We previously developed a pH-responsive copper-doxorubicin (CuDox) cargo in lysolipid-based temperature-sensitive liposomes (LTSLs). The CuDox complex is released from the particle by elevated temperature; however, full release of doxorubicin from CuDox requires a reduced pH, such as that expected in lysosomes. The primary goal of this study is to evaluate the cellular uptake and intracellular trafficking of the drug-metal complex in comparison with intact liposomes and free drug. We found that the CuDox complex was efficiently internalized by mammary carcinoma cells after release from LTSLs. Intracellular doxorubicin and copper were 6-fold and 5-fold greater, respectively, after a 0.5h incubation with the released CuDox complex, as compared to incubation with intact liposomes containing the complex. Total cellular doxorubicin fluorescence was similar following CuDox and free doxorubicin incubation. Imaging and mass spectrometry assays indicated that the CuDox complex was initially internalized intact but breaks down over time within cells, with intracellular copper decreasing more rapidly than intracellular doxorubicin. Doxorubicin fluorescence was reduced when complexed with copper, and nuclear fluorescence was reduced when cells were incubated with the CuDox complex as compared with free doxorubicin. Therapeutic efficacy, which typically results from intercalation of doxorubicin with DNA, was equivalent for the CuDox complex and free doxorubicin and was superior to that of liposomal doxorubicin formulations. Taken together, the results suggest that quenched CuDox reaches the nucleus and remains efficacious. In order to design protocols for the use of these temperature-sensitive particles in cancer treatment, the timing of hyperthermia relative to drug administration must be examined. When cells were heated to 42°C prior to the addition of free doxorubicin, nuclear drug accumulation increased by 1.8-fold in cancer cells after 5h, and cytotoxicity increased 1.4-fold in both cancer and endothelial cells. Endothelial cytotoxicity was similarly augmented with mild hyperthermia applied prior to treatment with released CuDox. In summary, we find that the drug-metal complex formed in temperature-sensitive particles can be internalized by cancer and endothelial cells resulting in therapeutic efficacy that is similar to free doxorubicin, and this efficacy can be enhanced by elevated temperature.

Previous studies in mice have shown that topical L-selenomethionine (SeMet) can prevent UVB-induced skin cancer when applied continuously before, during, and after the radiation exposure. With topical application of SeMet, selenium levels were shown to increase in the skin and liver, as well as in tumor tissue. Thus, possibly, the timing of SeMet application could affect the degree of inhibition of UVB-tumorigenesis (or maybe even enhance tumorigenesis at some stage). The goal of this research was to determine whether topical SeMet best inhibits UV-induced skin cancer if (a) begun before and continued during and after UVB exposure, (b) if begun before UVB-exposure and discontinued when tumors are first clinically detected, or (c) if begun only after tumors are first detected and continued thereafter. Groups of ten Skh: 1 hairless, non-pigmented mice were treated topically with vehicle lotion, or with SeMet (0.05%) in that vehicle lotion applied either (a) before, during, and after UV exposure, (b) before UV radiation and continued only until the first tumor was detected, or (c) only after the first tumor was detected. In all cases, UV irradiation was discontinued at the time of detection of the first tumor. Optimal inhibition of skin cancer was achieved by application of topical SeMet before, during, and after exposure; significant protection was also attained with application only after the onset of tumors. Notably, statistically significant protection was not seen with SeMet application only prior to tumor detection. These results suggest that even beginning SeMet supplementation late in the process of tumorigenesis can help protect from UV-induced photodamage and skin cancer.

Spatial imaging of cadmium (Cd) in the hyperaccumulator Sedum alfredii was investigated in vivo by laser ablation inductively coupled plasma mass spectrometry and x-ray microfluorescence imaging. Preferential Cd accumulation in the pith and cortex was observed in stems of the Cd hyperaccumulating ecotype (HE), whereas Cd was restricted to the vascular bundles in its contrasting nonhyperaccumulating ecotype. Cd concentrations of up to 15,000 μg g−1 were measured in the pith cells, which was many fold higher than the concentrations in the stem epidermis and vascular bundles in the HE plants. In the leaves of the HE, Cd was mainly localized to the mesophyll and vascular cells rather than the epidermis. The distribution pattern of Cd in both stems and leaves of the HE was very similar to calcium but not zinc, irrespective of Cd exposure levels. Extended x-ray absorption fine structure spectroscopy analysis showed that Cd in the stems and leaves of the HE was mainly associated with oxygen ligands, and a larger proportion (about 70% in leaves and 47% in stems) of Cd was bound with malic acid, which was the major organic acid in the shoots of the plants. These results indicate that a majority of Cd in HE accumulates in the parenchyma cells, especially in stems, and is likely associated with calcium pathways and bound with organic acid (malate), which is indicative of a critical role of vacuolar sequestration of Cd in the HE S. alfredii.

Perturbations in copper (Cu) metabolism are a characteristic of diabetes, for example, elevated plasma Cu and compromised oxidant defense related to diabetes-induced effects on Cu-containing enzymes. Herein, the redistribution of Cu in selected tissues is described in response to diabetic and nondiabetic states in rats that were fed diets adequate in (12 mg Cu/kg of diet) or deficient in (no added Cu) Cu. Diabetes was induced by intravenous administration of streptozotocin (40 mg/kg body weight). After 5 weeks, rats were gavaged with 67Cu (0.74 MBq per rat) using the Cu-deficient diet as a vehicle (suspended 1:3 in water) and killed at various time points. The use of 67Cu allowed for the assessment of short-term Cu distribution and its comparison to the steady-state Cu distribution, as determined by direct Cu analysis. In contrast to control rats, the adaptive mechanisms for Cu homeostasis in diabetic rats were impaired. In general, measures of Cu retention were reduced in diabetic rats compared to corresponding values for control rats. Moreover, diabetic rats had low copper, zinc superoxide dismutase activity that was reduced even further when diabetic rats were fed with low-Cu diets. However, liver and kidney metallothionein and plasma ceruloplasmin levels were elevated in diabetic rats compared to control rats. Such diabetes-related metabolic alterations were taken as measures of increased oxidative stress and inflammation, which may have implications in the progression of diabetes-related pathologies.

The Kuna Amerinds reside chiefly in the San Blas islands (Kuna Yala) on the Caribbean coast of Panama. The diet of this population has not previously been described in detail and composition data for certain foods consumed by this population does not exist or is lacking for certain important nutrients. The protein, fat, moisture, fiber, sugar, mineral and procyanidin content was determined in foods selected because of the frequency with which they are consumed by this population. For that reason, emphasis was given to Tule Masi, a typical Kuna stew-like dish, and cocoa-containing beverages. The foods analyzed were generally low in fat and protein. Potassium and magnesium are present in Tule Masi, common beverages and certain fruits and vegetables at significant levels when considering the preliminary reports on the daily intake of these foods. In addition, preliminary reports indicate that salt use is common, an observation that is supported by the sodium content of the Tule Masi. The cocoa and cocoa beans used in the preparation of beverages are rich in several minerals and procyanidins, as expected. This analysis will allow for the estimation of nutrient intake and subsequent investigations into the relationship between diet and health in this population.

A potentially high bioavailability of arsenic in gold mine tailings from a site in northern California has been suggested by solubility studies. To help address this issue, an in vivo dosing study was conducted using 12‐day‐old Swiss Webster mouse pups (n=8/group). A sample of size‐fractionated mine tailings from the site (<20 μm particle size, 691 μg g−1 arsenic) was prepared as an aqueous suspension and administered by gavage in a volume that provided 4 mg As/kg body weight. The control group received the same volume of a commercial soil (1 μg g−1 As) of similar particle size (<60 μm). No mortality or toxic signs were noted in either group. Tissue samples were collected 1 h after gavage, freeze‐dried, microwave‐digested and analysed for arsenic by ICP/MS (detection limit 2 ng As g−1 dry weight). Arsenic concentrations (ng As g−1 dry weight) in tissues from the pups who received mine tailings were significantly higher than in control tissues. The mean elevation in arsenic concentration was highest in the liver (3364% of control, p<0.0001), followed by blood (818 of control, p<0.0001), skin (207% of control, p=0.07), and brain (143% of control, p<0.0001). The carcass arsenic concentration (excluding the GI tract, liver, brain and skin) was 138 of control (p=0.02). The data indicate uptake of arsenic from weathered mine tailings by the immature mouse pups after oral exposure.

It has been hypothesized that the developmental toxicity of certain compounds is, in part, due to maternal toxicity resulting in alterations in zinc (Zn) metabolism that affects the developing conceptus. In the present work the effects of developmentally toxic doses of 2-ethylhexanoic acid (EHXA), 2-ethylhexanol (EHXO), and valproic acid (VPA) on Zn metabolism were investigated in the pregnant rat. In experiment 1, dams were intubated with EHXA (3.13, 6.25, 9.38 or 12.5 mmol/kg), EHXO (6.25, 9.38 or 12.5 mmol/kg), VPA (1.56, 3.13, 6.25 or 9.38 mmol/kg), or corn oil (control; 1.0 ml/kg) at 14:00 h on gestation day (GD) 11.5, intubated with 32 μCi 65Zn at 22:00 h, and then killed at 08:00 h on GD 12.5. At the higher dose levels of EHXA and EHXO, and at all dosages of VPA, the percentage of 65Zn retained in maternal liver was higher, while that in the embryos was lower, than in controls. Chemical-associated changes in 65Zn distribution were associated with increased maternal liver metallothionein (MT) concentrations. In experiment 2, dams were fed diets containing 1, 25 or 97 μg Zn/g from GD 0–16 and intubated with 3.5 mmol EHXA or 1.0 ml corn oil/kg/d from GD 8–15. Dams were killed on GD 16 or 19. High incidences of encephalocele and tail defects were noted in the GD 16 fetuses of EHXA-treated dams fed either the low or adequate Zn diet, the highest incidences being in the low Zn group. On GD 19 the incidence of tail defects tended to be higher in the EHXA groups than in oil-treated controls, the highest incidence occurring in the low Zn EHXA group. Encephalocele was only observed in the low Zn EHXA-treated group. Fetal weight and crown-rump lengths were decreased by EHXA treatment and low dietary Zn. The incidence of rib anomalies was higher in the EHXA-exposed groups than in their respective oil controls. In experiment 3, GD 10.5 embryos collected from control dams were cultured for 48 h in serum from control or EHXA-treated male rats fed 4.5 or 25.0 μg Zn/g diets. Embryos cultured in either EHXA or low Zn sera exhibited delayed development; the addition of Zn to these sera eliminated their developmental toxicity. These results support the hypothesis that certain chemicals which induce maternal toxicity act, in part, to influence embryonic Zn metabolism and trigger abnormal development. Importantly, the teratogenic effects of these chemicals can be modulated by dietary Zn intake.

BACKGROUND:Little data exist on the requirements of trace metals and minerals for endurance athletes. Changes in body status of these elements must be examined before specific nutritional recommendations can be made. This study was designed to determine whether a marathon run was associated with changes in serum and urine metal and mineral concentrations.METHODS:Forty subjects who planned to complete the 1996 Houston-Tennaco marathon were recruited. Subjects had blood and urine samples collected 2 weeks prior to the race and immediately following the race. Blood and urine specimens were analyzed for copper, iron, magnesium and zinc concentrations. Blood was also analyzed for calcium concentration and ceruloplasmin activity.RESULTS:Twenty-six subjects (24 male, 2 female) completed the marathon. Finish times varied between 2 hours 43 minutes and 5 hours 28 minutes. There was no significant change in serum calcium, copper or zinc concentrations or ceruloplasmin activity. Serum and urine magnesium concentration decreased significantly (19.55+/-1.73 to 16.55+/-1.53 ppm, p=0.00001; 34.02+/-8.64 to 21.80+/-12.24 ppm, p=0.003, respectively). Serum iron concentration increased significantly (1.06+/-0.48 to 1.35+/-0.42 ppm, p=0.006), while urine copper and iron concentrations were below the limits of detection, zinc concentration did not change.CONCLUSIONS:Serum and urinary magnesium concentrations decrease during endurance running, consistent with the possibility of magnesium deficiency. This may be related to increased demand in skeletal muscle. Serum iron concentration increases, possibly related to tissue injury. The exact etiology for these observations, as well as their clinical significance, requires further investigation.