Induction of Chromosome Aberrations in the Allium Cepa Test System Caused by the Exposure of Cells to Benzo(a)pyrene
ORIGINAL PAPER
Induction of Chromosome Aberrations in the Allium Cepa Test System Caused by the Exposure of Cells to Benzo(a) pyrene Mirsad Cabaravdic Institute for Occupational Health of Sarajevo Canton
igher plants haveINTRODUCTION been proposed as test organisms for the detec- sented in the Picture 1. There are numerous car(PAU) are reports ongenotoxic tion of genotoxicPolycyclic substances in thearomatic environment.hydrocarbons Several plant test cinogenicity of BaP as well as of certain systems are already in use and are found to be as sensitive and relicommon environment pollutants, present in By the use able as other short-term tests. Allium cepa is one of these plants, which has representatives of PAU group (tumor of urinary bladder, lactic glands andsubstance intestheto air, food andaberrations potable water. They are been used in different studies detect chromosome induced by tinal tumors in experimental animals) chemicals. The use of non-animal test methods, including in vitro studies, generated in the processes of (2,3). incomplete macrosco Many PAU, including BaP too, are provides importent tools to enhance our understanding of hazardous effect established to induce genetic mutation combustion of fossil fuels and by industrial appearanc of chemicals, and for predicting these effects in humans. In vitro systems in bacteria and cells of mammals, actprocesses, is considered majorand generatinggentotoxic are used principally for screening purposes,which and for generating toxicologi- to ing be genotoxically chrocal profiles. Numerous chemicals can generate the breakage or interchange mosome aberration, exchange of sisters sources of PAU in urban air. Presence of frequency of DNA segments between chromosomal structures. Allium test is used as of chromatids and generation of micromajor quantity ofdifferent PAUchemical is established in . the abnormal Researches of genotoxica screening method for genotoxicity evaluation of sub- nucleus (4,5) of BaP indicate that it induces mustances, including polycyclic aromatic hydrocarbons (PAHs). Benzo(a)pyrene smoke of cigarettes, too, as wellity as inAmes the limited tations in test system (6,7), umu(BaP), as representative member of PAHs, were investigated of genotoxicity food prepared by baking, grilling and genotoxic SOS test systemin (8) as well as aberraby using Allium roote chromosomes assay. The treatment with different tions of chromosomes in cultures of the food model of series of concentrations smoked of BaP, ranging from (1). 1.0-50.0 µg/ml respectively. mammals cells (9). For this particular Used BaP caused decreased in the Mitotic index (MI)(BaP) and increase Benzo(a)pyrene is frequency a representative of of genotoxicalintention reason, evaluation /carof abnormal mitosis when compared with the control. Key words: Benzo(a) cinogenic potential it has for humans group of PAU. Benzo(a)pyrene ’s genotoxic pyrene, Allium cepa test,chemical Chromosome aberration, Genotoxicity. is particularly important and worthy
H
attention. chemical formula is C H withofmolecular Allium cep Plant tests of gentoxicity are used as weight of 252.3, and it is characterized bytest system for screefficient and quick chemical substances in evalupractical insolubility in water, but itaning is ofsoluble MATERIA ating mutagenocity and clastogenicity 1. INTRODUCTION it is characterized in organicandsolvents (5).by practical in- (10,11). Allium cepa test is used for dePolycyclic aromatic hydrocarbons solubility in water, but it is soluble in termination of genotoxical effects of Chemical Structural formula of BaP is presented in the (PAU) are common environment pol- organic solvents (5). different substances. By the use of this lutants, present in the air, food and po- 1. Structural formula of BaP is pre- tests, mutagenic effects of substances used in th Picture table water. They are generated in the may be analyzed by monitoring macrofollowing processes of incomplete combustion of scopic parameters, like the appearance fossil fuels and by industrial processes, NO: 50-3 and growth of the roots or by gentotoxic which is considered to be major sources parameters, like type and frequency of Chemical of PAU in urban air. Presence of major chromosome aberrations and abnormal quantity of PAU is established in the ( cell division (10,11,13,14). There isethanol limsmoke of cigarettes, too, as well as in ited number of researches on genotoxScientific) the food prepared by baking, grilling icity of BaP by the use of plant model of and in smoked food (1,2,3,4). CAS NO genotoxicity, and, actually, the intention Benzo(a)pyrene (BaP) is a repreof this work itself is to evaluate Sigma genoC sentative of chemical group of PAU. toxic potential of BaP at the cells of AlPicture 1. Structural formula of benzo(a)pyrene. Benzo(a)pyrene ’s chemical formula is USA). Source:EPA 2007 Source:EPA 2007lium cepa test system. C H with molecular weight of 252.3, Test sys Picture 1 - Structural formula of benzo(a)pyrene treatment MED ARH 2010; 64(4) • ORIGINAL PAPER 215 There are numerous reports on research
20 12,Canton Corresponding author: Ass Mirsad Cabaravdic, MD, PhD. Institute for Occupational Health of Sarajevo Bulevar Mese Selimovica 2, 71000 Sarajevo Bosnia and Herzegovina Telephone: 387 33 714-825 Telefax: 387 33 656-246 E-mail:
[email protected]
20
12,
Induction of Chromosome Aberrations in the Allium Cepa Test System Caused by the Exposure of Cells to Benzo(a)pyrene
PAU
Conc. µg/ml
Treat.(hr)
Control (DMSO) BaP 1.0 Control (DMSO) BaP 10.0 Control (DMSO)
24
Mitotic distribution %
MI±SD
P±SD
M±SD
A±SD
T±SD
M/P
14.2(3.52) 11.3(1.95)a 13.7(1.06) 10.1(4.60)a
36.7(2.06) 46.3(1.29)b 35.1(2.12) 46.7(3.07)b
30.3(0.60) 34.1(2.34)c 30.0(1.11) 32.6(1.85)b
25.6.(0.44) 12.6(2.02)c 26.8(1.00) 12.8(3.55)a
7.4(0.56) 6.9(1.99) 8.1(0.99) 10.9(3.74)a
0.82 0.73c 0.85 0.69c
14.1(1.95)
36.0(2.89)
31.2(1.78)
25.7(1.43)
BaP 50.0 8.7(3.61)b 49.1(1.09)b 20.2(2.07)b 21.7(4.59)a Control (DMSO) 14.2(3.52) 36.7(2.06) 30.3(0.60) 25.6.(0.44) BaP 1.0 10.8(3.10)a 47.2(1.55)b 21.6(0.80)a 22.5(2.40)a Control (DMSO) 48 13.7(1.06) 35.1(2.12) 30.0(1.11) 26.8(1.00) BaP 10.0 8.18(2.24)a 56.1(2.20)b 24.8(0.52)b 10.8(3.97)a Control (DMSO) 13.9(1.95) 36.0(2.89) 31.2(1.78) 25.7(1.43) BaP 50.0 7.01(1.61)a 61.2(3.13)b 19.1(6.14)a 14.4(0.77)c a-P≤ 0.05, b- P≤ 0.01, c- P≤ 0.001; B(a)P – benzo(a)pyrene; Each value given in the table is mean ± SD; MI –mitotic index,P-prophase, M-metaphase, A-anaphase, T-telophase, M/P-metaphase/prophase ratio.
7.1(2.01)
0.86
a6.7(2.80)* 7.4(0.56) 8.7(1.15)a 8.1(0.99) 8.5(2.56) 7.1(2.01) 5.5(4.16)a
0.41b 0.82 0.45c 0.85 0.44c 0.86 0.31c
Table 1. Mitotic index (MI) and distribution of mitosis in cells of Allium cepa treated with B(a)P in three different concentrations and two periods of treatment (24 i 48 hours)
2. MATERIAL AND METHOD Chemical material–Chemical substances used in the research are obtained from the following sources: benzo(a)pyrene (CAS NO: 50-32-8; purity >/= 98%) from Sigma Chemical Co.(St. Louis, Missouri, USA), ethanol (CAS NO:64-17-5; 95%, Fisher Scientific), dimethyl sulphoxide (DMSO, CAS NO:67-68-5; purity > 99.9%) from Sigma Chemical Co.(St. Louis, Missouri, USA). Test system and Allimu cepa testtreatment–A. cepa test was used in the research (2n=16) to evaluate clastogenic effects of BaP, in accordance with Fiskesjö method (10,11) with minor modifications. Onion bulb, without growth factors added, of 15-25 mm size, were obtained in commercial sale. Bulbs were made germinated in common pottable water during the course of 2-3 days, before roots reaches 3cm length, and after that they were treated by a serie of concentration of BaP 1.0, 10.0 and 50.0 µg/ml during the period of 24 and 48 hours. Benzo(a)pyrene was dissolved in DMSO, therefore the control solution contains DMSO. The amount of test compound to be used in the experiment were selected based on the preliminary cytotoxicity assays, so that the concentrations that are not genotoxic are not used in the experiments. After treatment, roots were fixed in the mixture of absolute ethanol and acetic acid (3:1 ratio) (Carnoy reagent) for 24h at 5oC. Slides have been prepared by using Feulgen squash technique tor analyze mitotic index and chromosomal aberrations (12). Both 216
contrencation and control as well require five preparations each, with marking of each preparation. At least 500 visible metaphase cells per each concentration and control are determined. Cytogenetic parameters–Cytogenetical analysis encompasses the determination of the following: a) mitotic index (MI); b) mitosis distribution; c) type and frequency of chromosomes aberration (structural and numerical ones). Statistical analysis–Significance of observed variables is estimated by analysis of variance (ANOVA), for mitotic index (P
