| Heavy metal contamination has been of great concern because of its inherent toxicity, vast sources, persistence and non-degradability. Anthropogenic sources of heavy metals are from mining, smelting, agriculture, agrochemical industry, aquaculture, petrochemical and the electronic industries, from where they are ultimately discharged into the environment and eventually bio-accumulated by organisms and even biomagnified through the food chain resulting in elevated levels in predatory animals. The presence of these metals in the environment has been associated with numerous adverse effects on humans and animals.This study reports on the toxicity of four kinds of heavy metals on exposure to mice and zebrafish embryos. Mice and zebrafish embryos, were exposed to low concentration individual and mixtures of the heavy metals, based on the National Standard of The Peoples’Republic of China for Standards for Drinking Water Quality (GB5749-2006)(Pb-0.01 mg/L, Hg-0.001 mg/L, As-0.01 mg/L and Cd-0.005 mg/L). These metal concentrations are not supposed to adversely affect consumers. To our knowledge, they are the lowest to be used in assessing toxicological effects resulting from exposure to mixtures.The study offers contribution in five main ways. The procedures and contributions in all phases are briefly outlined below:(1) Toxicity assessment of individual and mixtures of four low concentration heavy metals on sub-chronic exposure to mice: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 exposed individually and as mixtures through drinking water, to 10 groups of 40 three-week old mice (20 males and 20 females), for 120 days. The study showed that low concentration heavy metals induced toxicity to the brain, liver, and kidney of mice. Metal mixtures showed higher toxicities compared to individual metals, as exposure to Pb+Hg+Cd reduced brain weight and induced structural lesions, such as neuronal degeneration after 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 malondialdehyde (MDA). These were accentuated by increases in alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (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. The findings of this study, underlines the importance of elucidating the toxicity of low concentration metal mixtures in the environment.(2) Interaction of four low concentration heavy metals with non-essential and essential metals in brain, liver and kidneys of mice on sub-chronic exposure:This phase of the study sought to assess the effect of the interaction of low concentration individual heavy metals on non-essential and essential metals present in the brain, liver and kidneys of ICR mice. Low concentrations of Pb, Hg, Cd and As were singly exposed to four groups of three-week old mice for 120 days. Mice were sacrificed monthly and organs such as the brain, liver, and kidneys were removed, rinsed in cold saline water, weighed, and used for metal analyses after wet digestion. It was observed that exposure to Pb significantly increased brain Pb by 61.2% after 120 days compared to control group. Hg increased brain Pb and Hg by 157% and 222%, respectively, after 30 days. Additionally, Pb exposure increased brain Mg and Cu by 55.5% and 266%, respectively. Increased brain Mg may have resulted from the metabolic activity of brain to combat insults; whiles Cu overload may be due to alteration and dysfunction of CTR1 and ATP7A molecules. Furthermore, it was observed that liver Ca was reduced by 56.0% and 31.6%, respectively, on exposure to As and Cd. Decreases in kidney Mg, Ca and Fe were associated with the uptake of complexes formed between toxic metals such as As and Cd with thiol groups from the proximal tubular lumen. This phase of the study established that, at considerably low concentrations, toxic metals disturb the homeostasis of non-essential and essential metals present in organs of young mice.(3) Interaction of low concentration heavy metal mixtures with non-essential and essential metals in brain, liver and kidneys of mice, on sub-chronic exposure:In this contribution, interactions between low concentrations of mixtures of Pb, Hg, As and Cd with non-essential and essential metals, were assessed in organs of mice. For 120 days, six groups of forty mice each were exposed to metal mixtures, while the control group was given distilled water. It was found that, exposure to Pb+Cd increased brain Pb by 479% after 30 days, whiles Pb+Hg+As+Cd reduced liver Hg by 46.5%, but increased kidney As by 130% in 30 days. Additionally, brain Cu, increased by 221% on Pb+Hg+As+Cd exposure, whiles liver Ca reduced by 36.1% on Pb+Hg exposure in 60 days. By using the combination index method it was found that interactions within metal mixtures were largely synergistic. Exposure to low concentration metal mixtures affected concentration of toxic and essential metals in tissues of mice.(4) Embryonic exposure to low concentration individual and mixtures of heavy metals in zebrafish (Daniorerid):Innate immune-related gene expression:The zebra fish model was employed to investigate the effect of low concentration individual and mixtures of heavy metals on aquatic organisms.The effect of low concentration heavy metals on innate immune and antioxidant-related gene expressions of zebra fish embryos was assessed. It was confirmed that, mRNA levels of IL1β, TNF-α, IFNy, Mx, Lyz, C3B and CXCL-Clc which are closely associated with the innate immune system, were affected after exposing zebra fish embryos to low concentrations of individual and mixtures of metals for 120 h post fertilization (hpf). IL1β genes were significantly up regulated on exposure to Pb+As and Pb+Hg+Cd by 99% and 88.1%, respectively. However, TNF-a was significantly inhibited on exposure to As (73.8%) and Pb+As (70.1%) compared to control group. Additionally, antioxidant genes were affected, as CAT and GPx gene expressions generally increased, whiles Mn-SOD and Zn/Cu-SOD reduced. Multivariate analysis showed that exposure to individual metals greatly influenced the modulation of innate immune genes, while metal mixtures influenced antioxidant gene expressions. This suggests that beside oxidative stress, there may be other pathways influencing gene expressions of innate immune and antioxidant-related genes.(5) Modulation of N-methyl-D-aspartate receptors (NMDAR), Bcl-2 and C-fos gene expressions on exposure to individual and mixtures of low concentration metals in zebra fish(Daniorerio):This part of the study reports on the neurological gene expressions, resulting after exposing.zebra fish’embryos to low concentration toxic heavy metals,120 h post fertilization. Embryos were exposed to low concentration individual and mixtures of Pb, Hg, As and Cd and quantitative real-time PCR was used to assess gene expressions. The findings of this study confirmed that exposure to such low concentration heavy metals up regulated certain N-methyl-D-aspartate (NMDA) receptor subunits (NR2A, NR2B and NR2D) and Bcl-2 genes. NR2A genes were significantly up regulated by 90% and 74%, respectively, on exposure to Pb+As and Pb+Cd. NR2A gene expressions from exposure to Pb+As,were observed to be higher than that exhibited on exposure to individual metals (Pb and As).Exposure to As, Pb+Cd and Pb+Hg+As significantly up regulated Bcl-2 genes by 2.01-,1.84-and 1.80-folds, respectively. NR1A and C-fos gene expressions were not significantly different from control, after exposure to both low concentration toxic metals. Principal component analysis (PCA) confirmed the influence of low concentration individual and mixtures of Pb, Hg, As and Cd on gene expression of NMDAR subunits and Bcl-2. These data suggests that altered expression of NMDA receptor subunits and Bcl-2 genes may explain toxicity of low concentration individual and mixtures of Pb, Hg, As and Cd. |
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