Effects of aluminum chloride on the serum protein, bilirubin and hepatic trace elements in chickens
Toxicology and Industrial Health 1–7 © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233715578035 tih.sagepub.com
Ben Wang1, Yanzhu Zhu2, Hongling Zhang1, Liming Liu1, Guojiang Li1, Yongli Song1 and Yanfei Li3 Abstract The aim of this study was to reveal the effects of aluminum chloride (AlCl3) on the hepatic metabolism function and trace elements’ distribution. Two hundred healthy male chickens (1 day old) were intraperitoneally administered with AlCl3 (0, 18.31, 27.47, and 36.62 mg kg1 day1 of Al3þ) consecutively for 3 days. Then the chickens were allowed to rest for 1 day. The cycle lasted four days. The cycle was repeated 15 times (60 days). The contents of serum total protein (TP), albumin (ALB), total bilirubin (TBI), direct bilirubin (DBI), hepatic aluminum (Al), copper (Cu), iron (Fe), and zinc (Zn) were examined. The results showed that the contents of serum TP and ALB and hepatic Fe and Zn decreased and the contents of serum TBI and DBI and hepatic Al and Cu increased in the chickens with AlCl3. This indicates that chronic administration of AlCl3 impairs the hepatic metabolism function and disorders the hepatic trace elements’ distribution. Keywords Al, liver, chickens, trace elements, protein, bilirubin
Introduction Aluminum (Al) is ubiquitous in the environment, so Al exposure is inevitable to increase a potential risk for humans and animals (Hewitt and Savory, 1990; Rengel, 2004). Livers from male Wistar rats were perfused in a recirculating system for 240 min, and at higher Al levels of 6535.3–16694.9 g l1 signs of toxicity toward isolated perfused livers were observed as indicated by an increased release of the enzymes aspartate aminotransferase and alanine transaminase into the perfusate, a pronounced reduction of bile flow rate and a 50% suppression of oxygen consumption (Wilhelm et al., 1996). Moreover, a systems biology approach in cultured hepatoblastoma cells (HepG2) was used to identify the molecular targets of Al toxicity and found that mitochondrial metabolism is the main site of the toxicological action of Al. Additionally, Al toxicity leads to an increase in intracellular lipid accumulation due to enhanced lipogenesis and a decrease in the -oxidation of fatty acids (Mailloux et al., 2011). Later Alemmari et al. (2011) found marked blunting of bile canaliculi microvilli in the pigs with aluminum chloride (AlCl3), serum total bile acids and hepatic Al concentration correlated with the
duration of Al exposure. Though the hepatic Al toxicity was observed, effects of Al on the hepatic metabolism function and trace elements’ distribution remain elusive. Total protein (TP) and albumin (ALB) were important indexes to response hepatic protein synthesis. Liver produces 90% of the proteins in an organism. TP and ALB were the dominant components of the proteins. Serum TP and ALB contents significantly decreased in rats that were orally administrated with AlCl3 (8.5 mg kg1 body weight (B.W.) for 8 weeks) (Fyiad, 2007). Al induced the reduction of TP and ALB, but Al dose was low. Serum ALB content was
College of Veterinary Medicine, Jilin Science and Technology Vocational College, Jilin, China 2 Institute of Special Animal and Plant Sciences of Chinese Academy of Agricultural Sciences, Changchun, China 3 College of Veterinary Medicine, Northeast Agricultural University, Harbin, China Corresponding author: Yanfei Li, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China. Email: [email protected]
decreased in rats that were administered with Al (32.5 mg kg1, 24 h) (Bhadauria, 2012). It revealed that Al induced the reduction of ALB without TP, and exposure time was short. Total bilirubin (TBI) and direct bilirubin (DBI) can reflect the injury of hepatic secretion and excretory function. The TBI released from senile erythrocytes transferred to the surface of hepatocytes by blood circulation. DBI was formed by the combination between TBI and glucuronic acid. Gao et al. (2007) found that serum TBI content decreased in the rats with AlCl3 by gavage. A significant increase of TBI and cholesterol was observed in rats that were orally administrated with AlCl3 (8.5 mg kg1 B.W. for 8 weeks) (Fyiad, 2007). In addition, TBI and DBI contents were significantly increased in the workers who were exposed to 4.6 mg m3 of Al dust and fumes in workplace air for 21.6 + 2.5 years (Bogdanovic´ and Bulat, 2008). The workers were present in complex conditions, and thus other factors might affect the results. Iron (Fe), zinc (Zn), and copper (Cu) are essential trace elements for the hepatic function. Fe regulates glucose homeostasis via AMP-activated protein kinase in liver of mice (Huang et al., 2013). Cu facilitates the formation of reactive oxygen species that can damage DNA and chromatin (Linder, 2012). Zn is essential for the activity of enzymes and transcription factors, and Zn deficiency was present in patients with liver disease (Prasad, 2013). In pregnant rats, the hepatic concentrations of Fe, Zn, and Cu were significantly higher in the Al-treated rats compared with the control (Belle´s et al., 2001). It focused on the hepatic distribution of Fe, Zn, and Cu with Al exposure, but the rats were pregnant and the data were recorded under complex conditions. Therefore, effects of Al (as AlCl3, measured as Al) on the serum protein (measured as TP and ALB), serum bilirubin (measured as TBI and DBI), and hepatic trace elements (measured as Fe, Zn, and Cu) were examined to find the effects of AlCl3 on the hepatic metabolism function and trace elements’ distribution.
Materials and methods Chickens The chickens were acclimatized for 1 week. Two hundred healthy male chickens (1 day old) were randomly allocated equally into control group (CG, 0), low-dose group (LG, 18.31 mg kg1 day1 of Al3þ), mid-dose
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group (MG, 27.47 mg kg1 day1 of Al3þ), and highdose group (HG, 36.62 mg kg1 day1 of Al3þ). Each group had 50 chickens. Throughout the experiment, chickens were intraperitoneally administered with AlCl3 consecutively for 3 days. Then the chickens were injected with 2500 IU of penicillin and streptomycin when the chickens were allowed to rest for 1 day. Four days was a cycle. The cycle was repeated 15 times (60 days). The chickens were given drinking water and food addiction ad libitum. Chickens were housed in the Biomedical Research Center, Jilin Science and Technology Vocational College, China. The housing conditions were maintained at a temperature of 24 + 1 C and a 12-h light/12-h dark cycle. The chickens were kept in plastic cages (five chickens per cage) with soft chip bedding. The size of all the cages was 600 625 280 mm3, large enough for the growth of five chickens. The health status of chickens was observed daily and body weight of the chickens was recorded every week.
Sample collection The experimental protocol was approved by the Ethics Committee on the Use and Care of Animals, Jilin Science and Technology Vocational College, China. After 60 days, the chickens were anesthetized with ether and were killed. Then the blood samples and livers were collected from each chicken. The blood sample was used to obtain serum to determine the contents of TP, ALB, TBI, and DBI. The liver was used to examine the contents of Al, Fe, Zn, and Cu.
The detection of TP, ALB, TBI, and DBI in the serum The serum concentrations of TP, ALB, TBI, and DBI were measured according to the study by Oyagbemi et al. (2013).
The detection of Al, Fe, Zn, and Cu in the liver The contents of hepatic Al were determined using a graphite furnace atomic absorption spectrophotometer, and the contents of hepatic Fe, Zn, and Cu were determined using a flame atomic absorption spectrophotometer according to Zhu et al. (2012).
Statistical analysis Statistical analyses were done using SPSS 15.0 package programmer (SPSS Inc., Chicago, Illinois, USA). One-way analysis of variance followed by Student’s
678.3. 673.1 669.5 667.1 0.073 0.463b 0.064b 0105b 552.2 + 546.8 + 547.5 + 544.2 + 0.033 0.583a 0.024b 0.063b + + + + 454.8 451.6 447.5 448.6 + 0.063 + 0.153a + 0.085b + 0.253b 366.9 363.2 358.6 358.1 + 0.053 + 0.113a + 0.303a + 0.635b 275.6 273.9 273.2 268.5 + 0.033 + 0.263b + 0.013b + 0.063b 195.8 187.5 185.6 186.5 + 0.133 + 0.113a + 0.563b + 0.033b 125.4 123.2 123.8 119.7
CG: control group; LG: low-dose group; MG: mid-dose group; HG: high-dose group. a p < 0.05 versus CG. b p < 0.01 versus CG.
Al pollutants are a global health risk due to their ability to cause a variety of diseases. Al accumulates in
+ + + +
CG LG MG HG
Contents of hepatic Al and Cu in chickens increased with AlCl3. The contents of Al in LG, MG, and HG were significantly higher than those in CG (p < 0.01). Cu contents in MG and HG were higher than those in CG (p < 0.05). Contents of hepatic Fe and Zn in chickens decreased with AlCl3. Fe contents in MG and HG were lower than those in CG (p < 0.05). Zn contents in MG (p < 0.05) and HG (p < 0.01) were lower than those in CG (Table 4).
21-Day old (g)
Effects of Al on hepatic Al, Fe, Zn, and Cu contents
14-Day old (g)
Contents of TBI and DBI in the serum of chickens increased with AlCl3. The contents of TBI in MG and HG were higher than those in CG (p < 0.01). The contents of DBI in MG (p < 0.05) and HG (p < 0.01) were higher than those in CG (Table 3).
7-Day old (g)
Effects of Al on serum TBI and DBI contents
Table 1. The body weight of chicken (means + SD, n ¼ 50 per group).
Contents of TP and ALB in the serum of chickens decreased with AlCl3. The contents of TP in MG (p < 0.05) and HG (p < 0.01) were lower than those in CG. The contents of ALB in LG (p < 0.05), MG (p < 0.01), and HG (p < 0.01) were lower than those in CG (Table 2).
Effects of Al on serum TP and ALB contents
28-Day old (g)
35-Day old (g)
42-Day old (g)
No chicken died during the experiment. The chickens with AlCl3 were less active than those in the control. The body weight of chickens in four groups increased continuously throughout the experimental period, and the body weight of AlCl3-treated chickens was significantly lower than that of CG (p < 0.05 and p < 0.01; Table 1).
75.6 74.2 75.1 76.2
Results Effects of Al on the body weight
0.011 0.033a 0.046 0.061a
49-Day old (g)
56-Day old (g)
t-test was used to compare the effects of different contents of AlCl3 exposure on the serum protein, bilirubin, and hepatic trace elements with CG. Data were shown as means and standard deviation. A p value of less than 0.05 was considered significant and a p value of less than 0.01 was considered markedly significant.
+ 0.153 + 0.052b + 0.523b + 0.634b
Wang et al.
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Table 2. Effects of Al on the TP and ALB in serum of chicken (means + SD, n ¼ 50 per group). Item CG LG MG HG
TP (g l1) 53.26 + 51.38 + 49.62 + 45.76 +
7.23 5.64 3.45a 4.98b
ALB (g l1) 15.12 13.23 11.54 10.58
+ 3.90 + 2.98a + 1.96b + 2.56b
CG: control group; LG: low-dose group; MG: mid-dose group: HG: high-dose group; TP: total protein; ALB: albumin; Al: aluminum. a p < 0.05 versus CG. b p < 0.01 versus CG.
Table 3. Effects of Al on the TBI and DBI in serum of chicken (means + SD, n ¼ 50 per group). Item
TBI (mol l1)
CG LG MG HG
7.10 + 8.43 + 10.13 + 11.36 +
0.75 090 2.23a 3.42a
DBI (mol l1) 6.25 + 7.77 + 8.20 + 9.00 +
0.89 1.02 0.56b 0.85a
CG: control group; LG: low-dose group; MG: mid-dose group; HG: high-dose group; TBI: total bilirubin; DBI: direct bilirubin; Al: aluminum. a p < 0.01 versus CG. b p < 0.05 versus CG.
the liver (Willhite et al., 2012), and liver participates in the metabolic function of an organism. The accumulation of hepatic Al would impair the metabolic function of the liver. This experiment focused on the effects of Al on the hepatic metabolism function and trace elements’ distribution. The contents of TP and ALB response the metabolism function of the liver. In the present experiment, AlCl3 decreased the contents of TP and ALB in the serum of chickens. The transportation, synthesis, and disintegration of protein were related to the function of liver. Al impaired the hepatic structure and that caused the reduction of TP and ALB in the serum of chickens. Mosoni et al. (1996) found that motion promoted protein synthesis. In the present experiment, the chickens with AlCl3 were less active. It might result in the reduction of TP and ALB in the serum of chickens. Oxidative stress was observed in the liver of rats with Al exposure (Chaitanya et al., 2012). Oxidative stress impaired proper protein synthesis (Ling and So¨ll, 2010). Thus, the hepatic oxidative stress induced by Al pollutants caused the reduction of TP and ALB. Zn regulated the synthesis of specific proteins in the liver of rats through the changes of the relative abundance of specific messenger RNA
(Kimball et al., 1995). In the present experiment, hepatic Zn content decreased in the chickens with AlCl3. Thus, the reduction of hepatic Zn content might reduce the contents of TP and ALB. The reduction of TP in the present experiment indicated that Al impaired the hepatic protein synthesis in the chickens. ALB could combine many toxins and attenuate the toxic effects of toxins. The decrease of ALB weakened the hepatic Al detoxification and indirectly enhanced the toxicity of Al. Gao et al. (2007) confirmed that serum TP content decreased when rats were exposed to AlCl3 through gavage for 3 weeks. After the usage of 1,2-dimethyl-3-hydroxy4-pyridone, hepatic Al concentration was significantly decreased and protein synthesis was recovered. It indicates that Al exerts the toxic effect by suppressing the hepatic protein metabolism. Bilirubin is produced when the senile erythrocytes are decomposited and destructed in liver, spleen, and bone marrow mononuclear phagocyte system. The high level of bilirubin is related to the disorder of hepatocytes, abnormal hemolysis, and biliary obstruction disease. In the present experiment, serum TBI and DBI contents increased in the chickens with AlCl3. The metabolism of TBI and DBI was mediated by the hepatocytes. The TBI and DBI contents would be increased when hepatocytes do not metabolize TBI and DBI completely. The blocked excretion of TBI and DBI would also result in the increase of serum TBI and DBI. Sinusoidal dilatation, congestion of central vein, lipid accumulation, and lymphocyte infiltration were observed in liver when the rats were orally administrated with 34 mg AlCl3 kg1 B.W. daily for 30 days (Tu¨rkez et al., 2010). The hepatic pathohistological change affected the function of hepatocytes. Thus, the increases of TBI and DBI contents were induced by hepatic pathohistological change through impaired hepatocytes. Al decreased the number of erythrocytes and induced anemia in the rats (Zhang et al., 2011). Hemoglobin was released from the damage of erythrocytes. Most TBI derived from hemoglobin of the splitting erythrocytes. TBI and DBI were the ultimate breakdown products of hemoglobin. This would result in the increase of serum TBI and DBI contents. Bilirubin is tightly bound to circulating ALB (Wang et al., 2006). In the present experiment, serum TBI and DBI contents increased and serum ALB contents decreased in chickens with AlCl3. It might be that the reduction of ALB content was not enough to conjugate all the bilirubin in serum and induced the
Wang et al.
Table 4. The contents of Al, Fe, Zn, and Cu in the liver of the chicken (means + SD, n ¼ 50 per group). Item CG LG MG HG
Al 7.559 16.534 65.129 69.307
+ 2.548 + 4.619a + 5.238a + 7.193a
Fe 154.676 + 127.051 + 93.566 + 69.166 +
Zn 16.519 31.937 9.545b 6.607b
43.667 41.067 36.935 34.985
+ 2.167 + 1.379 + 1.313b + 1.182a
Cu 4.831 + 6.674 + 7.221 + 7.821 +
1.449 0.510 0.336b 0.736b
CG: control group; LG: low-dose group; MG: mid-dose group; HG: high-dose group; Al: aluminum; Cu: copper; Fe: iron; Zn: zinc. a p < 0.01 versus CG. b p < 0.05 versus CG.
increase of TBI and DBI contents. The increased TBI and DBI contents would penetrate cell membrane and induce toxicity. Increase of bilirubin concentration caused serious nerve injury (Miao et al., 2010). The excessive bilirubin combined with the lipid in the nuclei basales of brain, which impaired the brain function. It indicates that Al exerts neurotoxicity by suppressing hepatic bilirubin metabolism. Bilirubin is an important pigment in the bile. Bile is an important route of Al excretion in male SpragueDawley rats (Sutherland et al., 1996). Serum total bile acid contents were increased and marked blunting of bile canaliculi microvilli was observed in domestic pigs that were intravenously administered with AlCl3 at 1500 g kg1 day1 for 1, 2, 3, or 4 weeks (Alemmari et al., 2012). The subclinical sign of cholestasis was manifested in the workers with impaired biliary secretion (Bogdanovic´ and Bulat, 2008). It indicates that AlCl3 induces the increase of TBI and DBI in the serum by blocking the bile acid excretion. Fe, Zn, and Cu are essential trace elements for protein synthesis and production of enzymes. In the present experiment, hepatic Fe content was decreased in the chickens with AlCl3. Fe was present in the liver with ferritin (Munro and Linder, 1978). Transferritin was mainly from hepatocytes (Mason and Taylor, 1978). Liver maintained the Fe homeostasis by regulation of the transferritin and transferritin receptor (Takami and Sakaida, 2011). Al impaired the hepatic structure and it might disorder Fe homeostasis and reduce Fe content. Moreover, the absorption of Fe and Al were mainly carried by transferritin, and Al competed with Fe in ferritin and transferritin (Cochran et al., 1984). Van Landeghem et al. (1994) confirmed that Al would compete with Fe for the Fe-binding protein. The decrease of hepatic Fe content in the present study may attribute to the competition of Al with Fe. Fe deficiency would decrease the activity of enzymes and disorder the metabolism of organism
(Boldt, 1999). The absence of Fe would block the metabolism of protein and induce abnormal function of the liver. Fe was an important component of catalase. The absence of Fe would result in the lower activity of catalase and inhibit the antioxidant ability. It showed that oxidative stress was observed in the liver with Al exposure (Chaitanya et al., 2012). So the hepatic oxidative stress might attribute to the Fe deficiency induced by AlCl3. Mammalian Fe absorption was primarily controlled by the hepatocyte-derived peptide hepcidin, encoded by the HAMP gene. Hepcidin regulated ferroportinmediated Fe export from enterocytes and macrophages, thus determined dietary Fe uptake as well as the amount of Fe available for erythropoiesis (Krijt et al., 2012). Al might injure the hepcidin and decrease Fe content in the liver. In our previous research, microsomal protein and nicotinamide adenine dinucleotide phosphate-cytochrome c reductase decreased in the rats with AlCl3 (Zhu et al., 2013). It indicates that Al exerts hepatic toxicity by the induction of apoptosis. In the present experiment, hepatic Zn content decreased and hepatic Cu content increased in the chickens with AlCl3. Zn and Cu had antagonism relationship. The increase of Cu might attribute to the decrease of Zn. Cu facilitated the formation of reactive oxygen species that damaged DNA and chromatin (Linder, 2012). The overload of Cu would induce lipid peroxidation in the mitochondria of hepatocytes (Sokol et al., 1990). Zn was also an antioxidant and anti-inflammatory agent (Linder, 2012). Zn deficiency would induce oxidative injury and impair the membrane of hepatocytes. The oxidative stress in liver induced by Al was observed (Viezeliene et al., 2011). It indicates that Al affects hepatic Cu and Zn contents and induces oxidative stress in the liver. The latest evidence showed that Al might mediate Alzheimer’s disease through liver toxicity and the excess free Cu caused brain oxidation and -amyloid aggregation
(Brenner, 2013). Zn was effective in attenuating the liver damage inflicted by Al toxicity (Bhasin et al., 2014). The reduction of Zn will decrease the function of hepatic detoxification. It suggests that AlCl3 induces oxidative stress by the reduction of Zn and the increase of Cu. In conclusion, the decreases of serum TP and ALB and hepatic Fe and Zn contents and the increases of serum TBI and DBI and hepatic Al and Cu contents indicate that the chronic administration of AlCl3 induces cumulative effect, damages hepatic metabolism function and disorders trace element distribution. Authors’ Note Ben Wang and Yanzhu Zhu contributed equally to this study. Jilin Science and Technology Vocational College and Institute of Special Animal and Plant Sciences of Chinese Academy of Agricultural Sciences, Changchun 130112, China, contributed equally to this study.
Funding The study was supported by a Grant from the National Science Foundation Project (31172375, 31302147), the Youth Station in Jilin Science and Technology Vocational College (01002) and Science Foundation for Young Scientists of Jilin Province (20130522091JH).
References Alemmari A, Miller GG, Arnold CJ, et al. (2011) Parenteral aluminum induces liver injury in a newborn piglet model. Journal of Pediatric Surgery 46: 883–887. Alemmari A, Miller GG, Bertolo RF, et al. (2012) Reduced aluminum contamination decreases parenteral nutrition associated liver injury. Journal of Pediatric Surgery 47: 889–894. Belle´s M, Albina ML, Sanchez DJ, et al. (2001) Effects of oral aluminum on essential trace elements metabolism during pregnancy. Biological Trace Element Research 79: 67–81. Bhadauria M (2012) Combined treatment of HEDTA and propolis prevents aluminum induced toxicity in rats. Food Chemical Toxicology 50: 2487–2495. Bhasin P, Singla N and Dhawan DK (2014) Protective role of zinc during aluminum-induced hepatotoxicity. Environmental Toxicology 29(3): 320–327. Boldt DH (1999) New perspectives on iron: an introduction. American Journal of the Medical Sciences 318: 207–212. Bogdanovic´ M, Bulat P (2008) Biliary function in workers occupationally exposed to aluminium dust and
Toxicology and Industrial Health fumes. Arhiv Za Higijenu radai Toksikologiju 59: 135–139. Brenner S (2013) Aluminum may mediate Alzheimer’s disease through liver toxicity, with aberrant hepatic synthesis of ceruloplasmin and ATPase7B, the resultant excess free copper causing brain oxidation, beta-amyloid aggregation and Alzheimer disease. Medical Hypotheses 80: 326–327. Chaitanya TV, Mallipeddi K, Bondili JS, et al. (2012) Effect of aluminum exposure on superoxide and peroxide handling capacities by liver, kidney, testis and temporal cortex in rat. Indian Journal of Biochemistry & Biophysics 49: 395–398. Cochran M, Coates J and Neoh S (1984) The competitive equilibrium between aluminum and ferric ions for the binding sites of transferrin. Febs Letters 176: 129–132. Fyiad AA (2007) Aluminium toxicity and oxidative damage reduction by melatonin in rats. Journal of Applied Science Research 3: 1210–1217. Gao JH, Liu P, Feng GC, et al. (2007) Effects of Al chelate agent on the hepatic zymogram and necessary elements of rats with Al exposure. Chinese Journal of Public Health 23: 316–317. Hewitt CD, Savory J and Wills MR (1990) Aspects of aluminum toxicity. Clinics in Laboratory Medicine 10: 403–422. Huang J, Simcox J, Mitchell TC, et al. (2013) Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice. Faseb Journal 27(7): 2845–2854. Kimball SR, Chen SJ, Risica R, et al. (1995) Effects of zinc deficiency on protein synthesis and expression of specific mRNAs in rat liver. Metabolism 44: 126–133. Krijt J, Fry´dlova´ J, Kukacˇkova´ L, et al. (2012) Effect of iron overload and iron deficiency on liver hemojuvelin protein. PLos One 7: e37391. Linder MC (2012) The relationship of copper to DNA damage and damage prevention in humans. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 733: 83–91. Ling J, So¨ll D (2010) Severe oxidative stress induces protein mistranslation through impairment of an aminoacyltRNA synthetase editing site. Proceedings of the National Academy of Sciences of the United States of America 107: 4028–4033. Mailloux RJ, Lemire J and Appanna VD (2011) Hepatic response to aluminum toxicity: dyslipidemia and liver diseases. Experimental Cell Research 317: 2231–2238.
Wang et al. Mason DY, Taylor CR (1978) Distribution of transferrin, ferritin, and lactoferrin in human tissues. Journal of Clinical Pathology 31: 316–327. Miao Q, Lu ZJ, Mou YR, et al. (2010) The neuro toxicity of bilirubin. Clinical Journal of Medical Office 38: 669–672. Mosoni L, Malmezat T, Valluy MC, et al. (1996) Muscle and liver protein synthesis adapt efficiently to food deprivation and refeeding in 12-month-old rats. Journal of Nutrition 126: 516–522. Munro HN, Linder MC (1978) Ferritin: structure, biosynthesis, and role in iron metabolism. Physiological Reviews 58: 317–396. Oyagbemi AA, Omobowale TO, Azeez IO, et al. (2013) Toxicological evaluations of methanolic extract of moringa oleifera leaves in liver and kidney of male Wistar rats. Journal of Basic and Clinical Physiology and Pharmacology 18: 1–6. Prasad AS (2013) Discovery of human zinc deficiency: its impact on human health and disease. Advances Nutrition 4: 176–190. Rengel Z (2004) Aluminum cycling in the soil-plantanimal-human continuum. BioMetals 17: 669–689. Sokol RJ, Devereaux M, Mierau GW, et al. (1990) Oxidant injury to hepatic mitochondrial lipids in rats with dietary copper overlond. Gastroenterology 99: 1061–1071. Sutherland JE, Radzanowski GM and Greger JL (1996) Bile is an important route of elimination of ingested aluminum by conscious male sprague-dawley rats. Toxicology 109: 101–109. Takami T, Sakaida I (2011) Iron regulation by hepatocytes and free radicals. Journal of Clinical Biochemistry and Nutrition 48: 103–106. Tu¨rkez H, Yousef MI and Geyikoglu F (2010) Propolis prevents aluminium-induced genetic and hepatic
7 damages in rat liver. Food Chemical Toxicology 48: 2741–2746. Van Landeghem GF, D’Haese PC, Lamberts LV, et al. (1994) Quantitative HPLC/ETAAS hybrid method with an on-line metal scavenger for studying the protein binding and speciation of aluminum and iron. Analytical Chemistry 66: 216–222. Viezeliene D, Jansen E, Rodovicius H, et al. (2011) Protective effect of selenium on aluminium-induced oxidative stress in mouseliver in vivo. Environmental Toxicology and Pharmacology 31: 302–306. Wang X, Chowdhury JR and Chowdhury NR (2006) Bilirubin metabolism: applied physiology. Current Pediatric 16: 70–74. Wilhelm M, Jaeger DE, Schu¨ll-Cablitz H, et al. (1996) Hepatic clearance and retention of aluminium: studies in the isolated perfused rat liver. Toxicology Letters 89: 257–263. Willhite CC, Ball GL and McLellan CJ (2012) Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water. Critical Reviews in Toxicology 42: 358–442. Zhang LC, Li XW, Gu QY, et al. (2011) Effects of subchronic aluminum exposure on serum concentrations of iron and iron-associated proteins in rats. Biological Trace Element Research 141: 246–253. Zhu YZ, Li XW, Chen CX, et al. (2012) Effects of aluminum trichloride on the trace elements and cytokines in the spleen of rats. Food Chemical Toxicology 50: 2911–2915. Zhu YZ, Han YF, Zhao HS, et al. (2013) Suppressive effect of accumulated aluminum trichloride on the hepatic microsomal cytochrome P450 enzyme system in rats. Food Chemical Toxicology 51: 210–214.