Genotoxic effects of aluminum chloride in cultured humanDocument Transcript
Food and Chemical Toxicology 45 (2007) 1154–1159 www.elsevier.com/locate/foodchemtox Genotoxic eﬀects of aluminum chloride in cultured human lymphocytes treated in diﬀerent phases of cell cycle P.D.L. Lima a, D.S. Leite a, M.C. Vasconcellos b, B.C. Cavalcanti b, R.A. Santos c, L.V. Costa-Lotufo b, C. Pessoa b, M.O. Moraes b, R.R. Burbano a,* a ´ ´ Human Cytogenetics Laboratory, Department of Biology, Center for Biological Sciences, Federal University of Para, Belem/PA, Brazil b ´ Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceara, Fortaleza/CE, Brazil c Department of Genetics, Faculty of Medicine of Sao Paulo, University of Sao Paulo, Ribeirao Preto/SP, Brazil ˜ ˜ ˜ Received 7 February 2006; accepted 25 December 2006Abstract Aluminum (Al) is the most abundant metal and the third common chemical element on earth. It is known that Al is toxic, especiallyits trivalent form (Al3+), that represents the its most soluble form. Al intoxication is related to some pathogenic disorders, principallyneurodegeneratives ones as Parkinson and Alzheimer diseases. The present study aimed to evaluate the mutagenic potential of aluminumchloride (AlCl3). Comet assay and chromosome aberrations analysis were applied to evaluate the DNA-damaging and clastogenic eﬀectsof AlCl3, respectively, in diﬀerent phases of the cell cycle. Cultured human lymphocytes were treated with 5, 10, 15 and 25 lM aluminumchloride during the G1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle. All tested concentrations were cytotoxic andreduced signiﬁcantly the mitotic index in all phases of cell cycle. They also induced DNA damage and were clastogenic in all phasesof cell cycle, specially in S phase. AlCl3 also induced endoreduplication and polyploidy in treatments performed during G1 phase.The presence of genotoxicity and polyploidy on interphase and mitosis, respectively, suggests that aluminum chloride is clastogenicand indirectly aﬀects the construction of mitotic fuse in all tested concentrations.Ó 2007 Elsevier Ltd. All rights reserved.Keywords: Aluminum chloride; Chromosome aberration; Comet assay1. Introduction itates the potential for human exposure and for causing harm (Berthon, 1996; Candura et al., 1998; Smith, 1996; Aluminum and its compounds are common food and Williams, 1996; Zhang and Zhou, 2005).water contaminants, are present in medicine, cosmetic No biological function of the element has beenand environmental products and aluminium chloride is identiﬁed, whereas some aspects of its toxicity have beenan important coagulant used in water treatment and puri- described (Bjorkstein et al., 1988; Berthon, 1996; Corainﬁcation. This wide distribution of the element clearly facil- et al., 1996; Suwalsky et al., 2001; Reinke et al., 2003). It has been suggested that there is a relationship between high levels of Al and increased risk of a number of pathogenic Abbreviations: Al, aluminum; AlCl3, aluminum chloride; CA, chro- disorders, such as microcytic anemia, osteomalacia and,mosomal aberration; COL, colchicine; DOX, doxorubicin; EMS, ethyl- possibly, neurodegenerative disorders including dialysismethanesulfonate; End, endoreduplication; FBS, fetal bovine serum; encephalopathy, Alzheimer’s disease and Parkinson’sHAR, harvest; MI, mitotic index; PHA, phytohaemaglutinin; Polyp, disease (Ganrot, 1986; Berthon, 1996; Corain et al., 1996;polyploid cells. * Corresponding author. Tel.: +55 091 3183 1102/8835 7972; fax: +55 Becaria et al., 2002; Yokel, 2002).091 3183 1601. The more toxic form of Al appears to be the trivalent E-mail address: email@example.com (R.R. Burbano). Al3+, which can easily bind to chlorine conﬁguring A better0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2006.12.022
P.D.L. Lima et al. / Food and Chemical Toxicology 45 (2007) 1154–1159 1155understanding of Al metabolism and its eﬀects on physio- Co. St. Louis, MO, USA) and 0.005 mg/ml of streptomycin (USB,logical process requires knowledge of its distribution in Cleveland, OH). The culture tubes were incubated at 37 °C in a humidiﬁed atmosphere composed of 5% CO2 atmosphere and 95% humidity.biological ﬂuids (Sorenson et al., 1974). There are few stud-ies relating the genotoxic activities of Al. Its mutagenic 2.4. Treatments and biological testspotential have been studied by micronucleus assay, sisterchromatid exchange, Ames and chromosomal aberration For cytogenetic analysis, AlCl3 was studied at four concentrations (5,assay (Pagano et al., 1996; Botchway et al., 1997; Moreno 10, 15 and 25 lM) at diﬀerent phases of the cell cycle. At G1, lymphocyteset al., 1997; Yumei et al., 1998; Dovgaliuk et al., 2001a,b; in complete culture medium were treated with a combination of 0.2 mlTrippi et al., 2001; Elmore, 2003; Synzynys et al., 2004; phytohaemaglutinin-M and AlCl3. The cells were ﬁxed following 52 h of incubation at 37 °C. At transition phase G1-S, the cultures were treatedVarella et al., 2004). with the diﬀerent concentrations of AlCl3 24 h after phytohaemaglutinin On the basis of this data, the present study investigated stimulation and were ﬁxed 52 h after the initiation of the culture. Tothe genotoxic, clastogenic and cytotoxic eﬀects of AlCl3 in determine the speciﬁc eﬀects of AlCl3 in the S phase, pulse treatments withdiﬀerent phases of cell cycle using temporary cultures of AlCl3 for 1 h and 6 h were performed 24 h after phytohaemaglutininhuman lymphocytes in vitro. It was analyzed the mitotic stimulation. Following each pulse treatment, cells were washed one time in serum-free medium, re-incubated in complete medium, and ﬁxed after 52 hindex (MI), chromosomal aberrations (CAs) and DNA of incubation. In the G2 treatments, 69-h cultures were treated with AlCl3damage index detected by comet assay. for 3 h, and then ﬁxed immediately (72 h total incubation) (Table 1). The objective of this study was evaluate the AlCl3 geno-toxicity in similar concentrations of those tested in studies 2.5. Cytogenetic studiesabout the toxicity of AlCl3 in human neural cells. Toimelaet al. (2004), Toimela and Tahti (2004) and Atterwill et al. In order to obtain a suﬃcient number of analyzable metaphases, col-(1992) investigated the toxicity of AlCl3 concentrations chicine was added at a ﬁnal concentration of 0.0016%, 2 h prior harvest- ing. The cells were harvested by centrifugation and treated with 0.075 Mranging 1–1000 lM, in human cells lines of neural origin. KCl at 37 °C for 20 min. The cells were then centrifuged and ﬁxed in 1:3Thus, the concentrations of AlCl3 used in this present study (v/v) acetic acid:methanol. Finally, slides were prepared, air-dried and(5–25 lM) are based in the literature references, adapted to stained with 3% Giemsa solution (pH 6.8) for 8 min (Moorhead et al.,lymphocyte cell cultures. 1960). Slides were analyzed with an optical microscope and structural and numerical CAs were examined in metaphases from the aluminum-treated2. Materials and methods cultures and from the respective controls. The frequency of CAs (in 100 metaphases per culture) and the MI (number of metaphases per 2.0002.1. Chemical agents lymphoblasts per culture) were determined. Stock solutions were made by dissolving AlCl3 (CAS No. 7446-70-0,Sigma Aldrich Co., St. Louis, MO, USA) in methanol (CAS No. 67-56-1, 2.6. Comet assayMerck-Schuchardt) immediately prior use. Doxorubicin hydrochloride (adriamycin, CAS No. 25316-40-9) and Peripheral venous blood was collected in heparinized vials as aboveEthylmethanesulfonate (EMS, CAS No. 62-50-0) were purchased from from four normal, healthy donors, two women and two men, aged 21–26Sigma Aldrich Co. (St. Louis, MO, USA). years, with no history of smoking/drinking or chronic use of medication. Phytohaemagglutinin (PHA) was obtained from Abbott Laboratories, Peripheral blood lymphocytes were isolated by Ficoll density gradientMaidenhead, UK. HAM F-10 growth medium and fetal calf serum were (Hystopaque 1077; Sigma Diagnostics, Inc., St. Louis) incubated for 3 hpurchased from GibcoÒ (Invitrogen, Carlsbad, CA, USA). Colchicine was with diﬀerent concentrations of AlCl3 (5, 10, 15 and 25 lM) and thenpurchased from Sigma Aldrich Co. (St. Louis, MO, USA). mixed with low-melting point agarose. The alkaline version of the comet assay (single-cell gel electrophoresis) was performed as described by Singh et al. (1988) with minor modiﬁca-2.2. Test controls tions (Hartmann and Speit, 1997). Slides were prepared in duplicate and 100 cells were screened per sample (50 cells from each duplicate slide) with Methanol was used as the vehicle since it did not induced chromo- a ﬂuorescence microscope (Zeiss) equipped with a 515–560 nm excitationsomal aberrations nor reduced the mitotic index when compared to the ﬁlter, a 590 nm barrier ﬁlter, and a 40· objective. Undamaged cellscultures without its presence, even in the highest concentration used to appeared as intact nuclei without tails, whereas damaged cells had thedissolve AlCl3 (data not shown). appearance of a comet. Comets were classiﬁed visually as belonging to one The cytotoxic and mutagenic agent doxorubicin was used as positive of ﬁve classes according to tail size and given a score of 0, 1, 2, 3 or 4 (fromcontrol for lymphocyte cultures at the concentration 0.01 lg/ml (Dhawanet al., 2003) and EMS used as positive control for comet assay at theconcentration 4 · 10À5 M (Wagner et al., 2003). Table 1 Treatment protocols of AlCl3 applied to short-term cultures of human2.3. Lymphocyte culture lymphocytes Treatment PHA (h) Al (h) Wash (h) COL (h) HAR (h) Peripheral blood was collected from four normal, healthy donors, twowomen and two men, aged 21–26 years, with no history of smoking/ G1 0 0 – 50 52drinking or chronic drug use. Venous blood (10 ml) was collected from G1/S 0 24 – 50 52 ´each donor into heparinized vials (5.000 IU/ml; Liquemine; Roche). S1 (1 h pulse) 0 24 24 50 52Short-term lymphocyte cultures were initiated according to a standard S1 (6 h pulse) 0 24 24 50 52protocol (Preston et al., 1987). The culture medium consisted of 5 ml G2 0 69 – 70 72HAM-F10 (78%), heat-inactivated fetal calf serum (20%), phytohaemag- PHA: phytohaemagglutinin; FBS: fetal bovine serum; COL: colchicine;glutinin-M (2%) and antibiotics 0.01 mg/ml of penicillin (Sigma Aldrich HAR: harvest.
1156 P.D.L. Lima et al. / Food and Chemical Toxicology 45 (2007) 1154–1159undamaged = 0, to maximally damaged = 4). Thus, the total damage Table 3score for 100 comets ranged from 0 (all undamaged) to 400 (all maximally Chromosome aberrations (CAs) and mitotic index (MI) in humandamaged) (Speit and Hartmann, 1999). lymphocytes treated with AlCl3 during the S phase of the cell cycle AlCl3 treatment MI (%) CAs Polyp End2.7. Statistical analysis Gaps Breaks Total The student’s t test was used to compare the frequencies of CAs 1h Control 5.4 2 0 2 0 0observed in cells exposed to the various concentrations of aluminum with 5 lM 2.3* 28 8 36** 0 0the respective controls. The F test (ANOVA) was used to detect signiﬁcant 10.0 lM 1.7* 35 11 46** 0 0diﬀerences in the MI. For comet assay, data were analyzed by one-way 15.0 lM 1.2* 30 14 44** 0 0ANOVA followed by Tukey test. The level for statistical signiﬁcance (p) 25.0 lM 1.0* 43 19 62** 0 0was established at 5% (Ayres et al., 2003). DOX 2.4* 10 7 17* 1 1 6h Control 4.9 2 0 2 0 03. Results 5 lM 1.4* 27 9 36** 0 0 10.0 lM 1.0* 23 12 35** 0 0 15.0 lM 0.5** 7 8 15* 0 03.1. Chromosome aberrations and mitotic index 25.0 lM 0.2** 10 13 23* 0 0 DOX 1.9* 14 10 24* 3 0 At G1 and G1/S, the frequency of CAs was signiﬁcantly Polyp: polyploid cells; End: endoreduplication; Doxorrubicin (positiveincreased with all the tested concentrations of AlCl3 (Table control).2). During G1 and G2 treatments, signiﬁcantly induced * p < 0.05. **endoreduplication and polyploidy were observed (Table 2). p < 0.01.AlCl3 treatment during S phase resulted in highly signiﬁcantincreases in the frequency of CAs in most concentrations(p < 0.01); however, there were no signiﬁcant diﬀerences Table 4between S phase treatments of 1 and 6 h (Table 3). The treat- Chromosome aberrations (CAs) and mitotic index (MI) in humanment at G2 also induced a signiﬁcant increase in the fre- lymphocytes treated with AlCl3 during the G2 phase of the cell cyclequency of CAs (Table 4). Chromatid gaps and chromatid AlCl3 treatment MI (%) CAs Polyp Endbreaks were the most frequent CAs. Gaps Breaks Total The cytotoxic eﬀects of AlCl3 were observed as decreases Control 5.8 8 0 8 0 1in the MI of lymphocyte in cultures treated during the G1, 5 lM 3.5* 15 4 19* 10* 2G1/S, S and G2 phases of the cell cycle (Tables 2–4). 10.0 lM 3.0* 17 6 23* 14* 0 15.0 lM 2.9* 23 8 31* 20* 43.2. Comet assay 25.0 lM 2.4* 33 14 47** 27* 5 DOX 5.2* 24 8 32* 7 4 All treatments resulted in signiﬁcant increases in DNA Polyp: polyploid cells; End: endoreduplication; DOX: doxorrubicin (positive control).damage (Fig. 1). * p < 0.05. All tested concentrations of AlCl3 were statistically dif- ** p < 0.01.ferent from the negative control (p < 0.01), however, theyTable 2Chromosome aberrations (CAs) and mitotic index (MI) in cultured humanlymphocytes treated with AlCl3 during the G1 and G1/S phases of the cellcycleAlCl3 treatment MI (%) CAs Polyp End Gaps Breaks TotalG1 Control 5.6 1 0 1 0 0 5 lM 3.4* 4 6 10* 6 10* 10.0 lM 2.4* 8 8 16* 10* 9* 15.0 lM 1.9* 8 9 17* 9* 9* 25.0 lM 1.8* 8 14 22* 14* 8* DOX 3.1* 5 4 9* 2 4G1/S Control 5.5 2 0 2 0 1 5 lM 2.8* 12 0 12* 0 0 10.0 lM 1.9* 15 9 24* 1 0 15.0 lM 1.5* 14 8 22* 0 1 25.0 lM 1.2* 18 10 28* 1 0 DOX 3.2* 17 6 23* 5 0Polyp: polyploid cells; End: endoreduplication; DOX: Doxorrubicin(positive control). Fig. 1. Damage index of comet assay in AlCl3-treated human lympho- * p < 0.05. cytes. * p < 0.05.
P.D.L. Lima et al. / Food and Chemical Toxicology 45 (2007) 1154–1159 1157were not diﬀerent from the positive control (EMS) the inclusion of gaps as true chromatid lesions. Some(p > 0.05). authors argue that the counting of gaps can be subjective, and they may be the result of technical artifacts, of4. Discussion variability within the same culture, and of variability in the culture conditions (Schinzel and Schmid, 1976; Brog- Metals are among the oldest toxic agents known by ger, 1982). Yet, Paz-y-Mino et al. (2002) obtained resultshumans (Ferrer, 2003). In an industrialized world, there indicating that gaps are indicative of DNA damage, whichare thousands of types of metals in use, and humans are supports their inclusion in the analysis of CAs.exposed to them at work, or as a result of contamination A considerable frequency of CAs was found when AlCl3of food, water and environmental. There is a lot of evi- exposures where conducted in all phases of the cell cycle.dence indicating increase of neurodegenerative disorders However, more breaks and gaps were seen in the S phasein industrialized countries (Veldman et al., 1998). The clin- than the other phases. This may be due to the fact thatical symptoms and the possible mutagenic eﬀects produced the S phase treatments were carried out using shortby acute poisoning and chronic exposure to metals are of ‘‘pulses’’, i.e., AlCl3 was in contact with the cells exclusivelyconsiderable interest (Ganrot, 1986; Ferrer, 2003). during this phase; immediately after treatment (1 or 6 h), Al was chosen for the present study because it is the the metal was removed from the culture, and the cell cyclemost abundant metal and the third common chemical ele- continued. Based on these ﬁndings, we believe that AlCl3ment (Corain et al., 1996; Suwalsky et al., 2001), existing a preferentially acts on S phase, when DNA synthesis occurs.relevant risk to population occupational disorders related Trippi et al. (2001) related that aluminum treatment into Al exposure (Bentes, 1992) once there are a considerable cells from Parkinson disease patients did not increasenumber of studies indicating relationship between Al expo- micronucleus frequency, indicating that aluminum do notsures and increased risk to neurodegenerative disorders as have an ampliﬁer mutagenic eﬀect on these patients.Alzheimer’s disease and Parkinson’s disease (Yasui et al., Elmore (2003) also observed absence of teratogenic eﬀects1992; Fahn, 1995; Altschuler, 1999). in mice fetus and genotoxic eﬀects detected by Ames assay The MI results indicated that AlCl3 is cytotoxic at all in cosmetic aluminum-contend formulations.tested concentrations during the G1, G1/S, S and G2 On the other hand, other studies demonstrated thephases of the cell cycle. This toxicity probably occurs due mutagenic potential of aluminum in human cells, for exam-to the inhibition of the cell cycle by this metal. Toimela ple, Varella et al. (2004) demonstrated the mutagenicet al. (2004) did not found cytotoxic eﬀects of AlCl3 at potential of garbage originated from aluminum factories1 lM for 24 h exposure in ARPE-19 and SH-SY5Y, epithe- by the mutagenic Salmonella assay.lial and neuronal cell lines, respectively. Yumei et al. (1998) examined the dust derived from an The cytotoxicity observed in the present study at 5, 10, 15 electrolytic aluminum plant and evaluated its genotoxicityand 25 lM, corroborate to Kopac et al. (2002) and Rein- by Ames assay, unscheduled DNA synthesis, sister chro-hardt et al. (1985) where AlCl3 was cytotoxic to Chinese matid exchanges and micronuclei frequency in human lym-hamster ﬁbroblasts. The present data are also in accord to phocytes. The results of these four experiments indicated aSynzynys et al. (2004) that observed immunotoxic eﬀects high genotoxicity of the dust organic extract.of 0,04 M AlCl3 in tumoral T lymphocytes of mice. Botchway et al. (1997) also observed chromosome The signiﬁcant increase of endoreduplications and poly- breaks in V79-4 Chinese hamster cells irradiated with alu-ploidy cells found only in treatments performed during the minum ions of low energy.G1 and G2 phases indicates that AlCl3 exposure has an As exposed below, there are a considerable number ofeﬀect on the mitotic apparatus along the other phases of evidences that reinforce the mutagenicity induced by Al;cell cycle. however more speciﬁc studies in human cells are necessary. A study reinforcing this notion was conducted in Mexi- The present study also evaluated the DNA-damagingcali, Mexico by Moreno et al. (1997) which showed the eﬀects of AlCl3 detected by the Comet assay in peripheralinduction of chromosomal aberrations, mostly numeric blood lymphocytes. This assay has been widely used to(anaphasic) alterations, in vitro in the Balb c 3T3 cell line detect single- and double-strand DNA breaks. The highexposed to Mexicali dust (20–80 mg/ml), a mixture of par- sensitivity of this test is due to the fact that it detectsticles of potassium aluminum silicates À98% and sodium DNA breaks, alkali-labile lesions and genomic lesions thatdioxide À2%. are subject to repair (Gontijo and Tice, 2003). All the AlCl3 Dovgaliuk et al. (2001a,b) studied the cytogenetic eﬀects treatments increased the levels of DNA damage, reinforc-of metal toxic salts including aluminum (Al[NO3]3) using ing the results found on chromosomal aberration assay.meristematic cells from Alium cepa, and demonstrated the The present study is the ﬁrst to include the analysis geno-clastogenic and aneugenic eﬀects (mitosis and cytokinesis mic lesions detected by comet assay.disturbs) in these cells. When compared to mercury, a heavy metal, aluminum Chromosome and/or chromatid breaks and gaps were presents higher level of genotoxicity using the same assaysscored as chromosome aberrations in this study. There (Amorim et al., 2000; Bahia et al., 2004; Silva-Pereira et al.,has been a great deal discussion in the literature regarding 2005); probably aluminum action in mitosis is similar to
1158 P.D.L. Lima et al. / Food and Chemical Toxicology 45 (2007) 1154–1159mercury, once these two metals induce polyploidy cells. Dhawan, A., Kayani, M.A., Parry, J.M., Parry, E., Anderson, D., 2003.However, other studies are necessary to evaluate the ability Aneugenic and clastogenic eﬀects of doxorubicin in human lympho- cytes. Mutagenesis 18, 487–490.of aluminum to bind to tubulin complexes. Dovgaliuk, A.I., Kaliniak, T.B., Blium, I.B., 2001a. Assessment of phyto- In conclusion, the present study indicates that AlCl3 and cytotoxic eﬀects of heavy metals and aluminum compounds usingproduces DNA damage and is cytotoxic during all phases onion apical root meristem. Tsitol. Genet. 35 (1), 3–9.of the cell cycle and at G1, treatment of cells resulted in Dovgaliuk, A.I., Kaliniak, T.B., Blium, I.B., 2001b. Cytogenetic eﬀects ofpolyploidy and endoreduplication, consistent with AlCl3 toxic metal salts on apical meristem cells of Allium cepa L. seed roots. Tsitol. Genet. 35 (2), 3–10.interacting with the mitotic spindle apparatus. These data, Elmore, A.R., 2003. Final report on the safety assessment of aluminumalong with the results of other studies reported in the liter- silicate, calcium silicate, magnesium aluminum silicate, magnesiumature, indicate that AlCl3 is genotoxic and should be used silicate, magnesium trisilicate, sodium magnesium silicate, zirconiumwith caution. silicate, attapulgite, bentonite, Fuller’s earth, hectorite, kaolin, lithium magnesium silicate, lithium magnesium sodium silicate, montmoril- lonite, pyrophyllite and zeolite. Int. J. Toxicol. 22 (1), 37–102.Acknowledgements Fahn, S., 1995. Parkinsonism. In: Rowland, L.P. (Ed.), Merrit’s Textbook of Neurology. In: Media. P.A. Williams & Wilkins, This work was supported by Financiadora de Estudos e pp. 713–730. Ferrer, A., 2003. Metal poisoning. An. Sist. Sanit. Navar. 26, 141–153.Projetos (FINEP CT-INFRA/FADESP) Grant No. 0927- Ganrot, P.O., 1986. 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