Evaluation of
in vivo
and
in vitro
toxicological and genotoxic potential
of aluminum chloride
Letícia Nazareth Fernandes Paz
a, Laís Mesquita Moura
a, Danielle Cristinne A. Feio
a,*,
Mirella de Souza Gonçalves Cardoso
a, Wagner Luiz O. Ximenes
a, Raquel C. Montenegro
b,
Ana Paula N. Alves
c, Rommel R. Burbano
b, Patrícia Danielle L. Lima
a aMolecular Biology LaboratoryePost Graduate Program of Amazon Parasitic Biology, Biological and Health Sciences Center, State University of Para, Belem,
Para, Brazil
bHuman Cytogenetics Laboratory, Institute of Biological Sciences, Federal University of Para, Bel em, Para, Brazil cDepartment of Clinical Dentistry
eHealth Sciences Center, Federal University of Ceara, Fortaleza, Ceara, Brazil
h i g h l i g h t s
We observe that Al, in the smallest concentrations, presents genotoxic actionin vitro.
In vivoindications of systemic toxicity with the presence of morphological alterations. Suggest the metal Al is potentially the cause of pathological disorders.
a r t i c l e
i n f o
Article history:
Received 22 July 2016 Received in revised form 31 January 2017 Accepted 3 February 2017 Available online 3 February 2017
Handling Editor: Frederic Leusch
Keywords:
Aluminum Cytotoxicity Genotoxic Micronucleus test Risk assessment Swiss mice
a b s t r a c t
Aluminum and its compounds are common contaminants of water and food, as well as medications and cosmetics. The wide distribution of the element facilitates the demand for detailed studies of its bio-logical and toxicobio-logical effects. This work aimed to evaluate the possible genotoxic and toxic activity resulting fromin vivoandin vitroexposure to Al. Forin vivoanalysis, 40 Swiss mice were used, various concentrations of hydrated aluminum chloride were administered orally. They were analyzed for possible genic activity and metal cytotoxicity using a micronucleus test (MN), and for toxicity through histopathological evaluation of the extracted organs. Forin vitro analysis, lymphocytes from the pe-ripheral blood of 3 healthy donors were used. These cells were exposed to the same chemical agent in various concentrations.In vivostudy revealed a significant increase in the number of MN in all Al con-centrations. Furthermore, significant alterations in all the organs evaluated were verified by the presence of irreversible lesions (such as necrosis). Corroborating these findings, a significant increase in the quantity of MN in all concentrations with lymphocytesin vitro. In light of this, we suggest that this metal presents genotoxic potential and is potentially a cause of pathological disorders.
©2017 Elsevier Ltd. All rights reserved.
1. Introduction
Aluminum (Al) is the most abundant metal and the third-most-common chemical element in the earth’s crust, with levels of its production reaching 36.9 million tons in 2009 (Geological Survey,
2010). In addition to this, Al and its compounds are common
contaminants of water and food, present in kitchen utensils, teas, spices, deodorants, food additives and medications, providing easy exposure to human beings (Kumar and Gill, 2009).
Aluminum chloride (AlCl3) is used as a coagulant in water
pu-rification (Ochmanski and Barabasz, 2000), and thus far there are no national or international limits on maximum concentrations for its use (Willhite et al., 2012). Despite its great abundance in the natural environment, no beneficial biological function has yet been found for Al (Lima et al., 2012).
The broad distribution of this element has led to increasing human contact with it, as much in their work environment as their
*Corresponding author. Laboratorio de Biologia Molecular, Centro de Ci ^encias Biologicas, Universidade Estadual do Para, Rua Perebebuí, 2623, Belem, PA, 66087-670, Brazil.
E-mail address:daniellefeio@yahoo.com.br(D.C.A. Feio).
Contents lists available atScienceDirect
Chemosphere
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c h e m o s p h e r e
http://dx.doi.org/10.1016/j.chemosphere.2017.02.011
0045-6535/©2017 Elsevier Ltd. All rights reserved.
macroenvironment (Ochmanski and Barabasz, 2000). This imposes an obligation upon researchers to develop detailed studies of its biological and toxicological effects. Brazil is the world’s third-largest producer of bauxite, the raw material from which aluminum is made. It accounts for 13.8% of the global production of this mineral the country’s most meaningful reserves are (94%) are located in the northern region, in the state of Para (Geological Survey, 2010).
Aluminum-based antacids, medications that include aspirin, drinking water and foods are the main sources of healthy people’s exposure to the metal. Its systemic absorption after the ingestion of monomeric salts is greater after the ingestion of water than (0.28%) after the ingestion of food (0.1%). Once absorbed, it will accumulate mainly in the bones, brain, liver and kidneys (Willhite et al., 2012).
Until recently this metal was considered harmless, since it presents itself in the trivalent oxidized form Al3þ, which easily at-taches itself to ions and forms colloidal polymeric particles. Because of the formation of insoluble particles of this kind, it was believed that the absorption of this metal would be limited (Bondy, 2010). However Al compounds have shown themselves to be toxic to animals (Garcia et al., 2009; Geyikoglu et al., 2012), and there is growing concern about their potential effect on human health (Willhite et al., 2012). A relationship between elevated levels of Al and an increased risk of a series of diseases has therefore been suggested. Among these are encephalopathy in individuals with renal deficiencies (dialysis encephalopathy), cognitive deficits in small children and hyperchromic microcytic anemia (Willhite et al., 2012).
In subacute and chronic doses, there are strong indications of its systemic toxic activity, with evidence of neural toxicity and enzy-matic alterations in organs such as the liver, testicles and kidneys (Geyikoglu et al., 2012; Newairy al, 2009andTurkez et al, 2010).
Despite evidence that the effect of aluminum on the immune system may be limited, it is reported that long-term exposure re-sults in elevated intercellular levels of this metal in lymphocytes, which can contribute to a condition of immunosuppression (Kamalov et al., 2011).
Epidemiological studies have demonstrated weak performance on cognitive tests and a higher prevalence of neurological symp-toms in workers exposed to aluminum. There are also reports of the metal inducing ventricular tachycardia in those acutely intoxicated and chronically poisoned by it (Kawahara and Kato-Negishi, 2011;
Yildiz et al., 2012).
There is also evidence suggesting possible genotoxic activity of Al, presented in studies conductedin vitro(Lima et al., 2007) and in vivo(Klingelfus et al., 2015; Turkez et al., 2010). Exposure to aluminum induces failures in the construction of mitotic spindles, which can lead to endoreduplications, polyploidy and clastogenic activity (chromosomal breakage) in human lymphocytes exposed to it (Lima et al., 2007). Studies have also proven that aluminum causes delayed DNA synthesis in human lymphocytes exposed to it, micro-nucleation, and immunotoxic and neurotoxic effects (Synzynys et al., 2004).
There is also know that Al Aluminum can be particularly toxic to the genetic apparatus, affecting the homeostatic and functional gene signaling, by mechanisms such as changes in the expression of micro-RNAs (miRNAs), becoming reasonably clear that aluminum disturbs genetic signaling programs in the CNS (Pogue and Lukiw, 2016).
Ghorbel et al. (2015)also related toxic effects of aluminum and co-exposure of aluminum and acrylamide, showing and effects such alterations on biochemical variables, genotoxic, and histo-pathological changes on kidney.
Despite the already documented and recognized toxic potential
of Al, especially it’s neurotoxic potential (Geological Survey, 2010; Lankoff et al., 2006), there has been little research on its actual ability to provoke DNA damage and the mechanisms of its systemic physiopathological effects in others tissues (Lima et al., 2007; Klingelfus et al., 2015; Turkez et al., 2010). The main objective of the present was to verify the genotoxic and toxicology potential of AlCl3usingin vivoandin vitroexperiments.In vivoexperiment was
performed by acute exposure of Swiss mice to AlCl3andin vitro
experiment by exposure of human lymphocytes to this metal. Both methods are well described and considered realistic models for assessing the effects of xenobiotic contamination.
2. Methodology
The study was performed in accordance with the Helsiniki Declaration and national legislation in force against animal vivi-section (Brazilian Federal Law 11794 of 2008), as well as being approved by an ethics committee on the use of animals and research with human beings at the State University of Para (Pro-tocol Nos. 01/11 and 02/2012).
2.1. Animals for in vivo tests
40 Swiss mice were used, all of which were healthy young al-bino adults (between six and eight weeks of age) of both sexes. The animals variation in weight did not exceed 20% of the median weight for each sex. The animals came from the Bioterium of the UEPA Laboratory of Experimental Surgery and were kept in an environment with a temperature of 22 C (± 3 C), with a 12-h
bright/12-h dark light cycle, receiving water and rations ad libi-tum. The sample size was based on international norms for the application of micronucleus tests for acute exposition (Cardoso, 2012; Igarashi et al., 2010; OECD, 1997; Takagi et al., 2011).
The 40 animals were randomly distributed, paired by sex and separated into 5 groups (three aluminum study groups, positive control and negative control), according to the following description:
- Aluminum Group: composed of three groups, each with 8 ani-mals (4 females and 4 males), corresponding to the three con-centrations of Al used (49, 98 e 161 mg/kg).
- Negative Control Group: composed of 8 animals (4 females and 4 males) that received distilled water.
- Positive Control Group: composed of 8 animals (4 females and 4 males) that received cyclophosphamide (50 mg/kg).
2.2. Samples for in vitro tests
For thein vitrostudy, human lymphocytes were analyzed after being taken from 3 individuals (2 men and 1 woman), ranging from 17 to 20 years of age, non-smokers and non-drinkers who were not exposed to radiation, infection or any type of medication for a period of less than one month from the date of collection. The in vitroexperiments were performed independently and duplicates.
2.3. Chemical agents
Forin vivoand in vitroexposure, aluminum chloride hexahy-drate (AlCl3$6H2O CAS nº7446-70-0) was used, diluted in distilled
water in concentrations of 49 mg/kg, 98 mg/kg e 161 mg/kg for in vivotests and 5
m
M, 10m
M and 20m
M forin vitrotests. AlCl3is themost common chemical form of Al (Bondy, 2010), and the doses used in our in experiments represent, respectively, 1/8, 1/4 and 1/2 of aluminum chloride LD50 for mice, according toKrasovskii et al.
(1979)andOndreicka et al. (1966). Distilled water was used as a negative control for the two experiments. The doses of AlCl3used
onin vitroexperiment were based on citotoxicity data for this metal on human lymphocytes (Lima et al., 2012). The positive control used for thein vivotests was cyclophosphamide (C7H15Cl2N2O2P/
CAS nº 50-18-0), diluted in 0.9% of physiological serum and
administered in a concentration of 50 mg/kg (OECD, 1997). Positive
control used for the in vitro experiment was Doxorubicin in a
concentration of 0.02
m
g/mL. All the solutions were preparedimmediately before use.
2.4. In vivo experimental procedures
The animals were exposed only once to the chemical agents used, at a maximum volume for 0.4 mL per animal. In the positive control group, a solution of cyclophosphamide was administered by intraperitoneally injection. In all other groups, the means of administration used was oral.
Animals were sacrificed 24 h after the administration of the substance. Soon afterward, dissection of the stomach, liver and kidneys was performed on the animals from the aluminum group and the negative control, as well as the removal of the animals’left femurs to obtain bone marrow cells. This long bone was dissected and its proximal epiphysis was cut for the extraction of bone marrow.
2.4.1. In vivo micronucleus test (MN)
Thein vivoMN test was performed according to the method-ology described in theOECD (1997)andSchmid (1975), thus, 0.5 mL of physiological serum was injected into the medullary canal of the femur. Once bone marrow cells were collected, their suspension was transferred to a centrifuge containing 2.5 mL of physiological serum. Next, the cell suspension was homogenized and centrifuged at 1000 rpm. Over the course of 10 min, 2 mL of supernatant were discarded. With the remaining suspension, 2 slides were prepared per animal. This was done using a rubbing method in which one slide was smeared on the other. After 24 h, with the slides totally dry, staining was performed with Leishman’s stain. This staining process used four tubs, in which the samples were immersed sequentially as follows: 1) Pure Leishman’s dye for 3 min; 2) So-lution of Leishman in distilled water (1:6) for 15 min, after which they were washed in running water; 3) With PA acetone for 10 min. Finally, after a drying period, the samples were protected with Entellen-mounted coverslips.
For genotoxic analysis, all the slides, including those from the positive and negative controls, were coded, and at least 2000 immature erythrocytes (polychromatic erythrocytes, known as PCEs) were counted per animal, assessing the presence of micro-nuclei. To evaluate the cytotoxic potential of the metal, the
pro-portion of PCEs among the total of erythrocytes
(PCEs þ normochromatics) was verified; this proportion was
determined as amounting to at least 200 total erythrocytes per animal. The decrease in the ratio of immature erythrocytes (PCE) reflects a decrease in the PCE/NCE ratio and is therefore considered a parameter of cytotoxicity (Ribeiro et al., 2003).
2.4.2. Micro- and macroscopic study (histopathological)
The organs removed from the animals (stomach, liver and kid-neys) were weighed and macroscopically analyzed with regard to coloring, presence of hemorrhages, consistency, and other alter-ations like erosions or nodules. The relative size of the extracted organs was calculated according to the following formula: Index¼weight of organ (mg)/weight of animal (g).
Next, these parts were preserved in 10% formaldehyde, to be processed 48 h later according to the standard histopathological
technique (dehydration with a progressively strongerdfrom 70% to
100%dseries of alcohol solutions, diaphanization in xylol,
impregnation and inclusion in paraffin). In a semiautomatic
rotating microtome, the tissue fragments, encased in paraffin, were sectioned to a thickness of 5
m
m, and then colored withhematox-ylin and eosin. One slide was produced per organ, with a minimum of three slices for histopathological analysis. The histopathological analysis was performed by Dr. Edvaldo Silveira (UEPA, pathologist).
2.5. In vitro experimental procedures
2.5.1. Obtainment of blood samples
10 mL of peripheral blood were collected from the individuals selected, using venous puncture after asepsis of the area to be punctured, using disposable needles and syringes previously hep-arinized with Liquemine (Heparin sodium). The sample size was based on recent articles that used the micronucleus test on human lymphocytes exposed to possibly genotoxic substances. Seeing as the most important factor is not number of individuals used, but rather the number of cells counted (Lu et al., 2012), the present work maintained a count of 1000 cells per culture. The collected material was kept for about one and a half hours in an antiseptic chamber and wrapped in aluminum foil to ensure faster sedi-mentation of blood globules.
2.5.2. Temporary lymphocyte culture technique (according to
Moorhead et al. (1960))
Resuspended lymphocytes of peripheral blood were cultivated in 5 mL of culture RPMI-1640, supplemented with antibiotics (penicilline5 UI/mL RPMI e streptomycine0.0125 mg/mL), 20%
sterile Fetal Bovine Serum and 0.2 mL (2%) of phytohemagglutinin (PHA), for a period of 72 h in a CO2-filled incubator at 37C.
The treatment with aluminum (AlCl3$6H2O) was performed
after 20 h of seeding the cultures, at concentrations of 5
m
M, 10m
M e20
m
M. The doses used for this experiment followed those reportedin the literature and used in otherin vitrotests with lymphocytes exposed to AlCl3(Lima et al., 2007). At this stage the treatment with
doxorubicin 0.02
m
g/mL was also performed on the positive controlgroup (1 man and 1 woman). The negative control group received 0.05 mL of distilled water, the same volume used for the treatments with Al and doxorubicin.
2.5.3. Micronucleus test (MN) with cytokinesis blocking (according toFenech and Morley (1985))
After 44 h of incubating the cultures, Cytochalasin-B was added at a concentration of 6
m
g/mL, which was active until the end of theculture time (72 h).
In the present study, the micronucleus test was performed ac-cording to the methodology described in OECD guidelines for the testing of chemicals, number 487, from 2007.
28 h after the treatment with Cytochalasin-B, the culture collection was performed. For this, the cultures were centrifuged at 1000 rpm for 5 min. After this procedure, the supernatant was removed, leaving a 1-mL pellet of content that was resuspended in 5 mL of frozen hypotonic solution (KCl 0.075 M); the content was centrifuged again at 1000 rpm for 5 min. Next, the supernatant was removed, leaving a 1-mL pellet resuspended in 5 mL of Carnoy fixative (5 methanol:1 acetic acid). In this phase, 3 drops of form-aldehyde were added to each tube, which were all then centrifuged at 1000 rpm for 5 min. After this procedure, two more washes with Carnoyfixative were performed (3 methanol:1 acetic acid), leaving, by the last wash, a pellet with afinal volume of only 0.3 mL.
The material obtained was used to create the slides, dropping three drops of the material, letting it dry at room temperature before staining it with Giemsa solution for 7 min. After drying at
room temperature, observation of the cells under an optical mi-croscope and a count of the MN was performed.
All the MN testing was performed blind, with coded samples under a backlit optical microscope at 400 magnification. The
criteria for identifying micronuclei were described bySarto et al. (1987)and byTolbert et al. (1991). They are structures that pre-sent chromatic distribution and coloration equal to (or occasionally darker than) the nucleus, which may be on the same plane as it.
They present defined boundaries and similarities to nuclei, and their size does not exceed 1/3 of the size of the nucleus. 1000 binucleated cells per prepared slide were tallied. The count was performed on cells with intact cytoplasm. Only structures with the characteristics describeddand which were found to be distinctly
separate from the main nucleusdwere considered micronuclei.
2.6. Statistical analysis
The data were submitted to statistical analysis using version 5.0 of the Bioestat®
program with a significance threshold of
a
¼0.05(5%), using the ANOVA test, followed by a Dunett’s test and a qui-quadrant test.
3. Results
3.1. In vivo experiments
Table 1 and Fig. 1 show our evaluation of the results of the micronucleus test on mice exposed to different concentrations of Aluminum chloride. According to this parameter we can observe that the metal referred to presents statistically significant genotoxic potential in all tested concentrations, in comparison to a negative
Table 1
Mean and standard deviation of the proportion of erythrocytes and frequency of micronuclei (MN) in mice exposed to Aluminum Chloride.
Treatment Animalsa PCE/(PCE
þNCE)b MN/2000 PCE
Negative Control 4 Males 0.54±0.04 4.5±1.9
4 Females 0.51±0.02 2.5±0.6
49 mg/kg AlCl3 4 Males 0.53±0.02 55.25±3.4*
4Females 0.53±0.01 63.25±6.4*
98 mg/kg AlCl3 4 Males 0.55±0.04 78.00±15.1*
4Females 0.55±0.004 76.5±7.2*
161 mg/kg AlCl3 4 Males 0.6±0.06 72.0±4.3*
4Females 0.62±0.03 79.0±5.0*
PCE: Polychromatic erythrocyte; NCE: Normochromatic erythrocyte; (*) p<0.01 (ANOVAeDunnett).
aHotelling test (p
¼0.193) no statistical difference between males and females. b ANOVA (p¼0.9948).
Fig. 1.Histopathologic Findings found onin vivoexperiments.A: 161 mg/kg AlCl3mouse stomach (), showing inflammatory infiltrate predominantly mononuclear (pink arrow) and ectatic vessel (white arrow).B: 98 mg/kg AlCl3mouse liver, with inflammatory infiltrate and necrosis (black arrow), sinusoidal hemorrhage (white arrow), and tumefaction of the hepatocyte (square).C: 49 mg/kg AlCl3mouse liver, especially centrilobular congestion (black arrow) and vacuolar degeneration (white arrow).D: 161 mg/kg AlCl3mouse liver especially hemosiderin pigment (black arrow) and vacuolar degeneration (red arrow).E: 98 mg/kg AlCl3kidney of mouse, especially glomerular and interstitial hemorrhage (black arrow), inflammatory infiltrate (yellow arrow) and cell tumefaction (pink arrow). (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)
control group (Table 1). The totals of 2000 cells were analyzed for each group evaluated.
The values presented refer to the means of the values found for all the animals in each group.
Table 2shows the effects of Aluminum chloride on the relative weight of the organs analyzed. According to this parameter the metal referred to caused significant shrinkage in the stomach and kidneys of 161 mg/kg AlCl3Group, in comparison with the control
group.
Macroscopic alterations in the organs of the treated and control groups were not verified. However, all the groups subjected to microscopic analysis were found to have histopathological alter-ations in the tissue integrity of their stomachs, kidneys anddmost
of alldtheir livers (Tables 3 and 4). Histopathological data
demonstrated that there is probably no dose/treatment relation-ship of the effects observed after Al treatment.
InFig. 1, the main microscopicfindings of altered tissue integrity in the stomachs, kidneys and livers of the mice treated with various concentrations of AlCl3are demonstrated.
3.2. In vitro experiments
In Table 5 are the results of our evaluation of the in vitro Micronucleus Test on human lymphocytes subjected to different concentrations of Aluminum chloride. According to this parameter we can observe that the metal referred to presented genotoxic potential and significant increase in all tested concentrations in comparison to a negative control group. Treatments with the 10 and 20
m
M concentrations presented the largest meaningfulin-crease in MNs. The totals of 2000 cells were analyzed for each group evaluated.
Table 6exhibit the quantity of micronuclei (MN) per binucleated cell in the three AlCl3concentrations evaluated. In comparison to
the negative control group, one can observe that there was a sig-nificant increase in the number of 1 MNs per binucleated cell. However, the number of 2 MNs per binucleated cell was only sig-nificant in concentrations of 10 and 20
m
M. For the smallestcon-centration (5
m
M) the increase was not statistically significant. Table 2Effect of Aluminum Chloride mean and standard deviation of the relative weight of the stomach, liver and kidneys of mice.
Organs Sex Negative Control 49 mg/kg AlCl3 98 mg/kg AlCl3 161 mg/kg AlCl3
Stomach Weight Male 12.54±1.26 14.07±1.8 13.79±5.32 7.4±1.93*
Female 14.55±3.59 13.36±2.32 14.14±2.77 10.6±3.04
Liver Weight Male 47.48±3.62 45.77±5.24 43.82±5.05 43.2±10.71
Female 46.29±11.28 41.30±6.9 42.18±3.43 42.98±2.42
Kidneys Weight Male 6.95±1.29 7.34±1.3 6.38±0.49 4.7±1.92**
Female 4.97±1.06 5.06±1.54 5.17±0.74 4.72±0.15
(*) p<0.05 (ANOVAeDunnett); (**) p<0.01 ANOVAeDunnett). The values presented refer to the means of the values found for all the animals in each group.
Table 3
Frequency of the main histopathologicalfindings in the stomach and liver of mice exposed to Aluminum Chloride.
Organ Main histopathologicalfindings Frequency
49 mg/kg AlCl3(N¼8) 98 mg/kg AlCl3(N¼7) 161 mg/kg AlCl3(N¼8) Positive Control (N¼8)
Stomach Mononuclear inflammatory cells 3 2 6 5
Eosinophilic and neutrophilic inflammatory cellsa 0 0 3* 0
Ectatic vessels 0 1 2 3
Liver Portal and centrilobular congestion 2 3 3 7
Sinusoidal hemorrhage 0 3 5* 3
Kupffer cell hyperplasia 8 6 6 7
Discreet tumefaction of hepatocytes 5 7 6 7
Mononuclear inflammatory cells 3 3 5 4
Focal necrosis 1 1 4 3
Microvesicular steatosis 3 0 2 1
Vacuolar degenerationb 4* 0 2 1
Hemosiderin pigmentation 0 2 2 0
ap¼0,0252 (Chi-square: Partition - * p value:0.0155). bp
¼0.0208 (Chi-square: Partition - * p value: 0.0066).
Table 4
Frequency of the main histopathologicalfindings in the kidneys of mice exposed to Aluminum Chloride.
Main histopathologicalfindings Frequency
49 mg/kg AlCl3(N¼8) 98 mg/kg AlCl3(N¼7) 161 mg/kg AlCl3(N¼8) Positive Control (N¼8)
Portal and centrilobular congestion 2 3 3 7
Sinusoidal hemorrhage 0 3 5* 3
Kupffer cell hyperplasia 8 6 6 7
Discreet tumefaction of hepatocytes 5 7 6 7
Mononuclear inflammatory cellsa 3 3 5 4
Focal necrosis 1 1 4 3
Microvesicular steatosis 3 0 2 1
Vacuolar degenerationb 4* 0 2 1
Hemosiderin pigmentation 0 2 2 0
ap¼0.0151 (Chi-square: Partition - * p value: 0.0023). bp
¼0.0001 (Chi-square: Partition - * p value<0.0001).
4. Discussion
Beginning in the last century, the use of chemical substances in industry and agriculture, has led humans to be exposed to them as much in their work environment as in their macroenvironment. Unfortunately, some of themdmetals especiallydcan lead to toxic
effects upon contact with living beings (Kuno, 2009). Aluminum is naturally present in the air, water and soil. It is the most widely distributed element in the environment and the third most com-mon element in Earth’s crust, as well as being extensively used in daily life, clearly exposing the human population to its acute and chronic effects (Kumar and Gill, 2009).
Toxicity of Al, such as other metals, is a complex matter (Klingelfus et al., 2015), related to many types of influences such as interacting with the performance of essential biomolecules, dis-placing other metals found in the system, and changing the struc-tures of proteins. Some ways or some environmental conditions, and individuals conditions, like genetic predisposition, may
influence the action of metals in inducing carcinogenic process or interacting with genetic materials (Costa et al., 1984;Kazantzis and Lilly, 1986; Woo et al., 1988). In light of these facts, it has become important to make detailed studies of its toxicology and genotox-icity potential in human cells andin vivomodels.
4.1. Genotoxicity and cytotoxicity in vivo and in vitro
In vivo exposure of mice to AlCl3 revealed genotoxicity in all
tested doses when evaluated using anin vivomicronucleus assay. The results of ourin vitroexperiments, in which human lympho-cytes were exposed to AlCl3in three concentrations also revealed
the genotoxicity of this metal (clastogenic or aneugenic potential) in all tested concentrations. These results corroborate with what has been described in the literature, where it is pointed out, through other tests and experimental models, that Al is proved to be a metal with the capacity to produce damages in the DNA molecule. The results thereby demonstrate that Al is a genotoxic substance that acts by breaking chromosomes or interfering with the formation of mitotic spindles, altering the balanced distribution of chromosomes during cellular division. There are some studies in the literature that relate to the genotoxic potential of aluminum metal, mainly as it has been demonstrated throughin vitro exper-iments (Pogue and Lukiw, 2016; Klingelfus et al., 2015; Lima et al., 2012, 2007; Octive et al., 1991).
According toLima et al. (2007), aluminum chloride is an agent
capable of inducing chromosomal aberrations in vitroin human
lymphocytes, generating endoreduplications and polyploidy during the G1 and G2 phases of mitosis, as well as presenting chromo-somal aberrations in all phases of the cell cycle. Their response demonstrates the clastogenic activity of this metal. And when evaluated using a test for chromosomal aberrations, it indirectly demonstrates Al’s capacity to compromise lymphocytic construc-tion of mitotic spindles. Finally, this study demonstrated the gen-otoxic activity of AlCl3 in human lymphocytes evaluated using a
comet test.
In a similar fashion, the study byIarmarcovai et al. (2005) per-formed on individuals occupationally exposed to various metals (metal welders), concluded that, among the metals studied, Al was among those that provoked the most damage to genetic material, as demonstrated by anin vitromicronucleus test with Cytochalasin-B. The current study demonstrated that Al treatments, even in low concentrations (5
m
Me in vitroand 49 mg/kg e in vivo) couldinduce damage to genetic material and thereby enable the forma-tion of micronuclei (MN). Thesefindings are in accord with those of
Kamalov et al. (2011), who demonstrated that low concentrations (10
m
M) of a chemical form similar to aluminum can also induceDNA damage detectible by micronucleus test.
In thein vivoandin vitroexperiments, the amount of damage found (number of micronuclei) was directly proportional to the concentration used, with frequency and mean MN values higher in treatments with higher concentrations of AlCl3, suggesting that
exposure to high concentrations of Al, or cumulative contact with this metal provokes significantly probable alterations in DNA. In the literature, studies directed toward tests of genotoxicity, performed with various concentrations of Al, point out that the greater the concentration of this metal is, the greater damage it does to DNA itself (Pogue and Lukiw, 2016; Lima et al., 2007; Paz, 2012).
There are yet other studies that have also evinced the potential genotoxicity of this metal in various other chemical forms (Balasubramanyam et al., 2009,Geyikoglu et al., 2012; Turkez et al., 2010).
The genotoxicity generated by Al seems to occur through different mechanisms such as: alteration of chromatic structure (Lima et al., 2007), induction of free radicals of oxygen (ROS)
Table 5
Mean and standard deviation in the frequency of micronuclei (MN) in human blood samples submitted to AlCl3.
Treatments Sample Number of MN Mean±SD
AlCl35Мm M1 96 103±11.3*
M2 97
W 116
AlCl310Мm M1 248 175.7±63.7**
M2 128
W 151
AlCl320Мm M1 198 232.7±48.4**
M2 212
W 288
Negative Control M1 24 27±9.8
M2 19
W 38
Positive Control M 87 87±0
W 87
M1: Man 1; M2: Man 2; W: Woman; MN: Micronuclei; (*) p<0.05 - ANOVA/ Dunnett (p¼0.0018); (**) p<0.01 - ANOVA/Dunnett test (p¼0.0018).
Table 6
Mean and Standard Deviation of the frequency and number of micronuclei (MN) by binucleated cell in human blood samples submitted to 3 different concentrations of Al chloride.
Treatments Number of MN Frequency of MN/ Binucleated cell
Mean±SD
M1 M2 W
AlCl35mM 1 82 79 91 84±6.2*
2 4 4 8 5.3±2.3
3 2 2 3 2.3±0.6
4 or more 0 1 0 0.3±0.6
AlCl310mM 1 78 90 112 93.3±17.2*
2 30 12 10 17.3±11**
3 30 2 5 12.3±15.4
4 or more 5 2 1 2.7±2.1
AlCl320mM 1 94 114 90 99.3±12.9*
2 30 17 26 24.3±6.7**
3 12 16 30 19.3±9.4
4 or more 2 4 14 6.7±6.4
Negative Control 1 16 13 20 16.3±3.5
2 4 3 6 4.3±1.5
3 0 0 2 0.7±1.1
4 or more 0 0 0 0±0
Positive Control 1 27 - 33 30±4.2
2 7 - 11 9±2.8
3 10 - 4 7±4.2
4 or more 4 - 5 4.5±0.7
M1: Man 1; M2: Men 2; W: Woman; MN: Micronuclei; (*) p<0.01 - ANOVA/ Dunnett (p¼0.0002).
(Kumar and Gill, 2009), free DNase from lysozymes (Banasik et al., 2005), and affecting the functional gene signaling (Pogue and Lukiw, 2016). Thus, it is conceivable that aluminum is genotoxic by one or all of these mechanisms (Balasubramanyam et al., 2009). Thefirst mechanism for these conclusions is that Al can infl u-ence chromatic structure and possibly cause damage to DNA (Bharathi et al., 2003; Lima et al., 2007). The second mechanism is that, supported by cellular interaction, Al can bring about the for-mation of free radicals of oxygen (Newairy et al., 2009; Turkez et al., 2010). The third mechanism indicates an increase in lysosomal membrane permeability with protein pump inhibition, leading to the DNase’s being freed into the cytoplasm and its passage into the nucleus, asZatta et al. (2000)have found. Finally, the last mecha-nism suggests that through changes in the expression of miRNAs molecules Al may alter the expression pattern of several genes, particularly affecting the function of genes related to the nervous system (Pogue and Lukiw, 2016).
In relation to possible cytotoxic activity, the statistical analysis of the number of erythrocytes in thein vivoexperiment did not show themselves to be significant in any experimental group compared to the control, suggesting that, at the amounts used, there was not a large amount of cellular death. There was, however, a slight in-crease in the proportion of erythrocytes in the groups treated with the highest concentrations of AlCl3(98 and 161 mg/kg), suggesting
that such a result could be justified by the short treatment time (24 h) used and/or by the relatively small dose of aluminum.
We did notfind statistically significant cytotoxic activity pro-voked by this metal in the present study, just asBalasubramanyam et al. (2009)did not. In this work too, only one exposure to metal was performed with the goal of understanding the biological ac-tivity of aluminum nanoparticles administered in an acute form.
Similar to thesefindings,Kamalov et al. (2011), in the course of evaluating in vitro cytotoxic activity of low concentrations of aluminum, did notfind elevated values for cellular death. However, they did identify damage to the integrity of the cell membrane, which by itself was insufficient to cause cell death owing to ne-crosis or apoptosis.
Differing from the result found in the present study, there are several studies in the literature (Klingelfus et al., 2015; Lima et al., 2007; Newairy et al., 2009; Turkez et al., 2010; Zhang et al., 2010) that evince cytotoxic activity in exposure to aluminum, in both in vitrodelineaments and in rat hepatocytes. In the study byZhang et al. (2010), it was evinced that aluminum chloride could induce cellular death by a combination of apoptosis and necrosis. However, elevated doses of aluminum were used in these studies and- spe-cifically in the work conductedin vivo-the animals were exposed to the metal for a long period.
In thein vitro experiments, it was found that the treatments with the highest concentration of AlCl3, had the highest frequency
of binucleated cells with DNA damage, which can be observed with the highest quantity of micronuclei found at that concentration (20
m
M). We know that damage to DNA is directly proportional tothe quantity of micronuclei formed in a binucleated cell, since these are biomarkers of genotoxic events (Fenech, 2000).Colognato et al. (2007)also report that the greater the concentration of a poten-tially genotoxic substance used the greater the quantity of micro-nuclei in binucleated cells.
Yet in the experiments performedin vitro, binucleated cells with 3, 4 or more nuclei per cells were evident in low frequency in all concentrations. We know that the greater the quantity of micro-nuclei, the more unstable the cell will be, owing to the greater damage it has sustained. This damage leads to a process of cell death (Fenech, 2000). Such a result justifies that low frequency in cases with 3, 4 or more micronuclei per nucleated cell, since they
have probably entered into apoptosis. Similar findings were
reported in the study conducted byLankoff et al. (2006), in which the cell reduction was accompanied by a high degree of apoptosis, indicating the selective elimination of damaged cells.
4.2. Toxicological analysis
In toxicological analysis, evaluation of the relative weight of organs revealed that the all the animals treated with the smallest concentrations of AlCl3(49 mg/kg) and (98 mg/kg), as well as the
females treated with the highest concentration (161 mg/kg), have not demonstrated significant alterations in any of the organs analyzed (stomach, liver and kidneys). However, in males from the group treated with the highest concentration of AlCl3(161 mg/kg),
the stomach and kidneys presented hypotrophy, with consequent decrease of weight of these organs (Table 2).
Macroscopically, the organs analyzed presented no apparent alterations. In histopathological evaluation, some of the findings probably happened during the animal’s death process, or arose from the usual problems associated with histopathological analysis, since they did not present a dose-response relationship, occurring equally in both the experimental and control groups.
5. Conclusion
Having the support of this work’sfindings, with a base of pre-vious publications and our study group, we observe that Al, even in the smallest concentrations, presents genotoxic actionin vitrowith the presence of DNA damage, andin vivoindications of systemic toxicity, with the presence of morphological alterations in all the organs researched. Considering the presence and importance of this compound for human beings, we therefore suggest that this metal presents genotoxic potential, and it is potentially the cause of pathological disorders found.
Acknowledgments
This work was principally supported by the Research Activities Support and Development Program at the State University of Para, number 061/2010.
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