Molecular Mechanisms of Liver Injury of Mice Caused by Lanthanides
|Keywords||Lanthanides Mice liver Oxidative stress Inflammatory response Enzymatic structure–function|
Rare earth elements （REEs） are comprised of scandium, yttrium and 15 lanthanides （Ln） of the IIIB family in the periodic table of chemical element, and possess unique physical and chemical properties. With their increasingly wide applications in agriculture, industry, culture, medicine and many fields in daily life, more and more REE compounds enter the ecological environment and human body through the food chain inevitably. As non-essential elements of the organisms, they are potential risks to human health. Extensive researches have been carried out on their toxicological effect, but the molecular mechanisms of liver injury caused by REEs are unclear, and they always focused on single REE. Thus, ICR mice were exposed to LaCl3, CeCl3, and NdCl3, respectively, we investigated the molecular mechanisms of liver damage, and the effects 4f shell and alterable valence properties of Ln on liver toxicity of mice. Our findings aroused the attention of Ce application and exposure effects especially on human liver for long-term and low-dose treatment.Three main aspects are involved:（1） In this study, LaCl3, CeCl3, and NdCl3 at a higher dose of 20 mg/kg body weight were injected into the abdominal cavity of ICR mice for 14 consecutive days, and the inflammatory responses and oxidative stress–mediated responses in the liver of mice were investigated by histopathological test, real–time quantitative reverse transcription polymerase chain reaction （RT–PCR）, and enzyme–linked immunosorbent assay （ELISA） methods. The results showed the significant accumulation of Ln in the liver results in the increase of coefficients of the liver to body weight, and liver histopathological changes (including basophilia for few hepatocytes and congestion of central veins in the 20 mg/kg BW La3+–treated group, hepatocyte necrosis and basophilia in the 20 mg/kg BW Ce3+–treated group, congestion of central veins and prominent vasodilatation in the 20 mg/kg BW Nd3+–treated group). Ln exposure could significantly elevate the activities of alanine aminotransferase （ALT）, alkaline phosphatase （ALP）, aspartate aminotransferase （AST）, lactate dehydrogenase （LDH）, pseudocholinesterase （PChE）, and decrease ratio of albumin to globulin in serum of mice, and Ce3+ treatment was the greatest, Nd3+ treatment medium, and La3+ treatment smallest, suggesting that Ln exposure damaged liver function of mice. The real–time quantitative RT–PCR and ELISA analysis showed that Ln could significantly alter the mRNA and protein expressions of several inflammatory cytokines, including nucleic factor–κB, macrophage migration inhibitory factor, tumor necrosis factor–α, interleukin–1β, interleukin–6, cross–reaction protein, interleukin–4, and interleukin–10 in a Ln which has a 4f shell and alterable valence properties–dependent manner, showing that the inflammation is able to induce an increase in the expression level of inflammatory cytokines by higher dose Ln. The increase of lipid peroxide in the liver produced by Ln suggested an oxidative attack that was activated by an obvious reactive oxygen species （ROS） accumulation, a reduction of antioxidative defense mechanisms as measured by analyzing the activities of superoxide dismutase, catalase, and ascorbate peroxidase, as well as antioxidant levels such as glutathione and ascorbic acid, which were greatest in Ce3+ treatment, medium in Nd3+, and least in La3+. Taken together, our results also implied that the inflammatory responses, oxidative stress and liver injury likely are caused by 4f shell and alterable valence properties of Ln–induced liver toxicity.（2） In order to investigate the molecular mechanism of inflammatory response in the mouse liver caused by exposure to CeCl3, we measured the liver indices, and cerium content, evaluated the liver histopathological section, detected serum biochemical parameters of liver function, and the immunoglobulin M （IgM） content, analyzed the liver mRNA and protein expression levels of Toll–like receptor 2, 4 （TLR2, TLR4） and inflammatory cytokines in liver using real–time quantitative RT–PCR and ELISA after gavage administration with CeCl3 for 45 days. The results showed that exposure to CeCl3 decreased body weight, and caused cerium accumulation in the mouse liver and histopathological changes of liver （such as inflammatory cell infiltration）. Furthermore, biochemical assays suggested that CeCl3 could promote the activities of alanine aminotransferase （ALT）, alkaline phosphatase （ALP）, aspartate aminotransferase （AST）, lactate dehydrogenase （LDH）, pseudocholinesterase （PChE）, and leucine aminopeptidase （LAP）, decrease serum IgM, upregulate the levels of TLR2, TLR4, nuclear factor–κB （NF–κB）, NF–κBP52, NF–κBP65, NF–κB–inducing kinase （NIK）, IκB kinaseα（IKK–α）, IκB kinaseβ（IKK–β）, and tumor necrosis factor–α（TNF–α） expression, and suppress NF–κB–inhibiting factor （IκB） and interleukin–2 （IL–2） expression in liver. Taken together, the inflammation of mice liver caused by exposure to CeCl3 might be closely associated with the alteration of inflammatory cytokine expressions in the mouse liver, the signal-transducing events happening in CeCl3–induced macrophages of liver sequentially might occur via activation of CeCl3→TNF–α↑→TLRs（TLR2、TLR4）↑→NIK↑→IκB kinase（including IKK–α, IKK–β）↑→IκB↓→NF–κB（including NF–κBP52, NF–κBP65）↑→inflammation→liver injury.（3） The mechanisms of interactions between La3+, Ce3+, Nd3+ and enzymes involved in liver function such as LDH and ALT.In this section, we investigated the mechanisms of LaCl3, CeCl3, and NdCl3 on LDH activity in vivo and in vitro. Our results showed that La3+, Ce3+, and Nd3+ could significantly activate LDH in vivo and in vitro; the order of activation was Ce3+>Nd3+>La3+>control. The affinity of LDH for Ce3+ was higher than Nd3+ and La3+; the saturated binding sites for Ce3+ on the LDH protein were 1.2 and for La3+ and Nd3+ 1.55. Ln3+ caused the reduction of exposure degree of cysteine or tryptophan/tyrosine of LDH, the increase of space resistance, and the enhancement ofα–helix in secondary structure of LDH, which was greatest in Ce3+ treatment, medium in Nd3+ treatment, and least in La3+ treatment. It implied that the changes of structure-function on LDH caused by Ln3+ were closely related to the characteristics of 4f electron shell and alternation valence in Ln.We also investigated the mechanisms of CeCl3 on ALT activity in vivo and in vitro. Our results showed that Ce3+ could significantly activate ALT in vivo and in vitro; the kinetics constant （Km） and Vmax were 0.018μM and 1,380 unit mg?1 protein min?1, respectively, at a low concentration of Ce3+, and 0.027μM and 624 unit mg?1 protein min?1, respectively, at a high concentration of Ce3+. By UV absorption and fluorescence spectroscopy assays, the Ce3+ was determined to be directly bound to ALT; the binding site of Ce3+ to ALT was 1.72, and the binding constants of the binding site were 4.82×108 and 9.05×107 L mol-1. Based on the analysis of the circular dichroism spectra, it was concluded that the binding of Ce3+ altered the secondary structure of ALT, suggesting that the observed enhancement of ALT activity was caused by a subtle structural change in the active site through the formation of the complex with Ce3+.