Determination of an acute no-observed-adverse-effect level (NOAEL) for copper in water.

A prospective, double-blind controlled study was designed to determine the acute no-observed-adverse-effect level (NOAEL) of nausea in an apparently healthy population of 179 individuals who drank copper-containing water as the sulfate salt. Subjects were recruited at three different international sites and given a blind, randomly selected dose (0, 2, 4, 6, or 8 mg Cu/L) in a bolus of 200 ml (final total copper dose was equivalent to 0, 0.4, 0.8, 1.2, and 1.6 mg) once weekly over a consecutive 5-week period. Gastrointestinal (GI) symptoms of nausea, abdominal pain, vomiting, or diarrhea were screened for a period of up to 24 h. Nausea was the most frequently reported effect and was reported within the first 15 min of ingestion. For the combined trisite population (n=179), 8, 9, 14, 25, and 44 subjects responded positively to one or more GI symptoms at 0, 2, 4, 6, and 8 mg Cu/L, respectively. Analysis of the data demonstrated a clear dose response to the combined positive GI effects and to nausea alone. Statistically significant greater reporting of effects occurred at 6 and 8 mg Cu/L. Therefore, an acute NOAEL and lowest-observed-adverse-effect level of 4 and 6 mg Cu/L (0.8 and 1.2 mg Cu), respectively, were determined in drinking water for a combined international human population.

Uncertainties in the reference dose for methylmercury.

This paper critically examines the National Academy of Sciences and the National Research Council report on the toxicological effects of methyl mercury and the recently published US Environmental Protection Agency Reference Dose (RfD) for Methylmercury. Particular scrutiny is placed on the choice of the critical study and the underlining assumptions utilized in the selection of specific uncertainty factors (UFs) and the rationale for using a less-than-default factor of 10. The UFs that were utilized or considered by other agencies and organizations are also critically examined, explained and compared to one another. Based on these analyses, the authors suggest research that could be performed that would ameliorate the uncertainty of choosing a more precise partial UFor that may even provide completeness of database to allow for selecting of a UF for unity, thus improving the precision of the current published RfD.

Alpha 2-macroglobulin is the major neutralizing inhibitor of influenza A virus in pig serum.

Horse, pig, and rabbit sera contain distinct glycoprotein inhibitors of influenza A viruses that inhibit hemagglutinating activity and neutralize viral infectivity. Although alpha 2-macroglobulin has been identified as the inhibitor in horse serum, the inhibitors in pig and rabbit sera have not been identified. As an initial step in elucidating the structural differences among inhibitor molecules, we sought to isolate the inhibitor in pig serum. The purified inhibitor decreased the hemagglutinating activity of influenza A virus, A/Los Angeles/2/87 (H3N2), and represented the majority of the virus-neutralizing activity in pig serum. The inhibitor corresponded in size to alpha 2-macroglobulin and cross-reacted antigenically with human alpha 2-macroglobulin. Characterization of the inhibitor's oligosaccharide moiety using linkage-specific lectins revealed the presence of N-acetylneuraminic acid-alpha 2,6-galactose but not N-acetylneuraminic acid-alpha 2,3-galactose. These data indicate that alpha 2-macroglobulin is the major neutralizing inhibitor of influenza A virus in pig serum.

Application of Directigen FLU-A for the detection of influenza A virus in human and nonhuman specimens.

Directigen FLU-A, a new enzyme immunoassay membrane test, rapidly detects influenza A virus antigen in specimens from patients. Nasopharyngeal washes and pharyngeal gargles were used to determine the effectiveness of the assay as applied to different types of routinely collected clinical samples. All specimens had been previously shown to contain influenza A virus by virus isolation in tissue culture. Directigen FLU-A was 90% sensitive (95% confidence interval, 56 to 99.7%) with nasopharyngeal washes but only 39% sensitive (95% confidence interval, 17 to 64%) with pharyngeal gargles (P = 0.018) when used with samples containing similar amounts of infectious virus (50% tissue culture infective dose, 1.0 to 4.5). The intensity of the positive reaction with Directigen FLU-A did not correlate with the amount of virus in the specimens. Directigen FLU-A was found to detect cell-associated antigen more readily than free virus; only 20 infected cells were required to identify cell-associated influenza A virus antigen, whereas the limit of detection for free virus was 1.63 x 10(3) infectious virus particles. These findings suggest that Directigen FLU-A detects the cell-associated antigen present in clinical specimens rather than free virus. In addition, Directigen FLU-A detected avian and swine influenza A viruses in both cloacal swabs (75% sensitivity) and swine lung homogenates (86% sensitivity), indicating its potential usefulness in the surveillance of nonhuman influenza A viruses.

Distinct glycoprotein inhibitors of influenza A virus in different animal sera.

Normal horse and guinea pig sera contain the glycoprotein inhibitor alpha 2-macroglobulin, which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. In the current study, the presence of inhibitors of influenza A virus in pig and rabbit sera was investigated. Variants of influenza virus type A/Los Angeles/2/87(H3N2) that were resistant to horse, pig, or rabbit serum were isolated. Analysis of the variant viruses with anti-hemagglutinin (HA) monoclonal antibodies revealed that antigenic changes occurred with the development of serum inhibitor resistance. Characterization of the inhibitors in pig and rabbit sera by using periodate and receptor-destroying enzyme demonstrated that carbohydrate is an important constituent of the active portion of both inhibitor molecules and that sialic acid is involved in the interaction of the inhibitors with influenza virus HA. Nucleotide sequence analysis of the HA molecule revealed that the serum-resistant variants each acquired a different set of amino acid alterations. The multiply resistant variants maintained the original amino acid changes and acquired additional changes. Sequence modifications in the HA involved the conserved amino acids within the receptor binding site (RBS) at position 137 and the second-shell RBS residues at positions 155 and 186. Amino acid changes also occurred within antigenic site A (position 145) and directly behind the receptor binding pocket (position 220). Amino acid alterations resulted in the acquisition of a potential glycosylation site at position 128 and the loss of potential glycosylation sites at positions 246 and 248. The localization of the amino acid changes in HA1 to the region of the RBS supports the concept of serum inhibitors as receptor analogs. The unique set of mutations acquired by the serum inhibitor-resistant variants strongly suggests that horse, pig, and rabbit sera each contain distinct glycoprotein inhibitors of influenza A virus.

Differential effects of selenium on normal and neoplastic canine mammary cells.

Four different canine mammary tumor (CMT) cell lines and a nonneoplastic primary culture of mammary cells were examined for their in vitro responsiveness to selenium supplementation. These cell lines were found to vary in their metabolic response to increasing concentrations of selenium. Sensitivity to selenium, as sodium selenite, increased with increasing concentrations of this trace element in all of the neoplastic lines. These data also suggest that increasing the plating density of tumor cells further increases the sensitivity to selenium. A relatively selenium-sensitive cell line (CMT-13) and relatively insensitive cell line (CMT-11) were characterized on the basis of reduced growth resulting from selenium supplementation. Increasing the concentration of selenium to 0.75 microgram/ml depressed the growth of CMT-13 and CMT-11 cells by 75% and 11%, respectively, while no inhibition was observed in nonneoplastic cells. These cell lines also varied in their sensitivity to different forms of selenium. Selenodiglutathione was the most effective form of selenium examined that inhibited tumor cell growth. The sensitivity of the neoplastic lines was selenodiglutathione much greater than sodium selenite much greater than selenocystine greater than selenomethionine. None of the forms of selenium examined inhibited the growth of the nonneoplastic mammary cells in culture. Supplementation with sodium selenite (1 microgram Se per ml) for 60 min resulted in a dramatic depression in RNA biosynthesis in CMT-13, but not CMT-11 or nonneoplastic cells.

Inhibition of human breast cancer cells by selenium.

The viability of human breast cancer cells (cell lines MCF-7 and MDA-MB 231) was inhibited in vitro in a dose-dependent manner by selenium supplementation. However, a normal diploid human cell line (MRC-5) was relatively resistant to selenium supplementation. The presence of selenium as Na2SeO3 at 1.1 X 10(-6) M reduced cancer cell viability by approximately 50%, whereas non-cancerous cells were not affected. Parenteral administration of sodium selenite also significantly inhibited the growth of the cancerous cell lines transplanted into nude mice. Selenium administration at 0.8 micrograms/g body wt resulted in an 80-93% reduction in the rate of tumor growth without apparent ill effects on the host.

Factors influencing the antitumorigenic properties of selenium in mice.

Sodium selenite (Na2SeO3) was administered at 2.0 micrograms selenium (Se) to Swiss ICR mice six times over a 9-day period by intraperitoneal (i.p.) injection or by gastric gavage. Survival time was significantly increased in Ehrlich ascites tumor (EAT)-bearing mice by 170 and 20%, respectively, compared to controls. In two separate studies, 5.0 micrograms Se as Na2SeO3 or selenodiglutathione (GSSeSG) administered i.p. was more effective in inhibiting EAT propagation in mice than either untreated (control) mice or mice receiving sodium selenide, dimethyl selenide [(CH3)2Se] or seleno-DL-cystine. In another study, EAT cells were preincubated with either 1 or 3 ppm Se as GSSeSG, Na2SeO3, or (CH3)2Se, washed, and reinoculated into mice. Only in mice inoculated with cells pretreated with GSSeSG was a significant increase in survival observed. The observed tumor inhibition was not limited to ascitic tumors since growth of solid Ehrlich tumors was also significantly inhibited by i.p. treatment of Na2SeO3. Following i.p. administration of Na2(75)SeO3, the solid tumors retained more selenium-75 than did blood, lung, kidney, or liver. Supplementation of a torula yeast diet with 2.5 or 5.0 ppm Se as Na2SeO3 also significantly increased the survival time of EAT-bearing mice. These data show that the form and mode of administration of selenium influence the antitumorigenic properties of this trace element. In addition, the data suggest that some intermediate in the normal pathway for selenium detoxification is probably responsible for this trace element's antitumorigenic properties.

The effect of various seleno-compounds on ehrlich ascites tumor cells.

The effect of various concentrations and forms of selenium on in vitro viability of Ehrlich Ascites Tumor Cells (EATC) was investigated. Sodium selenite, selenium dioxide, seleno-DL-cystine, and seleno-DL-methionine, dramatically decreased EATC viability as measured by dye exclusion. Sodium selenate only marginally decreased EATC viability. Cell viabilities decreased with increasing selenium in the incubation media and as a function of time. Viabilities determined by dye exclusion did not correlate with the inhibition of tumor growth observed after treatment with selenium. Intraperitoneal injections of selenite in mice previously inoculated with EATC significantly inhibited tumor development. Delaying intraperitoneal injections of selenite to 5 and 7 days after inoculation of mice with EATC reduced the effectiveness of this nutrient on the inhibition of EATC growth. Incubation of EATC in vitro with supplemental selenium prior to injection of mice completely inhibited EATC development in vivo before any appreciable alteration in cell viability was observed.