Neurobehavioral effects of the exposure to mercury vapor and methylmercury during postnatal period on mice.

In this study, we investigated the neurobehavioral alterations and modifications of gene expression in the brains of female mice exposed to low-level mercury vapor and/or methylmercury during postnatal development. The mice were exposed to low-level mercury vapor at a mean concentration of 0.094 mg/m3 and supplied with tap water containing 5 ppm methylmercury from postnatal day 11 to 12 weeks of age. Behavioral analyses were performed at 17 weeks of age. Total locomotor activity in the open field test and the retention trial performance in the passive avoidance test were significantly reduced in the combined exposure group compared with those in the control group. The differences in locomotor activity and performance in the retention trial at 17 weeks were no longer detected at 45 weeks. These results suggest that the effect of aging on the behavioral abnormalities resulting from postnatal exposure to mercury complexes are not significant. In the microarray analysis of brains in the combined exposure group, the gene expression levels of Ano2 and Sgk1 were decreased. Real-time RT-PCR analysis confirmed these changes caused by combined mercury exposure, showing significant downregulation of Ano2 and Sgk1 in the cerebrum. These genes play key roles in the brain as a calcium-activated chloride channel and as a kinase that responds to cellular stress, respectively. Our findings provide insight into the neurobehavioral changes caused by combined mercury exposure.

Long-Term Exposure to Cadmium Causes Hepatic Iron Deficiency through the Suppression of Iron-Transport-Related Gene Expression in the Proximal Duodenum.

Cadmium (Cd) is an environmental pollutant that damages various tissues. Cd may cause a depletion of iron stores and subsequently an iron deficiency state in the liver. However, the molecular mechanism of decreased iron accumulation in the liver induced by long-term exposure to Cd is unknown. In this study, we investigated the hepatic accumulation of iron and the proximal duodenal expression of the genes involved in iron transport using mice chronically exposed to Cd. Five-week-old female C57BL/6J mice were fed a diet containing 300 ppm Cd for 12, 15, 19 and 21 months. The iron concentration in the liver was markedly decreased by Cd. Among iron-transport-related genes in the proximal duodenum, the gene expression of HCP1 and Cybrd1 was significantly decreased by Cd. HCP1 is an influx transporter of heme iron. Cybrd1 is a reductase that allows non-heme iron to enter cells. The expression of iron-transport-related genes on the duodenal basolateral membrane side was hardly altered by Cd. These results suggest that long-term exposure to Cd suppresses the expression of HCP1 and Cybrd1 in the proximal duodenum, resulting in reduced iron absorption and iron accumulation in the liver.

Empirical single-cell tracking and cell-fate simulation reveal dual roles of p53 in tumor suppression.

The tumor suppressor p53 regulates various stress responses via increasing its cellular levels. The lowest p53 levels occur in unstressed cells; however, the functions of these low levels remain unclear. To investigate the functions, we used empirical single-cell tracking of p53-expressing (Control) cells and cells in which p53 expression was silenced by RNA interference (p53 RNAi). Here, we show that p53 RNAi cells underwent more frequent cell death and cell fusion, which further induced multipolar cell division to generate aneuploid progeny. Those results suggest that the low levels of p53 in unstressed cells indeed have a role in suppressing the induction of cell death and the formation of aneuploid cells. We further investigated the impact of p53 silencing by developing an algorithm to simulate the fates of individual cells. Simulation of the fate of aneuploid cells revealed that these cells could propagate to create an aneuploid cell population. In addition, the simulation also revealed that more frequent induction of cell death in p53 RNAi cells under unstressed conditions conferred a disadvantage in terms of population expansion compared with Control cells, resulting in faster expansion of Control cells compared with p53 RNAi cells, leading to Control cells predominating in mixed cell populations. In contrast, the expansion of Control cells, but not p53 RNAi cells, was suppressed when the damage response was induced, allowing p53 RNAi cells to expand their population compared with the Control cells. These results suggest that, although p53 could suppress the formation of aneuploid cells, which could have a role in tumorigenesis, it could also allow the expansion of cells lacking p53 expression when the damage response is induced. p53 may thus play a role in both the suppression and the promotion of malignant cell formation during tumorigenesis.

Cadmium Toxicity Is Regulated by Peroxisome Proliferator-Activated Receptor δ in Human Proximal Tubular Cells.

Cadmium (Cd) is a toxic heavy metal that is widely present in the environment. Renal proximal tubule disorder is the main symptom of Cd chronic poisoning. Our previous study demonstrated that Cd inhibits the total activities of peroxisome proliferator-activated receptor (PPAR) transcription factors in human and rat proximal tubular cells. In this study, we investigated the involvement of PPAR in Cd renal toxicity using the HK-2 human proximal tubular cell line. Among PPAR isoform genes, only PPARD knockdown significantly showed resistance to Cd toxicity in HK-2 cells. The transcriptional activity of PPARδ was decreased not only by PPARD knockdown but also by Cd treatment. DNA microarray analysis showed that PPARD knockdown changed the expression of apoptosis-related genes in HK-2 cells. PPARD knockdown decreased apoptosis signals and caspase-3 activity induced by Cd treatment. PPARD knockdown did not affect the intracellular Cd level after Cd treatment. These results suggest that PPARδ plays a critical role in the modification of susceptibility to Cd renal toxicity and that the apoptosis pathway may be involved in PPARδ-related Cd toxicity.

A Novel In Vitro Assay Using Human iPSC-Derived Sensory Neurons to Evaluate the Effects of External Chemicals on Neuronal Morphology: Possible Implications in the Prediction of Abnormal Skin Sensation.

Neuronal morphological changes in the epidermis are considered to be one of causes of abnormal skin sensations in dry skin-based skin diseases. The present study aimed to develop an in vitro model optimised for human skin to test the external factors that lead to its exacerbation. Human-induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) were used as a model of human sensory neurons. The effects of chemical substances on these neurons were evaluated by observing the elongation of nerve fibers, incidence of blebs (bead-like swellings), and the expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2). The nerve fiber length increased upon exposure to two common cosmetic preservatives-methylparaben and phenoxyethanol-but not to benzo[a]pyrene, an air pollutant at the estimated concentrations in the epidermis. Furthermore, the incidence of blebs increased upon exposure to benzo[a]pyrene. However, there was a decrease in the expression of NMNAT2 in nerve fibers, suggesting degenerative changes. No such degeneration was found after methylparaben or phenoxyethanol at the estimated concentrations in the epidermis. These findings suggest that methylparaben and phenoxyethanol promote nerve elongation in hiPSC-SNs, whereas benzo[a]pyrene induces nerve degeneration. Such alterations may be at least partly involved in the onset and progression of sensitive skin.

Changes in DNA-binding activity of transcription factors in the kidney of mice exposed to cadmium.

Cadmium (Cd) is a toxic heavy metal, long-term exposure to which causes renal damage associated with disruption in gene expression. Transcription factors whose activities were altered in the kidneys of mice exposed to Cd for 3 months were assessed using protein/DNA-binding assays. Female C57BL/6J mice were exposed to 300 ppm Cd in the diet for 3 months. Nuclear extracts of kidney were used for protein/DNA-binding assays. The concentration of Cd was approximately 100 ppm in mouse kidney, a level that did not induce renal toxicity. Among the 345 transcription factors evaluated, five transcription factors showed over a two-fold increase in their activities and 14 transcription factors showed a half-fold change in their activities after Cd exposure. These findings may provide new information about the causative transcription factors associated with Cd renal toxicity.

Cadmium toxicity mediated by the inhibition of SLC2A4 expression in human proximal Tubule cells.

Cadmium (Cd) is an environmental contaminant that causes renal toxicity. We have previously demonstrated that Cd induces renal toxicity by altering transcriptional activities. In this study, we show that Cd markedly inhibited the activity of transcription factor MEF2A in HK-2 human proximal tubule cells, which generated significant cytotoxicity in the cells. This reduction in the nuclear levels of MEF2A protein may be involved in the Cd-induced inhibition of MEF2A activity. We also demonstrate that one of the glucose transporters, GLUT4, was downregulated not only by Cd treatment but also by MEF2A knockdown. Knockdown of SLC2A4, encoding GLUT4, eliminated both cell viability and Cd toxicity. Cd treatment or SLC2A4 deficiency reduced the cellular concentration of glucose. Therefore, the suppression of SLC2A4 expression, which mediates the reduction in cellular glucose, is involved in Cd toxicity. The Cd toxicity induced by the reduction in GLUT4 may be associated with a reduction of cellular ATP levels in HK-2 cells. The levels of Slc2a4 mRNA in the kidney of mice exposed to Cd for 6 or 12 months were significantly lower than those in the control group. These results demonstrate that Cd exerts its cytotoxicity through the suppression in SLC2A4 expression and the subsequent inhibition of MEF2A transcriptional activity. Cd-induced suppression of SLC2A4 expression also reduces cellular ATP levels, partly by reducing glucose levels. This study suggests that the glucose transporter plays an important role in the renal toxicity of Cd, and provides a crucial breakthrough in our understanding of the mechanism of Cd toxicity.

Galectin-3 enhances neutrophil motility and extravasation into the airways during Aspergillus fumigatus infection.

Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian ß-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections.

Cadmium induces iron deficiency anemia through the suppression of iron transport in the duodenum.

Cadmium (Cd) is an environmental contaminant that triggers toxic effects in various tissues such as the kidney, liver, and lung. Cd can also cause abnormal iron metabolism, leading to anemia. Iron homeostasis is regulated by intestinal absorption. However, whether Cd affects the iron absorption pathway is unclear. We aimed to elucidate the relationship between the intestinal iron transporter system and Cd-induced iron deficiency anemia. C57BL/6J female and male mice, 129/Sv female mice, and DBA/2 female mice were given a single oral dose of CdCl2 by gavage. After 3 or 24 h, Cd decreased serum iron concentrations and inhibited the expression of iron transport-related genes in the duodenum. In particular, Cd decreased the levels of divalent metal transporter 1 and ferroportin 1 in the duodenum. In addition, human colon carcinoma Caco-2 cells were treated with CdCl2. After 72 h, Cd decreased the expression of iron transport-related factors in Caco-2 cells with a pattern similar to that seen in the murine duodenum. These findings suggest that Cd inhibits iron absorption through direct suppression of iron transport in duodenal enterocytes and contributes to abnormal iron metabolism.

Methylmercury toxic mechanism related to protein degradation and chemokine transcription.

Methylmercury is an environmental pollutant that causes neurotoxicity. Recent studies have reported that the ubiquitin-proteasome system is involved in defense against methylmercury toxicity through the degradation of proteins synthesizing the pyruvate. Mitochondrial accumulation of pyruvate can enhance methylmercury toxicity. In addition, methylmercury exposure induces several immune-related chemokines, specifically in the brain, and may cause neurotoxicity. This summary highlights several molecular mechanisms of methylmercury-induced neurotoxicity.

Transcription Factors and Downstream Genes in Cadmium Toxicity.

Cadmium (Cd) is a harmful heavy metal widely present in the environment which can cause severe kidney damage. The proximal tubular cells are the main target of renal Cd toxicity. The consequences of Cd cytotoxicity involve apoptosis and necrosis. Recently, we and others have focused on how Cd affects transcription factors and the regulation of their target genes. Those studies showed that transcription factors initiate numerous pathways upon Cd exposure, leading to apoptosis, autophagic cell death, disruption of cell-cell adhesion, and generation of mitochondrial reactive oxygen species. Of particular note, Cd induces endoplasmic reticulum stress, resulting in not only apoptosis but also autophagic dysregulation, which can trigger cell damage. In some cases, however, Cd-regulated transcription factors can induce cell survival signaling. This review centers on our own research to elucidate the transcription factor-downstream gene cascades that are central to Cd-induced renal toxicity.

Dose-dependent spatiotemporal responses of mammalian cells to an alkylating agent.

Cultured cell populations are composed of heterogeneous cells, and previous single-cell lineage tracking analysis of individual HeLa cells provided empirical evidence for significant heterogeneity of the rate of cell proliferation and induction of cell death. Nevertheless, such cell lines have been used for investigations of cellular responses to various substances, resulting in incomplete characterizations. This problem caused by heterogeneity within cell lines could be overcome by investigating the spatiotemporal responses of individual cells to a substance. However, no approach to investigate the responses by analyzing spatiotemporal data is currently available. Thus, this study aimed to analyze the spatiotemporal responses of individual HeLa cells to cytotoxic, sub-cytotoxic, and non-cytotoxic doses of the well-characterized carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although cytotoxic doses of MNNG are known to induce cell death, the single-cell tracking approach revealed that cell death occurred following at least four different cellular events, suggesting that cell death is induced via multiple processes. We also found that HeLa cells exposed to a sub-cytotoxic dose of MNNG were in a state of equilibrium between cell proliferation and cell death, with cell death again induced through different processes. However, exposure of cells to a non-cytotoxic dose of MNNG promoted growth by reducing the cell doubling time, thus promoting the growth of a sub-population of cells. A single-cell lineage tracking approach could dissect processes leading to cell death in a spatiotemporal manner and the results suggest that spatiotemporal data obtained by tracking individual cells can be used as a new type of bioinformatics data resource that enables the examination of cellular responses to various external substances.

Demonstration of Mitochondrial Damage and Mitophagy in Cisplatin-Mediated Nephrotoxicity.

Cisplatin is a chemotherapeutic widely used in the treatment of various types of solid tumors. Acute kidney injury is the most critical dose-limiting factor in cancer patients treated with cisplatin; mitochondrial dysfunction and resultant cell damage by reactive oxygen species released from damaged mitochondria are suspected to be involved in the kidney injury. Pathological features of mitochondrial damage in relation to cisplatin-mediated nephrotoxicity, however, is not fully described. The purpose of this study was to demonstrate mitochondrial damage and clearance of damaged mitochondria by mitophagy in cisplatin-mediated nephrotoxicity. Three groups of rats received a single intraperitoneal injection of cisplatin at 20 mg/kg and were sacrificed at 24, 48 and 72 hours after the treatment. A time-dependent increase in the number of damaged renal tubules and the serum levels of blood urea nitrogen, creatinine, and mitochondrial aspartate transaminase was observed in rats after the treatment. We showed the increased numbers of swollen and fragmented mitochondria, observed by electron microscopy, and of cytochrome c oxidase IV- and 8-nitroguanosine-positive intracytoplasmic granules, detected by immunohistochemistry, in the degenerated renal tubules of the treated animals. Moreover, activated autophagy process was indicated in the degenerated renal epithelial cells, based on the findings of immunohistochemistry of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and lysosomal-associated membrane protein 1 (LAMP-1), a lysosome marker, and swollen and fragmented mitochondria in autophagosomes. These results suggest that mitochondrial damage and clearance of damaged mitochondria by mitophagy is involved in cisplatin-mediated nephrotoxicity.

Effect of Metallothionein-III on Mercury-Induced Chemokine Gene Expression.

Mercury compounds are known to cause central nervous system disorders; however the detailed molecular mechanisms of their actions remain unclear. Methylmercury increases the expression of several chemokine genes, specifically in the brain, while metallothionein-III (MT-III) has a protective role against various brain diseases. In this study, we investigated the involvement of MT-III in chemokine gene expression changes in response to methylmercury and mercury vapor in the cerebrum and cerebellum of wild-type mice and MT-III null mice. No difference in mercury concentration was observed between the wild-type mice and MT-III null mice in any brain tissue examined. The expression of Ccl3 in the cerebrum and of Cxcl10 in the cerebellum was increased by methylmercury in the MT-III null but not the wild-type mice. The expression of Ccl7 in the cerebellum was increased by mercury vapor in the MT-III null mice but not the wild-type mice. However, the expression of Ccl12 and Cxcl12 was increased in the cerebrum by methylmercury only in the wild-type mice and the expression of Ccl3 in the cerebellum was increased by mercury vapor only in the wild-type mice. These results indicate that MT-III does not affect mercury accumulation in the brain, but that it affects the expression of some chemokine genes in response to mercury compounds.

Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), in which mutations in the dystrophin gene disrupts the firm adhesion. In patients with DMD, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in patients with DMD, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina and increases the efficiency of the myogenesis. Galectin-3 is an oligosaccharide-binding protein and is known to be involved in cell-cell interactions and cell-matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle, although its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the synthesis of binding partners (oligosaccharides) of galectin-3, promote myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased muscle-force production. The results suggest that treatment with N-acetylglucosamine might mitigate the burden of DMD.-Rancourt, A., Dufresne, S. S., St-Pierre, G., Lévesque, J.-C., Nakamura, H., Kikuchi, Y., Satoh, M. S., Frenette, J., Sato, S. Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

Glutathione has a more important role than metallothionein-I/II against inorganic mercury-induced acute renal toxicity.

Inorganic mercury is a harmful heavy metal that causes severe kidney damage. Glutathione (GSH), a tripeptide comprising L-glutamic acid, glycine and L-cysteine, and metallothionein (MT), a cysteine-rich and metal-binding protein, are biologically important protective factors for renal toxicity by inorganic mercury. However, the relationship between GSH and MT for the prevention of renal toxicity by inorganic mercury is unknown. We examined the sensitivity of the mice depleted in GSH by treatment with L-Buthionine-SR-sulfoximine (L-BSO), and MT-I/II null mice genetically deleted for MT-I and MT-II, to inorganic mercury (HgCl2). Kidney damage was not induced in the wild-type mice treated with HgCl2 (30 µmol/kg). In the MT-I/II null mice, renal toxicity was induced by HgCl2 at a dose of 30 µmol/kg but not 1.0 µmol/kg. All GSH-depleted mice of both strains were dead following the injection of HgCl2 (30 µmol/kg). GSH-depleted wild-type mice treated with HgCl2 (1.0 µmol/kg) developed kidney damage similar to MT-I/II null mice treated with HgCl2 (30 µmol/kg). Moreover, renal toxicity induced by HgCl2 (1.0 µmol/kg) was more severe in GSH-depleted MT-I/II null mice compared with GSH-depleted wild-type mice. The present study found that GSH and MT-I/II play cooperatively an important role in the detoxification of severe kidney damage caused by inorganic mercury. In addition, GSH may act as a primary protective factor against inorganic mercury-induced acute renal toxicity, because GSH-depleted mice were more sensitive to inorganic mercury than MT-I/II null mice.

Effects of long-term cadmium exposure on urinary metabolite profiles in mice.

Cadmium (Cd) is a common environmental pollutant with known toxic effects on the kidney. Urinary metabolomics is a promising approach to study mechanism by which Cd-induced nephrotoxicity. The aim of this study was to elucidate the mechanism of Cd toxicity and to develop specific biomarkers by identifying urinary metabolic changes after a long-term of Cd exposure and with the critical concentration of Cd in the kidney. Urine samples were collected from wild-type 129/Sv mice after 67 weeks of 300 ppm Cd exposure and analyzed by ultra performance liquid chromatography connected with quadrupole time of flight mass spectrometer (UPLC-QTOF-MS) based metabolomics approach. A total of 40 most differentiated metabolites (9 down-regulated and 31 up-regulated) between the control and Cd-exposed group were identified. The majority of the regulated metabolites are amino acids (glutamine, L-aspartic acid, phenylalanine, tryptophan, and D-proline) indicating that amino acid metabolism pathways are affected by long-term exposure of Cd. However, there are also some nucleotides (guanosine, guanosine monophosphate, cyclic AMP, uridine), amino acid derivatives (homoserine, N-acetyl-L-aspartate, N-acetylglutamine, acetyl-phenylalanine, carboxymethyllysine), and peptides. Results of pathway analysis showed that the arginine and proline metabolism, purine metabolism, alanine, aspartate and glutamate metabolism, and aminoacyl-tRNA biosynthesis were affected compared to the control. This study demonstrates that metabolomics is useful to elucidate the metabolic responses and biological effects induced by Cd-exposure.

Neurobehavioral effects of postnatal exposure to low-level mercury vapor and/or methylmercury in mice.

This study examined the effects on neurobehavioral function of exposure to low-level mercury vapor (Hg0), methylmercury (MeHg) in female mice and the combination of Hg0 and MeHg during postnatal development. Postnatal mice were exposed to Hg0 at a mean concentration of 0.188 mg/m3 Hg0 and supplied with food containing 3.85 µg/g of MeHg from day 2 to day 28 after delivery. The combined exposure group was exposed to both Hg0 and MeHg, using the same procedure. When their offspring reached the age of 11 weeks, behavioral analyses were performed. The behavioral effects in mice were evaluated based on locomotive activity and rate of center entries in the open field (OPF), learning activity in the passive avoidance response (PA) and spatial learning ability in the radial maze (RM). Total locomotive activity in the OPF significantly decreased in the Hg0, MeHg and combined exposure groups compared with the control group. The proportion of entries to central area in the OPF was significantly higher in the combined exposure group than in the control group, while those in the Hg0 or MeHg exposure group did not differ from the control group. Other behavioral tests did not reveal significant differences among the groups. Behavioral anomalies were more distinctive after combined exposure compared to Hg0 or MeHg exposure alone. The brain Hg concentration of offspring, immediately after exposure, was highest in the combined exposure group, exceeding 2 µg/g, followed by the MeHg and Hg0 exposure groups. Thus, the enhancement of neurobehavioral effects in the combined exposure group was associated with higher brain mercury concentration.

Effect of gestational cadmium exposure on fetal growth, polyubiquitinated protein and monoubiqutin levels in the fetal liver of mice.

Cadmium (Cd) is an environmental pollutant present in contaminated water, food and soil. Cd adversely affects fetal development. We exposed pregnant mice to daily oral doses of 5 and 10 mg/kg Cd and examined fetal growth. It was demonstrated that the exposure to Cd (10 mg/kg) during gestation caused fetal growth retardation (FGR). Investigation of the ubiquitin-proteasome system in fetal livers of mice exposed to gestational Cd revealed increased polyubiquitinated protein accumulation, contrasting with decreased levels of monoubiquitin protein. Moreover, the expression level of Ubc (encoding polyubiquitin C protein) was significantly decreased in 5 and 10 mg/kg Cd-treated groups in comparison with the control group. Therefore, we propose that decrease of monoubiquitin level and accumulation of polyubiquitinated protein in the fetal liver may be important factors in Cd-induced FGR.