Cadmium osteotoxicity in experimental animals: mechanisms and relationship to human exposures.

Extensive epidemiological studies have recently demonstrated increased cadmium exposure correlating significantly with decreased bone mineral density and increased fracture incidence in humans at lower exposure levels than ever before evaluated. Studies in experimental animals have addressed whether very low concentrations of dietary cadmium can negatively impact the skeleton. This overview evaluates results in experimental animals regarding mechanisms of action on bone and the application of these results to humans. Results demonstrate that long-term dietary exposures in rats, at levels corresponding to environmental exposures in humans, result in increased skeletal fragility and decreased mineral density. Cadmium-induced demineralization begins soon after exposure, within 24 h of an oral dose to mice. In bone culture systems, cadmium at low concentrations acts directly on bone cells to cause both decreases in bone formation and increases in bone resorption, independent of its effects on kidney, intestine, or circulating hormone concentrations. Results from gene expression microarray and gene knock-out mouse models provide insight into mechanisms by which cadmium may affect bone. Application of the results to humans is considered with respect to cigarette smoke exposure pathways and direct vs. indirect effects of cadmium. Clearly, understanding the mechanism(s) by which cadmium causes bone loss in experimental animals will provide insight into its diverse effects in humans. Preventing bone loss is critical to maintaining an active, independent lifestyle, particularly among elderly persons. Identifying environmental factors such as cadmium that contribute to increased fractures in humans is an important undertaking and a first step to prevention.

MicroRNAs-141 and 200a regulate the SVCT2 transporter in bone marrow stromal cells.

Vitamin C is a micro-nutrient which plays an important role in bone marrow stromal cell (BMSCs) differentiation to osteogenesis. This vitamin is transported into the BMSCs through the sodium dependent vitamin C transporter 2 (SVCT2). We previously reported that knockdown of the SVCT2 transporter decreases osteogenic differentiation. However, our understanding of the post-transcriptional regulatory mechanism of the SVCT2 transporter remains poor. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate the messenger RNAs of protein-coding genes. In this study, we aimed to investigate the impact of miR-141 and miR-200a on SVCT2 expression. We found that mouse BMSCs expressed miR-141 and miR-200a and repressed SVCT2 expression at the functional level by targeting the 3'-untranslated region of mRNA. We also found that miR-141 and miR-200a decreased osteogenic differentiation. Furthermore, miRNA inhibitors increased SVCT2 and osteogenic gene expression in BMSCs. Taken together, these results indicate that both miRNAs are novel regulators of the SVCT2 transporter and play an important role in the osteogenic differentiation of BMSCs.

The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis.

Vitamin C is an antioxidant that plays a vital role in various biological processes including bone formation. Previously, we reported that vitamin C is transported into bone marrow stromal cells (BMSCs) through the sodium dependent Vitamin C Transporter 2 (SVCT2) and this transporter plays an important role in osteogenic differentiation. Furthermore, this transporter is regulated by oxidative stress. To date, however, the exact role of vitamin C and its transporter (SVCT2) in ROS regulated autophagy and apoptosis in BMSCs is poorly understood. In the present study, we observed that oxidative stress decreased survival of BMSCs in a dose-dependent manner and induced growth arrest in the G1 phase of the cell cycle. These effects were accompanied by the induction of autophagy, confirmed by P62 and LC3B protein level and punctate GFP-LC3B distribution. The supplementation of vitamin C significantly rescued the BMSCs from oxidative stress by regulating autophagy. Knockdown of the SVCT2 transporter in BMSCs synergistically decreased cell survival even under low oxidative stress conditions. Also, supplementing vitamin C failed to rescue cells from stress. Our results reveal that the SVCT2 transporter plays a vital role in the mechanism of BMSC survival under stress conditions. Altogether, this study has given new insight into the role of the SVCT2 transporter in oxidative stress related autophagy and apoptosis in BMSCs.

Cadmium pathways during gestation and lactation in control versus metallothoinein 1,2-knockout mice.

Effects of metallothionein (MT) on cadmium absorption and transfer pathways during gestation and lactation in mice were investigated. Female 129/SvJ metallothionein-knockout (MT1,2KO) and metallothionein-normal (MTN) mice received drinking water containing trace amounts of (109)CdCl(2) (0.15 ng Cd/ml; 0.074 micro Ci (109)Cd/ml). (109)Cd and MT in maternal, fetal, and pup tissues were measured on gestation days 7, 14, and 17 and lactation day 11. In dams, MT influenced both the amount of (109)Cd transferred from intestine into body (two- to three-fold higher in MT1,2KO than MTN dams) and tissue-specific (109)Cd distribution (higher liver/kidney ratio in MT1,2KO dams). Placental (109)Cd concentrations in MT1,2KO dams were three- and seven-fold higher on gestation days 14 and 17, respectively, than in MTN dams. Fetal (109)Cd levels were low in both mouse types, but at least 10-fold lower in MTN fetuses. MT had no effect on the amount of (109)Cd transferred to pups via milk; furthermore, 85-90% of total pup (109)Cd was recovered in gastrointestinal tracts of both types, despite high duodenal MT only in MTN pups. A relatively large percentage of milk-derived intestinal (109)Cd was transferred to other pup tissues in both MT1,2KO and MTN pups (14 and 10%, respectively). These results demonstrate that specific sequestration of cadmium by both maternal and neonatal intestinal tract does not require MT. Although MT decreased oral cadmium transfer from intestine to body tissues at low cadmium exposure levels, MT did not play a major role in restricting transfer of cadmium from dam to fetus via placenta and to neonate via milk.

A Nest Box to Facilitate Excreta Collection From Mouse Dams Through Pregnancy, Parturition, and Lactation.

Standard metabolism cages are inadequate for collecting excreta from dams during parturition because newborn pups can fall through the grating into the excreta collection area and out of reach of the dam. A nest box was designed that facilitates excreta collection from mouse dams continuously housed in metabolism cages from conception, through parturition, and into lactation and provides a safe, warm environment for pups during their first week of life. The nest box was tested by using pregnant and lactating mice of two varieties of strain 129/SvJ, metallothionein-normal and metallothionein-knockout; non-pregnant mice were used as controls. Pregnant mice (with nest box) and non-pregnant mice (without nest box) each twice received a solution of 109CdCl2 by gavage. Dams with nest boxes fastidiously urinated and defecated outside the nest box. The percentage of gavage 109Cd dose recovered in dam feces was the same after the first gavage (mean6SE, with nest box through parturition, 95%66%; n=5) as after the second gavage (mean6SE, without nest box, 95%66%; n=5). Weights and percentage weight gain of mouse dams were independent of housing conditions (metabolic cage with next box vs. conventional polycarbonate caging). Furthermore, pup growth and survival were unaffected by the inclusion of the nest box or by its removal at 1 week after birth. Therefore, the described nest box provides for the first time a way to quantitatively collect excreta from mouse dams through pregnancy, parturition, and the early postnatal period. Additional experiments are needed to test its application to other animal species and strains of mice, including those with poor mothering behavior.

Knockdown of SVCT2 impairs in-vitro cell attachment, migration and wound healing in bone marrow stromal cells.

Bone marrow stromal cell (BMSC) adhesion and migration are fundamental to a number of pathophysiologic processes, including fracture and wound healing. Vitamin C is beneficial for bone formation, fracture repair and wound healing. However, the role of the vitamin C transporter in BMSC adhesion, migration and wound healing is not known. In this study, we knocked-down the sodium-dependent vitamin C transporter, SVCT2, the only known transporter of vitamin C in BMSCs, and performed cell adhesion, migration, in-vitro scratch wound healing and F-actin re-arrangement studies. We also investigated the role of oxidative stress on the above processes. Our results demonstrate that both oxidative stress and down-regulation of SVCT2 decreased cell attachment and spreading. A trans-well cell migration assay showed that vitamin C helped in BMSC migration and that knockdown of SVCT2 decreased cell migration. In the in-vitro scratch wound healing studies, we established that oxidative stress dose-dependently impairs wound healing. Furthermore, the supplementation of vitamin C significantly rescued the BMSCs from oxidative stress and increased wound closing. The knockdown of SVCT2 in BMSCs strikingly decreased wound healing, and supplementing with vitamin C failed to rescue cells efficiently. The knockdown of SVCT2 and induction of oxidative stress in cells produced an alteration in cytoskeletal dynamics. Signaling studies showed that oxidative stress phosphorylated members of the MAP kinase family (p38) and that vitamin C inhibited their phosphorylation. Taken together, these results indicate that both the SVCT2 transporter and oxidative stress play a vital role in BMSC attachment, migration and cytoskeletal re-arrangement. BMSC-based cell therapy and modulation of SVCT2 could lead to a novel therapeutic approach that enhances bone remodeling, fracture repair and wound healing in chronic disease conditions.

Calcium isolation from large-volume human urine samples for 41Ca analysis by accelerator mass spectrometry.

Calcium oxalate precipitation is the first step in preparation of biological samples for (41)Ca analysis by accelerator mass spectrometry. A simplified protocol for large-volume human urine samples was characterized, with statistically significant increases in ion current and decreases in interference. This large-volume assay minimizes cost and effort and maximizes time after (41)Ca administration during which human samples, collected over a lifetime, provide (41)Ca:Ca ratios that are significantly above background.

Cadmium intake and systemic exposure in postmenopausal women and age-matched men who smoke cigarettes.

Mean blood cadmium (B-Cd) concentrations are two- to threefold higher in smokers than in nonsmokers. The basis for this phenomenon is not well understood. We conducted a detailed, multifaceted study of cadmium exposure in smokers. Groups were older smokers (62±4 years, n = 25, 20% male) and nonsmokers (62±3 years, n = 16, 31% male). Each subject's cigarettes were machine smoked, generating individually paired measures of inhaled cadmium (I-Cd) versus B-Cd; I-Cd and B-Cd were each evaluated three times, at monthly intervals. Urine cadmium (U-Cd) was analyzed for comparison. In four smokers, a duplicate-diet study was conducted, along with a kinetic study of plasma cadmium versus B-Cd. Female smokers had a mean B-Cd of 1.21ng Cd/ml, with a nearly 10-fold range (0.29-2.74ng Cd/ml); nonsmokers had a lower mean B-Cd, 0.35ng Cd/ml (p < 0.05), and narrower range (0.20-0.61ng Cd/ml). Means and ranges for males were similar. Estimates of cadmium amounts inhaled daily for our subjects smoking ≥ 20 cigarettes/day were far less than the 15 µg Cd reported to be ingested daily via diet. This I-Cd amount was too low to alone explain the 3.5-fold elevation of B-Cd in our smokers, even assuming greater cadmium absorption via lungs than gastrointestinal tract; cadmium accumulated in smokers' lungs may provide the added cadmium. Finally, B-Cd appeared to be linearly related to I-Cd values in 75% of smokers, whereas 25% had far higher B-Cd, implying a possible heterogeneity among smokers regarding circulating cadmium concentrations and potentially cadmium toxicity.

Low-volume, high-sensitivity assay for cadmium in blood and urine using conventional atomic absorption spectrophotometry.

An assay for cadmium in whole blood and urine using deuterium background-correction electrothermal atomic absorption spectroscopy (D(2)-ETAAS) was developed. Cadmium (in a 1- to 2-ml sample) was bound to 15 mg anion-exchange resin, interfering ions were removed in a 2-ml Bio-Spin column, and cadmium was extracted into 100 microl 1M nitric acid for analysis. Cadmium in the sample extract was concentrated 7-fold for blood and 10-fold for urine over the starting material. These steps produced cadmium atomic absorption traces with high signal to background ratios and allowed analysis against aqueous standards. At approximately 0.1 ng Cd/ml, mean intra- and interassay coefficients of variation were 11-12%. Cadmium recovery for 0.1 to 0.6 ng added cadmium was 107+/-4% for blood and 94+/-4% for urine (mean+/-SE, n=3). The mean detection limit (mean + 3 x SD of blank) was 0.008 ng/ml for blood and 0.003 ng/ml for urine. Samples from "unexposed" animals including humans ranged from 0.051+/-0.000 to 0.229+/-0.035 ng/ml. Values were approximately 10-fold lower than those obtained by the method of Stoeppler and Brandt using Zeeman background-correction ETAAS. This new high-sensitivity, low-volume assay will be useful for epidemiological studies, even those involving children, and will provide a means to help determine the contribution of cadmium to disease incidence in the general population.

Role of fos and src in cadmium-induced decreases in bone mineral content in mice.

Cadmium decreases bone mineral in mice and stimulates osteoclast formation and activity in cell culture. Bones from fos-deficient mice contain very few osteoclasts; src-/- bones contain osteoclasts that fail to activate. Fos-/- and src-/- mice develop osteopetrotic bones and their teeth do not erupt. These mice were used to determine if cadmium requires c-Fos or c-Src and secondarily functional osteoclasts to decrease bone mineral content. Mice heterozygous for fos deficiency were mated to produce fos-/- and fos+/o (wild-type) offspring. Pups were divided into four groups: fos+/o, Cd-; fos+/o, Cd+; fos-/-, Cd-; and fos-/-, Cd+. Cd+ pups received daily sc injections (50 microg Cd/kg) on days 17-20 and Cd in drinking water thereafter (10 ppm, days 21-27; 20 ppm, days 27-50). An analogous protocol was followed mating mice heterozygous for src deficiency. For acute exposures, 50-day-old mice were placed on a low-calcium diet and given two sequential 100 microg Cd doses by gavage, and feces were monitored for excretion of bone calcium. Continuous exposure results demonstrate that cadmium (1) significantly decreased bone calcium content (14-15%) and concentration (12-13%) in both fos+/o and fos-/- mice, (2) doubled multinucleated osteoclast-like cell number in fos-/- bones, and (3) stimulated tooth eruption in 40% of fos-/- mice. Cd gavage stimulated bone calcium excretion in both fos+/o and fos-/- mice. In contrast, cadmium had no effect on bones or teeth in src-/- mice. Results indicate that cadmium can decrease bone mineral via a c-Fos-independent pathway; however, c-Src is required for cadmium to stimulate bone remodeling and tooth eruption pathways.

Microarray analysis of changes in bone cell gene expression early after cadmium gavage in mice.

We developed an in vivo model for cadmium-induced bone loss in which mice excrete bone mineral in feces beginning 8 h after cadmium gavage. Female mice of three strains [CF1, MTN (metallothionein-wild-type), and MT1,2KO (MT1,2-deficient)] were placed on a low-calcium diet for 2 weeks. Each mouse was gavaged with 200 microg Cd or vehicle only. Fecal calcium was monitored daily for 9 days, beginning 4 days before cadmium gavage, to document the bone response. For CF1 mice, bones were taken from four groups: +/- Cd, 2 h after Cd and +/- Cd, 4 h after Cd. MTN and MT1,2KO strains had two groups each: +/-Cd, 4 h after Cd. PolyA+ RNA preparations from marrow-free shafts of femura and tibiae of each +/- Cd pair were submitted to Incyte Genomics for microarray analysis. Fecal Ca results showed that bone calcium excreted after cadmium differed for the three mouse strains: CF1, 0.24 +/- 0.08 mg; MTN, 0.92 +/- 0.22 mg; and MT1,2KO, 1.7 +/- 0.4 mg. Gene array results showed that nearly all arrayed genes were unaffected by cadmium. However, MT1 and MT2 had Cd+/Cd- expression ratios >1 in all four groups, while all ratios for MT3 were essentially 1, showing specificity. Both probes for MAPK 14 (p38 MAPK) had expression ratios >1, while no other MAPK responded to cadmium. Vacuolar proton pump ATPase and integrin alpha v (osteoclast genes), transferrin receptor, and src-like adaptor protein genes were stimulated by Cd; other src-related genes were unaffected. Genes for bone formation, stress response, growth factors, and signaling molecules showed little or no response to cadmium. Results support the hypothesis that Cd stimulates bone demineralization via a p38 MAPK pathway involving osteoclast activation.