ObjectivesWe provide an update of the issues surrounding health risk assessment of exposure to cadmium in food.Data sourcesWe reviewed epidemiologic studies published between 2004 and 2009 concerning the bioavailability of cadmium in food, assessment of exposure, and body burden estimate, along with exposure-related effects in nonoccupationally exposed populations.Data extraction and synthesisBioavailability of ingested cadmium has been confirmed in studies of persons with elevated dietary exposure, and the findings have been strengthened by the substantial amounts of cadmium accumulated in kidneys, eyes, and other tissues and organs of environmentally exposed individuals. We hypothesized that such accumulation results from the efficient absorption and systemic transport of cadmium, employing multiple transporters that are used for the body’s acquisition of calcium, iron, zinc, and manganese. Adverse effects of cadmium on kidney and bone have been observed in environmentally exposed populations at frequencies higher than those predicted from models of exposure. Increasing evidence implicates cadmium in the risk of diseases that involve other tissues and organ systems at cadmium concentrations that do not produce effects on bone or renal function.ConclusionsPopulation data raise concerns about the validity of the current safe intake level that uses the kidney as the sole target in assessing the health risk from ingested cadmium. The data also question the validity of incorporating the default 5% absorption rate in the threshold-type risk assessment model, known as the provisional tolerable weekly intake (PTWI), to derive a safe intake level for cadmium.

Arsenic and cadmium (Cd؉2 ) are human carcinogens, and epidemiological studies have implicated both pollutants in the development of urinary bladder cancer. Despite this epidemiological base, it is unknown if either Cd ؉2 or arsenite (As ؉3 ) can directly cause the malignant transformation of human urothelial cells. The goal of this study was to determine if Cd ؉2 and/or As ؉3 are able to cause the malignant transformation of human urothelial cells. The strategy employed was to expose the nontumorigenic urothelial cell line UROtsa to long-term in vitro exposure to Cd ؉2 and As ؉3 , with the endpoint being the ability of the cells to form colonies in soft agar and tumors when heterotransplanted into nude mice. It was demonstrated that a long-term exposure to either 1 M Cd ؉2 or 1 M As ؉3 resulted in the selection of cells that were able to form colonies in soft agar and tumors when heterotransplanted into nude mice. The histology of the tumor heterotransplants produced by UROtsa cells malignantly transformed by Cd ؉2 had epithelial features consistent with those of a classic transitional-cell carcinoma of the bladder. The histology of the tumor heterotransplants produced by cells malignantly transformed by As ؉3 was unique in that the cells displayed a prominent squamoid differentiation.

The UROtsa cell line was isolated from a primary culture of normal human urothelium through immortalization with a construct containing the SV40 large T antigen. It proliferates in serum-containing growth medium as a cell monolayer with little evidence of uroepithelial differentiation. The working hypothesis in the present study was that this cell line could be induced to differentiate and express known features of in situ urothelium if the original serum-containing growth medium was changed to a serum-free formulation. We demonstrated that the UROtsa cells could be successfully placed into a serum-free growth medium consisting of a 1:1 mixture of Dulbeco's modified Eagle's medium and Ham's F-12 supplemented with selenium (5 ng/mL), insulin (5 microg/mL), transferrin (5 microg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). Under serum-free growth conditions, confluent UROtsa cells were shown by light microscopy to produce raised, three-dimensional structures. Routine ultrastructural examination disclosed these three-dimensional areas to consist of a stratified layer of cells that strongly resembled in situ urothelium. The cells displayed numerous desmosomal connections, complex interactions of the lateral membranes, and abundant intermediate filaments within the cytoplasm. Freeze fracture analysis demonstrated that the cells possessed tight-junction sealing strands and gap junctions. The overall morphology was most consistent with that found in the intermediate layers of in situ urothelium. The basal expression patterns of the metallothionein (MT) and heat shock proteins 27, 60, and 70 were determined in these cells, and expression was in agreement with that known to occur for in situ urothelium. The cells were also successfully tested for their ability to be stably transfected using expression vectors containing the MT-3 or MT-2A genes. The findings suggest that the UROtsa cells grown with a serum-free medium could be a valuable adjunct for studying environmental insult to the human urothelium in general and for the stress response in particular.

We provide an update of the issues surrounding health risk assessment of exposure to cadmium in food. Bioavailability of ingested cadmium has been confirmed in studies of persons with elevated dietary exposure, and the findings have been strengthened by the substantial amounts of cadmium accumulated in kidneys, eyes, and other tissues and organs of environmentally exposed individuals. We hypothesized that such accumulation results from the efficient absorption and systemic transport of cadmium, employing multiple transporters that are used for the body’s acquisition of calcium, iron, zinc, and manganese. Adverse effects of cadmium on kidney and bone have been observed in environmentally exposed populations at frequencies higher than those predicted from models of exposure. Population data raise concerns about the validity of the current safe intake level that uses the kidney as the sole target in assessing the health risk from ingested cadmium. The data also question the validity of incorporating the default 5% absorption rate in the threshold-type risk assessment model, known as the provisional tolerable weekly intake (PTWI), to derive a safe intake level for cadmium.

This laboratory has shown that arsenite (As+3) exposure can cause the malignant transformation of the UROtsa human urothelial cell line. This single isolate formed subcutaneous tumors with a histology similar to human urothelial cell carcinoma. The tumors also displayed areas of squamous differentiation of the urothelial cells, an infrequent, but known component of human bladder cancer. In the present study, five additional independent isolates of As+3 -transformed urothelial cells were isolated and each were shown to produce subcutaneous urothelial cell tumors with a characteristic histology very similar to those described in the initial report. That there were underlying phenotypic differences in the 6 independent isolates was demonstrated when they were assessed for their ability to form tumors within the peritoneal cavity. It was shown that two isolates could form hundreds of small peritoneal tumor nodules, one isolate a moderate number of tumor nodules, and three isolates no or only one tumor nodule. The peritoneal tumors were also characterized for their degree of squamous differentiation of the urothelial cells and, while areas of squamous differentiation could be found, such differentiation was substantially reduced compared to subcutaneous tumors. Immunostaining for keratin 6 was tested as a potential marker for malignant urothelial cells that had undergone squamous differentiation. Keratin 6 was shown to consistently stain only cells having some evidence of squamous differentiation. Keratin 16 was shown to follow the staining pattern of keratin 6. The isolates and tumor heterotransplants were all examined for keratin 6, 16 and 17 mRNA and protein expression.

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Parkinson's disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta, and in other sub-cortical nuclei associated with a widespread occurrence of Lewy bodies. The cause of cell death in Parkinson's disease is still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative and nitrative stresses have been proposed. We have studied control(wt) (C57B1/6), metallothionein transgenic (MTtrans), metallothionein double gene knock (MTdko), alpha-synuclein knock out (alpha-syn(ko)), alpha-synuclein-metallothionein triple knock out (alpha-syn-MTtko), weaver mutant (wv/wv) mice, and Ames dwarf mice to examine the role of peroxynitrite in the etiopathogenesis of Parkinson's disease and aging. Although MTdko mice were genetically susceptible to 1, methyl, 4-phenyl, 1,2,3,6-tetrahydropyridine (MPTP) Parkinsonism, they did not exhibit any overt clinical symptoms of neurodegeneration and gross neuropathological changes as observed in wv/wv mice. Progressive neurodegenerative changes were associated with typical Parkinsonism in wv/wv mice. Neurodegenerative changes in wv/wv mice were observed primarily in the striatum, hippocampus and cerebellum. Various hallmarks of apoptosis including caspase-3, TNFalpha, NFkappaB, metallothioneins (MT-1, 2) and complex-1 nitration were increased; whereas glutathione, complex-1, ATP, and Ser(40)-phosphorylation of tyrosine hydroxylase, and striatal 18F-DOPA uptake were reduced in wv/wv mice as compared to other experimental genotypes. Striatal neurons of wv/wv mice exhibited age-dependent increase in dense cored intra-neuronal inclusions, cellular aggregation, proto-oncogenes (c-fos, c-jun, caspase-3, and GAPDH) induction, inter-nucleosomal DNA fragmentation, and neuro-apoptosis. MTtrans and alpha-Syn(ko) mice were genetically resistant to MPTP-Parkinsonism and Ames dwarf mice possessed significantly higher concentrations of striatal coenzyme Q10 and metallothioneins (MT 1, 2) and lived almost 2.5 times longer as compared to control(wt) mice. A potent peroxynitrite ion generator, 3-morpholinosydnonimine (SIN-1)-induced apoptosis was significantly attenuated in MTtrans fetal stem cells. These data are interpreted to suggest that peroxynitrite ions are involved in the etiopathogenesis of Parkinson's disease, and metallothionein-mediated coenzyme Q10 synthesis may provide neuroprotection.

The third isoform (MT-3) of the metallothionein gene family is unique in that it has a limited tissue distribution, is not induced by metals, has a neuronal growth inhibitory activity, and sequesters zinc more effectively under zinc-depleted conditions. The goal of the present study was to determine whether MT-3 was absent in normal breast tissue, was overexpressed in breast cancers, and if MT-3 overexpression would be associated with disease outcome. A combination of immunohistochemistry and reverse-transcription polymerase chain reaction was used to demonstrate that the normal breast had no detectable expression of MT-3 mRNA or protein. Using immunohistochemistry, it was shown that MT-3 was overexpressed in 25 of 34 cases of breast cancer. In all cases of positive staining, MT-3 was diffusely localized to the cytoplasm. The tumors from these 34 cases were divided as to outcome based on known 5-year survival, with 20 patients being disease free at 5 years (good outcome) and the other 14 having recurring disease within 5 years (bad outcome). When analyzed for MT-3 staining, it was shown that there was a trend for increased MT-3 immunoreactivity in the group having bad outcomes. However, when the tumor subgrouping was further defined on the basis of carcinoma in situ (CIS), there was a marked significant difference in MT-3 staining between patients with good and bad outcomes. Limited to DCIS, MT-3 staining was significantly increased in patients with bad outcomes compared to those with good outcomes. Thus, these studies demonstrate that MT-3 is overexpressed in selected breast cancers and that overexpression is associated with tumors having a poor prognosis. (Am J Pathol 2001, 159:21-26) The metallothioneins (MTs) are a family of cysteine-rich, low molecular weight, intracellular proteins that bind transition metals.1 In both mice and humans, there are four classes of very similar MT proteins, designated MT-1 through -4, defined on the basis of small differences in sequence and charge characteristics.1,2 The MT-1 and MT-2 isoforms have been extensively studied, and are believed to serve an important role in the homeostasis of essential metals such as Zn 2ϩ or Cu 2ϩ during growth and development, as well as in the detoxification of heavy metals such as Cd 2ϩ and Hg 2ϩ , rendering the MTs important mediators and attenuators of heavy metal-induced toxicity, particularly hepato-and nephrotoxicity.