Changes in chromosome structure due to a break or a swapping of chromosomal material are termed as CAs. Most of the CAs in cells are lethal, but many of them are also viable and can cause genetic effects, either somatic or inherited [27]. These events can lead to the loss of chromosomal material at mitosis or to the inhibition of exact chromosome segregation at anaphase anaphase /ana·phase/ (an´ah-faz) the third stage of division of the nucleus in either meiosis or mitosis.

n. . The result of these changes is cell lethality [28]. In our experiment, we came across significant differences compared with control in the CA frequency at 40 and 60 mM, with or without S9 mix. SCE is usually a more sensitive indicator of genotoxic effects than CA [28]. There is a correlation between the carcinogenicity and SCE inducing ability of many chemicals. Moreover, the SCE induction mechanism is heterogeneous and very different from the mechanism of CA induction [8]. Androgenic steroids display teratogenic ter·a·to·gen·ic
Of, relating to, or causing malformations of an embryo or a fetus.



pertaining to or emanating from teratogen. effects in all species that have been studied so far, and do so in a very predictable and consistent way [13]. Various psychological and physiological effects have been reported in both males and females among frequent users of androgens [15]. There is little, if any, information available on the exact reasons for the genotoxic behavior of stanozolol and trenbolone. However, the present study is concurrent with the studies performed on synthetic steroids such as cyproterone cy·prot·er·one
A synthetic steroid that inhibits the secretion of androgens.



a synthetic steroid that inhibits the secretion of androgens. acetate, ethynodiol diacetate, chlormadinone acetate, medroxyprogesterone acetate, norgestrel norgestrel /nor·ges·trel/ (-jes´trel) a synthetic progestational agent used as an oral contraceptive..

n. and megestrol acetate that induced CAs and SCEs with or without metabolic activation system [1,10,21,22,23,25,26].


The outcome of this investigative experiment shows that stanozolol and trenbolone have the potential to be genotoxic and cytotoxic, especially at 40 and 60 mM, with or without metabolic activation, in cultured human lymphocytes. The evaluation of these genotoxicity tests is a useful tool for determining the toxic effects of potentially genotoxic chemicals, leading to identification of such carcinogenic agents. It is advisable to use the steroids studied here at their lowest effective dosage so that the risk to public health could be minimized. The risk of damage to human genetic material is very likely at higher doses of these drugs.


We gratefully acknowledge the indispensable laboratory facilities provided by the Chairman, Department of Zoology, AMU amu atomic mass unit.

atomic mass unit , for this experiment to be successfully conducted.


[1.] Ahmad, M.E., G.G.H.A. Shadab, M.A. Azfer and M. Afzal, 2001. Evaluation of Genotoxic Potential of Synthetic Progestins--Norethindrone and Norgestrel in Human Lymphocytes In Vitro. Mutation Research, 494: 13-20.

[2.] Boada, L.D., M. Zumbado, S. Torres, A. Lopez, B.N. Diaz-Chico, J.J. Cabrera and O.P. Luzardo, 1999. Evaluation of Acute and Chronic Hepatotoxic hep·a·to·tox·ic
Damaging or destructive to the liver.



causing liver damage. Effects Exerted by Anabolic-Androgenic Steroid Stanozolol in Adult Male Rats. Archives of Toxicology, 73(8-9): 465-472.

[3.] Carballo, M.A., S. Aluarez and A. Boveris, 1993. Cellular Stress by Light and Rose Bengal in Human Lymphocytes. Mutation Research, 288: 215-222.

[4.] Dewhurst, J. and R.R. Gordon, 1984. Fertility Following Change of Sex: a Follow-up. Lancet, 22; 2(8417-8418): 1461-1462.

[5.] Elashoff, J.D., A.D. Jacknow, S.G. Shain and G.D. Braunstein, 1991. Effects of Anabolic-Androgenic Steroids on Muscular Strength. Annals of Internal Medicine Annals of Internal Medicine (Ann Intern Med) is an academic medical journal published by the American College of Physicians (ACP). It publishes research articles and reviews in the area of internal medicine. Its current editor is Harold C. Sox. , 115(5): 387-393.

[6.] Fisher, R.A. and Y. Yates, 1963. Statistical Table for Biological, Agricultural and Medical Research, 6th edition. Oliver and Boyd, Edinburgh.

[7.] Gannon, T., 1994. Dermatologic Emergencies. When Early Recognition can be Lifesaving. Postgrad Medicine, 96(5): 28-30.

[8.] Gebhart, E., 1981. Sister Chromatid Exchange (SCE) and Structural Chromosome Aberrations in Mutagenicity mutagenicity /mu·ta·ge·nic·i·ty/ (-je-nis´it-e) the property of being able to induce genetic mutation.


the property of being able to induce genetic mutation. Testing. Human Genetics, 58: 235-254.

[9.] Haynes, R.C. Jr. and F. Murad, 1985. Adrenocorticotropic hormone; Adrenocortical adrenocortical /adre·no·cor·ti·cal/ (-kor´ti-k'l) pertaining to or arising from the adrenal cortex.

Of, relating to, or derived from the adrenal cortex. steroids and their synthetic analogs; Inhibitors of adrenocortical steroid biosynthesis Biosynthesis

The synthesis of more complex molecules from simpler ones in cells by a series of reactions mediated by enzymes. The overall economy and survival of the cell is governed by the interplay between the energy gained from the breakdown of compounds . In The pharmacological basis of therapeutics, Eds., Gilman, A.G., L.S. Goodman, T.W. Rall and F. Murad. New York: Macmillan Publishing Co., pp: 1459-1489.

[10.] Hundal, B.S., V.S. Dhillon and I.S. Sidhu, 1997. Genotoxic Potentials of Estrogens Estrogens
Hormones produced by the ovaries, the female sex glands.

Mentioned in: Acne, Polycystic Ovary Syndrome

estrogens (es´tr***333;jenz),
n. . Mutation Research, 389: 173-181.

[11.] International Programme on Chemical Safety, 1985. Environmental Health Criteria 46. Guidelines for the study of genetic effects in human population. WHO, Geneva, 25-54.

[12.] International Programme on Chemical Safety, 1993. Poisons Information Monograph 918, Pharmaceutical. WHO, Geneva. PIM G007.

[13.] Juchau, M.R., 1997. Chemicals recognized as teratogenic in humans. In Progress in drug research, Vol. 49, Eds., Jucker, E.. Boston: Birkhauser Verlag, pp: 25-92.

[14.] Maron, D.M. and B.N. Ames, 1983. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research, 113: 173-215.

[15.] McEvoy, G.K., 1995. AHFS 95 drug information. American Society of Health System Pharmacists, Bethesda, MD., pp: 2128.

[16.] Mooradian, A.D., J.E. Morley and S.G. Korenman, 1987. Biological Actions of Androgens. Endocrine Reviews, 8(1): 1-28.

[17.] Overly, W.L., J.A. Dankoff, B.K. Wang and U.D. Singh, 1984. Androgens and Hepatocellular Carcinoma in an Athlete. Annals of Internal Medicine, 100(1): 158-159.

[18.] Perry, P. and S. Wolff, 1974. New Giemsa Method for Differential Staining of Sister Chromatids. Nature, 251: 156-158.

[19.] Richold, M., 1988. The Genotoxicity of Trenbolone, a Synthetic Steroid. Archives of Toxicology, 61(4): 249-258.

[20.] Roberts, J.T. and D.M. Essenhigh, 1986. Adenocarcinoma of Prostate in a 40-year-old Body-builder. Lancet, 2: 742.

[21.] Siddique, Y.H. and M. Afzal, 2004 (a). Evaluation of Genotoxic Potential of Synthetic Progestin Chlormadinone Acetate. Toxicology Letters, 153: 221-225.

[22.] Siddique, Y.H. and M. Afzal, 2004 (b). Evaluation of Genotoxic Potential of Ethynodiol Diacetate in Human Lymphocytes In Vitro. Current Science, 86: 1161-1165.

[23.] Siddique, Y.H. and M. Afzal, 2004 (c). Induction of Chromosomal Aberrations and Sister Chromatidchromatid (kr***333;`m***601;t***601;d): see chromosome; crossing over.
..... Click the link for more information. Exchanges by Cyproterone Acetate in Human Lymphocytes. International Journal of Human Genetics, 4(3): 187-191.

[24.] Siddique, Y.H., G. Ara, T. Beg and M. Afzal, 2006. Genotoxic Potential of Medroxyprogesterone Acetate in Cultured 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. . Life Sciences, 80: 212-218.

[25.] Siddique, Y.H., T. Beg and M. Afzal, 2005 (a) Genotoxic Potential of Ethinylestradiol in Cultured Mammalian Cells. Chemico-Biological Interactions, 151: 141-144.

[26.] Siddique, Y.H., T. Beg and M. Afzal, 2005 (b) Antigenotoxic Effects of Ascorbic Acid Against Megestrol Acetate Induced Genotoxicity in Mice. Human and Experimental Toxicology, 24: 121-127.

[27.] Swierenga, S.H.H., J.A. Heddle hed·dle
One of a set of parallel cords or wires in a loom used to separate and guide the warp threads and make a path for the shuttle.


[Probably alteration of Middle English helde , E.A. Sigal, J.P.W. Gilman, R.L. Brillinger, G.R. Douglas and E.R. Nestmann, 1991. Recommended Protocols Based on a Survey of Current Practice in Genotoxicity Testing Laboratories, IV. Chromosome Aberration and Sister Chromatid Exchange in Chinese Hamster Ovary V79, Chinese Hamster Lung and Human Lymphocytes Cultures. Mutation Research, 246: 301-322.

[28.] Tucker, J.D. and R.J. Preston, 1996. Chromosome Aber rations, Micronuclei , 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. , Sister Chromatid Exchanges and Cancer Risk Assessment. Mutation Research, 365: 147-159.

[29.] Wikipedia, 2007. The Free Encyclopedia. Trenbolone - Wikipedia, the free encyclopedia.

Corresponding Author

Mohammad Afzal, Human Genetics and Toxicology Laboratory, Department of Zoology, Aligarh Muslim UniversityAligarh Muslim University was established by the Indian Muslims and the Act of Indian Parliament made it University. It is located in the city of Aligarh, Uttar Pradesh, India.
..... Click the link for more information., Aligarh (UP), 202 002, India.


Tanveer Beg, Yasir Hasan Siddique and Mohammad Afzal

Human Genetics and Toxicology Laboratory, Department of Zoology, Aligarh Muslim University, Aligarh (UP), 202 002, India.

Tanveer Beg, Yasir Hasan Siddique and Mohammad Afzal,: Chromosomal Damage Induced by Androgenic Anabolic Steroids, Stanozolol and Trenbolone, in Human Lymphocytes, Am.-Eurasian J. Sustain. Agric., 1(1): 39-43, 2007