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Aquatic Toxicology 88 (2008) 214219

Contents lists available atScienceDirect

Aquatic Toxicology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a q u a t o x

Action mechanisms of petroleum hydrocarbons present in waters impactedby an oil spill on the genetic material ofAllium ceparoot cells

Daniela Morais Leme a, Dejanira de Franceschi de Angelis b, Maria Aparecida Marin-Morales a,

a Departamento de Biologia, Instituto de Biociencias, Universidade Estadual Paulista (UNESP), Rio Claro, SP, Brazilb Departamento de Microbiologia, Instituto de Biociencias, Universidade Estadual Paulista (UNESP), Av. 24A, 1515, 13506-900, Rio Claro, SP, Brazil

a r t i c l e i n f o

Article history:

Received 28 January 2008

Received in revised form 17 April 2008

Accepted 29 April 2008

Keywords:

Allium cepa

Chromosomal aberrations

Aneugenic and clastogenic agents

Cell death

Polluted waters

Petroleum hydrocarbons

a b s t r a c t

Chromosomal aberration (CA) assays have been widely used, not only to assess the genotoxic effects

of chemical agents, but also to evaluate their action mechanisms on the genetic material of exposedorganisms. This is of particular interest, since such analyses provide a better knowledge related to the

action of these agents on DNA. Among test organisms, Allium cepa is an outstanding species due to its

sensitivity and suitable chromosomal features, which are essential for studies on chromosomal damageor disturbances in cell cycle. The goal of the present study was to analyze the action mechanisms of

chemical agents present in petroleum polluted waters. Therefore, CA assay was carried out in A. cepa

meristematic cellsexposed to the Guaeca river waters, located inthe city of Sao Sebastiao, SP, Brazil, which

had its waters impacted by an oil pipeline leak. Analyses of the aberration types showed clastogenic andaneugenic effects for theroots exposed to the polluted waters from Guaeca river, besides theinductionof

cell death. Probably all the observed effects were induced by the petroleum hydrocarbons derived fromthe oil leakage.

2008 Elsevier B.V. All rights reserved.

1. Introduction

Chromosomal aberrations (CA) are characterized by changes in

either chromosomal structure or in the total number of chromo-somes, which may occur both spontaneously and as a result fromthe exposure to physicalor chemical agents(Russel, 2002). Physical

and chemical agents can induce CA through different mechanisms,involving clastogenic and aneugenic actions. Clastogenic action ischaracterized by the induction of chromosomal breakage during

cell division, while aneugenic action comprises the inactivation ofa cell structure, such as themitotic spindle, leading tochromosomal

losses (Fenech, 200 0).Several CA types are derived from nuclear abnormalities, suchas nuclear buds, micronuclei (MN), mini cells, lobated nuclei andpolinucleated cells. The MN can result from acentric fragments

(aneugenic agent) or whole chromosomes (clastogenic agent) thatwere not incorporated to the main nucleus during the cell cycle(Fenech, 200 0). According toFernandes et al. (2007), nuclear buds

areindicative of an initial process of releasing the exceddingnuclearmaterial and, consequently, they can also be related to the MN for-mation, which might be further eliminated from cytoplasm as mini

Corresponding author. Tel.: +55 19 3526 4143, fax: +55 19 3536 0009.

E-mail address: [email protected](M.A. Marin-Morales).

cells. Lobate nuclei and polynucleated cells are resultant of CA, as aconsequence of multipolar anaphases, which are associated or notwith chromosomal adherence,making the cellsinviable(Fernandes

et al., 2007).Because of the increasing chemical dumping in the environ-

ment, several bioassays have been carried out in order to evaluate

the effects induced by these agents. The CA assay, one of the oldestand most used tests, has been considered as one of the few directmethods capable of measuring mutations in systems exposed to

putative mutagenic or carcinogenic substances (Rank et al., 2002).Moreover, suchtest, based on classical cytogenetics, is easily carried

out in a great number of organisms, making it useful for environ-mental monitoring.Among the test organisms used in this assay, higher plants,

including Allium cepa, are acknowledged as excellent bioindica-

tors of genotoxic and mutagenic effectsof environmental chemicals(Grant, 1994, 1999). Thus, CA test in A. cepahas been considered asan efficient short-term assay for environmental pollutant evalua-

tion(Fiskesjo, 1985, 1988; Cotelle et al., 1999) and,in particular, forwater pollutants (Smaka-Kincl et al., 1996; Rank and Nielsen, 1993,1994; Matsumoto et al., 2006; Fatima and Ahmad, 2006; Migid etal., 2007,Leme and Marin-Morales, 2008). Furthermore, the chro-

mosomal features of this species favor carrying out the CA test,not only for genotoxic effect assessment, but also for understand-ing the action mechanisms of the tested chemicals (Fiskesjo, 1985;

Rank and Nielsen, 1997).

0166-445X/$ see front matter 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquatox.2008.04.012


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D.M. Leme et al. / Aquatic Toxicology 88 (2008) 214219 215

Besides its sensitivity, some researchers have also selected the

Allium test becauseof the high correlation betweenit andother test

systems. These features are essential to an accurate assessment ofenvironmental risks, as well as to a successful extrapolation of testresults obtained in exposed organisms to other species. Regard-

ing these characteristics, Fiskesjo (1985)showed that the Alliumtest presents a similar sensitivity to that of some algal and humanlymphocyte test systems.Rank and Nielsen (1994)showed a cor-relation of 82% between Alliumtest and carcinogenicity assays in

rodents, besides demonstratingthat the former was more sensitivethan Ames or microscreen tests. Furthermore,studies of sensitivityamongst higher plants have also showed that A. cepais more sen-

sitive than some other species, such as Vicia faba(Ma et al., 1995;Migid et al., 2007).

Taking into account that petroleum hydrocarbons can cause a

harmful effect on the genetic material of exposed organisms andthat their action mechanisms on DNA need to be understood, theaim of the present study was to analyze the action mechanisms of

petroleum hydrocarbon polluted waters impacted by an oil pipeline

leak.

2. Material and methods

2.1. Material

Water sample from the Guaeca river, located in the Serra do MarState Park, next to the city of Sao Sebastiao, SP, Brazil, was used

as a tested material due to petroleum hydrocarbon contaminationderived from an oil pipeline leakage, in February 2004 (Fig. 1). Thisriver runs through a permanent preservation area, being character-ized by a high surrounding biodiversity and by the proximity to the

city water reservoir. The oil leakage was a result of a crack in thepipeline, which is located underground. Thus, the leaked oil first

affected the underground waters and later has arisen in the Guaecariver spring, affecting the waters along its entire system. However,the total volume of oil leaked was not estimated, since the day theleakage started could not be accurately determined.

The water sampling was carried out in July 2005 at the springof the Guaeca river (Fig. 1). This was the oil leak surface area andthe only site where the presence of petroleum hydrocarbons was

detected through previous Total Petroleum Hydrocarbons (TPHs)and Polycyclic Aromatic Hydrocarbons (PAHs) chemical analyses(Table 1). Forthe watersampling procedure,the method of superfi-

cial water collection wasused, according to CETESB protocol(1987).A brand new20 l plastic container was usedfor the watersampling.The container was first submerged in the water where sampling

was going to take place for a initial rinse. Afterwards, the container

was submergedagainin thecollection site, butthis timein the mid-dle of the river, until it was completely filled up.Then,the container

was immediatelysealed up and cooled at 42 C to be transportedto the laboratory where the bioassay was carried out.

2.2. Test organism

Seeds of A. cepa were used as test organism because they

are genetically and physiologically homogeneous, besides beingavailable all year roundfeatures that ensured a reliable assay per-formance.

2.3. Test procedure

The chromosomal aberration assay using A.cepameristematiccells was carried out according to a modified version of Grantsprotocol (1982).

Fig.1. Location of Sao Sebastiao city,collection site (S1) in theGuaeca river andthepipeline responsible for the oil impact.

Onion seeds were germinated, at room temperature (20 5 C),in several Petri dishes, each dishcovered with filter paper and indi-vidually wetted with the Guaeca river water and the negative and

positive control treatments. Ultra pure water (Milli-Q) was usedas a negative control, and 4104 M of methyl methanesulfonate(MMS, SigmaAldrich, CAS 66-27-3) was used as positive control.

When the roots reached about 2.0 cm in length, approximately 5days after the beginning of the assay, they were fixed in alcohol-acetic acid (3:1-v/v) for 24 h. Then, the fixed roots were stained

with Schiff reagent. To prepare the slides, the meristematic regionswere coatedwith coverslipsand carefullysquashed into a drop of2%

acetic carmine solution. The coverslips were removed using liquidnitrogen and the slides were mountedin synthetic resin (MountingMedia, Permount, Fisher Scientific) to further analysis.

Several types of CA were analyzed within different cell division

stages (prophase, metaphase, anaphase and telophase) and classi-fied according to the action mechanism (aneugenic or clastogeniceffects). The chromosomal breaks and bridges were considered to

result from clastogenic effect, whereas the chromosomal losses,laggards, adherence, multipolarity and c-metaphases were consid-ered to result from aneugenic effect, since the latter aberrations

derive from disturbances in the mitotic spindle. Nuclear abnormal-ities,such as the presence of lobated nucleiand polynucleated cells,were used as indicators of cell death processes. The micronucleus(MN) incidence, as well as its size, has also been visually analyzed

to evaluate clastogenic and aneugenic effects.The analysis was done by scoring 5000 cells per treatment, 500

cells perslide, comprising a totalof 10 slides.Statistical analysiswas


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216 D.M. Leme et al. / Aquatic Toxicology 88 (2008) 214219

Table 1

TPHs and PAHs detected through chemical analysis in Guaeca river water sample

Sampling Site Chemical analysis Types of hydrocarbons Concentration (g/l) Detection limit (g/l)

July 2005 S1 TPHs C12 148.34 50.00

C13 148.34 50.00

C14 310.40 50.00 C15 271.53 50.00

C16 261.53 50.00

C17 966.30 50.00

C18 509.27 50.00

Total of n-alcanos 2615.71

MCNR 79896.62

PAHs Naphthalene 7.08 0.25

Acenaphthylene 4.56 0.25

Acenaphthene 1.91 0.25

Fluorene 15.12 0.25

Phenanthrene 78.78 0.25

Pyrene 9.43 0.25

Benzo(a)anthracene 3.82 0.25

Chrysene 18.88 0.25

Total of PAHs 139.58 MCNR: Complex mixture not resolved.

performed using the KruskalWallis test, accepting the probabilityof 0.05 to show a significant effect.

2.4. Chemical analysis

The analyses of the Total Petroleum Hydrocarbons and the Poly-

cyclic Aromatic Hydrocarbons (PAHs) in the Guaeca river watersample wereconducted by the Analytical Technology Company, SaoPaulo, SP, Brazil.

The TPHs analyses were performed according to the U.S.

EPA 8015 method, involving gase chromatography with flameionization detector (Gcfid). The PAHs analyses followed the

U.S. EPA 8270 method, using also the gas chromatography,but with mass detector (Gcms). This approach was able todetect the following PAHs: naphthalene, acenaphthylene, ace-

naphthene, fluorene, phenanthrene, anthracene, fluoranthene,pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene,benzo(k)fluoranthene, benzo(a)pyrene, indene(123-cd)pyrene,

dibenzo(a,h)anthracene, and benzo(g,h,i)perilene.

3. Results

TheresultsfromCAassayinA. cepa root cellsexposed to pollutedwater of Guaeca river are shown below inTable 2andFig. 2.

The CA and nuclear abnormalitiesobserved in the present studywere visualized in all stages of the cell cycle: interphase, prophase,metaphase, anaphase, and telophase (Fig. 2).

The cells in interphase showed the following abnormalities:

nuclear buds, MN, mini cells, lobate nuclei and polynucleatedcells (Fig. 2a1a6). In prophase, MN was the only type of abnor-mality observed (Fig. 2b1b3). In metaphase, we observed cells

with MN, chromosomal breaks, chromosomal losses, chromoso-mal adherence, and C-metaphases (Fig. 2c1c3). Cells in anaphaseshowed chromosomal bridges, chromosomal losses, multipolarity

and laggards in the chromosome segregation (Fig. 2d1d3). Buds,MN, chromosomal bridges and multipolarity were also observedin telophase cells (Fig. 2e1e3). Moreover, the MN observed in therootsexposed to Guaeca river water showed different sizes,ranging

from small to large MN (Fig. 2a2, a5, a6, b1b3, c3, e2, and e3).In the meristematic cells of A. cepa exposed to Guaeca river

waters, all the CA and nuclear abnormality frequencies were found

Table 2

CA and nuclear abnormalities in Allium cepa meristematic cells after exposure to

the polluted waters of Guaeca river and the negative (Milli-Q water) and positive

control (MMS) treatments

Milli-Q water MMS Guaeca river water

sample

Chromosomal aberrations

Chromosome breaks 0.01 0.31 0.16 0.87 0.05 0.67

Chromosome bridges 0.10 0.84 0.50 1.99* 0.38 2.45

Chromosome losses 0.01 0.31 0.14 1.13 0.31 1.45*

Laggards 0.01 0.31 0.07 0.51 0.10 0.70

Adherence 0.40

1.33 1.20

4.22

*

0.47

2.64Multipolarity 0.01 0.31 0.02 0.31 0.20 0.70

C-metaphases 0.08 0.70 0.07 0.51 0.38 2.82

Nuclear abnormalities

Lobated nuclei 0 0 0 0 1.05 6.65*

Polynucleated cells 0 0 0.29 3.37 0.01 0.31

Nuclear buds 0,07 0.96 0.48 1.71 1.86 8.37*

Micronucleus 0,12 0.95 0.53 2.28 2.68 9.10*

Mini cells 0 0 0.02 0.31 1.96 8.13*

5000 cells analyzed per treatment. MeanS.D.* Significantly different from negative control (p


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Fig. 2. Meristematic cells ofAllium cepaexposed to polluted water of Guaeca river. (a) Normal interphasic nucleus; (a1 ) nuclear bud; (a2) MN; (a3) mini cell; (a4) lobated

nucleus; (a5 and a6) polynucleated cells; (b) normal prophase; (b1) mini cellbeginning of the formation; (b2) prophase with large MN; (b3) prophase with a small MN; (c)

normal metaphase; (c1) metaphase with chromosomal break; (c2) metaphase with chromosomal loss; (c3) metaphase with MN; (d) normal anaphase; (d1) anaphase with

chromosomal bridge; (d2) anaphase with chromosomal losses; (d3) multipolar anaphase with bridge and chromosomal loss; (e) normal telophase; (e1) telophase with bud;

(e2) telophase with MN; (e3) telophase with MN and bridge.

and fluorescentin situhybridization (FISH), must be carried out to

corroborate the proposed hypothesis.According toFenech (2002), MN are determined from acentric

fragments (clastogenic agent) or whole chromosomes (aneugenic

agent) that were not incorporated to the main nucleus during thecell division cycle. Yamamoto and Kikuchi (1980) showed that,through measuring the MN diameter, it would be possible to deter-

mine if the agent tested was clastogenic or aneugenic. The authorsshowed that the MN derived from clastogenic agents were, in gen-

eral, smaller than MN derived from aneugenic action. Although thismethod has been satisfactory for a great number of MN, it does notshowreliableevidence in manyother cases.However,other authors(Combes et al., 1995; Fenech, 2000) concluded that the utilization

of MN size analysis as a parameter to determine xenobiotic clas-togenic or aneugenic actions would not be reliable enough, since,depending on the species, there are differences in relation to the

chromosome size in karyotypes, e.g. in human.Oppositeto man, some higherplantspecies,suchasA. cepa,have

a symmetric karyotype, which is homogeneous in relation to chro-

mosomal size, with large and few chromosomes (2n =16) (Grant,1982; Fiskesjo, 1985). Thus,the methodproposedby Yamamoto andKikuchi (1980), which considers the MN size to determine whether

an agent is clastogenic and/or aneugenic, can be effective for the A.

cepatest system.Chromosomal fragments can be derived from chromosomal

breaks in anaphase bridges, which can originate from cohesive

chromosomal translocations (Fiskesjo, 1993). Our data showed no

significant value for chromosomal breaks in A. cepameristematiccells exposed to Guaeca river waters. However, they showed, asmentioned above,significant valuefor MN withboth different sizes

and condensation levels (Table 2andFig. 2a2, a5, a6 , b1b3, c3, e2,and e3). Thus, the smaller MN can indicate the occurrence of chro-mosomal breaks, due to the clastogenic action of the petroleum

hydrocarbons present in the sample tested.Accordingto Fiskesjo andLevan(1993), Marcano andDel Campo

(1995),Marcano et al. (1999) and Turkoglu (2007), chromosomaladherence is a common sign of toxic effects on the genetic mate-rial and maycauseirreversible effectson the cell, triggering the celldeath process. Marcano et al.(2004) showed that the chromosomal

adherence can lead to chromosomal bridges and, thereby, chromo-somal breaks. The chromosomal bridges derived from adherencecan be multiple and can persist until telophase (Giacomelli, 1999).

Our data showed no significant chromosomal adherence value forthe roots exposed to Guaeca river waters (Table 2). However, thepresence of this abnormality can explain the presence of chromo-

somal bridges and breaks in these roots, as well as the aneugeniceffects of petroleum hydrocarbons.

C-metaphases are evidence of aneugenic agents, since they pro-

vide the complete inactivation of the cell mitotic spindle ( Fiskesjo,

1985, 1993). The spindles are inactivated when no equatorialplate is organized and, consequently, the centromere division is

blocked. According toKrisch-Volders et al. (2002) and Fernandes


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218 D.M. Leme et al. / Aquatic Toxicology 88 (2008) 214219

et al. (2007), the presence of C-metaphases can result in multi-nuclear cells, although the most frequent result is the induction

of a great amount of MN. The present data showed no significantC-metaphase value in A. cepa meristematic cells exposed to theGuaeca river water sample (Table 2). However, these results are

consistent with the data ofKrisch-Volders et al. (2002), since agreat number of MN on the cells exposed to polluted waters wasobserved, indicating the aneugenic effect induced by the presenceof petroleum hydrocarbons.

According to Rank and Nielsen (1998), multipolar anaphasesresult from a misfunction of the mitotic spindle, which leads tounbalanced chromosome distribution, heading them for more than

two poles onto the cells, opposite to what occurs in the normaldivision cycle. In the present study, no significant multipolarityvalue was observed for theA. cepa cells exposed to the water tested

(Table 2,Fig. 2a4).Fernades (2005) showed that lobated nuclei can result from

multipolar anaphases with chromosomal bridges. According to this

author, the presence of multipolarity during the nuclear division

seems not to avoid the reorganization of the nuclear envelope andthe membrane would follow the unbalanced distribution of the

genetic material within the cell, resulting in the lobated nuclei. Ourdata showed a significant number of lobated nuclei in the rootsexposed to the Guaeca river waters (Table 2, Fig. 2a4). We con-

clude that the presence of such nuclear abnormality leads to theinduction of cell death process, once they were not observed inthe F1 cells (non-meristematic region) ofA. cepa roots (Leme and

Marin-Morales, 2008).Some authors report that chromosomal losses and breaks, as

well as the excess material, promoted by the DNA replication, can

induce MN, which can be eliminated from the cell in the form ofmini cells, i.e., small cytoplasm portions with a reduced fractionof nuclear material (Fernandes et al., 2007). In the present study,

we observed significant numbers of mini cells in the meristematiccells ofA. cepaexposed to the Guaeca river water sample (Table 2andFig. 2a3). The presence of these mini cells seems to be a resultfrom the MN elimination of the multimicronuclei cells, which is

consistent with the data ofFernandes et al. (2007), studying effectsof aneugenic agents.

Taking into account that the A. cepa test system evaluates the

environmental risks present due to its high sensitivity and goodcorrelation with tests using other organisms (Fiskesjo, 1985; Rankand Nielsen, 1994; Fatima and Ahmad, 2006), we can infer that

the pollution caused by the oil leakage still affected Guaeca riverwaters, even oneyearafterthe accident,revealing that thepresenceof the petroleum hydrocarbons,althoughat lowconcentrations,can

causeharmful effectsin the exposed organisms.However, although

A. cepa has a good correlation with other test systems (Fiskesjo,

1985; Rank and Nielsen, 1994; Ma et al., 1995; Migid et al., 2007 ),the extrapolationof the present results requirescaution,since livingorganisms might have many biological differences, such as distinctmetabolic systems, which can lead to a differential action response

to chemical agents.Therefore, we can conclude that petroleum hydrocarbons can

show both clastogenic and aneugenic effects, as well as induce cell

death process, on the genetic material of exposed organisms. Inaddition, we can also conclude that these effects are probably aresult from the mixture of PAHs detected by the chemical analysis

of the Guaeca river water sample.In summary, we suggest that the CA assay in A. cepa, asso-

ciated with the MN size analysis, can be an efficient test toassess the action mechanisms of different chemical agents, includ-

ing petroleum hydrocarbons. However, it is important to carryout other cytogenetic techniques to accomplish more reliableresults.

Acknowledgements

We would like to thank Dr. Joao Carlos, C. Milanelli and InstitutoFlorestal, Sao Sebastiao, SP, Brazil, for his collaboration during fieldworks, and the Programa de Recursos Humanos ANP/FINEP/MCT-

CTPETRO, PRH-05 of Universidade Estadual Paulista (UNESP), RioClaro, SP, Brazil, for the financial support.

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