Genotoxic and cytotoxic effects of iron sulfate in cultured


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Genotoxic and cytotoxic effects of iron sulfate in cultured

  1. 1. Available online at Toxicology in Vitro 22 (2008) 723–729 Genotoxic and cytotoxic effects of iron sulfate in cultured human lymphocytes treated in different phases of cell cycle P.D.L. Lima a, M.C. Vasconcellos b, R.A. Montenegro b, C.M.L. Sombra b, M.O. Bahia a, L.V. Costa-Lotufo b, C.O. Pessoa b, M.O. Moraes b, R.R. Burbano a,* a ´ Human Cytogenetics Laboratory, Institute of Biological Sciences, Federal University of Para, Av. Augusto Correa, ´ 01, CEP 66075-110 Belem/PA, Brazil b ´ Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceara, Rua Coronel Nunes de Mello, ´ 1127, Rodolfo Teofilo, P.O. Box 3157, CEP 60431-970 Fortaleza/CE, Brazil Received 13 August 2007; accepted 19 November 2007 Available online 28 November 2007Abstract Iron (Fe) is a common chemical element that is essential for organisms as a co-factor in oxygen transport, but that in high amountspresents a significant risk of neurodegenerative disorders. The objective of this study was to evaluate the mutagenic potential of ironsulfate. The comet assay and chromosome aberration (CA) analysis were applied to determine the DNA-damaging and clastogeniceffects of iron sulfate. Human lymphocytes were treated in the quiescent phase for the comet assay and proliferative phase during theG1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle for CA analysis, with 1.25, 2.5 and 5 lg/mL concentrations ofFeSO4 Á 7H2O. All tested concentrations were cytotoxic and reduced significantly the mitotic index (MI) in all phases of the cell cycle.They also induced CA in G1, G1/S and S (pulses of 1 and 6 h) phases. Iron sulfate also induced polyploidy in cells treated during G1. Inthe comet assay, this metal did not induce significant DNA damage. Our results show that Fe causes alteration and inhibition of DNAsynthesis only in proliferative cells, which explain the concomitant occurrence of mutagenicity and cytotoxicity, respectively, in the lym-phocytes studied.Ó 2007 Elsevier Ltd. All rights reserved.Keywords: Iron sulfate; Chromosome aberration; Comet assay 1. Introduction Abbreviations: Fe, iron; FeSO4 Á 7H2O, iron sulfate heptahydrate; In an industrialized world, there are thousands of typesFeSO4, iron sulfate; CA, chromosomal aberration; COL, colchicine; of metals in use, and humans are exposed to them at work,DOX, doxorubicin; End, endoreduplication; FBS, fetal bovine serum; or as a result of contaminated food, water and environmentHAR, harvest; MI, mitotic index; PHA, phytohaemaglutinin; Polyp, (Ferrer, 2003). One feature of the normal human diet is thepolyploid cells; ROS, reactive oxygen species; AlCl3, aluminum chloride; simultaneous presence of both essential and toxic metals56Fe, high-energy iron ions; Fe-NTA, ferric-nitrilotriacetate; Fe2O5, ironoxide; SCE, sister chromatid exchange; FeCl3, ferric chloride; FeCl2, (Rojas et al., 1999).ferrous chloride. There is considerable evidence indicating an increase in * ´ Corresponding author. Present address: Laboratorio de Citogenetica´ neurodegenerative disorders in industrialized countries ´ ´ ˆHumana e Genetica Toxicologica, Instituto de Ciencias Biologicas, ´ (Veldman et al., 1998). The clinical symptoms and the pos- ´ ´Universidade Federal do Para, Campus Universitario do Guama, Av. ´ ´ ´Augusto Correa, 01, CEP 66075-110, Belem, Para. Tel.: +55 091 3183 sible mutagenic effects produced by acute poisoning by and1102/8802 7972; fax: +55 091 3183 1601. chronic exposure to metals are of significant interest (Fer- E-mail address: (R.R. Burbano). rer, 2003).0887-2333/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.tiv.2007.11.013
  2. 2. 724 P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 723–729 Populations are exposed to Fe mainly through foods 2. Materials and methodsand beverages. Good dietary sources of Fe include dryfruits, general grains, nuts, green vegetables, seeds, soy, 2.1. Chemical agentsmilk, coffee, teas, fish, red meat, eggs, chocolate and molas-ses (Souci et al., 2000), where Fe is also present in the Stock solutions were made by dissolving FeSO4 Á 7H2Odrinking water (WHO, 1996). (CAS No. 7782-63-0, Sigma Aldrich Co., St. Louis, MO, Fe is an essential trace element used by almost all living USA) in double-distilled water immediately prior to use.organisms, where it is often incorporated into the heme The concentrations of FeSO4 Á 7H2O used in the presentcomplex. Heme is a necessary component of cytochrome study (1.25, 2.5 and 5 lg/mL) are based on preliminaryproteins, which mediate redox reactions, and of oxygen experiments indicating that cells treated with FeS-transport proteins such as hemoglobin in red cells and O4 Á 7H2O concentrations equal to or higher than 10 lMmyoglobin in muscle cells. It can also be found in the bone produced total inhibition of cell division (data not shown).marrow, liver and spleen and is required in the immune sys- Doxorubicin hydrochloride (Adriamycin, CAS No.tem response and in the production of energy (Ganong, 25316-40-9) was purchased from Sigma Aldrich Co. (St.1993; Nelson and Cox, 2002). Louis, MO, USA). Although intake of this metal is regulated, large Phytohemagglutinin (PHA) was obtained from Abbottamounts of ingested Fe can cause excessive levels of Fe Laboratories, Maidenhead, UK. HAM F-10 growth med-in the blood, because high Fe levels can cause damage ium and fetal bovine serum were purchased from GibcoÒto the cells of the gastrointestinal tract which prevents (Invitrogen, Carlsbad, CA, USA). Colchicine was pur-them from regulating Fe absorption. The corrosive nat- chased from Sigma Aldrich Co. (St. Louis, MO, USA).ure of Fe seems to further increase its absorption, leadingto poisoning. In human beings, several alterations have 2.2. Test controlsbeen related to high Fe intake, especially in the pulmon-ary tract leading to cancer caused by inhalation of iron All drugs were dissolved in double-distilled water since itoxide (Chau et al., 1993), skin rashes by inhalation of did not induce chromosomal aberrations and/or reduce theFe salts (NIOSH, 1996), heart, kidney, liver and gastroin- mitotic index (data not shown). Furthermore, double-dis-testinal tract alterations and also diabetes mainly because tilled water was used as a negative control for all experiments.of the ingestion of high concentrations of iron sulfate The cytotoxic and mutagenic agent doxorubicin was(FeSO4) (0.5–2.5 g) found currently in drugs (Lima, used as positive control for lymphocyte cultures and comet2001). assay at a concentration of 0.01 lg/mL (Dhawan et al., Fe is moreover toxic to neural tissue leading to neurode- 2003).generative disorders (Montgomery, 1995; Campbell andBondy, 2000). It has been postulated that free Fe reacts 2.3. Lymphocyte culturewith peroxides to produce free radicals, which are highlyreactive and can damage DNA, proteins, lipids, and other Peripheral blood was collected from four normal,cellular components. Thus, Fe toxicity occurs when there is healthy donors, two women and two men, aged 19–30free Fe in the cell, which generally occurs when Fe levels years, with no history of smoking/drinking or chronic drugexceed the capacity of transferrin to bind Fe (Willmore use. A sample of 10 mL of venous blood was collected fromand Rubin, 1984). each donor into heparinized vials (5.000 IU/mL; Lique- ´ Several studies have been conducted to demonstrate mine; Roche). Short-term lymphocyte cultures were initi-the potential induction of DNA aberrations by Fe and ated according to a standard protocol (Preston et al.,also by drugs and compounds containing this metal. 1987). The culture medium consisted of 5 mL HAM-F10However, the results are inconclusive, and the mutagenic (78%), heat-inactivated fetal bovine serum (20%), phyto-effect of Fe is yet to be elucidated(Heidelberger et al., hemagglutinin-M (2%) and antibiotics (0.01 mg/mL of1983; Tucker et al., 1993; Abalea et al., 1999; Dunkel penicillin (Sigma Aldrich Co. St. Louis, MO, USA) andet al., 1999; Anderson et al., 2000a,b; Barbouti et al., 0.005 mg/mL of streptomycin (USB, Cleveland, OH).2001; Evans et al., 2001; Kostoryz and Yourtee, 2001; The culture tubes were incubated at 37 °C in a humidifiedPagano et al., 1996; Durante et al., 2002; Glei et al., atmosphere composed of 5% CO2 atmosphere with 95%2002; Evans et al., 2003; Garry et al., 2003; Franke al., 2006). The protocol of this study was approved by the Ethics On the basis of this data, the aim of the present study Committee of CNPq (Conselho Nacional de Desenvolvi-was to investigate the genotoxic, clastogenic and cytotoxic ´ mento Cientıfico e Tecnolo ´gico) – Brazil.effects of FeSO4 in different phases of the cell cycle usingshort-term cultures of human lymphocytes in vitro. The 2.4. Treatments and biological testsbioactivity parameters tested were the mitotic index (MI),chromosomal aberrations (CAs) and DNA damage index For cytogenetic analysis, FeSO4 Á 7H2O was studied atdetected by the comet assay. three concentrations (1.25, 2.5 and 5 lg/mL) at different
  3. 3. P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 723–729 725phases of the cell cycle. At G1, lymphocytes in complete 2.6. Comet assayculture medium were treated with a combination of0.2 mL phytohemagglutinin-M and FeSO4 Á 7H2O. The Peripheral venous blood was collected in heparinizedcells were fixed following 52 h of incubation at 37 °C. At vials as above from four normal, healthy donors, twotransition phase G1-S, the cultures were treated with the women and two men, aged 23–27 years, with no historydifferent concentrations of FeSO4 Á 7H2O 24 h after phyto- of smoking/drinking or chronic use of medication. Periph-hemagglutinin stimulation and were fixed 52 h after the ini- eral blood lymphocytes were isolated by Ficoll density gra-tiation of the culture. To determine the specific effects of dient (Hystopaque 1077; Sigma Diagnostics, Inc., St.iron sulfatein the S phase, pulse treatments with this metal Louis), incubated for 3 h with different concentrations offor 1 h and 6 h were performed 24 h after phytohemagglu- FeSO4 Á 7H2O (1.25, 2.5 and 5 lg/mL) and then mixed withtinin stimulation. Following each pulse treatment, cells low-melting point agarose.were washed once in serum-free medium, re-incubated in The alkaline version of the comet assay (single-cell gelcomplete medium, and fixed after 52 h of incubation. In electrophoresis) was performed in quiescent cells asthe G2 treatments, 69-h cultures were treated with iron sul- described by Singh et al. (1988) with minor modificationsfate for 3 h, and then fixed immediately (72 h total incuba- (Hartmann and Speit, 1997). Slides were prepared in dupli-tion) (Table 1). cate and 100 cells were screened per sample (50 cells from each duplicate slide) with a fluorescence microscope (Zeiss)2.5. Cytogenetic studies equipped with a 515–560 nm excitation filter, a 590 nm bar- rier filter, and a 40Â objective. Undamaged cells appeared In order to obtain a sufficient number of analyzable as intact nuclei without tails, whereas damaged cells hadmetaphases, colchicine was added at a final concentration the appearance of a comet. Comets were classified visuallyof 0.0016%, 2 h prior to harvesting. The cells were har- as belonging to one of five classes according to tail size andvested by centrifugation and treated with 0.075 M KCl at given a score of 0, 1, 2, 3 or 4 (from undamaged = 0, to37 °C for 20 min. The cells were then centrifuged and fixed maximally damaged = 4). Thus, the total damage scorein 1:3 (v/v) acetic acid:methanol. Finally, slides were pre- for 100 comets ranged from 0 (all undamaged) to 400 (allpared, air-dried and stained with 3% Giemsa solution maximally damaged) (Speit and Hartmann, 1999).(pH 6.8) for 8 min (Moorhead et al., 1960). Slides were analyzed with a light microscope, and struc- 2.7. Statistical analysistural (chromosome/chromatid gaps and breaks) andnumerical CAs were examined in metaphases from the iron Student’s t test was used to compare the frequencies ofsulfate-treated cultures and from the respective controls. CAs observed in cells exposed to the various concentra-The frequency of CAs (in 100 metaphases per culture) tions of Fe with the respective controls. The F testand the MI (number of metaphases per 2000 lymphoblasts (ANOVA) was used to detect significant differences in theper culture) were determined. MI results. For the comet assay, data were analyzed by The evaluation of gaps in chromosome aberrations tests one-way ANOVA followed by Tukey’s test. The level foris controversial since their exact biological meaning is statistical significance (p) was established at 5% in relationunknown. However, a study by Paz-y-Mino et al. (2002) ˜ to the negative control (Ayres et al., 2000).on peripheral blood lymphocytes exposed to low radiationdoses showed a high correlation between the comet assay 3. Resultsand the analysis of chromosome aberrations when chroma-tid and chromosome gaps were included in the analysis. 3.1. Chromosome aberrations and mitotic indexAccording to these investigators, this increased correlationsupports the hypothesis that gaps constitute a kind of chro- At G1 (12, 15 and 24 CAs at 1.25, 2.5 and 5 lg/mL,mosome aberration and should always be counted and respectively) and G1/S (18, 14 and 21 CAs at 1.25, 2.5evaluated in this kind of analysis. and 5 lg/mL, respectively), the frequency of CAs was sig- nificantly increased with all the concentrations of iron sul- fate tested (Table 2). During G1 (8, 14 and 12 polyploids atTable 1 1.25, 2.5 and 5 lg/mL, respectively), significantly inducedTreatment protocols of iron sulfate applied to short-term cultures ofhuman lymphocytes polyploidy was observed (Table 2). Iron sulfate treatment during S phase also resulted in significant increases in theTreatment PHA (h) Fe (h) Wash (h) COL (h) HAR (h) frequency of CAs at most concentrations; however, thereG1 0 0 – 50 52 were no significant differences between S-phase treatmentsG1/S 0 24 – 50 52S1 (1 h pulse) 0 24 24 50 52 of 1 (12, 18 and 24 CAs at 1.25, 2.5 and 5 lg/mL, respec-S6 (6 h pulse) 0 24 24 50 52 tively) and 6 h (14, 19 and 29 CAs at 1.25, 2.5 and 5 lg/G2 0 69 – 70 72 mL, respectively) (Table 3). The treatment at G2 (3, 2PHA: phytohemagglutinin; FBS: fetal bovine serum; COL: colchicine; and 5 CAs at 1.25, 2.5 and 5 lg/mL, respectively) didHAR: harvest. not induce a significant increase in the frequency of CAs
  4. 4. 726 P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 723–729Table 2 Table 5Chromosome aberrations (CAs) and mitotic index (MI) in cultured human Effect of iron sulfate or doxorubicin (DOX) on G0 quiescent cell damagelymphocytes treated with iron sulfate during the G1 and G1/S phases of index in comet assaythe cell cycle Substance Treatment Damage Damage Frequency Iron sulfate MI CAs Polypb Endc (lg/ml) Indexc (%)c treatment (%) Gaps Breaks Total Doxa 0.3 102 ± 28.54* 32.7 ± 10.01* a NCb – 19.50 ± 0.95 6.7 ± 0.47G1 NC 4.8 4 1 5 1 0 FeSO4 Á 7H2O 1.25 20.00 ± 2.3 10.00 ± 0.3 5 lg/mL 2.2* 16 8 24* 12* 0 2.5 14.25 ± 1 4.5 ± 0.64 2.5 lg/mL 2.6* 10 5 15* 14* 0 5.0 30.50 ± 8.2 7.0 ± 1.0 1.25 lg/mL 2.9* 8 4 12* 8* 1 a Dox 3.6* 6 3 9* 2 4 Doxorubicin, positive control. b a Negative control.G1/S NC 4.8 4 1 5 1 0 c Mean values and standard deviation obtained from average of 100 cells 5 lg/mL 2.2* 15 6 21* 0 0 per experiment – total of four experiments per dose for each substance. 2.5 lg/mL 2.4* 11 3 14* 0 0 * Data significant in relation to NC at: * p < 0.001. 1.25 lg/mL 2.6* 13 5 18* 0 0 DOXd 3.6* 13 8 21* 4* 0 a Negative Control. (Table 4). Chromatid gaps and chromatid breaks were the b Polyploid cells. most frequent CAs. c Endoreduplication. d Doxorubicin (positive control). The cytotoxic effects of iron sulfate were observed as * p < 0.05 in relation to the negative control. decreases in the MI of cultured lymphocytes treated during the G1, G1/S, S and G2 phases of the cell cycle (Tables 2–4).Table 3 3.2. Comet assayChromosome aberrations (CAs) and mitotic index (MI) in humanlymphocytes treated with iron sulfate during the S phase of the cell cycle None of the concentrations of iron sulfate tested differedIron sulfate treatment MI (%) CAs Polypb Endc statistically from the negative control (p > 0.05) (Table 5). Gaps Breaks Total Thus, the comet assay showed that exposure to iron sulfate did not cause significant increases in DNA damage.1h NCa 4.8 4 1 5 1 0 5 lg/mL 2.5* 15 9 24* 0 0 2.5 lg/mL 3.0* 14 4 18* 0 0 4. Discussion 1.25 lg/mL 3.5* 8 4 12* 0 0 DOX 3.2* 14 7 21* 1 1 Metal ions can generate DNA damage directly or induce6h NCa 4.8 4 1 5 1 0 the formation of reactive oxygen species (ROS), leading to 5 lg/mL 1.6* 18 11 29* 0 0 2.5 lg/mL 2.3* 15 4 19* 0 0 DNA damage indirectly probably via Fenton-like reactions 1.25 lg/mL 2.6* 8 6 14* 0 0 (Linder, 2001; De Freitas and Meneghini, 2001). Recently, DOXd 3.0* 21 5 26* 2 0 our research team showed that aluminum chloride (AlCl3) a Negative control. was genotoxic in human lymphocytes, at all concentrations b Polyploid cells. and phases of the cell cycle tested (Lima et al., 2007). In c Endoreduplication. addition, Pagano et al. (1996) demonstrated a high muta- d Doxorubicin (positive control). * genic and teratogenic potential of bauxite factory samples, p < 0.05 in relation to the negative control. where high levels of metals are found, including Fe. Low concentrations of Fe are not able to induce geno- toxic effects, since this metal is a common chemical elementTable 4 of cells, and is essential for organisms as a co-factor in oxy-Chromosome aberrations (CAs) and mitotic index (MI) in human gen transport (Ganong, 1993; Nelson and Cox, 2002).lymphocytes treated with iron sulfate during the G2 phase of the cell cycle However, at high concentrations, this metal presents a sig-Iron sulfate treatment MI (%) CAs Polypb Endc nificant risk for the development of population disorders. Gaps Breaks Total Neoplasias and cardiac, pulmonary, hepatic, gastrointesti-NCa 4.8 4 1 5 1 0 nal and renal alterations are related to Fe exposure. This5 lg/mL 3.8* 3 2 5 0 0 metal is also toxic to neural tissue and it is related to an2.5 lg/mL 4.0* 2 0 2 0 0 increased risk for the development of neurodegenerative1.25 lg/mL 4.3* 3 0 3 0 0DOXd 3.7* 14 6 20* 2 0 disorders (Willmore and Rubin, 1984; Lima, 2001; Chau a et al., 1993). Negative control. b Polyploid cells. Organic Fe may increase the genotoxic effects of other c Endoreduplication. compounds when they are combined. For example, the d Doxorubicin (positive control). mutagenic activity of doxorubicin is significantly increased * p < 0.05 in relation to the negative control. by this metal when evaluated by the Ames test (Kostoryz
  5. 5. P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 723–729 727and Yourtee, 2001). In addition Barbouti et al. (2001) dem- tion, including iron sulfate, by the comet assay. Theonstrated in Jurkat cells that simultaneous treatment with authors reported a genotoxic effect of this metal in mousedesferrioxamine (Fe chelator) and hydrogen peroxide, inhib- blood cells after 24 h treatment at all concentrations used.ited significantly the DNA damage induced by hydrogen Genotoxic effects of Fe were also reported by Garry et al.peroxide, indicating that intracellular Fe, which is redox- (2003) in rats treated with iron oxide (Fe2O5) for 24 metal, plays a role in the induction of DNA breaks These authors reported that this metal only showed ainduced by hydrogen peroxide. In our experiments, we used mutagenic potential when a simultaneous benzopyreneiron sulfate alone since we aimed to determine the concentra- treatment was carried out.tion where this metal begins to exert its genotoxic effects. We performed the comet assay with nonproliferating Our results showed that in the G1, G1/S and S phases of cells because these cells may be less prone to false-positivethe cell cycle, the frequency of CAs was significantly responses potentially associated with agents that interfereincreased, at all concentrations of iron sulfate tested with DNA synthesis by affecting cellular metabolism. The(1.25, 2.5 and 5 lg/mL). It is possible that Fe acts on consensus decision of an expert panel was that cells in sus-DNA synthesis, since the chromosomal alterations are evi- pension or monolayer culture should be exposed to the testdent when the cells are treated before and during the DNA substance for 3–6 h (Tice et al., 2000).replication. This hypothesis is based on the fact that in G2, In our study, the cytotoxic effects of Fe (detected by athe genotoxic effect of Fe is not evident at the concentra- decrease in MI) was observed at all concentrations testedtions tested. and all phases of the cell cycle, with a significant relationship In our study, as expected, there was a significant differ- between toxicity and increasing iron sulfate concentration.ence in the number of CAs produced between the lowest The presence of significant polyploid cells in G1 phase(1.25 lg/mL) and highest concentrations (5 lg/mL) of iron suggests that Fe has an effect on tubulin synthesis, whichsulfate. The occurrence of CAs in cells treated with Fe has occurs at high levels before S phase. Our results are inalso been demonstrated in studies using alternative Fe agreement with the study of Hasan et al. (2005) whocompounds. High levels of chromosomal and chromatid reported that ferritin, a ubiquitously distributed iron stor-aberrations were found in human lymphocytes and TK6 age protein, interacts with microtubules in vitro. In otherlymphoblast cells exposed to high-energy iron ions (56Fe) cell cycle phases, treatments showed the absence of poly-(Evans et al., 2001; Durante et al., 2002; Evans et al., ploid cells, and therefore, the majority of tubulin was syn-2003). Glei et al. (2002) detected a significant DNA dam- thesized in G1. Except in G1 phase, cytotoxicity wasage, determined by microgelelectrophoresis, in differenti- demonstrated throughout the cell cycle, probably relatedated human colon tumor cells (HT29 clone 19A) to factors that exclude the binding of Fe with the mitoticincubated with ferric-nitrilotriacetate (Fe-NTA). Muta- spindle.genic activity was also found in elemental and salt forms In conclusion, our results showed that Fe induces alter-of Fe, evaluated with the tests for mutagenicity in Salmo- ations and inhibition of DNA synthesis which explain,, thenella typhimurium and L5178Y mouse lymphoma cells concomitant occurrence of mutagenicity and cytotoxicity,(Dunkel et al., 1999). respectively, in the lymphocytes studied. It has also been reported that iron compounds are muta- Iron sulfate is a common chemical element that is pres-genic in cultured mammalian cells, as detected by Syrian ent in foods and beverages and that is used to treat iron-hamster embryo cell transformation/viral enhancement deficiency anemia (Nelson and Cox, 2002). Despite this,assay (Heidelberger et al., 1983), sister chromatid exchange in humans, several alterations have been related to high(SCE) in hamster cells (Tucker et al., 1993) and base tauto- Fe intake, especially neurodegenerative diseases (Chaumerization in rat hepatocyte cultures (Abalea et al., 1999). et al., 1993), and as found in this study, it can also cause Our comet assay results where the cells are treated in G0 genotoxic damage. Based on these facts, the intake of thisquiescent state showed that there are no genotoxic effects of metal must be regulated. In accordance with WHO (1996)iron sulfate in such experiments. These data confirm our the recommended Fe intake varies in accordance with age.notion that it is necessary that cells be cycling for Fe to For children of up to 3 months an intake of 1.7 mg/kg/dayexert its genotoxic effects. Anderson et al. (2000a, b) also is indicated, and for adults 18 mg/kg/day is indicated.reported little or no DNA damage occurred after treatment Other experiments must be carried out to prove theof human lymphocytes with the iron compounds ferric genotoxic action of this metal. The use of the comet assaychloride (FeCl3) and ferrous chloride (FeCl2); though sig- with enzymes that recognize oxidative damage in the DNAnificant DNA damage induced by ferrous sulfate was molecule would allow a better evaluation of the associationobserved only at high concentrations of ferrous sulfate, this of the genotoxic and neurotoxic activities of Fe.was probably a consequence of chemical contamination ofthe metal salt. Acknowledgements The proposed mechanism of action for Fe in proliferat-ing cells can be observed by the comet assay in in vivo treat- This work was supported by Financiadora de Estudos ements. Franke et al. (2006) investigated the mutagenic Projetos (FINEP CT-INFRA/FADESP) Grant No. 0927-potential of metallic agents used in dietary supplementa- ´ 03; Conselho Nacional de Desenvolvimento Cientıfico e
  6. 6. 728 P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 723–729 ´Tecnologico (CNPq) and by Coordenacao de Aperfeicoa- ߘ ß Glei, M., Latunde-Dada, G.O., Klinder, A., Becker, T.W., Hermann, U., ´mento de Pessoal de Nıvel Superior (CAPES). We thank Voigt, K., Pool-Zobel, B.l., 2002. Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A.Dr. A. Leyva English editing of the manuscript. Mutation Research 519, 151–161. Hartmann, A., Speit, G., 1997. The contribution of cytotoxicity to DNA-References effects in the single cell gel test (comet assay). Toxicology Letters 90, 183–188.Abalea, V., Cillard, J., Dubos, M.P., Sergent, O., Cillard, P., Morel, I., Hasan, M.R., Morishima, D., Tomita, K., Katsuki, M., Kotani, S., 2005. 1999. Repair of iron-induced DNA oxidation by the flavonoid Identification of a 250 kDa putative microtubule-associated protein as myricetin in primary rat hepatocyte cultures. Free Radical Biology & bovine ferritin. Evidence for a ferritin–microtubule interaction. The Medicine 26, 1457–1466. FEBS Journal 272, 822–831.Anderson, D., Yardley-Jones, A., Hambly, R.J., Vives-Bauza, C., Heidelberger, C., Freeman, A.E., Pienta, R.J., Sivak, A., Bertram, J.S., Smykatz-Kloss, V., Chua-Anusorn, W., Webb, J., 2000a. Effects of Casto, B.C., Dunkel, V.C., Francis, M.W., Kakunaga, T., Little, J.B., iron salts and haemosiderin from a thalassaemia patient on oxygen Schechtman, L.M., 1983. Cell transformation by chemical agents – a radical damage as measured in the comet assay. Teratogenesis, review and analysis of the literature. A report of the US Environmental Carcinogenesis and Mutagenesis 20, 11–26. Protection Agency Gene-Tox Program. Mutation Research 114, 283–Anderson, D., Yardley-Jones, A., Vives-Bauza, C., Chua-Anusorn, W., 385. Cole, C., Webb, J., 2000b. Effect of iron salts, haemosiderins, and Kostoryz, E.L., Yourtee, D.M., 2001. Oxidative mutagenesis of doxoru- chelating agents on the lymphocytes of a thalassaemia patient without bicin–Fe(III) complex. Mutation Research 490, 131–139. chelation therapy as measured in the comet assay. Teratogenesis, Lima, I.V., 2001. Ecotoxicologia do ferro e seus compostos. In: Martins, I. Carcinogenesis and Mutagenesis 20, 251–264. ˆ Lima, I.V. (Eds.), Cadernos de referencia ambiental, Salvador. pp. 112.Ayres, M., Ayres Jr., M., Ayres, D.L., Santos, A.S., 2000. Bioestat 2.0. Lima, P.D.L., Leite, D.S., Vasconcellos, M.C., Cavalcanti, B.C., Santos, ´ ´ Belem. Sociedade Civil Mamiraua, p. 272. R.A., Costa-Lotufo, L.V., Pessoa, C., Moraes, M.O., Burbano, R.R.,Barbouti, A., Doulias, P.T., Zhu, B.Z., Frei, B., Galaris, D., 2001. 2007. Genotoxic effects of aluminum chloride in cultured human Intracellular iron, but not copper, plays a critical role in hydrogen lymphocytes treated in different phases of cell cycle. Food and peroxide-induced DNA damage. Free Radical Biology & Medicine 31, Chemical Toxicology 45, 1154–1159. 490–498. Linder, M.C., 2001. Copper and genomic stability in mammals. MutationCampbell, A., Bondy, S.C., 2000. Aluminum induced oxidative events and Research 475, 141–152. its relation to inflammation: a role for the metal in Alzheimer’s disease. Montgomery, E.B., 1995. Heavy metals and the etiology of Parkinson’s Cellular and Molecular Biology 46, 721–730. disease and other movement disorders. Toxicology 97, 3–9.Chau, N., Benamghar, L., Pham, Q.T., Teculescu, D., Rebstock, E., Mur, Moorhead, P.S., Nomell, P.C., Mellman, W.J., Battips, D.M., Hunger- J.M., 1993. Mortality of iron miners in Lorraine (France): relations ford, D.A., 1960. Chromosome preparations of leucocytes cultured between lung function and respiratory symptoms and subsequent from human peripheral blood. Experimental Cell Research 20, 613– mortality. British Journal of Industrial Medicine 50, 1017–1031. 616.De Freitas, J.M., Meneghini, R., 2001. Iron and its sensitive balance in the ´ ´ Nelson, D.L., Cox, M.M., 2002. Lehninger princıpios basicos de bioquı- ´ cell. Mutation Research 475, 153–159. mica. In: Simoes, A.A., Lodi, W.R.N. (trads), 3ª ed. SARVIER, Sao ˜ ˜Dhawan, A., Kayani, M.A., Parry, J.M., Parry, E., Anderson, D., 2003. Paulo, p. 975. Aneugenic and clastogenic effects of doxorubicin in human lympho- NIOSH (National Institute of Occupational and Safety), 1996. Docu- cytes. Mutagenesis 18, 487–490. mentation for immediately dangerous to life or health concentration.Dunkel, V.C., San, R.H., Seifried, H.E., Whittaker, P., 1999. Genotoxicity <>. of iron compounds in Salmonella typhimurium and L5178Y mouse Pagano, G., His, E., Beiras, R., De Biase, A., Korkina, L.G., Iaccarino, lymphoma cells. Environmental and Molecular Mutagenesis 33, 28–41. M., Oral, R., Quiniou, F., Warnau, M., Trieff, N.M., 1996. Cytoge-Durante, M., Gialanella, G., Grossi, G., Pugliese, M., Scampoli, P., netic, developmental, and biochemical effects of aluminum, iron, and Kawata, T., Yasuda, N., Furusawa, Y., 2002. Influence of the their mixture in sea urchins and mussels. Archives of Environmental shielding on the induction of chromosomal aberrations in human Contamination and Toxicology 31, 466–474. lymphocytes exposed to high-energy iron ions. Radiation Research 43, ´ ´ ´ Paz-y-Mino, C., Davalos, M.V., Sanchez, M.E., Arevalo, M., Leone, P.E., ˜ 107–111. 2002. Should gaps be included in chromosomal aberration analysis?Evans, H.H., Horng, M.F., Ricanati, M., Diaz-Insua, M., Jordan, R., Evidence based on the comet assay. Mutation Research 516, 57–61. Schwartz, J.L., 2001. Diverse delayed effects in human lymphoblastoid Preston, R.J., San Sebastian, J.R., McFee, A.F., 1987. The in vitro human cells surviving exposure to high-LET (56)Fe particles or low-LET lymphocyte assay for assessing the clastogenicity of chemical agents. (137)Cs gamma radiation. Radiation Research 156, 259–271. Mutation Research 189, 175–183.Evans, H.H., Horng, M.F., Ricanati, M., Diaz-Insua, M., Jordan, R., Rojas, E., Herrera, L.A., Poirier, L.A., Ostrosky-Wegman, P., 1999. Are Schwartz, J.L., 2003. Induction of genomic instability in TK6 human metals dietary carcinogens?. Mutation Research 443 157–181. lymphoblasts exposed to 137Cs gamma radiation: comparison to the Singh, N.P., Mccoy, M.T., Tice, R.R., Schneider, E.L.A., 1988. Single induction by exposure to accelerated 56Fe particles. Radiation technique for quantitation of low levels of DNA damage in individual Research 159, 737–747. cells. Experimental Cell Research 175, 184–191.Ferrer, A., 2003. Metal poisoning. Anales del Sistema Sanitario de Souci, S.W., Fachman, W., Kraut, H., 2000. Food Composition and Navarra 26, 141–153. Nutrition Table, sixth ed. CRC Press, Boca Raton. ´Franke, S.I.R., Pra, D., Giulian, R., Dias, J.F., Yoneama, M.L., Silva, J., Speit, G., Hartmann, A., 1999. The comet assay (single-cell gel test). A Erdtmann, B., Henriques, J.A.P., 2006. Influence of orange juice in the sensitive genotoxicity test for the detection of DNA damage and levels and in the genotoxicity of iron and copper. Food and Chemical repair. Methods in Molecular Biology 113, 203–212. Toxicology 44, 425–435. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., ´Ganong, W.F., 1993. Fisiologia medica, 14ª ed. Prentice Hall do Basil, Rio Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F., 2000. de Janeiro (pp. 342–343). Single cell gel/comet assay: guidelines for in vitro and in vivo geneticGarry, S., Nesslany, F., Aliouat, E., Haguenoer, J.M., Marzin, D., 2003. toxicology testing. Environmental and Molecular Mutagenesis 35, Hematite (Fe(2)O(3)) enhances benzo[a]pyrene genotoxicity in endot- 206–221. racheally treated rat, as determined by Comet Assay. Mutation Tucker, J.D., Auletta, A., Cimino, M.C., Dearfield, K.L., Jacobson- Research 538, 19–29. Kram, D., Tice, R.R., Carrano, A.V., 1993. Sister-chromatid
  7. 7. P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 723–729 729 exchange: second report of the Gene-Tox Program. Mutation WHO (World Health Organization), 1996. Guidelines for Drinking-Water Research 297, 101–180. Quality Recommendations, second ed. Geneva.Veldman, B.A., Widn, A.M., Knoers, N., Pramstra, P., Horstink, M.W., Willmore, L.J., Rubin, J.J., 1984. The effect of tocopherol and dimethyl 1998. Genetic and environmental risk factors in Parkinson’s disease. sulfoxide on focal edema and lipid peroxidation induced by isocortical Journal of Neurochemistry 71, 295–301. injection of ferrous chloride. Brain Research 296, 389–392.