Journal of Hazardous Materials 294 (2015) 109–120

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Toxicity assessment due to sub-chronic exposure to individual and mixtures of four toxic heavy metals Samuel J. Cobbina a,1 , Yao Chen a,1 , Zhaoxiang Zhou b,1 , Xueshan Wu b,1 , Ting Zhao b , Zhen Zhang a , Weiwei Feng c , Wei Wang c , Qian Li d , Xiangyang Wu a,∗ , Liuqing Yang b,∗∗ a

School of the Environment, Jiangsu University, Xuefu Rd. 301, Zhenjiang 212013, Jiangsu, China School of Chemistry and Chemical Engineering, Jiangsu University, Xuefu Rd. 301, Zhenjiang 212013, China c School of Food and Biological Engineering, Jiangsu University, Xuefu Rd. 301, Zhenjiang 212013, Jiangsu, China d School of Pharmacy, Jiangsu University, Xuefu Rd. 301, Zhenjiang 212013, Jiangsu, China b

h i g h l i g h t s • • • •

Low dose single and mixtures of toxic metals had adverse effect on mice. Metal mixtures exhibited higher toxicities compared to individual metals. Mixtures of low dose Pb + Hg + Cd induced neuronal degeneration in brain of mice. Exposure to Pb + Hg + As + Cd showed renal tubular necrosis in kidney.

a r t i c l e

i n f o

Article history: Received 18 July 2014 Received in revised form 2 March 2015 Accepted 26 March 2015 Available online 28 March 2015 Keywords: Metal mixture Low dose Multivariate Oxidative stress Toxicity

a b s t r a c t Humans are exposed to a cocktail of heavy metal toxicants in the environment. Though heavy metals are deleterious, there is a paucity of information on toxicity of low dose mixtures. In this study, lead (Pb) (0.01 mg/L), mercury (Hg) (0.001 mg/L), cadmium (Cd) (0.005 mg/L) and arsenic (As) (0.01 mg/L) were administered individually and as mixtures to 10 groups of 40 three-week old mice (20 males and 20 females), for 120 days. The study established that low dose exposures induced toxicity to the brain, liver, and kidney of mice. Metal mixtures showed higher toxicities compared to individual metals, as exposure to low dose Pb + Hg + Cd reduced brain weight and induced structural lesions, such as neuronal degeneration in 30-days. Pb + Hg + Cd and Pb + Hg + As + Cd exposure induced hepatocellular injury to mice evidenced by decreased antioxidant activities with marginal increases in MDA. These were accentuated by increases in ALT, AST and ALP. Interactions in metal mixtures were basically synergistic in nature and exposure to Pb + Hg + As + Cd induced renal tubular necrosis in kidneys of mice. This study underlines the importance of elucidating the toxicity of low dose metal mixtures so as to protect public health. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Heavy metals are a group of environmental chemicals which are ubiquitous and non- biodegradable. Though adverse effects emanating from their exposure are widely known, their usage and concentrations in the environment is increasing [1–3]. Numerous studies have highlighted the toxicity of individual metals to living systems [4–7], however, these metals do not only exist as

∗ Corresponding author. Tel.: +86 511 88791200; fax: +86 511 88791200. ∗∗ Corresponding author. Tel.: +86 511 88791800; fax: +86 511 88791800. E-mail addresses: [email protected] (X. Wu), [email protected] (L. Yang). 1 Contributed to this article equally and are co-first authors. http://dx.doi.org/10.1016/j.jhazmat.2015.03.057 0304-3894/© 2015 Elsevier B.V. All rights reserved.

individuals but also as mixtures in the environment [8–10]. Heavy metal exposures are linked to conditions such as embryogenesis [11], neurotoxicity [12], bladder cancer [13,14], cytogenicity [15] and genetic alteration of cells [16]. The few studies that have been done involve moderately high toxic metal concentrations. Rai et al. [12] exposed developing rat astrocytes to mixtures of human relevant doses of Pb (0.22 mg/L), Cd (0.098 mg/L) and As (0.38 mg/L) and observed the modulation in levels of myelin and axon proteins in the brain of developing rats. In their study, they noted that exposure to toxic metals induced a reduction in myelin thickness and axon- density of the optic nerve and a decrease in thickness of nerve fiber, plexiform layer and retinal ganglion cell counts of the retina [12].

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Jadhav et al. [17] observed that exposure to eight watercontaining metals (As, Cd, Hg, Cr, Ni, Mn, Fe) in drinking water adversely affected the general health of male rats. They noted that the functional and structural integrity of kidneys, liver and brain of rats were altered when metal concentrations 10 and 100 times the mode concentrations of individual metals were exposed to rats [17]. Another study by Whittaker et al. [18], further established that exposure to mixtures of Pb, Cd, and As increased delta aminolevulinic acid (ALA), iron and copper levels in male Sprague–Dawley rats. They further observed that increases in ALA were followed by statistically significant increases in kidney copper. Several other studies have established toxicities from exposures to joint mixtures of toxic heavy metals [19–23]. The study of exposure to drinking water containing low concentrations of heavy metals individually and as mixtures, to our minds, is very significant, as there is a paucity of information on their toxicities. There is currently a global interest in effects induced by exposure to mixtures at low metal concentrations. The few studies that have been conducted were done using concentrations that take into consideration the lowest-observed-effect-level (LOEL) [18,24,25] and also use the most frequently occurring concentration (mode concentration) [26]. This paper reports on toxicity of low dose individual and joint mixtures of Pb, Hg, Cd and As after sub-chronic exposure to Institute of Cancer Research (ICR) mice. Low doses used in this study are the maximum permissible limits (MPL) stipulated in the National Standard of The Republic of China for Municipal Water Standards (GB5749-2006). These concentrations, to our knowledge, are the lowest to be used in assessing toxicological effects resulting from exposure to mixtures of Pb, Hg, As and Cd. MPLs are set using single metals with the assumption that there is little interaction among metals in mixed solutions; however, few studies have tested this assertion. Furthermore, the toxicity of low dose mixtures of these metals on the environment and human health have rarely been investigated. Given the widespread water quality challenges and associated health impacts faced by China, and many developing countries around the world, this study is timely. Toxic heavy metals used in the study were selected due to their public health importance, environmental abundance and common mechanisms shared in their toxicities (e.g., generation of reactive oxygen species, interaction with essential metals) [27].

Medicine Center of the Yangzhou University in China. Mice were allowed to acclimatize to their new environment for a period of 3 days before commencement of the experiment. During the study, mice were fed under controlled environmental conditions, which included a temperature of 22 ± 2 ◦ C and humidity of 55–60%. Both male and female mice were kept separately in plastic cages padded with adsorbents at a 12-h light/dark cycle. Mice were fed with basal diet and given free access to drinking water. All experimental procedures conformed to The Code of Ethics of the World Medical Association for experiments involving humans (EC Directive 86/609/EEC for animal experiments). They were approved by the Jiangsu University Committee on Animal Care and Use (license number SYXK (SU) 2013-0036). 2.2. Experimental design and exposure

2. Material and methods

Ten of the groups were made up of forty mice (20 males and 20 females kept separately). Each were exposed to low concentration heavy metals individually (Pb-0.01 mg/L; Hg-0.001 mg/L; Cd-0.005 mg/L; As-0.01 mg/L) and as mixtures (Pb + Hg; Pb + As; Pb + Cd; Pb + Hg + As; Pb + Hg + Cd; Pb + Hg + Cd + As) through free drinking water which was available ad libitum (Table 1). The eleventh group (control) was given distilled water and all animals were fed twice a day with basal diet (made up of carbohydrate 60%; protein 22%; fat 10% and others 8%). Test chemicals and food were administered to mice for a period of 120 days with daily recording of water and food consumption. Mice were further, observed daily for adverse physical signs resulting from administration of test solutions. Signs such as deaths, deformation, rashes, and hair loss were examined. At the end of each month, 5 males and 5 females were selected randomly from each group for analysis. The Morris water maze test was used to assess the spatial learning abilities of the mice. At the end of the experimental period (monthly), the selected mice were weighed and, anesthetized with sodium pentobarbital and blood samples collected through retroorbital venous plexus. Blood samples were centrifuged at 3000 rpm for 15 min and the supernatant stored at a temperature of 4 ◦ C. The supernatant and a part of blood samples were sent to the Analysis and Testing Center (Zhenjiang, Jiangsu, China) for blood analysis. The brain, liver and kidneys of the mice were removed, rinsed in cold saline water, weighed and used for metal and various biochemical analyses. Relative organ weights were calculated using Eq. (1) below:

2.1. Chemicals and animals

Relative organ weight =

Analytical grade lead acetate, cadmium chloride, mercury chloride and sodium arsenite were purchased from Sinopharm Chemical Reagent Co., Ltd. Eleven groups of three-week old ICR mice were comprised of twenty males (13.18 ± 1.80 g) and twenty females (11.98 ± 1.40 g), purchased from the Comparative

Absolute organ weight (g) × 100% Body weight (g)

(1)

2.3. Biochemical assay Red blood cell count (RBC), white blood cell count (WBC), mean corpuscular hemoglobin concentration (MCHC), haemoglobin (Hb), glutamic-pyruvic transaminase (ALT), glutamic oxalacetic

Table 1 Experimental design for concentration of heavy metal administered to each group. Group

Concentration (mg/L) of trace metal in drinking water

Control Lead (Pb) Mercury (Hg) Arsenic (As) Cadmium (Cd) Lead + Mercury Lead + Arsenic Lead + Cadmium Lead + Mercury+Arsenic Lead + Mercury + Cadmium Lead + Mercury + Cadmium + Arsenic

0 0.01 0.001 0.01 0.005 0.01 (Pb) + 0.001 (Hg) 0.01 (Pb) + 0.01 (As) 0.01 (Pb) + 0.005 (Cd) 0.01 (Pb) + 0.001 (Hg) + 0.01 (As) 0.01 (Pb) + 0.001 (Hg) + 0.005 (Cd) 0.01 (Pb) + 0.001 (Hg) + 0.005 (Cd) + 0.01 (As)

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transaminase (AST), blood urea nitrogen (BUN), alkaline phosphatase (ALP), and creatinine (Cr) activities were determined using commercial diagnostic kits (Biosino Bio-technology and Science Inc., Nanjing, China). Malondialdehyde (MDA) was assayed calorimetrically in the brain, liver and kidney tissue homogenate according to the method of Ohkawa et al. [28]. Concentrations of Nitric oxide synthetase (NOS) was assayed using a NOS detection kit A014-1 (Institute of Biological Engineering of Nanjing Jianchen, Nanjing, China). Reduced glutathione, glutathion peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (TAOC) activity were determined using GPx Assay kit A005, SOD Assay Kit A001, CAT Assay Kit A007 and T-AOC Kit A015, respectively (Institute of Biological Engineering of Nanjing Jianchen, Nanjing, China). 2.4. Histopathological analyses Sections of kidneys and brain of each group were excised, fixed in 10% formalin and hydrated in ascending grades of ethanol, cleared in xylene and embedded in paraffin. The cut sections were stained with hematoxylin and eosin (H&E) and viewed under a light microscopy (Axioskop 40, Zeiss, Germany) by an experienced and board-certified veterinary pathologist who was blind to the animals assigned to each experimental group. 2.5. Statistical analysis Statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, USA) statistical software. Results were expressed as mean ± SD number of observations. Means were compared using one-way analysis of variance (ANOVA) and Duncan’s multiple range test. Differences among groups were considered statistically significant when p < 0.05 unless otherwise stated. Multivariate statistical analysis of the data was done using STATISTICA 10 software (Statsoft Inc., USA). The toxicity data associated with brain, liver and kidney were investigated using Principal Component and Cluster Analysis (PCA and CA) to explain variability of the data. In PCA, the maximization of the loading values of variables located in a component and reduction of variables with low loadings were done using the Varimax rotation method. Data was tested for normality using Shapiro–Wilk’s test, whiles the Box and Cox test was used to transform data that were not normally distributed. 3. Results 3.1. General observation All mice generally tolerated individual and mixtures of metals in drinking water as they did not show any physical signs as a result of toxicity. There was, however, one death in the female group exposed to Pb + As, after 90 days of exposure; yet the cause of death remained unknown. In addition, one mouse in the Pb + Hg female group developed deformed hind legs in the 90-day study period. 3.2. Body and organ weight The average monthly changes in body and organ weights of male and female mice during the study are presented in Tables S1a and S1b. More male groups experienced significant weight losses compared to females groups. More groups (7 female groups and 5 male groups) in the 30-day study experienced significant weight reductions followed by groups in the 120-day study, where three female (Hg, As and Pb + Hg) and four male (Cd, Pb + As, Pb + Hg + Cd and Pb + Hg + As + Cd) groups recorded significant weight reductions.

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Low dose metal mixtures seem to induce significant weight loss in early days of exposure (30- and 60-day) in both males and females compared to single metals (Tables S1a and S1b). Exposure to individual and mixtures of toxic metals did not significantly change absolute and relative organ weights in this study (Tables S2a–f). However, 30-day exposure to Pb + Hg + Cd reduced absolute brain weight by 14%, while similar reductions were recorded in Pb + Hg [14%] and Pb + Hg + Cd [12%] groups after 60-days (Table S2a). Significant liver weight reductions were recorded among 60-day As [32%] and Pb + Hg [23%] exposed groups (Table S2b). Relative liver weight reductions were recorded in As (0.034 ± 0.005 g) and Pb + Hg (0.038 ± 0.003 g) 60-day groups, compared to those in the control group (0.046 ± 0.002 g), however, the Pb + Cd (0.039 ± 0.002 g) exposed mice showed significant relative liver weight losses after 90-days (control 0.045 ± 0.003). No significant absolute or relative weight change was recorded in the kidneys of mice in the study (Tables S2c and f). Generally, drinking water and food intake in most groups were comparable to those in control. There were however, a few groups which recorded decreased water and food consumption during the study (Table S3a and b). After 30-days the water consumption in the Hg, Pb + As and Pb + Hg + Cd-exposed male groups reduced by 30%, 38% and 34%, respectively. After 120-days similar reductions of 55% and 50%, were recorded in male groups exposed to Pb + Hg and Pb + Hg + As + Cd, respectively (Table S3a). Reduced food intake were also recorded in Pb [22%], Pb + Hg [21%], Pb + As [30%] and Pb + Hg + As + Cd [29%] male groups after 30 days exposure. Similar reductions in food consumption were recorded in male groups exposed to Cd, Pb + Hg + Cd and Pb + Hg + As + Cd after 120 days (Table S3a). Some female groups were also observed to experience reduced water and food intake during the study (Table S3b). 3.3. Morris water maze test The effects of exposure to heavy metals individually and as mixtures on spatial learning and memory of mice in the Morris water maze test was also conducted. Generally, there were no significant differences in escape latencies and swimming speeds; however, mice exposed to Pb + Hg + Cd in the 30-day study, recorded a marginal increase in escape latency (48 ± 15 s; control: 44 ± 12 s) and lower swimming speeds (22 ± 4 m/s; control: 26 ± 6 m/s). 3.4. MDA, SOD and GPx Malondialdehyde (MDA) is basically an end product of the free radical-initiated oxidative decomposition of polyunsaturated fatty acids and therefore, it is often used as an indicator of oxidative stress [29,30]. In this study significant increases in brain, liver and kidney MDA compared to control group were observed (Table 2). Superoxide dismutase (SOD), are important antioxidant defense enzymes that catalyze the dismutation of superoxides into oxygen and hydrogen peroxides, and act as the first line of defense against oxidative stress [31]. In this study, the brain superoxide dismutase (SOD) activities were significantly reduced in mice exposed to Pb + Hg + Cd in both the 90- and 120-day studies (Table 3). Significantly reduced liver and kidney SOD activities were recorded during study (Table 3). Liver GPx activities were significantly reduced after 120 days, while marginal reductions in kidney GPx were recorded in all groups except the Cd exposed group (120days), though they were not significant (Table 4). 3.5. CAT and T-AOC Brain catalase (CAT) levels reduced significantly in all groups in both 30- and 60-day studies (except Pb + Hg) (Table S3). Also activities of liver CAT in the 60- (except Hg, As and Pb + As groups) and

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Table 2 Effect of metals on activities of MDA (nmol/gprot) after exposure to ICR mice. Group

Brain

Liver

90-Day Control Pb Hg As Cd Pb + Hg Pb + As Pb + Cd Pb + Hg + As Pb + Hg + Cd Pb + Hg + Cd + As

19.93 27.33 26.73 24.98 24.80 23.74 26.19 22.95 21.62 26.71 24.11

120-Day

± ± ± ± ± ± ± ± ± ± ±

0.99 3.57a 4.52a 4.64 2.39 2.68 0.59a 1.47 6.47 2.07a 3.96

20.25 30.91 24.95 27.23 23.91 23.45 23.82 19.98 20.44 21.30 23.91

± ± ± ± ± ± ± ± ± ± ±

Kidney

60-Day 0.99 9.97b 0.62 3.53a 2.06 1.01 2.50 2.59 2.58 0.58 3.53

11.82 27.32 22.71 13.76 14.13 17.56 12.42 22.24 15.24 18.55 16.87

± ± ± ± ± ± ± ± ± ± ±

90-Day 4.59 8.13b 8.30 1.36 8.31 7.31 0.68 2.54 8.29 3.43 5.46

16.61 23.68 23.66 24.46 26.16 31.11 19.61 25.60 26.60 22.87 26.60

± ± ± ± ± ± ± ± ± ± ±

120-Day 2.99 2.77 2.74 1.15 6.63 2.33b 4.98 9.77 4.56a 6.40 4.32a

13.81 13.71 23.78 18.97 12.57 26.20 16.24 17.83 18.61 19.35 13.30

± ± ± ± ± ± ± ± ± ± ±

30-Day 0.73 3.89 0.68b 4.78 4.41 4.08a 3.52 2.49 4.45 4.25 0.69

8.23 16.53 19.69 10.86 13.44 13.93 12.35 11.71 13.18 14.85 16.98

± ± ± ± ± ± ± ± ± ± ±

90-Day 0.89 4.48 5.26b 6.24 3.29 3.30 3.71 1.64 2.47 3.10 2.10

7.66 7.92 7.66 7.63 8.36 10.10 14.22 8.12 9.81 7.76 8.21

± ± ± ± ± ± ± ± ± ± ±

0.45 1.17 1.57 0.34 1.49 4.60 7.63a 1.53 2.80 1.21 1.46

Statistically significant at a p < 0.05 and b p < 0.01 from control.

Table 3 Effect of metals on activities of SOD (U/mgprot) after exposure to ICR mice. Group

Brain

Liver

90-Day Control Pb Hg As Cd Pb + Hg Pb + As Pb + Cd Pb + Hg + As Pb + Hg + Cd Pb + Hg + Cd + As

22.70 22.66 22.82 22.35 22.19 21.84 21.14 21.79 21.55 19.37 21.35

± ± ± ± ± ± ± ± ± ± ±

120-Day 0.46 0.84 3.16 0.20 0.51 0.70 0.37 0.55 1.08 2.96a 1.40

20.70 19.66 19.33 19.35 19.19 18.54 18.85 18.76 18.05 18.17 19.53

± ± ± ± ± ± ± ± ± ± ±

Kidney

30-Day 0.46 0.54 0.82 0.42 0.15 0.47 0.21 0.41 1.11 1.06a 1.03

13.47 3.26 2.35 2.14 5.21 3.63 3.37 4.81 5.59 4.68 4.95

± ± ± ± ± ± ± ± ± ± ±

60-Day 0.88 3.18b 1.49b 1.35b 1.95b 2.05b 2.52b 0.62b 1.44b 2.30b 2.32b

14.43 12.26 13.32 11.63 12.76 12.98 14.49 13.36 13.53 13.65 12.54

± ± ± ± ± ± ± ± ± ± ±

120-Day 0.60 0.48 1.00 2.93a 1.93 0.87 0.90 0.31 1.34 0.37 1.58

14.85 10.87 12.07 10.62 10.94 10.61 10.47 8.04 12.44 10.90 10.01

± ± ± ± ± ± ± ± ± ± ±

30-Day 0.17 2.11 1.45 0.41 1.24 1.08 1.28 5.58a 0.49 1.63 0.98

13.54 4.72 7.34 6.61 12.50 8.07 11.58 13.03 11.29 12.88 8.98

± ± ± ± ± ± ± ± ± ± ±

3.07 2.51b 1.42b 1.62b 1.03 3.18b 1.80 1.39 0.96 1.01 1.26b

Statistically significant at a p < 0.05 and b p < 0.01 from control.

90-day studies were significantly reduced compared to the control groups. Significant reductions in kidney CAT were recorded in As and Pb + Cd exposed groups in 60 days and in all groups in both 90- (except Pb + As) and 120-day studies (Table S4). Total antioxidant capacities (T-AOC) of mice generally reduced in many groups during the study (Table S5). 3.6. Nitric oxide synthase (NOS) The levels of iNOS recorded after 90-days of exposure to low dose metals were not significantly different from that of mice in control groups. There was, however, an 86% elevation in iNOS (7.27 ± 1.19 U/mgprot, p < 0.05) in mice exposed to only Hg in 120 day study (control; 1.01 ± 0.43 U/mgprot). Total nitric oxide synthase (TNOS) was significantly increased Table 4 Liver and kidney GPx levels in 120-day study. Group

Liver-GPx

Control Pb Hg As Cd Pb + Hg Pb + As Pb + Cd Pb + Hg + As Pb + Hg + Cd Pb + Hg + Cd + As

115.06 74.83 92.55 77.66 71.52 80.78 68.96 64.03 87.75 54.06 70.98

± ± ± ± ± ± ± ± ± ± ±

Kidney-GPx 8.77 6.78b 6.74a 10.52b 4.82b 5.29b 8.16b 14.87b 6.12a 2.69b 8.82b

Statistically significant at a p < 0.05 and b p < 0.01 from control.

98.27 71.45 78.20 7.38 100.00 79.44 82.66 83.18 84.79 67.39 80.74

± ± ± ± ± ± ± ± ± ± ±

7.27 6.52 6.61 13.96 5.73 14.49 14.78 5.12 14.15 3.61 13.19

in Pb + Cd (6.54 ± 1.10 U/mgprot, p < 0.01) and Pb + Hg + As (6.58 ± 3.46 U/mgprot, p < 0.01) groups in the 30-day study compared to the control [3.11 ± 1.47] group. After 60 days, exposure to Pb + Hg (6.47 ± 0.25 U/mgprot, p < 0.05), Pb + Hg + Cd (6.66 ± 1.54 U/mgprot, p < 0.05) and Pb + Hg + As + Cd (6.07 ± 1.42 U/mgprot, p < 0.05) increased TNOS markedly compared to the control group (3.24 ± 0.48 U/mgprot). There were no significant differences in both 90 and 120 day studies. 3.7. Biochemical assay Activities of ALT and AST are clinically used in the evaluation of hepatocellular injuries, to determine liver health. Injured hepatocytes release enzymes into the blood stream leading to elevation in ALT and AST [32]. Significant increases in ALT, AST and ALP on exposure to low dose toxic heavy metals were recorded during the study (Table 5). 3.8. BUN, creatinine and LDL Blood urea nitrogen (BUN) is an indication of renal health and high levels are associated with high protein in diet, along with a decrease in glomerular filtration rate and in blood volume. BUN levels were significantly reduced in all groups during study (Table 6). Creatinine (Cr) levels were, however, significantly increased in blood of mice in As, Cd, Pb + Cd, Pb + Hg + As and Pb + Hg + Cd + As groups after 30-day exposure. Levels of Cr, however, reduced markedly in the 60- and 120-day studies compared to control group (Table 6). Low density lipoprotein (LDL) molecules facilitate the movement of lipids, such as triglycerides, cholesterol, and phospholipids

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Table 5 The levels of plasma liver function related enzymes of mice during study. Group

30-Day

60-Day

ALT(IU/L)

AST (IU/L)

ALP (IU/L)

Control Pb Hg As Cd Pb + Hg Pb + As Pb+Cd Pb + Hg + As Pb + Hg + Cd Pb + Hg + Cd + As

25.20 ± 30.20 ± 33.40 ± 25.60 ± 30.80 ± 25.30 ± 30.20 ± 29.10 ± 30.00 ± 45.60 ± 30.80 ± 90-Day

0.96 2.00 1.68b 1.60 2.84a 0.79 3.10 4.88 5.10 2.36b 3.76a

79.90 90.10 94.20 86.40 105.00 93.10 81.90 83.50 100.50 134.70 115.40

± ± ± ± ± ± ± ± ± ± ±

2.00 6.60 4.25 6.67 7.90a 1.05 6.17 13.76 20.75 29.23b 9.41a

Control Pb Hg As Cd Pb + Hg Pb + As Pb + Cd Pb + Hg + As Pb + Hg + Cd Pb + Hg + Cd + As

28.03 19.47 23.03 23.10 41.86 49.90 26.37 27.97 28.40 22.73 27.12

± ± ± ± ± ± ± ± ± ± ±

9.30 2.46a 1.69 3.08 8.25b 1.74b 1.55 3.75 1.50 2.18 5.55

111.50 88.53 91.73 125.53 101.23 145.77 94.67 106.07 106.43 90.73 88.03

± ± ± ± ± ± ± ± ± ± ±

17.11 19.5 11.36 52.03 13.28 17.02b 15.18 20.65 13.65 12.45 12.86

89.00 114.00 124.00 112.00 103.00 92.00 101.00 100.00 112.00 112.00 97.00

± ± ± ± ± ± ± ± ± ± ±

4.04 12.77a 11.15b 6.25a 2.08 1.53 14.18 15.17 9.00a 12.76a 16.56

75.33 ± 12.22 70.33 ± 23.29 64.00 ± 5.57 68.67 ± 10.69 61.33 ± 6.66 55.67 ± 3.51 80.00 ± 6.00 66.00 ± 9.64 72.33 ± 30.06 61.33 ± 50.03 83.00 ± 4.58

ALT(IU/L)

AST (IU/L)

22.77 ± 1.32 26.67 ± 1.56 32.20 ± 5.31b 27.77 ± 2.40 25.23 ± 3.15 24.77 ± 2.05 36.23 ± 2.67b 28.00 ± 3.83 24.80 ± 2.59 33.60 ± 1.57b 28.90 ± 1.26a 120-Day

93.17 100.90 134.4 100.43 90.20 82.27 107.57 94.80 90.43 119.73 105.77

± ± ± ± ± ± ± ± ± ± ±

3.54 5.26 18.37b 1.76 1.61 7.93 1.26 13.62 15.28 4.31a 19.48

95.00 84.67 79.33 86.33 76.67 85.67 84.33 75.33 85.00 76.67 91.33

± ± ± ± ± ± ± ± ± ± ±

25.24 1.53 17.62 10.60 7.64 9.50 9.07 9.02 1.00 4.16 18.61

± ± ± ± ± ± ± ± ± ± ±

134.80 164.57 170.23 110.73 89.00 188.43 94.57 101.97 99.23 103.90 134.53

± ± ± ± ± ± ± ± ± ± ±

20.30 7.59b 24.61b 18.49a 2.31b 4.82b 4.90b 5.84b 12.00b 5.28b 0.35

81.67 65.67 67.67 54.00 60.33 72.00 63.67 66.00 60.67 52.33 62.33

± ± ± ± ± ± ± ± ± ± ±

25.42 5.86 18.82 7.81b 13.83a 3.61 2.08 8.72 0.57a 3.06b 1.53a

35.03 30.07 46.03 25.07 22.73 54.00 26.13 28.73 26.23 23.23 26.00

4.02 3.01 18.17a 1.93 2.15a 1.64a 1.72 2.76 3.10 3.85a 1.87

ALP (IU/L)

ALT – alanine transaminase, AST – aspartate transaminase, ALP – alkaline phosphatase. Statistically significant at a p < 0.05 and b p < 0.01from control.

within water around cells and are associated with cardiovascular diseases [33]. In this study, significant increases in LDL were recorded in groups exposed to Hg (1.00 mmol/L), As (0.91 mmol/L), Cd (0.91 mmol/L), Pb + Hg + As (0.87 mmol/L) and Pb + Hg + Cd (0.97 mmol/L) compared to the control group (0.57 mmol/L) in the 30 day study period. There were, however, reductions in LDL levels in subsequent months.

Heavy metal exposure to mice significantly increased WBC by 36%, 71%, 39%, 50%, 53% and 45% after exposure to As, Pb + As, Pb + Cd, Pb + Hg + As, Pb + Hg + Cd and Pb + Hg + As + Cd, respectively in 30 days compared to those in the control group. Significant decreases in RDW were observed throughout the study (Table 7).

3.9. Hematopoietic parameters

Histopathological evidence showed that Pb + Hg + Cd-exposed mice exhibited brain induced structural lesions as seen under a light microscope, which is known as neuronal degeneration especially after 30 days of exposure (Fig. 1A2 ). However, brain histology from the control, 60, 90 and 120-day groups on exposure to Pb + Hg + Cd showed normal architecture (Fig. 1A1 –D1 and B2 –D2 ). Exposure to Pb + Hg + As + Cd produced renal tubular necrosis in kidneys of mice in 30 days (Fig 2A1 ). Though effect of Pb + Hg + As + Cd exposure to kidney in 60, 90 and 120-day was not very clear, there seem to be slightly noticeable differences, especially in 60 (Fig 2B2 ) and 120 (Fig 2D2 ) days compared to control groups.

Hematological parameters, such as red blood cells (RBC), packed cell volume (PCV), haemoglobin (Hb), platelets (PLT), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), and platelet distribution width (PDW) measured during study were not significantly different from that of the control group. However, significant changes in white blood cells (WBC) and red cell distribution width (RDW) were observed in a number of groups during the study (Table 7).

3.10. Histopathological

Table 6 Statistically significant blood urea nitrogen (BUN) and creatinine (Cr) levels in blood of mice during study. Group

BUN

Creatinine (Cr)

30 Days

60 Days

90 Days

120 Days

30 Days

60 Days

120 Days

Control Pb Hg As Cd Pb + Hg Pb + As Pb + Cd Pb + Hg + As Pb + Hg + Cd Pb + Hg + Cd + As

6.29 ± 0.02 5.53 ± 0.20a ns ns ns ns ns ns ns ns ns

6.33 ± 0.33 ns ns ns ns ns ns ns ns 5.35 ± 0.56a ns

5.74 ± 0.76 4.46 ± 0.70b ns ns 4.75 ± 0.58a ns 4.65 ± 0.24b 4.63 ± 0.40b 4.47 ± 0.33b 4.06 ± 0.21b 4.09 ± 0.13b

5.07 ± 0.75 ns ns ns ns ns 3.57 ± 0.07b ns ns ns ns

12.40 ± 0.78 ns ns 15.10 ± 1.15b 15.40 ± 0.82b ns ns 14.70 ± 2.06a 15.10 ± 0.70b ns 17.20 ± 0.74b

10.30 ± 0.46 ns ns ns ns ns ns ns ns ns 5.57 ± 0.85b

33.23 ± 2.05 27.17 ± 0.38b 27.83 ± 2.99a 27.60 ± 1.51b 26.07 ± 4.91b 25.17 ± 1.26b 25.40 ± 0.10b ns 25.47 ± 3.44b 28.20 ± 1.15a ns

Statistically significant at a p < 0.05 and b p < 0.01 from control. ns – not significant.

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Fig. 1. Histopathology of brain tissue of ICR mice after exposure to Pb + Hg + Cd in 30, 60, 90 and 120-day study. A1 –D1 are control groups (250×) for the four months, showing normal brain architecture whiles A2 showed neuronal degeneration (arrowed). B2 , C2 and D2 (250×) which are for 60, 90 and 120 days seem to show normal architecture, comparable to control. Cut sections of brain were stained with hematoxylin and eosin (H&E) and viewed under a light microscopy.

S.J. Cobbina et al. / Journal of Hazardous Materials 294 (2015) 109–120

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Fig. 2. Histopathology of kidney tissue of ICR mice after exposure to Pb + Hg + As + Cd in a 30, 60, 90 and 120-day studies. A1 –D1 are control group (250×) showing normal kidney architecture; A1 is Pb + Hg + As + Cd-treated group (250×) showing renal tubular necrosis (arrowed), whiles there are slight effects noticeable, especially in 60 (B2 ) and 120 (D2 ) day studies. Cut sections of kidney were stained with hematoxylin and eosin (H&E) and viewed under a light microscopy.

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Table 7 Results of white blood cells (109 /L) and red cell distribution width (fL). Group

WBC

RDW

30-Day Control Pb Hg As Cd Pb + Hg Pb + +As Pb + Cd Pb + Hg + As Pb + Hg + Cd Pb + Hg + Cd + As

3.22 3.37 3.75 4.39 2.94 4.22 5.52 4.46 4.82 4.92 4.67

± ± ± ± ± ± ± ± ± ± ±

30-Day 0.32 0.80 0.42 1.14a 0.57 0.32 1.24b 0.35a 0.45b 0.66b 0.45a

37.8 ± 36.7 ± 38.2 ± 36.3 ± 35.4 ± 36.3 ± 34.5 ± 34.5 ± 34.4 ± 33.7 ± 30.9 ±

60-Day 0.67 0.61 1.03 1.65 0.71a 1.68 1.77b 0.70b 0.68b 0.61b ± 0.95b

36.50 35.23 35.53 32.97 34.17 35.37 32.13 32.87 33.33 32.93 35.20

± ± ± ± ± ± ± ± ± ± ±

90-Day 1.44 1.50 1.40 1.65a 1.00 2.98 0.81b 2.21a 1.65a 2.55a 0.60

34.37 33.60 31.73 32.40 30.80 33.07 32.20 33.00 31.30 32.10 32.12

± ± ± ± ± ± ± ± ± ± ±

120-Day 1.18 0.26 2.18a 0.46 0.66a 1.63 0.80 2.61 0.95a 2.51 1.01

35.33 34.13 34.30 31.56 34.60 37.00 34.40 32.80 33.53 32.46 31.26

± ± ± ± ± ± ± ± ± ± ±

0.81 2.15 0.72 0.76b 1.55 3.05 1.30 0.72 1.45 1.49a 1.78b

Statistically significant at a p < 0.05 and b p < 0.01 from control.

3.11. Correlation between variables in blood plasma and organs Correlation between variables in blood plasma and organs show that in the brain, strong mutual correlations (r > 0.70) existed between MDA–CAT (r = 0.80), MDA–TAOC (r = 0.72), MDA–LDL (r = −0.81), SOD–LDL (r = −0.72), CAT–LDL (r = 0.71), MDA–MCHC (r = 0.74) and MDA–TP (r = 0.72), which were all significant at p < 0.01. Correlations associated with blood plasma and liver variables were SOD–TAOC (r = 0.81), GPx–TAOC (r = 0.75), AST–ALT (r = 0.81) and RDW–MCHC (r = −0.83) all of which were significant at p < 0.01. For the kidney, associations observed were AKW–RKW (r = 0.73), SOD–MDA (r = −0.77), MDA–CAT (r = −0.71), AKW–TAOC (r = 0.80), RKW–TAOC (r = 0.74), RKW–BUN (r = −0.82), RKW–Cr (r = 0.86), CAT–LDL (r = −0.76), SOD–MCHC (r = 0.70) and RDW–MCHC (r = −0.70), which were all significant at p < 0.01. Significant negative correlation was found between BUN–Cr (r = −0.66) at p < 0.01. 3.12. Multivariate analysis To further investigate and characterize correlations between measured parameters associated with toxicities to brain, liver, and kidneys of mice, PCA and CA were applied. Associations between weight of organs, serum chemistry, activities of enzymes and hematopoietic parameters were considered (Tables S6, S7 and S8). Boldfaced values are loadings that represent the importance of the variables to the component. Correlation coefficients ≥0.7 were considered highly correlated. Generally, exposure of low dose toxic metals to mice induced oxidative stress in the brain and kidneys, as PCA and CA showed high loadings on variables such as MDA, SOD, CAT, LDL and TP (Figs. 3 and 4, S1 & S3). For exposure to the liver, high loadings were

Fig. 3. Score plot illustrating the differentiation of parameters associated with toxicity to brain of mice, with high loadings on PC 1 accounting for 36% of total variance.

Fig. 4. Score plot illustrating the differentiation of parameters associated with toxicity to kidney of mice, with high loadings on PC1 accounting for 43% of total variance.

observed on variables such as ALT, AST and TAOC, depicting injury to the liver (Fig. 5). PCA of variables associated with brain, kidney and liver of mice all showed a three component system accounting for 66%, 72% and 68% of total variance, respectively (Tables S6–S8). For brain, PC1 accounted for 35.87% with high loadings on

Fig. 5. Score plot illustrating the differentiation of parameters associated with toxicity to liver of mice, with high loadings on PC1 accounting for 32% of total variance.

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MDA, SOD, CAT, T-AOC, LDL, MCHC and TP. For kidneys, PC1and PC2 accounted for more than 60% of total variance with high loadings on MDA, SOD, CAT, T-AOC, TP, LDL, RKW, BUN and Cr (Table S7). PC1 for liver accounted for 32% of total variance with high loadings on T-AOC, ALT and AST (Table S8). CA also showed similar groupings among variables associated with oxidative stress (Figs. S1–S3).

4. Discussion The aim of this study was to assess toxicities of low dose toxic metals individually and as mixtures on ICR mice. The results showed significant reductions in body weights of mice in many groups after exposure to toxic metals. Joint mixtures induced significant body weight loss in more groups in the 30- and 60-day study compared to individual metals. Body weight reduction is an indication of the general health status of mice [34], and depicts retarded growth as a result of exposure to toxic metals. Metal exposure causes retardation of enzymatic activities, increased degradation of lipids and proteins, and degeneration of vital organs [35] which ultimately slow down the growth of mice [36,37]. Weight reductions during the 30- and 60-day studies could also be attributed to the effect of low dose toxic metals on immature and developing organs of the mice. Immature and developing brain, liver and kidneys are vulnerable to the influence of toxic heavy metals, as they are not developed enough to combat toxicity [38,39]. These findings corroborate work done by Jedhav et al. [17] and Antonio Garcia et al. [40]. Reduced body weights were also linked to consumption of water and food by mice during study. In 30 days, male mice exposure to Pb + As and Pb + Hg + As + Cd recorded corresponding reductions in body weight, water and food intake. Similar observations were made in females exposed to Cd and Pb + Hg + Cd in 30 days. Several studies link exposure to chemicals to reductions in weight, water and food intake [41,42]. Though there were no significant changes in absolute liver weights in many groups, mice exposed to As and Pb + Hg recorded significantly reduced absolute liver weights in 60-days. Arsenicinduced reduction was associated with significant decreases in antioxidants like SOD, GPx, and CAT with marginal increases in MDA (Tables 2–4 and S4). Studies link As exposure to hepatocellular injury and weight loss [43,44]. Though the mechanism involved is not well understood, emerging evidence link As-induced hepatotoxicity and inflammation to increased oxidative stress and production of pro-inflammatory cytokines [44,45]. Toxicity of Pb + Hg mixture on the liver was more pronounced than that of As, as MDA levels were higher on exposure to joint mixture than to individual As. Generally exposure to low dose toxic metals in this study induced oxidative stress in brain and kidneys of the mice in the study. Principal component analysis of variables associated with brain and kidneys showed high loadings (PC1) on MDA, SOD, CAT and TAOC (Tables S6 and S8, Figs. S1 and S3). Cluster analysis (CA) corroborated these findings, as variables associated with oxidative stress were mostly found in the same group; thus, implying a common relationship (Figs. S1 and S3). Strong correlations were found among MDA, SOD, TAOC and CAT, implying an association of these in brain and kidneys of mice in the study. Oxidative stress is basically an imbalance between the production of reactive oxygen species and the body’s ability to detoxify them through neutralization of antioxidants [46]. Elevated ROS results in oxidative damage to cellular macromolecules and altered signal transduction [47] . In this study, more groups exposed to low does mixtures showed toxicity compared to groups exposed to individual metals. For example, exposure to Pb + Hg + Cd induced structural lesions to the

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brain, as seen under a light microscope (Fig. 1), resulted in neuronal degeneration in 30 days. This was associated with significant reductions in activities of the brain SOD, CAT (p < 0.01), T-AOC (p < 0.01) and marginal increases in MDA in both 30- and 60-day studies. These findings depicts early signs of oxidative stress in the brain of mice and were consistent with studies by Zhang et al. [48]. Exposure to Pb + Hg + Cd further affected the learning and memory abilities of mice, evidenced by marginally high escape latency and low swimming speeds in the Morris water maze test. Similar trends were observed on exposure of low dose Pb + Hg mixture to the brain in the 60-day study. Adverse effects to the brain was further confirmed by elevated levels of brain NOS which was recorded after exposure to Pb + Hg + Cd and Pb + Hg to the mice in the 30-day study. Nitric oxide (NO) is produced when nitric oxide synthase (NOS) converts arginine to NO and citrulline in the nervous system. NO is unstable but vital in the brain, as it is involved in morphogenesis, blood pressure regulation, neurotransmission and also in immune responses [49] . Studies suggests that NOS levels measured in different parts of the brain is an indicator of NO production. High concentrations of NOS or NO has toxic effects whiles low levels perform protective or regulatory functions [50]. Mixtures such as Pb + Hg + Cd + As, Pb + Hg + As and Pb + Cd equally produced significantly elevated NOS in the brain. As expected, the concentration of toxic heavy metals in organs of ICR mice generally increased with time as a result of accumulation (Table 8). The toxic effect of Pb, Hg, Cd and As in mixtures were generally found to be antagonistic compared to their individual toxicities. Levels of MDA, SOD and TAOC were generally reduced on exposure to mixtures in this study compared to the individual metals (Tables 2, 3 and S5). Interactions of metals on brain weight was largely antagonistic in the 30-day study, however, some mixtures containing As showed synergistic interactions in 60-days (Table S2a). CAT also showed mixtures exhibiting both antagonistic and synergistic interactions (Table S4). In this study, exposure to Pb + Hg + As + Cd produced renal tubular necrosis in mice (Fig. 2). The toxic nature of low dose Pb + Hg + As + Cd mixture on the kidneys of mice in the study were facilitated by synergism among metals in mixture (Fig. 3). Exposure to heavy metals such as Pb, Hg and Co have been linked to renal tubular necrosis and the collapse of glomeruli [51,52]. Kidney dysfunction was corroborated by significantly high creatinine (Cr) recorded in 30-days of exposure to low dose Pb + Hg + As + Cd. High Cr level is a reflection of the degree of damage to glomerular filtration and is a more sensitive indicator for predicting kidney dysfunction than BUN [17,53]. More mixture groups recorded significantly high Cr compared to mice exposed to single metals in the 30-day study. Elevated Cr were also recorded on exposure to Pb + Cd and Pb + Hg + As while adverse effects from As and Cd were also observed, corroborating studies by Wang and Fowler [27]. Cr levels, however, decreased with time as animals grew and developed better defensive systems. This finding was consistent with work conducted by Hambach et al. [54] where co-exposure of Pb increased the renal response to low levels of Cd. PCA and CA showed high loadings and associations on variables associated with oxidative stress in kidneys. The effect of mixture exposure on kidney MDA activity was largely synergistic. However, there were instances in which antagonistic interactions were observed. All four metals showed synergistic interactions in mixtures during the 90-day study. Kidney SOD, GPx and Cr mixture interactions were mostly synergistic. The liver serves as the main storage site and one of the essential organs targeted in heavy metal toxicity [55]. More groups exposed to joint mixtures of low dose metals showed significant toxicities compared to groups exposed to single metals. In 30 days, mice exposed to mixtures of Pb + Hg + Cd and Pb + Hg + As + Cd recorded significantly high ALT, AST and ALP enzyme activities, which may

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Table 8 Measured concentrations of trace metals (␮g/g) in organs of ICR mice. Group

30-Day

120-Day

Pb

Hg

As

Pb

Hg

Brain Control Pb Hg As Cd

0.81 1.91 2.08 1.95 2.54

± ± ± ± ±

0.22 0.52 0.34 0.41 0.45

1.30 1.54 1.61 1.70 0.83

± ± ± ± ±

0.35 0.43 0.16 0.39 0.40

0.76 1.15 1.14 0.59 1.00

± ± ± ± ±

0.33 0.99 0.47 0.10 0.36

1.88 3.03 2.96 2.68 2.38

± ± ± ± ±

Liver Control Pb Hg As Cd

0.93 2.65 1.88 1.70 1.51

± ± ± ± ±

0.36 0.33 0.73 1.00 1.02

0.43 0.48 0.66 0.06 0.45

± ± ± ± ±

0.23 0.18 0.27 0.37 0.23

0.37 0.41 0.30 0.65 0.53

± ± ± ± ±

0.22 0.05 0.15 0.29 0.26

1.32 3.32 1.34 1.53 1.26

Kidney Control Pb Hg As Cd

1.82 3.20 4.36 1.30 4.36

± ± ± ± ±

0.59 0.95 0.67 0.81 0.75

0.64 0.44 1.40 1.21 0.69

± ± ± ± ±

0.18 0.22 0.21 0.34 0.50

0.53 0.73 0.81 0.42 1.04

± ± ± ± ±

0.03 0.35 0.50 0.19 0.57

1.98 4.02 4.17 4.38 4.42

a

As

0.43 0.52 0.80 0.69 0.69

± ± ± ± ±

0.16 0.30 0.34 0.25 0.49

3.13 3.60 3.11 4.82 1.98

± ± ± ± ±

1.99 1.83 1.18 2.19 0.86

± 0.5 ± 1.0a ± 0.5 ± .8 ± 0.2

0.03 0.03 0.03 0.04 0.06

± ± ± ± ±

0.01 0.02 0.01 0.02 0.04

1.12 2.06 2.05 2.38 1.55

± ± ± ± ±

0.10 0.68 1.15 1.46 0.82

± ± ± ± ±

0.07 0.13 0.10 0.15 0.21

± ± ± ± ±

0.03 0.07 0.05 0.01 0.15a

1.88 2.33 3.22 3.46 2.35

± ± ± ± ±

0.25 1.27 0.52 1.14 1.62

0.8 0.5 0.8 0.5 1.0

0.3 1.2a 1.6a 0.7a 1.1a

Statistically significant at p < 0.05 from control.

suggest injuries to hepatocytes [56]. Liver SOD, GPx and T-AOC were decreased (p < 0.05) with marginal increases in liver MDA (Tables 2 and 3, S3 and S4) affirming the incidence of hepatocellular injury. Strong significant correlations (r = 0.81, p < 0.01) were observed between ALT and AST the during study. Further, there were significant increases in WBC and LDL (Pb + Hg + Cd only) during the 30-day study. Elevated WBC was due to the immune system’s response to combat insults from causative agent’s [57,58]. High LDL, which may suggest abnormal lipid metabolism, could be oxidized in the form of cholesteryl-linoleate in conditions such as atherosclerosis or inflammatory diseases such as rheumatoid arthritis in humans [59]. Generally, toxicity, due to exposure to low dose heavy metals, affected the liver of young mice in the first two months of exposure in this study, because of immature organs and developing defensive systems for metal detoxification. This is consistent with studies by Feng et al. [60]. With regards to toxicity from low dose single metals, exposure to Hg induced hepatic injuries to mice during study, as ALT, AST and ALP were significantly increased throughout the (30-, 60- and 120day) study. Oxidative stress indices such as SOD, GPx, CAT, TAOC were significantly decreased during the study, with MDA increasing significantly after 20 days. Exposure to Hg induces generation of ROS and alters defense from antioxidants by inhibiting selenol ( SeH) or sulfhydryl ( SH) groups [61]. Activities of SOD and CAT aids in the metabolism of hydrogen peroxide [62], and therefore, serve as a defense line against generated ROS which can injure the liver. Reduction of SOD increases levels of • O2 - which is known to inactivate catalase activity. However, there is inactivation of SOD if H2 O2 is not removed by catalase [26]. 5. Conclusion Exposure to low dose toxic heavy metals individually and as mixtures, induced toxicity to the brain, liver and kidneys of young ICR mice. Adverse effects were, reductions in absolute brain and liver weights, neuronal degeneration, and renal tubular necrosis in kidneys. Toxicities observed in the 30-day studies primarily occurred due to immature and developing organs of mice and less developed defensive systems to detoxify toxic metals. The outcome of this study adds to a new sense of urgency for research to examine the mechanisms associated with toxicities of low dose toxic metals mixtures, so as to safe guard public health.

Acknowledgment This work was supported financially by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Collaborative Innovation Center of Technology and Material of Water Treatment, Specialized Research Fund for the Doctoral Program of Chinese Universities from the Ministry of Education (20113227110020), Open Fund Project from State Key Laboratory of Environmental Chemistry and Ecotoxicology (KF2011–20) and Graduate Innovative Projects in Jiangsu Province (KYLX 1067). We will like to thank Prof. J. E. Cobbina of Michigan State University for editing the language of this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2015.03.057. References [1] Q. Zhao, Y. Wang, Y. Cao, A. Chen, M. Ren, Y. Ge, Z. Yu, S. Wan, A. Hu, Q. Bo, Potential health risks of heavy metals in cultivated topsoil and grain including correlations with human primary liver, lung and gastric cancer, in Anhui province, Eastern China, Sci. Total Environ. 470 (2014) 340–347. [2] Z. Huang, X.-D. Pan, P.-G. Wu, J.-L. Han, Q. Chen, Heavy metals in vegetables and the health risk to population in Zhejiang, China, Food Control 36 (2014) 248–252. [3] P. Olmedo, A. Pla, A. Hernández, F. Barbier, L. Ayouni, F. Gil, Determination of toxic elements (mercury cadmium, lead, tin and arsenic) in fish and shellfish samples. Risk assessment for the consumers, Environ. Int. 59 (2013) 63–72. [4] A.L. Gollenberg, M.L. Hediger, P.A. Lee, J.H. Himes, G.M.B. Louis, Association between lead and cadmium and reproductive hormones in peripubertal US girls, Environ. Health Perspect. 118 (2010) 1782. [5] A. Basile, S. Sorbo, B. Conte, R.C. Cobianchi, F. Trinchella, C. Capasso, V. Carginale, Toxicity, accumulation, and removal of heavy metals by three aquatic macrophytes, Int. J. Phytoremediat. 14 (2012) 374–387. [6] P. Bhattacharjee, M. Banerjee, A.K. Giri, Role of genomic instability in arsenic-induced carcinogenicity. A review, Environ. Int. 53 (2013) 29–40. [7] V.F. Taylor, D. Bugge, B.P. Jackson, C.Y. Chen, Pathways of CH3 Hg and Hg ingestion in benthic organisms: an enriched isotope approach, Environ. Sci. Technol. 48 (2014) 5058–5065. [8] Y. Cui, Y.-G. Zhu, R. Zhai, Y. Huang, Y. Qiu, J. Liang, Exposure to metal mixtures and human health impacts in a contaminated area in Nanning, China, Environ. Int. 31 (2005) 784–790. [9] E. Smith, D. Gancarz, A. Rofe, I.M. Kempson, J. Weber, A.L. Juhasz, Antagonistic effects of cadmium on lead accumulation in pregnant and non-pregnant mice, J. Hazard. Mater. 199 (2012) 453–456.

S.J. Cobbina et al. / Journal of Hazardous Materials 294 (2015) 109–120 [10] H.-L. Ge, S.-S. Liu, B.-X. Su, L.-T. Qin, Predicting synergistic toxicity of heavy metals and ionic liquids on photobacterium Q67, J. Hazard. Mater. 268 (2014) 77–83. [11] S. Fathallah, M.N. Medhioub, M.M. Kraiem, Combined Toxicity of lead and cadmium on embryogenesis and early larval stages of the European clam Ruditapes decussatus, Environ. Eng. Sci. 30 (2013) 357–364. [12] N.K. Rai, A. Ashok, A. Rai, S. Tripathi, G.K. Nagar, K. Mitra, S. Bandyopadhay, Exposure to As Cd and Pb-mixture impairs myelin and axon development in rat brain, optic nerve and retina, Toxicol. Appl. Pharm. 273 (2013) 242–258. [13] M. Feki-Tounsi, P. Olmedo, F. Gil, R. Khlifi, M.-N. Mhiri, A. Rebai, A. Hamza-Chaffai, Cadmium in blood of Tunisian men and risk of bladder cancer: interactions with arsenic exposure and smoking, Environ. Sci. Pollut. Res. 20 (2013) 7204–7213. [14] Q. Pasha, S.A. Malik, M.H. Shah, Statistical analysis of trace metals in the plasma of cancer patients versus controls, J. Hazard. Mater. 153 (2008) 1215–1221. [15] T.-H. Le, E.-S. Lim, N.-H. Hong, S.-K. Lee, Y.S. Shim, J.R. Hwang, Y.-H. Kim, J. Min, Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers, Chemosphere 93 (2013) 2341–2348. [16] P. Koedrith, H. Kim, J.-I. Weon, Y.R. Seo, Toxicogenomic approaches for understanding molecular mechanisms of heavy metal mutagenicity and carcinogenicity, Int. J. Hyg. Environ. Health 216 (2013) 587–598. [17] S. Jadhav, S. Sarkar, R. Patil, H. Tripathi, Effects of subchronic exposure via drinking water to a mixture of eight water-contaminating metals: a biochemical and histopathological study in male rats, Arch. Environ. Contam. Toxicol. 53 (2007) 667–677. [18] M.H. Whittaker, G. Wang, X.-Q. Chen, M. Lipsky, D. Smith, R. Gwiazda, B.A. Fowler, Exposure to Pb, Cd, and As mixtures potentiates the production of oxidative stress precursors: 30-day 90-day, and 180-day drinking water studies in rats, Toxicol. Appl. Pharm. 254 (2011) 154–166. [19] R. Altenburger, S. Scholz, M. Schmitt-Jansen, W. Busch, B.I. Escher, Mixture toxicity revisited from a toxicogenomic perspective, Environ. Sci. Technol. 46 (2012) 2508–2522. ´ [20] T. Horvat, Zˇ . Vidakovic-Cifrek, V. Oreˇscˇ anin, M. Tkalec, B. Pevalek-Kozlina, Toxicity assessment of heavy metal mixtures by Lemna minor L, Sci. Total Environ. 384 (2007) 229–238. [21] N. Ince, N. Dirilgen, I. Apikyan, G. Tezcanli, B. Üstün, Assessment of toxic interactions of heavy metals in binary mixtures: a statistical approach, Arch. Environ. Contam. Toxicol. 36 (1999) 365–372. [22] J. Wu, L.Y. Mo, L.T. Qin, Z.Q. Zhu, Y.N. Zhu, Research advancement of the joint toxicity of heavy metals mixture, Appl. Mech. Mater. 522 (2014) 532–536. [23] N. Van Praet, M. De Jonge, R. Blust, R. Stoks, L. Bervoets, Behavioural, physiological and biochemical markers in damselfly larvae (Ischnura elegans) to assess effects of accumulated metal mixtures, Sci. Total Environ. 470 (2014) 208–215. [24] B.A. Fowler, M.H. Whittaker, M. Lipsky, G. Wang, X.-Q. Chen, Oxidative stress induced by lead, cadmium and arsenic mixtures: 30-day, 90-day, and 180-day drinking water studies in rats: an overview, Biometals 17 (2004) 567–568. [25] K. Mahaffey, S. Capar, B. Gladen, B. Fowler, Concurrent exposure to lead, cadmium, and arsenic. Effects on toxicity and tissue metal concentrations in the rat, J. Lab. Clin. Med. 98 (1981) 463–481. [26] S. Jadhav, S. Sarkar, M. Aggarwal, H. Tripathi, Induction of oxidative stress in erythrocytes of male rats subchronically exposed to a mixture of eight metals found as groundwater contaminants in different parts of India, Arch. Environ. Contam. Toxicol. 52 (2007) 145–151. [27] G. Wang, B.A. Fowler, Roles of biomarkers in evaluating interactions among mixtures of lead, cadmium and arsenic, Toxicol. Appl. Pharm. 233 (2008) 92–99. [28] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [29] E. Guney, M. Fatih Ceylan, A. Tektas, M. Alisik, M. Ergin, Z. Goker, G. Senses Dinc, O. Ozturk, A. Korkmaz, S. Eker, Oxidative stress in children and adolescents with anxiety disorders, J. Affect Disorders 156 (2014) 62–66. [30] T.-C. Yang, Y.-J. Chen, S.-F. Chang, C.-H. Chen, P.-Y. Chang, S.-C. Lu, Malondialdehyde mediates oxidized LDL-induced coronary toxicity through the Akt-FGF2 pathway via DNA methylation, J. Biomed. Sci. 21 (2014) 1–12. [31] Y. Fang, H. Yang, B. Liu, L. Zhang, Transcriptional response of lysozyme, metallothionein, and superoxide dismutase to combined exposure to heavy metals and bacteria in Mactra veneriformis, Comp. Biochem. Phys. C 157 (2013) 54–62. [32] S. Gao, X. Duan, X. Wang, D. Dong, D. Liu, X. Li, G. Sun, B. Li, Curcumin attenuates arsenic-induced hepatic injuries and oxidative stress in experimental mice through activation of Nrf2 pathway, promotion of arsenic methylation and urinary excretion, Food Chem. Toxicol. 59 (2013) 739–747. [33] B.J. Arsenault, J.S. Rana, E.S. Stroes, J.-P. Després, P.K. Shah, J.J. Kastelein, N.J. Wareham, S.M. Boekholdt, K.-T. Khaw, Beyond low-density lipoprotein cholesterol respective contributions of non-high-density lipoprotein cholesterol levels, triglycerides, and the total cholesterol/high-density lipoprotein cholesterol ratio to coronary heart disease risk in apparently healthy men and women, J. Am. Coll. Cardiol. 55 (2009) 35–41. [34] M. Messarah, F. Klibet, A. Boumendjel, C. Abdennour, N. Bouzerna, M.S. Boulakoud, A. El Feki, Hepatoprotective role and antioxidant capacity of selenium on arsenic-induced liver injury in rats, Exp. Toxicol. Pathol. 64 (2012) 167–174.

119

[35] K. Mitra, S. Kundu, A. Khuda Bukhsh, Efficacy of a potentized homoeopathic drug (Arsenicum Album-30) in reducing toxic effects produced by arsenic trioxide in mice: II. On alterations in body weight, tissue weight and total protein, Complement. Ther. Med. 7 (1999) 24–34. [36] Q. Wang, W. Luo, W. Zheng, Y. Liu, H. Xu, G. Zheng, Z. Dai, W. Zhang, Y. Chen, J. Chen, Iron supplement prevents lead-induced disruption of the blood–brain barrier during rat development, Toxicol. Appl. Pharm. 219 (2007) 33–41. [37] S. Xi, Y. Jin, X. Lv, G. Sun, Distribution and speciation of arsenic by transplacental and early life exposure to inorganic arsenic in offspring rats, Biol. Trace Elem. Res. 134 (2010) 84–97. ˙ nska, ´ ˛ [38] L. Struzy B. Dabrowska-Bouta, K. Koza, G. Sulkowski, Inflammation-like glial response in lead-exposed immature rat brain, Toxicol. Sci. 95 (2007) 156–162. [39] A.M. Favero, C.S. Oliveira, C. Franciscato, V.A. Oliveira, J.S. Pereira, C.M. Bertoncheli, S.C. Luz, V.L. Dressler, É.M. Flores, M.E. Pereira, Lactating and nonlactating rats differ to renal toxicity induced by mercuric chloride: the preventive effect of zinc chloride, Cell Biochem. Funct. 32 (2014) 420–428. [40] T. Antonio Garcia, L. Corredor, Biochemical changes in the kidneys after perinatal intoxication with lead and/or cadmium and their antagonistic effects when co-administered, Ecotoxicol. Environ. Saf. 57 (2004) 184–189. [41] M. De Santis, B. Pan, J. Lian, X.-F. Huang, C. Deng, Different effects of bifeprunox, aripiprazole, and haloperidol on body weight gain food and water intake, and locomotor activity in rats, Pharmacol. Biochem. Behav. 124 (2014) 167–173. [42] I. Butt, A. Hong, J. Di, S. Aracena, P. Banerjee, C.-H. Shen, The effects of serotonin1A receptor on female mice body weight and food intake are associated with the differential expression of hypothalamic neuropeptides and the GABAA receptor, Neuropeptides 48 (2014) 313–318. [43] M.F. Naujokas, B. Anderson, H. Ahsan, H.V. Aposhian, J.H. Graziano, C. Thompson, W.A. Suk, The broad scope of health effects from chronic arsenic exposure: update on a worldwide public health problem, Environ. Health Perspect. 121 (2013) 295. [44] F. Parvez, G.A. Wasserman, P. Factor- Litvak, X. Liu, V. Slavkovich, A.B. Siddique, R. Sultana, R. Sultana, T. Islam, D. Levy, Arsenic exposure and motor function among children in Bangladesh, Environ. Health Perspect. 119 (2011) 1665. [45] H.-J. Lin, T.-I. Sung, C.-Y. Chen, H.-R. Guo, Arsenic levels in drinking water and mortality of liver cancer in Taiwan, J. Hazard. Mater. 262 (2013) 1132–1138. [46] L. Miller, A.M. Shapiro, J. Cheng, P.G. Wells, The free radical spin trapping agent phenylbutylnitrone reduces fetal brain DNA oxidation and postnatal cognitive deficits caused by in utero exposure to a non-structurally teratogenic dose of ethanol: a role for oxidative stress, Free Radical Bio. Med. 60 (2013) 223–232. [47] B. Halliwell, J.M. Gutteridge, Free Radicals in Biology and Medicine, Oxford university press, Oxford, 1999. [48] Y.-M. Zhang, X.-Z. Liu, H. Lu, L. Mei, Z.-P. Liu, Lipid peroxidation and ultrastructural modifications in brain after perinatal exposure to lead and/or cadmium in rat pups, Biomed. Environ. Sci. 22 (2009) 423–429. [49] A. Levinsson, A.-C. Olin, L. Björck, A. Rosengren, F. Nyberg, Nitric oxide synthase (NOS) single nucleotide polymorphisms are associated with coronary heart disease and hypertension in the INTERGENE study, Nitric Oxide 39 (2014) 1–7. [50] A.E.A. Moneim, M.A. Dkhil, S. Al-Quraishy, Effects of flaxseed oil on lead acetate-induced neurotoxicity in rats, Biol. Trace Elem. Res. 144 (2011) 904–913. [51] T. Coccini, E. Roda, S. Barni, L. Manzo, Morphological and cytohistochemical evaluation of renal effects of cadmium-doped silica nanoparticles given intratracheally to rat, in: J Phys: Conference Series, IOP Publishing, 2013, pp. 12033. [52] S. Hanafy, M.E. Soltan, Effects of Vitamin E pretreatment on subacute toxicity of mixture of Co, Pb, and Hg nitrate-induced nephrotoxicity in rats, Environ. Toxicol. Pharm. 17 (2004) 159–167. [53] A.E. Abdel Moneim, M.A. Dkhil, S. Al-Quraishy, The protective effect of flaxseed oil on lead acetate-induced renal toxicity in rats, J. Hazard. Mater. 194 (2011) 250–255. [54] R. Hambach, D. Lison, P. D, ’, Haese, J. Weyler, E. De Graef, A. De Schryver, L. Lamberts, M. van Sprundel, Co-exposure to lead increases the renal response to low levels of cadmium in metallurgy workers, Toxicol. Lett. 222 (2013) 233–238. [55] L.M. Gaetke, C.K. Chow, Copper toxicity, oxidative stress, and antioxidant nutrients, Toxicology 189 (2003) 147–163. [56] M. Cave, S. Appana, M. Patel, K.C. Falkner, C.J. McClain, G. Brock, Polychlorinated biphenyls, lead, and mercury are associated with liver disease in American adults: NHANES 2003–2004, Environ. Health Perspect. 118 (2010) 1735. [57] W.-C. Lee, L.-C. Kuo, Y.-C. Cheng, C.-W. Chen, Y.-K. Lin, T.-Y. Lin, H.-L. Lin, Combination of white blood cell count with liver enzymes in the diagnosis of blunt liver laceration, Am. J. Emergency Med. 28 (2010) 1024–1029. [58] Y.-J. Lee, H.-R. Lee, J.-Y. Shim, B.-S. Moon, J.-H. Lee, J.-K. Kim, Relationship between white blood cell count and nonalcoholic fatty liver disease, Digest. Liver Dis. 42 (2010) 888–894. [59] W. Jira, G. Spiteller, W. Carson, A. Schramm, Strong increase in hydroxy fatty acids derived from linoleic acid in human low density lipoproteins of atherosclerotic patients, Chem. Phys. Lipids 91 (1998) 1–11. [60] W. Feng, M. Wang, B. Li, J. Liu, Z. Chai, J. Zhao, G. Deng, Mercury and trace element distribution in organic tissues and regional brain of fetal rat after in

120

S.J. Cobbina et al. / Journal of Hazardous Materials 294 (2015) 109–120

utero and weaning exposure to low dose of inorganic mercury, Toxicol. Lett. 152 (2004) 223–234. [61] M. Pal, M. Ghosh, Prophylactic effect of ␣-linolenic acid and ␣-eleostearic acid against MeHg induced oxidative stress, DNA damage and structural changes in RBC membrane, Food Chem. Toxicol. 50 (2012) 2811–2818.

[62] P.A. Mitozo, L.F. de Souza, G. Loch-Neckel, S. Flesch, A.F. Maris, C.P. Figueiredo, A.R.S. dos Santos, M. Farina, A.L. Dafre, A study of the relative importance of the peroxiredoxin-, catalase-, and glutathione-dependent systems in neural peroxide metabolism, Free Radical Bio. Med. 51 (2011) 69–77.