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Genotoxic and cytotoxic effects of manganese chloride in

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    Genotoxic and cytotoxic effects of manganese chloride in Genotoxic and cytotoxic effects of manganese chloride in Document Transcript

    • Available online at www.sciencedirect.com Toxicology in Vitro 22 (2008) 1032–1037 www.elsevier.com/locate/toxinvit Genotoxic and cytotoxic effects of manganese chloride in cultured human lymphocytes treated in different phases of cell cycle P.D.L. Lima a, M.C. Vasconcellos b, M.O. Bahia a, R.C. Montenegro b, C.O. Pessoa b, L.V. Costa-Lotufo 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, Para, 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, Ceara, Brazil Received 2 September 2007; accepted 20 December 2007 Available online 31 December 2007Abstract Manganese (Mn) has a natural occurrence and is necessary during the initial periods of the development. However, in high concen-trations, Mn can be related to neurodegenerative disorders. The aim of the present study was to evaluate the mutagenic potential of man-ganese chloride (MnCl2 Á 4H2O). Comet assay and chromosome aberrations analysis were applied to determine the DNA-damaging andclastogenic effects of MnCl2 Á 4H2O. Cultured human lymphocytes were treated with 15, 20 and 25 lM manganese chloride during theG1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle. All tested concentrations were cytotoxic and reduced significantly themitotic index in G1, G1/S and S (1 and 6 h) treatments, while in G2 treatment only the higher concentrations (20 and 25 lM) showedcytotoxic effects. Clastogenicity and DNA damage were found only in treatments with the highest concentration (25 lM). Chromosomeaberrations were found exclusively in the G2 phase of the cell cycle. The absence of polyploidy in mitosis, suggests that manganese doesnot affect the formation of the mitotic spindle with the concentrations tested. The genotoxicity found in G2 phase and in the comet assaycan be related to the short time of treatment in both cases.Ó 2007 Elsevier Ltd. All rights reserved.Keywords: Manganese chloride; Chromosome aberration; Comet assay1. Introduction deficiency can affect agriculture worldwide (Gerber et al., 2002). In animals, Mn acts in physiological processes such Manganese (Mn) is the 5th most abundant metal and as the regulation of reproduction, formation of connectivethe 12th most abundant element of the terrestrial crust, tissues and bone marrow, lipid and carbohydrate metabo-where its minerals (especially oxides, silicates and carbon- lism and in the maintenance of the brain (Keen et al., 1999;ates) are widely distributed. Mn is an essential nutrient that Pittman, 2005). This metal is also an important co-factorplays a role in plant and animal physiological processes. In for numerous enzymes involved in the biosynthesis ofplants, it participates in the respiratory process, where its DNA and neurotransmitters and in transduction signals (Roth and Garrick, 2003; Pittman, 2005). Abbreviations: Mn, manganese; MnCl2 Á 4H2O, manganese chloride te-trahydrate; MnCl2, manganese chloride; MnSO4, manganese sulfate; In one way, Mn has a natural occurrence, where the ero-KMnO4, potassium permanganate; CA, chromosomal aberration; COL, sion of soil is one of its most important natural sources. Incolchicine; DOX, doxorubicin; EMS, ethylmethanesulfonate; End, endo- another way, Mn can be found as a result of contaminationreduplication; FBS, fetal bovine serum; HAR, harvest; MI, mitotic index; in the ground, sediments and/or water by aerosols, insecti-PHA, phytohaemaglutinin; Polyp, polyploid cells. cides and fertilizers (Gerber et al., 2002). Despite the fact * Corresponding author. Tel.: +55 091 3183 1102/8802 7972; fax: +55091 3183 1601. that the absorption of the Mn is only 3–5%, food is the pri- E-mail address: rommel@ufpa.br (R.R. Burbano). mary sources of the metal ingested by human beings and0887-2333/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.tiv.2007.12.011
    • P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 1032–1037 1033animals. Other sources of Mn exposure is related to work Doxorubicin hydrochloride (Adriamycin, CAS no.conditions, where Mn particles can be inhaled or absorbed 25316-40-9) and ethylmethanesulfonate (EMS, CAS no.(WHO, 1999; Gerber et al., 2002). 62-50-0) were purchased from Sigma–Aldrich Co. (St. Deficiencies of Mn during the initial periods of develop- Louis, MO, USA).ment can induce skeletal muscle abnormalities and irrevers- Phytohemagglutinin (PHA) was obtained from Abbottible ataxia, in addition to fertility problems (Keen et al., Laboratories, Maidenhead, UK. HAM F-10 growth med-1999). Poisoning by high levels of Mn can lead to some ium and fetal bovine serum were purchased from GibcoÒalterations in organisms, where the lungs and the central (Invitrogen, Carlsbad, CA, USA). Colchicine was pur-nervous system (CNS) are the main target organs (Gerber chased from Sigma–Aldrich Co. (St. Louis, MO, USA).et al., 2002). Chronic exposure to high levels of Mn canalso induce a syndrome known as manganism, character- 2.2. Test controlsized by extrapyramidal dysfunction (bradykinesia, rigidityand dystonia) and neuropsychiatric symptoms that resem- Double-distilled water was used as the vehicle since itble idiopathic Parkinson’s disease (Calne et al., 1994; Barc- neither induced chromosomal aberrations nor reducedeloux, 1999; Gerber et al., 2002). the mitotic index when compared to the cultures without The neurotoxicity induced by Mn is related to the phys- its addition (data not shown).iological dichotomy between its oxidation states, Mn2+ and The cytotoxic and mutagenic agent doxorubicin wasMn3+. The first one is necessary for the normal cerebral used as positive control for lymphocyte cultures at a con-activities since is essential for the catalytic activity of super- centration of 0.018 lM (Dhawan et al., 2003) and EMSoxide dismutase that eliminates superoxide radicals (Ose used as positive control for the comet assay at a concentra-and Fridovich, 1976), while the second one enhances the tion of 40 lM (Wagner et al., 2003).oxidation of dopamine, thereby producing neurotoxic bio- ´products (Gutierrez and Salsamendi, 2001). The association between Mn and the risk for the devel- 2.3. Lymphocyte cultureopment of neurodegenerative processes can be related toDNA damage. There are few studies related to the geno- Peripheral blood was collected from four normal,toxic activities of Mn. Its mutagenic potential has been healthy donors, two women and two men, aged 20–27studied by in vitro tests in bacteria and in vivo/in vitro tests years, with no history of smoking/drinking or chronic drugin insect and mammalian cells, showing that some chemical use. A volume of 10 mL of venous blood was collectedforms of this metal have mutagenic potential (De Meo from each donor into heparinized vials (5.000 IU/mL; Liq-et al., 1991; Timchenko et al., 1991; Ogawa et al., 1994; ´ uemine; Roche). Short-term lymphocyte cultures were setBrega et al., 1998; WHO, 1999; Gerber et al., 2002). up according to a standard protocol (Preston et al., Therefore, the aim of this study was to investigate the 1987). The culture medium consisted of 5 mL HAM-F10genotoxic, clastogenic and cytotoxic effects of MnCl2 Á (78%), heat-inactivated fetal bovine serum (20%), phyto-4H2O in different phases of cell cycle using short-term cul- hemagglutinin-M (2%) and antibiotics (0.028 lM of peni-tures of human lymphocytes in vitro. These effects were cillin, Sigma–Aldrich Co., St. Louis, MO, USA;determined by the mitotic index (MI), chromosomal aberra- 0.0068 lM of streptomycin, USB, Cleveland, OH). Thetions (CAs) and DNA damage index detected by the comet culture tubes were incubated at 37 °C in a 5% CO2 atmo-assay. The Mn used in the present study was the tetrahy- sphere with 95% relative humidity.drate form of manganese chloride (MnCl2 Á 4H2O), one ofthe most common forms of Mn (WHO, 1999). 2.4. Treatments and biological tests The concentrations of MnCl2 Á 4H2O used in the presentstudy (15, 20 and 25 lM) are based on literature references For cytogenetic analysis, MnCl2 Á 4H2O was studied at(Eriksson and Heilbronn, 1983; Gerber et al., 2002; Goldoni three concentrations (15, 20 and 25 lM) in different phaseset al., 2003; Pifl et al., 2004; Isaac et al., 2006) adapted to of the cell cycle. In G1, lymphocytes in complete culture med-lymphocyte cell cultures. Besides, preliminary experiments ium were treated with a combination of 0.2 mL phytohemag-indicated that cells treated with MnCl2 Á 4H2O concentra- glutinin-M and MnCl2 Á 4H2O. The cells were fixedtions higher than 25 lM showed inhibition of cell division, following 52 h of incubation at 37 °C. In the transition phasepreventing the cytogenetic and cell cycle kinetics studies. G1–S, the cultures were treated with the different concentra- tions of MnCl2 Á 4H2O 24 h after phytohemagglutinin stim-2. Materials and methods ulation and were fixed 52 h after the initiation of the culture. To determine the specific effects of MnCl2 Á 4H2O in the S2.1. Chemical agents phase, pulse treatments with MnCl2 Á 4H2O for 1 and 6 h were performed 24 h after phytohemagglutinin stimulation. Stock solutions were made by dissolving MnCl2 Á 4H2O Following each pulse treatment, cells were washed once in(CAS no. 13446-34-9 Sigma–Aldrich Co., St. Louis, MO, serum-free medium, re-incubated in complete medium, andUSA) in double-distilled water, immediately prior to use. fixed after 52 h of incubation. In the G2 treatments, 69-h
    • 1034 P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 1032–1037cultures were treated with MnCl2 Á 4H2O for 3 h, and then 2.7. Statistical analysisfixed immediately (72 h total incubation) (Table 1). Student’s t-test was used to compare the frequencies of2.5. Cytogenetic studies CAs observed in cells exposed to the various concentra- tions of manganese with those of respective controls. The In order to obtain a sufficient number of metaphases to F-test (ANOVA) was used to detect significant differencesbe analyzed, colchicine was added at a final concentration in the MI. For comet assay, data were analyzed by one-of 0.0016%, 2 h prior to harvesting. The cells were har- way ANOVA followed by Tukey’s test. The level for statis-vested by centrifugation and treated with 0.075 M KCl at tical significance (p) was established at 5% (p < 0.05). Bioe-37 °C for 20 min. The cells were then centrifuged and fixed statÒ was the statistical software used in this study (Ayresin 1:3 (v/v) acetic acid:methanol. Finally, slides were pre- et al., 2000).pared, air-dried and stained with 3% Giemsa solution(pH 6.8) for 8 min (Moorhead et al., 1960). 3. Results Slides were examined with an optical microscope, andstructural and numerical CAs were determined in metapha- 3.1. Chromosome aberrations and mitotic indexses from the manganese-treated cultures and from therespective controls. The frequency of CAs (in 100 metapha- At G1, G1/S and S-phase treatments of 1 and 6 h,ses per culture) and the MI (number of metaphases per marked cytotoxic effects of manganese chloride were2000 lymphoblasts per culture) were determined. observed as decreases in the MI of cultured lymphocytes treated in all the tested concentrations (p < 0.01) (Tables2.6. Comet assay 2 and 3). In G2 phase, only the concentrations of 20 lM (p < 0.05) and 25 lM (p < 0.01) were cytotoxic to the lym- Peripheral venous blood was collected in heparinized phocyte cultures (Table 4).vials as above from four normal, healthy donors, two The frequency of CAs was significantly increased onlywomen and two men, aged 23–28 years, with no history during G2 treatment, with the high concentration ofof smoking/drinking or chronic use of medication. Periph- 25 lM (p < 0.01) (Table 4). Chromatid gaps and chromatideral blood lymphocytes were isolated by Ficoll density gra- breaks were the most frequent CAs. With the other treat-dient (Histopaque 1077; Sigma Diagnostics Inc., St. Louis) ments, no genotoxic effects of MnCl2 Á 4H2O were observedincubated for 3 h with different concentrations of (Tables 2 and 3). No significant endoreduplication or poly-MnCl2 Á 4H2O (15, 20 and 25 lM) and then mixed with ploidy was observed (Tables 2–4).low-melting point agarose. The alkaline version of the comet assay (single-cell gelelectrophoresis) was performed as described by Singh 3.2. Comet assayet al. (1988) with minor modifications (Hartmann andSpeit, 1997). Slides were prepared in duplicate and 100 cells Only the concentration of 25 lM showed statisticallywere screened per sample (50 cells from each duplicate different (p < 0.05) results in relation to the negative con-slide) with a fluorescence microscope (Zeiss) equipped witha 515–560 nm excitation filter, a 590 nm barrier filter, and a Table 2 Chromosome aberrations (CAs) and mitotic index (MI) in cultured human40Â objective. Undamaged cells appeared as intact nuclei lymphocytes treated with MnCl2 Á 4H2O during the G1 and G1/S phaseswithout tails, whereas damaged cells had the appearance of the cell cycleof a comet. Comets were classified visually as belonging MnCl2 treatment MI (%) CAs Polyp Endto one of five classes according to tail size and given a score Gaps Breaks Totalof 0, 1, 2, 3 or 4 (from undamaged = 0, to maximally dam-aged = 4). Thus, the total damage score for 100 comets G1 NC 6.0 2 0 2 0 0ranged from 0 (all undamaged) to 400 (all maximally dam- 15.0 lM 2.4** 2 0 2 1 0aged) (Speit and Hartmann, 1999). 20.0 lM 1.3** 0 0 0 0 0 25.0 lM 1.2** 3 1 4 1 0Table 1 Dox 3.1** 5 4 9* 2 4*Treatment protocols of MnCl2 Á 4H2O applied to short-term cultures ofhuman lymphocytes G1/S NC 6.0 2 0 2 0 0Treatment PHA (h) Mn (h) Wash (h) COL (h) HAR (h) 15.0 lM 4.0** 5 0 5 0 0G1 0 0 – 50 52 20.0 lM 3.0** 6 1 7 1 0G1/S 0 24 – 50 52 25.0 lM 3.0** 6 2 8 0 0S1 (1 h pulse) 0 24 25 50 52 DOX 3.2** 17 6 23* 5* 0S1 (6 h pulse) 0 24 30 50 52 NC: negative control; Polyp: polyploid cells; End: endoreduplication;G2 0 69 – 70 72 DOX: doxorubicin (positive control). *PHA: phytohaemagglutinin; FBS: fetal bovine serum; COL: colchicine; p < 0.05. **HAR: harvest. p < 0.01.
    • P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 1032–1037 1035Table 3 Table 5Chromosome aberrations (CAs) and mitotic index (MI) in human Effects of MnCl2 Á 4H2O or doxorubicin (DOX) on cell damage index inlymphocytes treated with MnCl2 Á 4H2O during the S phase of the cell comet assaycycle Substance Treatment Damage Damage frequencyMnCl2 treatment MI (%) CAs Polyp End (lM) indexa (%)a Gaps Breaks Total DOXb 151 ± 2.8** 49.80 ± 0.25** NCc – 24.70 ± 5.3 16.25 ± 1.551hNC 6.0 2 0 2 0 0 MnCl2 Á 4H2O 15 52.30 ± 6.9 25.70 ± 4.1715.0 lM 1.8** 1 0 1 0 0 20 51.75 ± 12.0 27.67 ± 5.2520.0 lM 1.9** 1 0 1 0 0 25 82.00 ± 14.0** 33.25 ± 2.17*25.0 lM 1.6** 4 0 4 0 0 Data significant in relation to NC at: *p < 0.01 **p < 0.001.DOX 2.4** 10 7 17* 1 1 a Mean values and standard deviation obtained from average of 100 cells6h per experiment À total of four experiment per dose for each substance. bNC 6.0 2 0 2 0 0 Doxorubicin, positive control.15.0 lM 0.8** 0 0 0 0 0 c Negative control.20.0 lM 0.8** 1 0 1 0 025.0 lM 0.6** 3 0 3 0 0DOX 1.9** 14 10 24* 3* 0 toxic for human lymphocytes at all the concentrationsNC: negative control; Polyp: polyploid cells; End: endoreduplication; and phases of the cell cycle tested (Lima et al., 2007).DOX: doxorubicin (positive control). * p < 0.05. Mn was chosen for the present study because it is a** p < 0.01. metal of natural occurrence, and a common chemical ele- ment, necessary for the initial periods of development (Keen et al., 1999; WHO, 1999; Gerber et al., 2002). SinceTable 4 there are studies indicating a probably relationship betweenChromosome aberrations (CAs) and mitotic index (MI) in human Mn exposure and increased risk of neurodegenerative dis-lymphocytes treated with MnCl2 Á 4H2O during the G2 phase of the cellcycle orders witch symptoms are similar to Parkinson’s disease (Calne et al., 1994; Barceloux, 1999; Gerber et al., 2002).MnCl2 treatment MI (%) CAs Polyp End The results of the present study showed a high cytotoxic Gaps Breaks Total potential of Mn in several phases of the cell cycle, as dem-NC 6.0 2 0 2 0 0 onstrated by decreases on MI. The cytotoxicity of a chem-15.0 lM 5.4 5 2 7 1 0 ical mutagen can be estimated from several endpoints and20.0 lM 5.0* 5 3 8 0 025.0 lM 4.9** 8 4 12* 0 0 one of the most used is MI (Galloway, 2000). When usingDOX 5.2* 24 8 32** 7* 4 lymphocyte cultures, the use of MI is acceptable as a tox-NC: negative control; Polyp: polyploid cells; End: endoreduplication; icity measure, especially when other toxicity measurementsDOX: doxorubicin (positive control). are cumbersome or impractical (Galloway et al., 1994). * p < 0.05. Chromosome aberrations (CAs) were observed only at** p < 0.01. the highest dose (25 lM) at G2 phase after 3 h treatment. Cytogenetic results corroborated the comet assay findings, where genotoxicity was also observed only at the highesttrol (p < 0.01), but no significant differences were seen dose after 3 h of drug exposure. It is possible that the repairbetween the different manganese concentrations tested. system in the other phases of the cell cycle protects cells All concentrations of manganese tested were shown to from CAs since, after treatment, the cells are kept in cul-differ from that of the positive control (dox) (p < 0.001) ture until the next cell cycle occurs. In cells treated in G2,(see Table 5). this did not happen mainly because the cells were exposed to colchicine treatment. We suggest as a main mechanism of manganese toxicity4. Discussion by DNA synthesis blocks, since the cytotoxicity was observed on small doses on phases G1, G1/S an S of the In an industrialized world, there are many types of met- cell cycle, on this phases, this metal was not clastogenic,als in use (such as manganese, iron, aluminum, mercury, but interferes on cell replication. Mn does not presentcopper, lead, zinc and others) and humans are exposed to direct genotoxicity; we believe that CAS found when cellsthem at work, or as a result of contamination of food, were treated on G2 is due to the blockage of the repairwater or environment (Veldman et al., 1998; Purdey, of DNA, thus, Mn does not relate directly to DNA. We2004; Shi et al., 2004). The clinical symptoms and the pos- can prove our hypotheses noticed that the cells treated withsible mutagenic effects produced by acute poisoning and Mn in the other phases even in high doses, do not exhibitchronic exposure to metals are of considerable interest CAs.(Ganrot, 1986; Ferrer, 2003). Recently, our research group From metabolic, the presence of the microsomal liverdemonstrated that aluminum chloride (AlCl3) was geno- fraction S9 in treatments with the same concentrations
    • 1036 P.D.L. Lima et al. / Toxicology in Vitro 22 (2008) 1032–1037cited in this work did not show significant differences with 0.5 mM) (WHO, 1999). In vivo studies have demonstratedthe previous results (data not shown), which reinforces that that oral doses of manganese sulfate or potassium perman-in general heavy metals do not suffer influence of metabolic ganate induces CAs in the bone marrow of animals,activation in the induction of CAs (Chen et al., 2006; De whereas no CAs have been observed after oral doses ofMeo et al., 1991). With respect to cytotoxicity, the S9 frac- manganese chloride on concentrations over than 12 lMtion not alters the effect of the presence of the metal in cul- (WHO, 1999). These results show that the mutagenicture, regardless of the phase of the cycle. Our results potential of Mn may be different between permanganateindicates that despite Mn be a toxic agent to liver cells salts and manganese salts.(Khan et al., 1997; Rovetta et al., 2006), your genotoxic De Meo et al., 1991 evaluated the genotoxicity of potas-action do not need metabolic activation. sium permanganate (KMnO4), manganese sulfate and New studies must be conducted to verify the relationship manganese chloride using the Ames test with the testerof intoxication with Mn and other metals present physio- strains TA97, TA98, TA100 and TA102 with and withoutlogically in the body. Chen et al., 2006 showed that Mn metabolic activation. The presence of direct-acting muta-induces an increase in the iron level on rat brain. gens was detected in all the samples of Mn with the tester It is important to point out that polyploidy and endore- strain TA102 without metabolic activation. Only manga-duplicated cells were not observed at any of the doses nese chloride induced DNA damage in human lymphocytestested, suggesting that Mn does not affect mitotic spindle with a dose–response relationship as determined by theformation. These observations show that Mn does not have comet assay. The mutagenic potential was 2.4 revertant/an antipolymerizing effect on tubulin dimers. nmol. This effect was also found in this work using the Gerber et al. (2002) demonstrated that high dose comet assay with the higher concentration of 25 lM.(0.05 M) of various compounds of Mn can affect DNA rep- According to the WHO (1999), other chemical forms oflication and repair in bacteria. As for mammalian cells, Mn have mutagenic potential in vitro and in vivo. Morehigh doses of Mn (compared with respect to concentration studies must be done to elucidate the probable mutagenic-of manganese recommended for daily consumption).can ity of Mn and its chemical forms in human health.affect fertilization and is toxic to the embryo and fetus, In conclusion, MnCl2 Á 4H2O shows strong cytotoxicitydemonstrating its teratogenic potential (Gerber et al., in all phases of the cell cycle. The genotoxicity observed2002). Manganese chloride (MnCl2) was also subjected to at G2 phase and in the comet assay, may be related tothe wing spot test of Drosophila melanogaster and was the lack of time for the cellular repair system to act or toshown to be clearly effective in inducing spots with one some other mechanism not studied in this work. Theor two mutant hairs (small spots) on concentrations over absence of CAs in the other phases of the cell cycle suggeststhan 12 lM (Ogawa et al., 1994). These findings, at high that Mn-mediate damage may be repaired in vitro.Mn concentrations, are in agreement with our results,where only treatment with the high concentration showed Acknowledgmentsgenotoxic potential. Previous studies by Brega et al. (1998) demonstrated This work was supported by Financiadora de Estudos ethat farm workers exposed to pesticides containing Mn, Projetos (FINEP CT-INFRA/FADESP) (Grant No. 0927-even at a low level, increased the mutagenic potential of ´ 03), Conselho Nacional de Desenvolvimento Cientıfico ethose pesticides, as evidenced by an increased number of ´ Tecnologico (CNPq) and by Coordenacao de Aperfeicoa- ߘ ßCAs. It is possible that chronic exposure to low doses of ´ mento de Pessoal de Nıvel Superior (CAPES). We thankMn induces CAs over years, since they are not acutely cyto- Dr. A. Leyva for English editing of the manuscript.toxic. The assay used in this work did not reproduce along-term exposure, thus the cytotoxic effects may havehindered the appearance of CAs in the cells. References It is possible that Mn causes genotoxic effects at low Ayres, M., Ayres, Jr., M., Ayres, D.L., Santos, A.S., 2000. Bioestat 2.0.doses only with long-term exposure, and this may be rea- ´ ´ Belem. Sociedade Civil Mamiraua, p. 272.son why Timchenko et al. (1991) did not find CAs in nasal Barceloux, D.G., 1999. Manganese. Journal of Toxicology: Clinicalmucosa of mammals exposed to Mn dioxide aerosol (40– Toxicology 37, 293–307.12,000 Hz, 80–100 dB). Brega, S.M., Vassilieff, I., Almeida, A., Mercadante, A., Bissacot, D., Cury, P.R., Freire-Maia, D.V., 1998. Clinical, cytogenetic and We agree that the genotoxic effects of Mn can be influ- toxicological studies in rural workers exposed to pesticides inenced by the presence of other chemical agents such as that Botucatu, Sao Paulo, Brazil. 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