Effects of molybdate and pentachlorophenol on the sulfation of acetaminophen.

Pentachlorophenol (PCP) is an inhibitor of phenol-sulfotransferases and has been used to ascertain the role of sulfation in toxicology. Recently, molybdate has been shown to inhibit the sulfation of various chemicals by decreasing hepatic concentrations of the cosubstrate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The purpose of this study was to compare the effectiveness of these two chemicals in inhibiting the sulfation of various doses of acetaminophen (AA) in the rat. PCP (40 micromol/kg) decreased the 2-h combined biliary and urinary excretion of AA-sulfate by 78, 83, 84, and 47% of the 0.1, 0.3, 1, and 3 mmol/kg doses of AA, respectively. Molybdate (7.5 mmol/kg) decreased the sulfation of these same doses of AA by 50, 65, 62, and 81%, respectively. These data indicate that PCP is more effective in decreasing the sulfation of low than high doses of AA, which may result from less AA, at lower doses, to compete with PCP for sulfotransferases. Conversely, molybdate is more effective in decreasing sulfation of high rather than low doses of AA because molybdate decreases sulfate availability and decreases PAPS synthesis. More PAPS is required for the sulfation of high than low doses of AA. Therefore, PCP inhibits sulfation more effectively at low doses of AA when sulfation is limited by sulfotransferases, and molybdate inhibits sulfation more effectively at high doses of AA when sulfation is limited by PAPS.

Effects of molybdate on the sulfation of harmol and alpha-naphthol.

Sulfation requires the activity of sulfotransferases to transfer the sulfuryl group to the substrate from the activated form of sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Inhibition of the sulfate reaction is an important aspect in evaluating the role of sulfation in toxicology. Molybdate decreases hepatic PAPS and its precursor, inorganic sulfate, and could be used as a tool to inhibit the sulfation reaction. The present study was designed to determine the effect of molybdate on the sulfation of two compounds (harmol and alpha-naphthol), for which sulfation is the predominate pathway of biotransformation. Molybdate (7.5 mmol/kg; p.o.) given 4 h prior to the start of harmol infusion (2.5 micromol/kg/min) decreased serum harmol-sulfate concentrations by 32 and 45%, at 45 and 60 min, respectively. This was paralleled by decreases in the cumulative biliary excretion of harmol-sulfate of 37 and 43%, at 45 and 60 min, respectively. Molybdate (5.0 mmol/kg, p.o.) decreased the 24-h cumulative urinary excretion of naphthyl-sulfate by 55% for a 125-micromol/kg i.p. dose of alpha-naphthol, with the greatest decrease (63%) occurring during the first 4 h. These results suggest that molybdate can inhibit the sulfation of compounds that are highly sulfated. Thus, molybdate may prove useful in future studies to examine the pharmacological and toxicological significance of sulfation of xenobiotics.

Effects of molybdate and pentachlorophenol on the sulfation of alpha-naphthol.

Sulfation is the conjugation of chemicals with sulfate, which usually decreases, but occasionally increases, their biological effects. The phenol-sulfotransferase inhibitor pentachlorophenol (PCP) is often used to distinguish the biological effects of a chemical from its sulfate conjugate. Recently, molybdate has been shown to decrease the hepatic concentration of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the cosubstrate for sulfation. Therefore, the present study was designed to compare the effectiveness and specificity of molybdate and PCP as inhibitors of sulfation. Alpha-naphthol (125 and 250 micromol/kg, i.p.) was administered to rats and the sulfate and glucuronide conjugates excreted into urine were quantified for this comparison. Molybdate (5.0, 7.5, and 10 mmol/kg) decreased the 24-h cumulative urinary excretion of the sulfate conjugate of the lower dose of alpha-naphthol by 54, 53, and 55%, respectively, with corresponding compensatory increases in glucuronide excretion at the two lower doses of molybdate. PCP (20, 40, and 80 micromol/kg) similarly decreased the sulfation of alpha-naphthol by 48, 38, and 41%, respectively, but prevented compensatory increases in glucuronide excretion. Molybdate (2.5, 5.0, and 7.5 mmol/kg) decreased the sulfation of the higher dose of alpha-naphthol by 21, 30, and 44%, respectively, again with corresponding compensatory increases in glucuronide excretion. In contrast, PCP did not decrease significantly the sulfation of the higher dose of alpha-naphthol. These data suggest that molybdate is equally or more effective than PCP at inhibiting sulfation of alpha-naphthol, and appears to be more specific.

Telemetric evaluation of body temperature and physical activity as predictors of mortality in a murine model of staphylococcal enterotoxic shock.

BACKGROUND AND PURPOSE: Hypothermia and death are used as experimental markers in murine models of staphylococcal enterotoxic shock. This study determined whether body temperature and physical activity, monitored telemetrically, could predict impending death and provide an earlier, more humane experimental endpoint. METHODS: The study consisted of two iterations (experiments 1 and 2) to determine reproducibility of the model. Each experiment consisted of 24 BALB/c mice surgically implanted with intra-abdominal telemetry transmitters and then injected intraperitoneally with sublethal or lethal doses of staphylococcal enterotoxin B (SEB) and/or lipopolysaccharide (LPS). Core body temperature and physical activity were continuously monitored in all mice for 10 days before, and 5 days after, injections. Additionally, in experiment 2, subcutaneous temperatures were compared with core body temperatures obtained by telemetry. RESULTS: Body temperature and physical activity were reduced in mice after administration of SEB and LPS, or LPS alone, but not SEB only. There was a significant (P < 0.05) correlation between mortality and body temperature (P = 0.0077), but not physical activity (P = 0.97). CONCLUSION: Body temperature proved to be an early indicator of mortality in this murine model of staphylococcal enterotoxic shock.

Sulfation and sulfotransferases 5: the importance of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the regulation of sulfation.

Sulfation is the transfer of a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a substrate that is catalyzed by a family of sulfotransferase enzymes. Many different endogenous and xenobiotic molecules are substrates for the sulfotransferases; sulfation affects many different physiological processes, including: 1) deactivation and bioactivation of xenobiotics, 2) inactivation of hormones and catecholamines, 3) structure and function of macromolecules, and 4) elimination of end products of catabolism. PAPS is the obligate cosubstrate that is synthesized in tissues to make available an "activated form" of sulfate for the sulfation reaction. PAPS participation in the reaction is dependent on its availability, which in turn is dependent on its synthesis, degradation, and ultimately its utilization in the sulfation reaction itself. PAPS synthesis is dependent on the availability of sulfate and on the activity of the two enzymes of its synthesis, ATP-sulfurylase and APS-kinase. Although the kinetic properties of these two enzymes are well described, their in vivo regulation is not fully understood. Sulfation is a high-affinity, low-capacity enzymatic process in which the entire liver content of PAPS can be consumed in less than 2 min. ATP-sulfurylase and APS-kinase can rapidly synthesize additional PAPS. The low capacity of sulfation in rats is due to the limited availability of sulfate, whereas in mice the sulfotransferases appear to limit sulfation capacity. Sulfation rates are not readily enhanced, but they can be decreased. 2,6-Dichloro-4-nitrophenol inhibits phenolsulfotransferases, but not hydroxysteroid-sulfotransferases. However, the sulfation of phenols and hydroxysteroids can be decreased by factors that decrease sulfate availability such as a low-sulfate diet, other xenobiotics that are sulfated, and molybdate, which inhibits sulfate intestinal absorption, renal reabsorption, and sulfate incorporation into PAPS.

Effects of molybdate and pentachlorophenol on the sulfation of dehydroepiandrosterone.

Pentachlorophenol (PCP) and molybdate have been shown to inhibit the sulfoconjugation of various chemicals in rats and therefore are useful to examine the role of sulfoconjugation on the toxicity of a chemical. PCP inhibits sulfation by competing with substrates for phenol-sulfotransferases, but not hydroxysteroid-sulfotransferases. In contrast, molybdate decreases sulfation by limiting sulfate availability and thereby decreasing the synthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which is the obligate cosubstrate for sulfation. Therefore, it was of interest to determine whether PCP or molybdate is effective in decreasing the in vivo sulfation of dehydroepiandrosterone (DHEA), which is a substrate for hydroxysteroid-sulfotransferases. PCP (40 micromol/kg ip) or molybdate (7.5 mmol/kg po) was given 45 min and 4 h, respectively, prior to the start of DHEA infusion. The effects of these two sulfation inhibitors on DHEA sulfation were dependent on the rate of DHEA infusion in rats. PCP had different effects on the sulfation of various infusion rates of DHEA in rats. PCP had little effect on the sulfation after the two lowest infusion rates of DHEA (12.5 and 25 mg/kg) and actually increased (233%) DHEA-sulfate serum concentrations with the highest DHEA infusion rate (50 mg/kg). Although molybdate had little affect on the sulfation of the lowest DHEA infusion rate, it significantly decreased (50-85%) DHEA-sulfate serum concentrations with the two higher DHEA infusion rates. These data indicate that molybdate, unlike PCP, decreases the sulfation of DHEA and may be a useful tool to decrease the sulfation of other substrates of hydroxysteroid-sulfotransferases.

Mucosal vaccination with recombinantly attenuated staphylococcal enterotoxin B and protection in a murine model.

Previous work in our laboratory revealed that mice parenterally vaccinated with recombinantly attenuated staphylococcal enterotoxin (SE) or toxic shock syndrome toxin 1 develop protective antibodies against a lethal intraperitoneal (i.p.) toxin challenge. This study investigated the efficacy of nasal and oral immunizations with an SEB vaccine (SEBv) toward an i.p. or mucosal (via an aerosol) toxin challenge. Both vaccination routes, with the immunoadjuvant cholera toxin (CT), elicited comparable SEB-specific immunoglobulin A (IgA) and IgG levels in saliva. Nasal or oral inoculations also generated SEB-specific IgA, IgG, and IgM in the serum, but the nasal route yielded higher specific IgG titers. SEBv alone, when given nasally or orally, did not induce any detectable SEB-specific antibody. Mice vaccinated mucosally were protected against a 50% lethal dose of wild-type SEB given i.p. or mucosally, thus demonstrating that nasal or oral administration of this SEBv, with CT, elicits systemic and mucosal antibodies to SEB that protect against SEB-induced lethal shock.

Efficacies of BCG and vole bacillus (Mycobacterium microti) vaccines in preventing clinically apparent pulmonary tuberculosis in rabbits: a preliminary report.

Tuberculosis (TB) kills more people in the world today than any other infectious disease, and the number of drug-resistant Mycobacterium tuberculosis isolates is increasing. Vaccines, better than most of the currently available strains of bacille Calmette-Guérin (BCG), are urgently needed to control this disease. TB in rabbits resembles human TB more closely than TB in any other common laboratory animal and a most pertinent method of assessing vaccine efficacy is Lurie's tubercle count method in this species. Vaccinated and control rabbits were infected by aerosol with virulent human-type tubercle bacilli (H37Rv). At necropsy 5 weeks thereafter, the grossly visible primary tubercles in the entire lung were counted. A decrease in the number of such tubercles is a quantitative measure of vaccine efficacy: An effective vaccine prevents microscopic tubercles from growing to grossly visible (clinically apparent) size. The Pasteur substrain of BCG and two substrains of Mycobacterium microti (the vole bacillus) reduced the number of visible primary tubercles an average of 75%, whereas three other substrains of BCG and three other substrains of vole bacilli only reduced the number an average of 40%. These initial studies indicate that Lurie's tubercle-count method in rabbits is a precise way to choose the best available tuberculosis vaccines.

Virulence of Mycobacterium tuberculosis CDC1551 and H37Rv in rabbits evaluated by Lurie's pulmonary tubercle count method.

The virulence of the CDC1551 strain of Mycobacterium tuberculosis was compared to that of H37Rv in a rabbit inhalation model. While rabbits that inhaled the two strains produced equal numbers of grossly visible primary tubercles, CDC1551 tubercles were smaller and contained fewer bacilli than H37Rv tubercles. These findings suggest that a miniepidemic near the Kentucky-Tennessee border caused by CDC1551 was due not to increased virulence but to increased transmissibility.