Printer Friendly

Attempt at the determination of aluminum nitrate [LD.sub.50] and the study of its neurotoxicological effect in Wistar rat.

Introduction

Aluminum is the third most abundant element, after oxygen and silicon in the earth's crust. It is widely distributed and constitutes approximately 8% of the earth's surface layer (Kabata-Pendias and Pendias, 1993). Due to their several chemical and physical properties, aluminum and its compounds are used in different fields. They are used in many diverse and important industrial applications such as alums in water treatment and alumina in abrasives and furnace linings. They are found in consumption products such as antacids, astringents, buffered aspirin, food additives, vaccines, and antiperspirants (aluminum profiles). Aluminum has several inorganic compounds (aluminum bromide, chloride, acetate, nitrates, and so on) that differ by their physicochemical characteristics and toxicity, identified by their [LD.sub.50] (Llobet et al., 1987; Yellamma et al., 2010).

Aluminum was considered, for a long time, as nontoxic element and completely excreted out of the body by renal way. However, nowadays, it is a well-known fact that Al affects several organs (brain, liver, kidney, etc.) and gets accumulated in them, and it specifically targets the nervous system (Julka et al., 1996; Azzaoui et al., 2008; Rawy et al., 2012). The hippocampus is most affected by aluminum toxicity. Santos et al. (1987) demonstrated a preferential accumulation of Al in the hippocampus in rat and Abd El-Rahman (2003) reported spongiform changes in the neurons of the hippocampus, nuclear deformity, neurofibrillary degeneration, and foci of demyelination in Al-intoxicated rats. Indeed, it is admitted that aluminum impairs the cholinergic system; some studies found that aluminum increases the AChE activity (Bilkei-Gorzo, 1993; Zatta et al., 2002). However, others showed that Al decreases the AChE activity (Kaizer et al., 2008; Yellamma et al., 2010). Gulya et al. (1990) proved that the effect of Al on AChE is biphasic: it increases AChE at low concentrations of Al and decreases it at higher concentrations of Al.

The aim of the present study is to determine an oral [LD .sub.50] of aluminum nitrate from which the rare values found in the literature are extremely different (542 mg Al/kg BW (National Research Council, 1981); 261 mg Al/kg (BW) (Llobet et al., 1987); and 3,671 mg Al/kg (BW) in some material safety data sheet of the same product. By corollary, ss another aim is to evaluate the effect of all aluminum doses used on rats' organs, on AChE activity, and on ACh level in rats' hippocampus.

Materials and Methods

Animals and treatment

Male Wistar rats, 6 months of age and 197.05 [+ or -] 0.66 g in weight (mean [+ or -] SEM, n = 28) at the beginning of the treatment, are used in this study. They were reproduced in colony room of Biology Department, Faculty of Sciences, Kenitra, Morocco. The rats are put in propylene cages under standard conditions (20[degrees]C, 50-70% humidity, and 12L:12D cycle). They are given free access to food and tap water. The control rats (n = 7) are given tap water and the tested rats (n = 21) receive three different doses: [Al .sub.1] = 2,500 mg/kg (n = 7), [Al.sub.2] = 3,500 mg/kg (n = 7), and [Al.sub.3] = 4,500 mg/kg (n = 7) of aluminum nitrate (Farco Chemical Supplies) diluted in distilled water, once by gavage. Experimental procedures are also examined and approved by the internal ethical committee for animal welfare.

Observations

All rats were examined twice on a daily basis for mortality during the 2-week experiment.

Body and organs' weight

Bodyweight is recorded at the beginning of the test and at the end of the experiment. The weight of the brain, liver, spleen, and kidneys is also taken at the end of the experiment, after anesthesia by the chloral 7%. All of these organs are subjected to detailed internal examination.

Determination of AChE levels

The specific activity of AChE is determined as described by Ellman et al. (1961). The reaction mixture contained 3.0 ml of 0.1 M phosphate buffer (pH 8.0), 20 ml of 0.075 M acetylthiocholine iodide, and 100 ml of 0.01 M 5,5-dithiobis-2-nitrobenzoic acid. The reaction was initiated with the addition of 100 ml of synaptosomal fraction. The color absorbance was measured at 412 nm in spectrophotometer (Reddy et al., 2007).

Determination of ACh levels

ACh levels were determined as described by Augustinsson (1963). The synaptosomal fractions of hippocampus were placed in boiling water for 5 min to terminate the AChE activity and also to release the bound ACh. To the synaptosomal fractions, 1 ml of alkaline hydroxylamine hydrochloride followed by 1 ml of 50% HCl was added. The contents were mixed thoroughly and centrifuged. To the supernatant 0.5 ml of 0.37 M ferric chloride was added and the intensity of the color developed was read at 540 nm against a reagent blank in a spectrophotometer (Reddy et al., 2007). Both results (of AChE and ACh) are expressed as percentage of control results.

Statistical analysis

The group data are expressed as mean [+ or -] SEM. The statistical tests used are analysis of variance (ANOVA1) and the least significant difference (LSD) post-hoc test. Differences between groups are considered significant at p < 0.05, 0.01 and 0.001.

Results

The objective of the experiment is to determine the [LD.sub.50] of aluminum nitrate; however, the used doses are very high, and the 50% of death is not reached--even at the last dose of 4,500 mg/kg of aluminum nitrate. At this dose, only 30% of rats died.

Gross pathology

In the sacrificed rats, all the organs are normal in the four groups. However, in the rats receiving [Al.sub.3] (4,500 mg/kg) and that died before the end of the experiment, a dark discoloration of the spleen is observed.

Bodyweight

The obtained results show no significant difference in BW between control and all treated groups, even at the beginning [(BW.sub.0]) or at the end of the experiment [(BW.sub.14]) (Figure1).

Organ weight

The three administered doses of Al [(Al.sub.1], [Al.sub.2], and [Al.sub.3]) had no effect on the weight of the brain, liver, and kidneys. However, they cause a significant decrease of spleen weight (F(3,16) = 7.48; p < 0.01). The post-hoc statistical study demonstrates that the high significant decrease is obtained after the administration of [Al .sub.2] and [Al.sub.3] (p < 0.01 and p < 0.001, respectively; Table 1).

AChE levels in the brain

The administration of aluminum nitrate decreases the AChE activity in hippocampus of intoxicated rats compared to the ones under control (F = 4.11; p < 0.01). The LSD post-hoc statistical test demonstrates that the [Al.sub.2] and [Al.sub.3] doses decrease significantly (p < 0.01) the AChE activity by 35.3% and 44.17%, respectively (Figure 2).

[GRAPHIC OMITTED]

[GRAPHIC OMITTED]

ACh levels in the brain

The administration of aluminum nitrate increases the ACh levels in hippocampus of intoxicated rats compared to the ones under control (F = 9.89; p < 0.001). The LSD post-hoc statistical test demonstrates that the [Al.sub.2] and [Al.sub.3] doses increase significantly the ACh levels ( p < 0.01 and p < 0.001, respectively) (Figure 3).

[ILLUSTRATION OMITTED]

Discussion

The experiment focused to determine an [LD.sub.50] of aluminum nitrate whose data are old, rare, and divergent in the literature. Some old studies reported that the oral [LD.sub.50] in the rats is 542 mg/kg (National Research Council, 1981), 264 mg/kg (US Coast Guard, 1984), and 261 mg/kg of aluminum nitrate (Llobet et al., 1987). However, most recent [LD.sub.50] value published in some Material Safety Data Sheet of societies producing aluminum nitrate as Mallinckrodt Chemicals, Sigma Aldrich, and Ficher Scientific, is 3,671 mg/kg of aluminum nitrate by oral route. In this study, the higher dose used [(Al.sub.3] = 4,500 mg/kg) caused the death of only 30% of the rats under study. So, it is impossible for us, to increase the dose more than 4,500 mg/kg), to determine the [LD.sub.50] because the OCDE (2001) sets the dose of 5,000 mg/kg as maximum testing dose of acute study tests.

The current findings show that aluminum does not affect the BW of intoxicated rats. In previous study, it was found that aluminum decreased the BW of intoxicated rats, but at the end of the intoxication experiment (90 days; Azzaoui et al., 2008). Other research found that exposition to aluminum salts did not affect the weight gain of rats (Muller et al., 1990; Gonda and Lehotzky, 1996).

Indeed, high acute doses of Al affect significantly the rat organs. The spleen of rats receiving the high dose of Al, and dying before the end of experiment, shows dark discoloration. A significant decrease of spleen weight in group receiving high dose [(Al.sub.2] and [Al.sub.3]) is registered, but the other observed organs show impairments. Few studies related to the effect of aluminum on spleen are published. Those we have come across evoked high aluminum accumulation in this organ. This accumulation could perturb the normal functioning of this organ (Llobet et al., 1987; Julka et al., 1996).

It was reported that the accumulation of Al in the brain, following acute and chronic intoxication by aluminum, causes biochemical changes leading to damage in the cholinergic system (Kosik et al., 1983; Julka et al., 1995; Meyer et al., 1996; Kaizer et al., 2005).

The cholinergic system is essential in mediating cognitive processes. Thus, any dysfunction in this system will induce impairments in all neurocognitive performance especially in learning and memory (Miu et al., 2003; Azzaoui et al., 2008; Voss et al., 2010; Abu-Taweel et al., 2012). The measure of AChE activity and ACh levels in the hippocampus of intoxicated rats shows a significant decrease in the AChE activity and significant increase in ACh levels, in rats receiving the acute high doses of Al (3,500 and 4,500 mg/kg). This result is consistent with others who found that the high concentrations of aluminum inhibit the AChE activity (Marquis and Black, 1984; Gulya et al., 1990). Indeed, Moraes and Leite (1994) report the in vitro inhibitory effect of very low concentrations of aluminum salts [(IC.sub.50] = 4.1 x [10.sup.-12] M) on bovine brain AChE. Moreover, acute toxicity of aluminum chloride at 3.7 g/kg BW, administered per o.s. to gerbil, decreases the activity of AChE in the mitochondrial and microsomal fractions of hippocampus (Micic and Petronijevic, 2000). An in vitro study by Jankowska et al. (2000) demonstrates that an excessive ACh release, evoked by Al, is likely to increase acetyl-CoA utilization for re-synthesis of the neurotransmitter pool and cause deficit of this metabolite in differentiated cells. Recently, Yellamma et al. (2010) have proved that AChE activity is inhibited by 700 mg/kg (BW) of aluminum acetate in hippocampus of orally intoxicated rats and their results also reveal that while AChE activity is inhibited, ACh level is elevated differentially in the studied area of the brain under aluminum toxicity.

Even several studies about the neurotoxic effect of aluminum are conducted (Jankowska et al., 2000; Kaizer et al., 2005; Nayak, 2006; Azzaoui et al., 2008; Yellamma et al., 2010; Abu-Taweel et al., 2012), its pathway is still discussed. Some studies suggest that Al interferes with the metabolism of glucose leading to the reduction of the synthesis of the precursors of the ACh. Other research shows that it could interact with [[Na.sup.+]/K.sup.+] ATPase and [[Ca.sup.2+]Mg.sup.2+] ATPase affecting the system of neurotransmission at the level of the neuronal presynaptic membrane (Nayak, 2002). Also, it is found that Al interferes with iron and magnesium (Crichton et al., 2002) and with calcium in extra and intracellular compartments, leading to an alteration in acetyl-CoA metabolism (Bielarczyk et al., 1998).

In this study, the high doses used (3,500 and 4,500 mg/kg) of aluminum nitrate affect the spleen (the principal organ of immunity), decrease the AChE activity and increase the ACh levels in hippocampus. More investigations are needed to understand well the neurotoxic effect of aluminum nitrate in acute toxicity.

Ethical Approval

The study was approved by the institutional ethical committee of the Department of Biology, Faculty of Sciences, Ibn Tofail University, Kenitra, Morocco.

Conflict of Interests

There is no conflict of interest.

Acknowledgement

This research was supported by Program of Support for Scientific Research (PROTARS III: D63/01), CNRST, Ministry of Higher Education and Scientific Research, Morocco. The authors thank Professor Hakim Hassan, Mohammed Premier University, Oujda, Morocco, for valuable comments and advice regarding English language.

References

Abd El-Rahman SS, 2003. Neuropathology of aluminum toxicity in rats (glutamate and GABA impairment). Pharmacological Research, 47(3): 189-194.

Abu-Taweel GM, Ajarem JS, Ahmad M, 2012. Neurobehavioral toxic effects of perinatal oral exposure to aluminum on the developmental motor reflexes, learning, memory and brain neurotransmitters of mice offspring. Pharmacology Biochemistry and Behavior, 101(1): 49-56.

Augustinsson KB, 1963. Cholinesterase and Anticholinesterase Agents, Vol. 5. Ed, Koelle GB, Berlin: Springer Verlag, pp. 89-128.

Azzaoui F-Z, Ahami AOT, Khadmaoui A, 2008. Impact of aluminum sub-chronic toxicity on body weight and recognition memory of Wistar rat. Pakistan Journal of Biological Sciences, 11(14): 1830-1834.

Bielarczyk H, Tomaszewicz M, Szutowicz A, 1998. Effect of aluminum on acetyl-CoA and acetylcholine metabolism in nerve terminals. Journal of Neurochemistry, 70: 1175-1181.

Bilkei-Gorzo A, 1993. Neurotoxic effect of enteral aluminum. Food and Chemical Toxicology, 31(5): 357-361.

Crichton RR, Florence A, Ward RJ, 2002. Aluminum and iron in the brain--prospects for chelation. Coordination Chemistry Reviews, 228: 365-371.

Ellman GL, Courtney KD, Andres V, Feather-Stone RM, 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7: 88-95.

Gonda Z, Lehotzky K, 1996. Effect of prenatal aluminum lactate exposure on conditioned taste aversion and passive avoidance task in the rat. Journal of Applied Toxicology, 16: 529-532.

Gulya K, Rakonczay Z, Kasa P, 1990. Cholinotoxic effects of aluminum in rat brain. Journal of Neurochemistry, 54: 1020-1026.

Jankowska A, Madziar B, Tomaszewicz M, Szutowicz A, 2000. Acute and chronic effects of aluminum on acetyl-CoA and acetylcholine metabolism in differentiated and non differentiated SN56 cholinergic cells. Journal of Neuroscience Research, 62(4): 615-622.

Julka D, Vasishta RK, Gill KD, 1996. Distribution of aluminum in different brain regions and body organs of rat. Biological Trace Element Research, 52(2): 181-192.

Julka D, Sandhir R, Gill KD, 1995. Altered cholinergic metabolism in rat CNS following aluminum exposure: implications on learning performance. Journal of Neurochemistry, 65: 2157-2164.

Kabata-Pendias A, Pendias M, 1993. Biogeochemia Pierwiastkow Sladowych. PWN Warszawa.

Kaizer RR, Corra MC, Spanevello RM, Morsch VM, Mazzanti CM, et al., 2005. Acetylcholinesterase activation and enhanced lipid peroxidation after long-term exposure to low levels of aluminum on different mouse brain regions. Journal of Inorganic Biochemistry, 99: 1865-1870.

Kaizer RR, Corra MC, Gris LRS, da Rosa CS, Bohrer D, et al., 2008. Effect of long-term exposure to aluminum on the acetylcholinesterase activity in the central nervous system and erythrocytes. Neurochemical Research, 33(11): 2294-2301.

Kosik KS, Bradley WG, Good PF, Rasool CG, Selkoe DJ, 1983. Cholinergic function in lumbar aluminum myelopathy. Journal of Neuropathology and Experimental Neurology, 42: 365-375.

Llobet JM, Domingo JL, Gmez M, Toms JM, Corbella J, 1987. Acute toxicity studies of aluminum compounds: antidotal efficacy of several chelating agents. Pharmacology and Toxicology, 60: 280-283.

Marquis JK, Black EE, 1984. Aluminum activation and inactivation of bovine caudate acetylcholinesterase. Bulletin of Environmental Contamination and Toxicology, 32: 704-710.

Meyer JJ, Allen DD, Yokel RA, 1996. Hippocampal acetylcholine increases during eye blink conditioning in the rabbit. Physiology and Behavior, 60: 1199-1203.

Micic DV, Petronijevic ND. 2000. Acetylcholinesterase activity in the Mongolian gerbil brain after acute poisoning with aluminum. Journal of Alzheimer's Disease, 2(1): 1-6.

Mi AC, Andreescu CE, Renata V, Olteanu A, 2003. A behavioral and histological study of the effects of long-term exposure of adult rats to aluminum. The International Journal of Neuroscience, 113: 1197-1211.

Moraes MS, Leite SR, 1994. Inhibition of bovine brain acetylcholinesterase by aluminum. Brazilian

Journal of Medical and Biological Research, 27(11): 2635-2638.

Muller G, Bernuzzi V, Desor D, Hutin MF, Burnel D, et al., 1990. Developmental alteration in offspring of female rats orally intoxicated by aluminum lactate at different gestation periods. Teratology, 42: 253-261.

Nayak P, 2006. Perinatal toxicity of aluminum. The Internet Journal of Toxicology, 3(1).

Nayak P, 2002. Aluminum: impacts and disease. Environmental Research, Section A, 89: 101-115.

National Research Council, 1981. Drinking Water & Health, vo., Washington, DC: National Academy Press, p. 164.

Organisation for Economic Co-operation and Development (OECD), 2001. OECD Guideline for Testing of Chemicals, Acute Oral Toxicity -- Acute Toxic Class Method, 423 p 1-14, adopted 17th December 2001.

Rawy SM, Morsy GM, Elshibani MM, 2012. Lethality, accumulation and toxicokinetics of aluminum in some tissues of male albino rats. Toxicology and Industrial Health, Feb 8. [Epub ahead of print]

Reddy GR, Devi BC, Chetty CS, 2007. Developmental lead neurotoxicity: Alterations in brain cholinergic system. Neurotoxicology, 28: 402-407.

Santos F, Chan JC, Yang MS, Savory J, Wills MR, 1987. Aluminum deposition in the central nervous system. Preferential accumulation in the hippocampus in weanling rats. Medical Biology, 65(1): 53-55.

US Coast Guard, Department of Transportation. CHRIS -- Hazardous Chemical Data, Vol. II, Washington, DC: U.S. Government Printing Office, pp. 1984-1985.

Voss B, Thienel R, Reske M, Habel U, Kircher T, 2010. Cognitive performance and cholinergic transmission: influence of muscarinic and nicotinic receptor blockade. European Archives of Psychiatry and Clinical Neuroscience, 260(S2): S106-S110.

Yellamma K, Saraswathamma S, Kumari BN, 2010. Cholinergic system under aluminum toxicity in rat brain. Toxicology International, 17: 106-112.

Zatta P, Ibn-Lkhayat-Idrissi M, Zambenedetti P, Kilyen M, Kiss T, 2002. In vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase. Brain Research Bulletin, 59(1): 41-45.
Table 1: Effect of Al on the brain, liver, spleen, and kidney
weights (g/100 g b.w.) [+ or -] SEM).
           Control (C)      [Al.sub.1]      [Al.sub.2]
Brain    1.89 [+ or -]   1.80 [+ or -]   1.79 [+ or -]
          [0.07.sup.a]    [0.07.sup.a]    [0.07.sup.a]
Liver    5.16 [+ or -]   5.14 [+ or -]   4.84 [+ or -]
          [0.34.sup.a]    [0.26.sup.a]    [0.42.sup.a]
Spleen   0.44 [+ or -]   0.35 [+ or -]   0.30 [+ or -]
          [0.04.sup.a]  [0.03.sup.a,b]  [0.02.sup.b,c]
Kidneys  0.81 [+ or -]   0.79 [+ or -]   0.82 [+ or -]
          [0.05.sup.a]    [0.04.sup.a]    [0.03.sup.a]
            [Al.sub.3]
Brain    1.73 [+ or -]
          [0.08.sup.a]
Liver    4.83 [+ or -]
          [0.55.sup.a]
Spleen   0.22 [+ or -]
          [0.02.sup.c]
Kidneys  0.91 [+ or -]
          [0.04.sup.a]
Note: Results are represented as mean [+ or -] SEM.
Weight is expressed in g/100 g of BW Values that do not
have the same letters (a, b, c) are significantly different
from (p < 0.01)**. C: control rats, [Al.sup.1]: rats receiving
2,500 mg/kg of Al nitrate, [Al.sup.2]: rats receiving 3,500 mg/kg of
Al nitrate, and [Al.sup.3]: rats receiving 4,500 mg/kg of Al nitrate.


F-Z Azzaoui (1),*, H Hami (2), M El-Hioui (1), S Boulbaroud (3), A Ahami (1)

(1) Unit for Clinic and Cognitive Neuroscience and Health, Laboratory of Biology and Health, Department of Biology, Faculty of Sciences, Ibn Tofail University, Kenitra, Morocco.

(2) Laboratory of Genetics and Biometry, Faculty of Sciences, Ibn Tofail University, Kenitra, Morocco.

(3) Unit for Neuroendocrine Physiology, Laboratory of Genetics and Neuroendocrine Physiology, Department of Biology, Faculty of Sciences, Ibn Tofail University, Kenitra, Morocco.

* Corresponding Author: azzaouifz@hotmail.com

Accepted: 13th Jun 2012, Published: 1stt Jul 2012
COPYRIGHT 2012 HATASO Enterprises, LLC
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2012 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Azzaoui, F-z; Hami, H.; El-Hioui, M.; Boulbaroud, S.; Ahami, A.
Publication:Biology and Medicine
Article Type:Report
Geographic Code:6MORO
Date:Apr 1, 2012
Words:3286
Previous Article:In vitro antimicrobial activity of some medicinal plants used by tribes in warangal district (andhra pradesh), india.
Next Article:Study of TORCH profile in patients with bad obstetric history.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters