Available online at www.sciencedirect.com Toxicology in Vitro 22 (2008) 723–729 www.elsevier.com/locate/toxinvit 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: firstname.lastname@example.org (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
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 humidity.et 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
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
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
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 h.active 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
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