Aluminium and its Derivatives Causing Genotoxicity and its Underlying Mechanism

INTRODUCTION

Aluminium(Al) occurs naturally in the soil, which constitutes the third most abundant element and makes up about 8% of the Earth’s crust. Free state of aluminium is not found, it is found in combination with other elements as aluminium oxide, aluminium silicate, aluminium borate, etc. They have special physicochemical properties hence aluminium compounds and their derivatives are used in the preparation of various commercial products, including therapeutic agents, water purifiers, and as food additives.¹ Nanotechnology is progressing rapidly and we attempt to explore the new horizons of it which constantly keep us guessing if the potential advantages outweigh the disadvantages or vice versa. Therefore, the responsibility lies on interventions and exploring the possibilities of the nanomaterials (NMs) in human health before they are pervasively dealt.² Currently, the important NMs are simple metal oxides, such as aluminium oxide (Al₂ O₃ ), silica dioxide (SiO₂ ), titanium dioxide (TiO₂ ), iron oxide (Fe₃ O₄ , Fe3 O₄ ) and zinc oxide (ZnO) which are most commercially available. Among them, aluminium compounds are the most abundantly produced NM, estimated to account for 20% of the 2005 world market of NMs.² Aluminium on exposure to humans leads to toxic effects where most studies ofthe genotoxic and cytotoxic potentials of aluminium have been performed in vitro on cultured cells. Various studies have shown that Al induces micronuclei and chromosomal aberrations in human cells.³ The dietary intake of aluminium may lead to chronic contamination results in exceeding human natural capacities of accommodation. Patients treated with a higher dose of aluminium may lead to acute intoxication. on average, the amount of ingested aluminium ranges from 5 mg to 3 g per day in humans. When aluminium is administered the fraction of it reaches the systemic circulation and is not excreted in the urine rapidly, accumulates in peripheral tissues, where it is strongly bound. Aluminium distributes intracellular in various organelles, but the distribution varies in lysosomes, mitochondria and nuclei which are reported to be the major sites of binding. Salts of Al may bind to DNA and RNA.⁴ However, there were a few considerable studies in which they explored whether exposure to Al may be involved in neurodegenerative disorders such as Alzheimer’s disease, dialysis and Parkinson’s dementias, peptic ulcer disease, gastrointestinal toxicity and hepatotoxicity. Al compounds have a very high affinity for DNA, RNA and many mononucleotides and found to make complexes with DNA which leads to DNA damage. Various studies have made an implication that oxidative stress as one of the major molecular mechanisms that result in the Al toxicity. A recent study report suggested that Al compounds could lead to induction of morphological and functional alterations in erythroid cells. It was found that it acts on circulating erythrocytes and erythrocyte fragility which may be due to oxidative damage.⁵ It was also found that Al also results in cardiotoxicity, nephrotoxicity, and neurotoxicity. Furthermore, Al-induced hepatic dysfunctions, DNA cross-linking in rat ascites hepatoma cells, MN and sister chromatid exchange (SCE) formations in human peripheral blood lymphocytes.⁶

Mechanism

Aluminium and its compounds have various applications commercially and it is frequently used by the consumers, particularly it is used as a therapeutic agent and the evaluation of its genotoxic effect is practically relevant to arrive at a safer mode of exposure, where in a few studies the doses selected were higher than normal human exposure levels. In recent years, the use of aluminium has increased dramatically not only in the therapeutic field but also in various other fields. Aluminium is corrosion resistant and also has low weight hence is used widely as a building material, thereby increasing occupational exposure. Besides, aluminium also has excellent heat conductivity, it is used for the manufacture of various types of cooking utensils,⁶ which may also lead to increased human exposure.⁷ Aluminium compounds are observed by passive diffusion through paracellular pathways. It is found to have binding affinity to various ligands in the blood and distributes to the different organ through out the body, with highest concentrations found in bone and lung tissues. Aluminium which is been absorbed is majorly excreted through urine and partly is found to excrete through bile.⁸ In general, aluminium NMs may lead to DNA damage through various direct and indirect mechanisms. Once it has gained access into the cell via diffusion mechanism through the nuclear membrane transport through the nuclear pore complexes or during mitosis. Alternatively, they may trigger the formation of reactive oxygen species (ROS), inflammation and aberrant signaling processes that could lead to genotoxicity.² The mechanisms of genotoxic activity of Al are not well understood but It can be hypothesized that Al could induce DNA damage through three possible mechanisms i.e.,modification of chromatin structure, induction of reactive oxygen species and liberation of DNase from the lysosomes.⁹

Chromatin structure

It is the material that is composed of the chromosomes which constitute the genetic material such as DNA and RNA. It is found that aluminium is known to act on the chromatin structure and inflict the damage. McGill et al found that the mitotic cells exhibited chromosome stickiness employing an electron microscope, which resulted in the inter-chromosomal and inter-chromatid connections both in metaphase and anaphase. This led the authors to give a mechanistic explanation, which states that, ‘When the chromosome fibres fail to condense properly in the preparation of mitosis, they may be trapped and tangled with the fibres of other chromosomes, thus becoming physically connected. Such abnormal sub-chromatid connections cause many chromosomes to adhere to one another resulting in stickiness’. Extreme stickiness leads to clumping and disintegration of chromosomes resulting in cell death¹⁰ or reduced mitotic division.¹¹

Reactive oxygen species:

Reactive oxygen species (ROS) are highly reactive chemical molecules that posses oxygen ions and peroxides that include hydrogen peroxide, singlet oxygen, nitric oxide, peroxynitrite, and superoxide free radicals. These free radicals are highly toxic and act directly on the cell which may lead to cell death.¹²

The exact mechanism about how aluminium and its compounds exert the cell damage is not known but it could probably act via oxidative stress mechanism which is suggested by a few authors. In recent studies, it was demonstrated that Al promotes the generation of iron-induced reactive oxygen species (ROS).⁴ Oxidative stress is found to develop especially when there are low levels of antioxidants, such as glutathione, and this leads to the production of reactive oxygen species (ROS) which exceeds the capacityof the cell to dispose them, which results in the several irreversible modifications of a biologically fundamental macromolecule which includes oxidation of purine nucleotides of nucleic acids and that of protein SH group.⁵ Similarly, Garcia-Medina et al. reported that Al is found to alter the activity of antioxidant enzymes and elicited higher levels of lipid peroxidation and oxidized proteins.¹³ Lipid peroxidation is one of the main alterations that is linked with the rapid generation of reactive oxygen species where both the exogenous and endogenous sources exist their contribution.¹⁴ xenobiotics induce Oxidative stress which is a major cause of sperm abnormality.¹⁵ It is known that free radicals/oxidative stress induces DNA damage in meiotic chromosomes¹⁶ and also interfere with spermatogenesis differentiation process,¹⁷ both mechanisms are involved in the production of abnormal sperms. Al and its compounds induced oxidative stress-mediated by free radicals have been demonstrated by different authors. The testicular toxicity may be involved with a similar mechanism. The head abnormalities are found to have a dominating effect among the abnormal sperms. The head shape of the sperm is been determined by the Y-chromosome and any damage here may lead to malformations of the sperm head.^18,19 But the specific Y-chromosome damage induced by aluminium and/or its salts has not been reported. The genotoxicity observed with Al NM may be also due to pro-inflammatory effects which are mediated through reactive oxygen species (ROS)-mediated mechanism. New findings have been made which support the hypothesis that the size of the Al NM may be the reason forsignificant genotoxicity. The liver is the chief site of iron storage, containing 98% of total iron and a large abundance of transferrin receptors. It produces toxic effects by modifying iron homeostasis and interfering with iron regulatory proteins. Al is also known to be bound by the Fe3þ carrying protein transferrin, thus reducing the binding of Fe2þ. The increasing intracellular Fe2þ causes the peroxidation of membrane lipids and thus membrane damage.²⁰

Lysosome damage:

Lysosomes are the membrane-bound organelles that consist of the hydrolytic enzymes which release the enzymes when required, and its works on the proton pump mechanism which triggers the release of the enzymes. Aluminium has been found to have the capability to influence the permeability of the lysosomal membrane²¹ and to inhibit the lysosomal proton pump.²² The release of the DNAse is based on both these actions, which lead to the DNA fragmentation by getting released into the nucleus. Aly et al.²³ demonstrated that there is major damage to the genetic material on the bases of the release of DNase into the cytoplasm by electroporation mechanism.¹ The aluminium compounds enhance the permeability of the lysosomal membrane this probably could lead to DNase being liberated into the cytoplasm and its passage into the nucleus, where it would break down the DNA. It has been shown that DNase is released into the cytoplasm by electroporation mechanism which is a potent inducer of cytogenetic damage.²⁴

Zinc fingers:

Al ions are also known to inhibit the inhibit proteins with zinc finger domains. These domains have been identified in several DNA repair enzymes during any damage. They include the members of the poly ADP-ribose polymerase (PARP) family. It is believed that a PARP increase is one of the earliest nuclear events following DNA strand break.²⁵ It is known that al compounds are potent inducers of PARP and causes the DNA strand breakage.

Aluminium derivatives

The exact mechanism of aluminium-induced genotoxicity is not completely understood. However, aluminium and its derivatives are known to form complexes with the chromatin structure²⁶ and induce fragmentation of chromatin in cultured cells.²⁷ Various aluminium derivatives or salts have been studied for their mutagenicity in bacterial systems,⁹ in vitro^28,29 in vivo models,^23,30 and in plant cells³¹ here are the few aluminium compounds that have reported to have the genotoxic effect.

Aluminium acetate:

It is one of the aluminium derivatives that is known to have the genotoxic effect where studies support the following reasons for the chromosomal abnormalities. The major type of Aluminium acetate(AA)-induced chromosomal abnormality by the probable reason for chromosomal stickiness. According to Heddle,³² during mitosis, the first chromosomal abnormality that was observed was chromosome stickiness, whereas other abnormalities occur only later. The reason for this is not exactly known.1 AA is reported to induce a toxic effect on proliferation of the cells in the bone marrow which was confirmed by the significant reduction in MI and P/N ratio in CA and MN tests, respectively. It was reported that the higher doses of AA i.e., 100 and 150 mg/kg bw only showed thismito-depressive effect whereas at the lower dose (50 mg/kg bw) this effect was not that significant any time interval. However, on frequent dosing of AA at a lower dose induces a mito-depressive effect which can be supported by the significant reduction in MI and P/N ratio. These studies report that the observations are known to cause cytotoxic effect due to aluminium compounds in different cell types.^33,34 It was quite interesting to note that in case of adult animals AA did not cause any micronuclei (MN)formation in bone-marrow erythroblasts even when the dose was increased dose, but it was significant that it induces MN in fetal erythroblasts even at a low dose which was observed where there was drastically increased. This was made as an indication of its high sensitivity to fetal cells and observed to possess the genotoxic effect.^35,36 As specified earlier, aluminium acetate is known to develop ROS upon biotransformation, and one of the toxic effects of ROS is cytotoxicity/met depression. Further, it is well known that cytotoxicity is a direct consequence of DNA/chromosomal damage. The transplacental micronucleus assay study was done by Cole et al. like it a recommended method for assessment of prenatal genotoxicity. And from a few other studies found that there was a significant dose-dependent increase in the frequency of micronucleated fetal erythrocytes which indicated the transplacental genotoxic potency of AA. This observation proves that some aluminium compounds displayed genotoxicity, including the induction of MN in the fetus.^37,38,39

Aluminium chloride:

Aluminium chloride(AlCl₃ ) causes a significant increase in brain MDA and leads to lowering of cerebral GPx and SOD activities. ElDemerdashhas observed that during the exposure to the aluminium chloride there occurs rapid lipid peroxidation which is the major marker in case of aluminium toxicity.¹² Onthe other hand, Nayak et al⁴⁰ found that there was marked decrease in the antioxidant activity as the levels ofGPx and SOD was decreasing significantly, indicating that AlCl₃ decreases antioxidant defense system. Few studies have shown that the resveratrol administration lowers the MDA elevation in rats receiving both Al and resveratrol. Resveratrol is also known to alter the SOD and GPx suppression by Al administration significantly¹² which could probably be one of the reasons for the genotoxicity induce in the cells. One of the main reason for the release of the ROS is Al toxicity which causes the oxidative stress-induced cytogenic and genotoxicity. Earlier studies support that interactions between oxidative stress and hepatic damage may increase the probability of enhanced chronic hepatic degenerative disorders, which also include the increase in enzymes induced by Al. On the other hand, Al causes the induction of polynuclear ions which lead to the production of changes in the membrane and aggregate chromatin.Chemicals such as AlCl₃ and other aluminium compounds trigger lipid peroxidation in the cell membrane, which is a principal cause of activation of other mediators which could damage the DNA.⁴¹ Lipid peroxidation may be due to the excess generation of reactive oxygen species (ROS), which may be contributed by the exogenous or the endogenous sources.²⁰ Previous studies have reported that AlCl₃ may also lead to the damage of the cells in the kidney and surrounding tissues. Somova et al. have experimented the effect of aluminium chloride byadministering it in a dose of 20 mg/kg b.w./day to rats and they observed changes. Haematological data of male SpragueDawley rats treated with AlCl₃ for 10 weeks were collected and analysed which resulted in wellmarked dose-dependent morphological changes in the kidney and also the CA and MN assay was showing the positive results. The changes were interstitial fibrosis around the damaged tubules and partial sclerosis in the glomeruli. In the case of the kidney.²⁰ AlCl₃ is also known to be bound by the Fe3þ carrying protein transferrin, thus reducing the binding of Fe2þ. The increasing intracellular Fe2þ causes the peroxidation of membrane lipids and thus membrane damage.

Aluminium oxide:

Aluminium oxide(Al₂ O₃ ) is known to cause genotoxicity and neurotoxicity in rat cells.^22,42 Data acquired revealed that maximum accumulation of AL₂ O₃ in the tissues. The aluminium nanoparticles such as Al2 O3 -30 nm and AL₂ O₃ revealed that there was an increased amount of retention of these compounds in kidneys, out of which AL₂ O₃ -30 was majorly found, followed by Al₂ O₃ -40 nm. These retained compounds may lead to the attribution of Al₂ O₃ NM and get entrapped in the reticular endothelial system which is excreted by the kidneys in vivo. Al₂ O₃ also mainly gets piled up in the brain and the reason for this may be the presence of certain sites in the brain that circumvent blood-brain barrier (BBB) and this may be the way for which the aluminium compounds reaching the brain vicinity. Al oxide can also lead to increase the permeability of the lysosomal membrane by inhibiting the lysosomal proton pump.This may probably lead to the release of the DNase into the cytoplasm and which forms apassage into the nucleus and results in the fragmentation of the DNA. Studies have been reported that DNase is released from the lysosome into the cytoplasm by electroporation is a potent inducer of cytogenetic damage.²³ The ROS generation is also one of the mechanisms for causing genetic damage. A by-product of oxidative phosphorylation causes damage in mitochondrial macromolecules including the mitochondrial DNA leading to mutation and cancer in humans. The AL₂ O₃ cause chromosomal aberrations and DNA damage, causing arresting of the cell cycle and finally leading to cell death.⁴³ When there is an exposure towards the aluminium oxide there will be an imbalance in the ROS production which causes DNA damage by the oxidation of purine molecules. The study shows that an aluminium-oxide NP-treated cells showed mitotic arrest, chromosomes break, and deletion in human lymphocytes.^44,45 It was shown that AL₂ O₃ does not induce apoptosis of BJ and L929 cells, although it can penetrate cells, which was initially seen in microscopic observation and then confirmed by ICP-OES.³

Aluminium sulphate:

The compound aluminium sulphate was known to be the cause the genotoxicity in human lymphocytes where the exact mechanism is not known. It is reported that the generation of the ROS may be the probable reason for the toxic effect through the induction of the oxidative stress. Abubakar et al 2003 have noted that lipid peroxidation was one of the cause for the genotoxicity. Al₂ (SO)₄ leads to the functional and morphological changes in the circulating erythrocytes due to the direct action and induction of the oxidative stress where the fragility is been lost in the cells. It is known that Al₂ (SO)₄ leads to the alteration of certain genes expression and protein phosphorylation, which also inhibits certain cellular enzymes. It is reported that aluminium sulphate also causes an increase in the SCE formation and reduce the SOD and other antioxidants such as catalase and glutathione in blood this lead to increase in the free radical formation and damage the cells. Al and its compounds exhibit both genotoxic and cytotoxic activity in human peripheral blood lymphocytes. In addition to inducing oxidative DNA damage in manner non-specific to the cell cycle phase, exposure to Al induces a cell cycle delay that was apparent as the accumulation of cells in the S-phase. It should be stated that Al and its derivatives inhibit the repair of radiation-induced DNA damage in human lymphocytes.⁴

CONCLUSION

The cytotoxicity and genotoxicity studies revealed that the aluminium and its derivatives were found to be more toxic to human lymphocytes and also other smaller animals. The least toxicity was known to have been reported in the case of Al₂ O₃ whereas the other compounds such as AlCl₃ , Al₂ (SO)₄ , aluminium acetate was known to cause the major toxicity. When exposed to aluminium and its derivatives the possible mechanism of action was known to be because of oxidative stress and lipid peroxidation. The other mechanism was also known to cause severe damage to the DNA. The human lymphocytes exhibited an increase in genotoxicity when the increase in the doses and also in the chronic cases of aluminium administration. The cytotoxicity and oxidative stress lead to DNA damage and chromosomal aberrations in human lymphocytes and other organisms. The toxicity associated with the exposure of the aluminium and its derivatives suggests that proper attention needs to be paid regarding the use of them since it can cause harmful effects to humans.

Acknowledgements

Authors are thankful to the authorities of Karnataka College of Pharmacy, Bangalore for providing the facilities for conducting this study.

Conflicts of interest

The authors declare no conflicts of interests.