Subchronic oral toxicity and cardiovascular safety pharmacology studies of resveratrol, a naturally occurring polyphenol with cancer preventive activity.

To characterize the subchronic oral toxicity of resveratrol, CD rats received daily gavage doses of 0, 200, 400, or 1000 mg resveratrol/kg/day, and beagle dogs received daily capsule doses of 0, 200, 600, or 1200 mg resveratrol/kg/day for 90 days. Resveratrol induced only minimal toxicity, consisting of dose-related reductions in body weight gain in female rats and both sexes of dogs, and a statistically significant increase in bilirubin levels in rats at the 1000 mg/kg/day dose. Clinical observations, hematology, ophthalmology, neurotoxicity evaluations (functional observational batteries), organ weights, and gross pathology provided no biologically significant evidence of resveratrol toxicity in either species. In rats, the high dose of resveratrol reduced the incidence of cardiomyopathy; no other microscopic changes were seen. Histopathologic changes in dogs were limited to minimal inflammatory infiltrates in the kidney and urinary bladder, which were not considered toxicologically significant. A cardiovascular safety pharmacology (telemetry) study in dogs revealed no evidence of resveratrol toxicity. Based on body weight effects, the No Observed Adverse Effect Level (NOAEL) for resveratrol was 200mg/kg/day in rats and 600 mg/kg/day in dogs. The apparent cardioprotective activity of resveratrol in rats demonstrates that its potentially beneficial activities may extend beyond efficacy in cancer prevention.

Subchronic oral toxicity studies of Se-methylselenocysteine, an organoselenium compound for breast cancer prevention.

Se-methylselenocysteine (MSC) is an organoselenium compound being developed for breast cancer chemoprevention. To characterize MSC toxicity, CD rats received daily gavage doses of 0, 0.5, 1.0, or 2.0 mg/kg/day (0, 3, 6, or 12 mg/m(2)/day), and beagle dogs received daily gavage doses of 0, 0.15, 0.3, or 0.6 mg/kg/day (0, 3, 6, or 12 mg/m(2)/day) for 28 days. In rats, MSC induced dose-related hepatomegaly in both sexes; mild anemia, thrombocytopenia, and elevated liver enzymes were observed in high dose females only. Microscopic pathology included hepatocellular degeneration (high dose males, all doses in females); arrested spermatogenesis (high dose males); and atrophy of corpora lutea (middle and high dose females). In dogs, MSC induced mild anemia in middle and high dose males, and in high dose females. Toxicologically significant microscopic lesions in dogs were seen only in the liver (peliosis and vacuolar degeneration in high dose males, midzonal necrosis in males in all dose groups). Based on liver pathology seen in female rats in all dose groups, the no observed adverse effect level (NOAEL) for MSC in rats is <0.5mg/kg/day. Based on alterations in hematology parameters and liver morphology in male dogs in all dose groups, the NOAEL for MSC in dogs is <0.15 mg/kg/day.

Oncogenicity evaluation of resveratrol in p53(+/-) (p53 knockout) mice.

A six-month study was conducted in p53(+/-) mice to evaluate the possible oncogenicity of resveratrol (3,5,4'-trihydroxy-trans-stilbene), a cancer chemopreventive agent present in grapes and other foods. p53(+/-) mice (25/sex/group) received daily gavage exposure to vehicle only (negative control), resveratrol doses of 1000, 2000, or 4000 mg/kg/day, or p-cresidine (400 mg/kg/day; positive control). No mortality was seen in mice receiving the low dose of resveratrol. However, the mid and high doses induced mortality associated with impaction of the test article in the gastrointestinal tract. Resveratrol had no effect on body weight, food consumption, or clinical signs in surviving mice in any dose group, but induced dose-related increases in liver weight and serum cholesterol in both sexes. Mild anemia was seen in male mice at the high dose only; hematologic effects were not seen in females. Histopathology identified the kidney (hydronephrosis) and urinary bladder (epithelial hyperplasia) as target tissues for resveratrol toxicity. The incidences of both benign and malignant tumors in mice exposed to resveratrol were comparable to those in vehicle controls. By contrast, the positive control article, p-cresidine, induced urinary bladder cancer in both sexes. When administered to p53(+/-) mice at its maximum tolerated dose, resveratrol demonstrates no evidence of oncogenicity.

Ornithine decarboxylase activity in tissues from rats exposed to 60 Hz magnetic fields, including harmonic and transient field characteristics.

Ornithine decarboxylase (ODC) activity is used widely as a biomarker for tumor promotion in animal model systems. Several previous studies have reported increases in ODC activity in tissues of rats exposed to 60 Hz magnetic fields. The goals of this study were to confirm these findings and to determine whether ODC activity is increased in tissues of animals exposed to magnetic fields containing complex metrics. Three experiments were conducted in male F344 rats. Each study included a sham control group and a group exposed to pure continuous 60 Hz fields (0.2 mT). Additional groups included animals exposed to randomly time-varying 60 Hz fields (range of 0.02 to 0.2 mT); intermittent 60 Hz fields (2 mT) with on-off cycles ranging from 5 s to 5 min; pure continuous 180 Hz fields (2 mT); 60 Hz fields with a superimposed 3rd harmonic (total field strength, 2 mT); 60 Hz fields with superimposed third, fifth, and seventh harmonics (total field strength, 2 mT); 60 Hz fields (2 mT) with superimposed transients; and randomly time-varying 60 Hz fields (range of 0.02 to 0.2 mT) with superimposed transients. After 4 weeks of exposure (18.5 h/day), eight animals per group were euthanized within 1 h of magnetic field deactivation. Homogenates of liver, kidneys, spleen, and brain were prepared from each animal, quick-frozen, and shipped for analysis by four independent laboratories. No consistent pattern of differences in the ODC activity among experimental groups was found either within a laboratory or among laboratories. The results do not support the hypothesis that exposure to extremely low frequency magnetic fields stimulates ODC activity.

Magnetic fields and mammary cancer in rodents: a critical review and evaluation of published literature.

Epidemiological data suggesting a possible increase in breast cancer risk in male electricians have raised concerns about the relationship between exposure to power-frequency magnetic fields and breast cancer. In this paper, we review the results of animal studies that are relevant to identifying possible increases in breast cancer risk resulting from exposure to 50 or 60 Hz magnetic fields. Three large-scale chronic bioassays of carcinogenesis in rats or mice exposed to magnetic fields for 2 years demonstrated no increases in the incidence of mammary cancer; it is generally accepted that power-frequency magnetic fields have little or no activity as a complete carcinogen in the rodent mammary gland. Findings from one laboratory, though inconsistent, suggest that magnetic fields may stimulate mammary neoplasia in rats treated with a chemical carcinogen. However, studies conducted in two other laboratories failed to confirm these findings; rats exposed to magnetic fields demonstrated patterns of tumor incidence, multiplicity, size and latency that were generally similar to those in sham-exposed controls. Where differences were seen, the groups exposed to magnetic fields generally had fewer mammary tumors than did sham-exposed controls. On this basis, evaluations of the activity of 50 or 60 Hz magnetic fields in models of multistage mammary cancer in rodents have generally been negative; positive findings have been reported from only one laboratory. The totality of rodent data does not support the hypothesis that power-frequency magnetic-field exposure enhances mammary cancer in rodents, nor does it provide experimental support for possible epidemiological associations between magnetic-field exposure and increased breast cancer risk.

Evaluation of the developmental toxicity of 60 Hz magnetic fields and harmonic frequencies in Sprague-Dawley rats.

Experimental data suggest that exposure to the 50 and 60 Hz sinusoidal components of power-frequency magnetic fields (MFs) does not have an adverse impact on fetal development. However, the possible developmental toxicity of MF harmonics has not been investigated. This study was designed to determine whether exposure to 180 Hz MFs (third harmonic), alone or in combination with 60 Hz MFs, induces birth defects in Sprague-Dawley rats. Groups of sperm-positive dams (> or =20/group) were exposed for 18.5 h per day from gestation days 6 through 19 to (1) ambient MFs only (<0.0001 mT; sham controls); (2) 60 Hz MFs at 0.2 mT; (3) 180 Hz MFs at 0.2 mT; or (4) 60 Hz + 180 Hz MFs (10% third harmonic; total field strength = 0.2 mT). Litter size, litter weight, percentage live births, sex ratio, and number of resorption sites were determined for each dam, and gross external, visceral, cephalic and skeletal examinations were performed on all fetuses. MF exposure had no significant effects on litter size, litter weight, or fetal development. With the exception of common rib variants, the incidence of fetal anomalies was comparable in all groups. A small increase in the incidence of rib variants was seen in the group exposed to 60 Hz + 180 Hz MFs; however, the incidence of rib variants in this group was similar to that in historical controls from our laboratory. These data extend the existing database on developmental toxicity of MFs by demonstrating that exposure to 180 Hz MFs, either alone or superimposed on an underlying 60 Hz signal, does not induce biologically significant developmental toxicity. These data do not support the hypothesis that exposure to power-frequency MFs is an important risk factor for fetal development.

60 Hz magnetic fields do not enhance cell killing by genotoxic chemicals in Ataxia telangiectasia and normal lymphoblastoid cells.

Ataxia telangiectasia (AT) is an inherited autosomal recessive disease characterized by increased risk of cancer, immune deficiency, and neurodegeneration. Cells cultured from AT patients are highly sensitive to genotoxic agents and are deficient in cell cycle arrest after exposure to ionizing radiation. In consideration of their sensitivity to both ionizing and nonionizing radiation, AT cells may provide a sensitive model system to study the biological activity of other components of the electromagnetic spectrum. To characterize the effects of power-frequency (60 Hz) magnetic fields (MFs) in AT cells, we compared responses of AT and normal lymphoblast cells to sinusoidal MFs at 1.0 mT, either alone or in combination with the genotoxic agents mitomycin C or streptonigrin. The MF alone had no effect on cell growth or survival in a clonogenic assay in either AT or normal cells. The MF also had no effect on induction of cell death by mitomycin C or streptonigrin in either cell type. AT cells do not demonstrate differential sensitivity to MF exposure. These results do not support the hypothesis that MFs interact with genotoxic agents to induce adverse biological effects in either normal or genetically susceptible human cells.

Expression of cancer-related genes in human cells exposed to 60 Hz magnetic fields.

Exposure to 60 Hz magnetic fields (MFs) may be a risk factor for human cancer. One mechanism through which MFs could influence neoplastic development is through alterations in the expression of cancer-related genes. Previous molecular studies of the action of MFs have measured effects on a limited number of genes. In the present studies, arrays containing cDNAs for 588 cancer-related genes were used to approach the hypothesis that the biological activity of MFs is mediated by alterations in gene expression. Cultures of normal (HME) and transformed (HBL-100) human mammary epithelial cells and human promyelocytic leukemia (HL60) cells were exposed to MFs at field strengths of 0, 0.01 or 1.0 mT for 24 h. Several genes were identified in MF-exposed cells whose expression was increased by at least twofold or decreased by 50% or more. However, no gene was found to be differentially expressed in each of three independent exposures for any cell type, and no relationship between exposure intensity and differential gene expression was found. These studies failed to identify a plausible genetic target for the action of MFs in human cells, and they provide no support for the hypothesis that MF exposure alters the expression of genes that are involved in cancer development.

Modulation of natural killer cell function after exposure to 60 Hz magnetic fields: confirmation of the effect in mature B6C3F1 mice.

In a previous study, we demonstrated that subchronic exposure to pure, linearly polarized 60 Hz magnetic fields (MFs) at flux densities ranging from 0.002 to 1.0 mT induced a modest but statistically significant and reproducible suppression of NK cell activity in young adult B6C3F(1) mice. NK cell activity in mice declines with age and is known to be suboptimal in older animals. The present study was designed to determine if the same MF exposure regimens will suppress NK cell activity in mature (i.e. more than 1 year old) animals. Extending our previous findings, a modest suppressive effect of MFs on NK cell activity in B6C3F(1) mice was found when subchronic exposure was initiated in animals held in quarantine for 1 year prior to exposure. These data demonstrate that MF exposure suppresses NK cell activity in both young and mature adult B6C3F(1) mice. However, because chronic exposure to the same MF parameters used in the NK function studies does not increase the incidence of neoplasia in B6C3F(1) mice, this statistically significant inhibition of NK cell function appears to be of limited biological significance.

Cell viability and growth in a battery of human breast cancer cell lines exposed to 60 Hz magnetic fields.

Epidemiological data suggest that exposure to power-frequency (50/60 Hz) magnetic fields (MFs) may be a risk factor for breast cancer in humans. To determine whether MFs affect human breast cancer cells, we measured viability, growth and cytotoxicity in a battery of breast cancer cell lines after in vitro MF and sham exposure. Cells of three estrogen receptor-positive human breast cancer cell lines (MCF-7, ZR-75-1 and T-47D) and one estrogen receptor-negative human breast cancer cell line (MDA-MB-231) and normal (nontransformed) human breast epithelial cells were exposed to MFs (1 mT) or sham fields (<0.0001 mT) for 72 h. Cell viability was determined using the sulforhodamine B (SRB) assay at 0 and 72 h after the MF exposure period. Cell growth was measured as the change in SRB dye uptake over 72 h after MF exposure. MF exposure had no effect on cell viability or growth in any cell type examined. Similarly, MF exposure had no effect on cytotoxicity induced by exposure to the retinoid N-(4-hydroxyphenyl)retinamide. These data do not support the hypothesis that MF exposure stimulates growth of breast cancer cells.