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.

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.

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.

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.

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.

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.

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.

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.

Identification of ARNT-regulated BIRC3 as the target factor in cadmium renal toxicity.

Cadmium (Cd) is an environmental contaminant that exhibits renal toxicity. The target transcription factors involved in Cd renal toxicity are still unknown. In this study, we demonstrated that Cd decreased the activity of the ARNT transcription factor, and knockdown of ARNT significantly decreased the viability of human proximal tubular HK-2 cells. Microarray analysis in ARNT knockdown cells revealed a decrease in the expression of a number of genes, including a known apoptosis inhibitor, BIRC3, whose gene and protein expression level was also decreased by Cd treatment. Although the BIRC family consists of 8 members, Cd suppressed only BIRC3 gene expression. BIRC3 is known to suppress apoptosis through the inhibition effect on caspase-3. Knockdown of BIRC3 by siRNA as well as Cd treatment increased the level of active caspase-3. Moreover, knockdown of BIRC3 not only triggered cell toxicity and apoptosis but also strengthened Cd toxicity in HK-2 cells. Meanwhile, the activation of caspase-3 by suppression of BIRC3 gene expression was mostly specific to Cd and to proximal tubular cells. These results suggest that Cd induces apoptosis through the inhibition of ARNT-regulated BIRC3 in human proximal tubular cells.

Different Regulation of p53 Expression by Cadmium Exposure in Kidney, Liver, Intestine, Vasculature, and Brain Astrocytes.

Chronic exposure to cadmium (Cd) is known to adversely affect renal function. Our previous studies indicated that Cd induces p53-dependent apoptosis by inhibiting gene expression of the ubiquitin-conjugating enzyme (Ube) 2d family in both human and rat proximal tubular cells. In this study, the effects of Cd on protein expression of p53 and apoptotic signals in the kidney and liver of mice exposed to Cd for 12 months were examined, as well as the effects of Cd on p53 protein levels and gene expression of the Ube2d family in various cell lines. Results showed that in the kidney of mice exposed to 300 ppm Cd for 12 months, there was overaccumulation of p53 proteins in addition to the induction of apoptosis, which was triggered specifically in the proximal tubules. Interestingly, the site of apoptosis was the same as that of p53 accumulation in the proximal tubules. In the liver of mice chronically exposed to Cd, gene expression of the Ube2d family tended to be slightly decreased, together with slight apoptosis without the accumulation of p53 protein. In rat small intestine epithelial (IEC-6) cells, Cd decreased not only the p53 protein level but also gene expression of Ube2d1, Ube2d2 and Ube2d4. In human brain microvascular endothelial cells (HBMECs), Cd did not suppress gene expression of the Ube2d family, but increased the p53 protein level. In human brain astrocytes (HBASTs), Cd only increased gene expression of UBE2D3. These results suggest that Cd-induced apoptosis through p53 protein is associated with renal toxicity but not hepatic toxicity, and the modification of p53 protein by Cd may vary depending on cell type.

Accumulation of p53 via down-regulation of UBE2D family genes is a critical pathway for cadmium-induced renal toxicity.

Chronic cadmium (Cd) exposure can induce renal toxicity. In Cd renal toxicity, p53 is thought to be involved. Our previous studies showed that Cd down-regulated gene expression of the UBE2D (ubiquitin-conjugating enzyme E2D) family members. Here, we aimed to define the association between UBE2D family members and p53-dependent apoptosis in human proximal tubular cells (HK-2 cells) treated with Cd. Cd increased intracellular p53 protein levels and decreased UBE2D2 and UBE2D4 gene expression via inhibition of YY1 and FOXF1 transcription factor activities. Double knockdown of UBE2D2 and UBE2D4 caused an increase in p53 protein levels, and knockdown of p53 attenuated not only Cd-induced apoptosis, but also Cd-induced apoptosis-related gene expression (BAX and PUMA). Additionally, the mice exposed to Cd for 6 months resulted in increased levels of p53 and induction of apoptosis in proximal tubular cells. These findings suggest that down-regulation of UBE2D family genes followed by accumulation of p53 in proximal tubular cells is an important mechanism for Cd-induced renal toxicity.

Involvement of ubiquitin-coding genes in cadmium-induced protein ubiquitination in human proximal tubular cells.

Cadmium (Cd) is a toxic heavy metal with a long half-life in humans. It causes disorders of various tissue systems, including the kidney, and is associated with protein aggregation. Our previous study demonstrated Cd-induced suppression of the UBE2D gene family, one of the ubiquitin-conjugating enzyme families. However, the precise role of ubiquitin-coding genes in Cd toxicity remains to be understood. In this study, we investigated the effect of Cd on expression of the ubiquitin-coding genes UBB, UBC, UBA80, and UBA52 in HK-2 human proximal tubular cells. Prior to the appearance of Cd toxicity, the UBB, UBC, and UBA80 expression levels increased following Cd treatment. Knockdown of UBB by siRNA transfection significantly decreased Cd cytotoxicity. Notably, Cd induces ubiquitinated protein levels in HK-2 cells, and knockdown of UBB blocked this process. These results suggest that UBB is involved in Cd-induced increase of protein ubiquitination, and that accumulation of ubiquitinated proteins through increased UBB expression may contribute to Cd toxicity in HK-2 cells.

Alteration of DNA binding activity of transcription factors in NRK-52E rat proximal tubular cells treated with cadmium.

Cadmium is a toxic heavy metal that causes severe clinical symptoms in various tissues including the kidney. In this study, activities of transcription factors were measured to identify what type of transcription factor was affected by cadmium in rat proximal tubular cells (NRK-52E cells) using the protein/DNA binding assay. After treatment of NRK-52E cells with 5 µM CdCl2 for 3 hr, nuclear extracts were used for the protein/DNA binding assay. Among 65 transcription factors, cadmium increased the activities of 6 transcription factors by more than 2.0-fold and decreased those of 15 transcription factors by less than 0.5-fold. These findings may provide new information about novel transcription factors associated with the mechanism of cadmium toxicity.

Gene expression differences in the duodenum of 129/Sv and DBA/2 mice compared with that of C57BL/6J mice.

We compared the cadmium (Cd) concentration in the liver and kidney of different strains of mice after exposure to 50 ppm Cd for 30 days via drinking water. Cd concentration in the liver and kidney of C57BL/6J mice were higher than those of 129/Sv and DBA/2 mice. Since orally ingested heavy metals are absorbed in the small intestine and then widely distributed to target tissues, microarray analyses were performed to compare the expression levels of transport-related genes in the duodenum between C57BL/6J mice and 129/Sv or DBA/2 mice. The expression levels of 9 and 11 genes were elevated more than 2.0-fold and 13 and 12 genes were reduced less than 0.5-fold in 129/Sv mice and DBA/2 mice, respectively. Among these low expressed genes, 10 genes (Slc2a2, Slc5a1, Slc16a2, Slc22a13, Slc22a18, Slc25a11, Slc36a1, Slco6c1, Abca3 and Abcd1) were common between the two types of strains. These results suggest that some of those genes might be involved in Cd absorption and its toxicity.

Arsenic (+3 oxidation state) methyltransferase is a specific but replaceable factor against arsenic toxicity.

Inorganic metalloids, such as arsenic (As), antimony (Sb), selenium (Se), and tellurium (Te), are methylated in biota. In particular, As, Se, and Te are methylated and excreted in urine. The biomethylation is thought to be a means to detoxify the metalloids. The methylation of As is catalyzed by arsenic (+3 oxidation state) methyltransferase (AS3MT). However, it is still unclear whether AS3MT catalyzes the methylation of the other metalloids. It is also unclear whether other factors catalyze the As methylation instead of AS3MT. Recombinant human AS3MT (rhAS3MT) was prepared and used in the in vitro methylation of As, Se, and Te. As, but not Se and Te, was specifically methylated in the presence of rhAS3MT. Then, siRNA targeting AS3MT was introduced into human hepatocarcinoma (HepG2) cells. Although AS3MT protein expression was completely silenced by the gene knockdown, no increase in As toxicity was found in the HepG2 cells transfected with AS3MT-targeting siRNA. We conclude that AS3MT catalyzes the methylation of As and not other biomethylatable metalloids, such as Se and Te. We speculate that other methylation enzyme(s) also catalyze the methylation of As in HepG2 cells.

Inorganic arsenic induces apoptosis through downregulation of Ube2d genes and p53 accumulation in rat proximal tubular cells.

Ube2d ubiquitin-conjugating enzymes promote p53 ubiquitination and proteasomal degradation. We previously showed that cadmium induced p53-dependent apoptosis through the suppression of expression of Ube2d family genes (Ube2d1, Ube2d2, Ube2d3 and Ube2d4) in normal rat proximal tubular cells. Here we examined the effects of inorganic arsenic and inorganic mercury, which induce apoptosis in proximal tubular cells, on cellular protein level of p53 and gene expression of Ube2d family. Inorganic arsenic induced apoptosis with p53 accumulation, and suppressed Ube2d1, Ube2d2 and Ube2d4 expression, but not Ube2d3. On the other hand, although apoptosis was induced in response to inorganic mercury in proximal tubular cells, protein level of p53 was not elevated by inorganic mercury. These results suggest that inorganic arsenic, but not inorganic mercury, may induce p53-dependent apoptotic pathways through downregulation of gene expression of Ube2d family in proximal tubular cells.

Gene expression analysis using DNA microarray in HK-2 human proximal tubular cells treated with cadmium.

We examined the alteration of gene expression in HK-2 human proximal tubular cells exposed to cadmium (Cd) using DNA microarray analysis. Cd increased the expression of 30 genes, including 7 genes coding for heat shock proteins, more than 2.0-fold and decreased the expression of 21 genes, including transcription-related genes, such as AP2B1, HOXA7, HOXA9 and TCEB2, less than 0.5-fold prior to the appearance of cytotoxicity in HK-2 cells.

DNA microarray expression analysis of mouse kidney following cadmium exposure for 12 months.

Cadmium (Cd) is a toxic heavy metal and chronic exposure causes kidney injury. This study used DNA microarray analysis to examine gene expression in the kidney of mice chronically exposed to Cd. Female C57BL/6J mice were fed a 300 ppm Cd-containing diet or a control diet for 12 months. In comparison with control mice, the expression levels of 32 genes, including Hmox1 and Mt2, were elevated more than 2.0-fold, whereas 113 genes, including transport- and ubiquitination-related genes, were reduced less than 0.5-fold.