Biologic effects of delta-8-estrone sulfate in postmenopausal women.

OBJECTIVE: To determine in postmenopausal women the biological effects of delta-8-estrone sulfate, a novel estrogen component of Premarin (Wyeth-Ayerst, Philadelphia, PA). METHODS: An open-label, nonrandomized study of six healthy postmenopausal women was conducted. Each subject took 0.125 mg of delta-8-estrone sulfate daily for 8 weeks. Blood samples were collected at day 0 (baseline) and once a week for 8 weeks. Urine was collected on day 0 and at weeks 2, 3, 5, 6, 7, and 8. Serum gonadotropins (follicle-stimulating hormone/luteinizing hormone), plasma binding proteins (corticosteroid-binding globulin/sex hormone-binding globulin), a marker for bone turnover (urinary n-telopeptide), and markers for cardiovascular effects (cholesterol, low-density lipoprotein, high-density lipoprotein(c), low-density lipoprotein oxidation, and rate of diene formation) were measured. RESULTS: Follicle-stimulating hormone levels decreased from 84.0 +/- 8.5 to 67.0 +/- 8.5 mlU/mL (P = .02), whereas luteinizing hormone levels did not change. Corticosteroid-binding globulin levels increased from 3.30 +/- 0.16 to 4.10 +/- 0.16 mg/dL (P = .02), and no change in sex hormone-binding globulin was noted. The n-telopeptide levels decreased an average of 31% from 40.7 +/- 4.9 to 28 +/- 7.0 nmol/L bone collagen equivalents/mmol/L creatinine (P = .03). Plasma diene concentration and diene production rate decreased by 34% and 40%, respectively; these changes were not significantly different from baseline values. In contrast, a significant (P = .03) 68% increase in the lag time for low-density lipoprotein(c) oxidation (38.5 +/- 5.5 minutes versus 64.8 +/- 8.5 minutes) was observed. No significant change occurred in total cholesterol, high-density lipoprotein(c), and low-density lipoprotein(c). CONCLUSION: Small doses (0.125 mg) of delta-8-estrone sulfate have profound estrogenic effects in postmenopausal women. The changes observed in n-telopeptide levels and the lag-time delay in oxidation of low-density lipoprotein(c) indicate that this estrogen contributes toward the overall beneficial effects on bone and cardiovascular system associated with Premarin therapy.

Transgenic models for the study of lung antioxidant defense: enhanced manganese-containing superoxide dismutase activity gives partial protection to B6C3 hybrid mice exposed to hyperoxia.

To investigate the role of manganese-containing superoxide dismutase (MnSOD) in lung antioxidant defense, lines of transgenic B6C3 hybrid mice carrying human MnSOD transgenes under the transcriptional control of a human beta-actin promoter were established. Expression studies demonstrated that the human MnSOD transgene in line TgHMS66 is expressed and functional. The cellular distribution of the transgene product in the lungs was further examined by immunocytochemical analysis. Increased immunoreactive MnSOD was found in mitochondria of lung type I epithelial cells, type II epithelial cells, capillary endothelial cells, and fibroblasts. Furthermore, the magnitude of increase in mitochondrial labeling density of type II cells of nontransgenic, hemizygous, and homozygous transgenic littermates was proportional to the increased lung activity of MnSOD found in these mice. Transgenic mice over-expressing MnSOD did not have enhanced survival relative to controls when exposed to > 99% oxygen. However, when exposed to 90% oxygen, the transgenic mice had a small but statistically significant increase in survival time. Our results indicate that when the beta-actin promoter is used to increase activity of MnSOD it provides modest protection to B6C3 mice against hyperoxic lung injury.

Antioxidant enzyme expression in rat lungs during hyperoxia.

To understand the molecular mechanisms that upregulate the activities of pulmonary antioxidant enzymes in adult rats during exposure to 85% oxygen, the relative contents of corresponding mRNA in normal and hyperoxic lungs were determined. Hyperoxic exposure drastically induced the expression of lung manganese-containing superoxide dismutase (MnSOD) mRNA. Maximal induction of MnSOD mRNA occurred at days 3 and 5 of exposure to hyperoxia, reaching a 600 and a 340% increase over the levels of air-exposed rats, respectively. In addition, hyperoxia induced lung mRNA for glucose-6-phosphate dehydrogenase, glutathione peroxidase, glyceraldehyde-3-phosphate dehydrogenase, alpha-tubulin, and gamma-actin to different extends at various days of exposure. Hyperoxia had little or no effect on the levels of mRNA for copper/zinc-containing superoxide dismutase (CuZnSOD), catalase, heat shock protein (HSP70), and creatine kinase. Nuclear run-on experiments showed that the transcriptional rate of the MnSOD gene is enhanced in hyperoxic rat lungs by approximately 400% at day 3 of exposure compared with that of controls. The specific activities of CuZnSOD and MnSOD in these lung samples per unit of lung protein or DNA were also determined. The activity of CuZnSOD in hyperoxic lungs was found to be unchanged compared with controls, except a 20% decrease at day 7 of exposure when standardized against protein content of lung homogenate. Changes of CuZnSOD activity were more dramatic in hyperoxic lungs (a 40% increase at days 3, 5, 7, and 14 of exposure) when enzyme activity was normalized using lung DNA content. Surprisingly, no proportional increase of lung MnSOD enzyme activity was observed at days 3 and 5 of oxygen exposure. The increase of MnSOD activity per unit of lung protein also did not parallel the increase in MnSOD protein content at days 5, 7, and 14 of exposure. These data suggest that, in addition to transcriptional activation, translational and/or posttranslational regulation of the MnSOD gene expression may play a critical role in controlling lung MnSOD activity on hyperoxic exposure.

Detection of Mycobacterium tuberculosis in respiratory specimens by strand displacement amplification of DNA.

A total of 294 clinical respiratory specimens, including 75 with culture-positive results, were tested for the presence of Mycobacterium tuberculosis by strand displacement amplification (SDA) of DNA. A region of the IS6110 insertion element and an internal control sequence were amplified and then detected by a chemiluminescence assay. Receiver operator-characteristic curves were used to evaluate three methods for declaring specimens positive for M. tuberculosis. By the preferred method, SDA chemiluminescence results were converted to theoretical numbers of M. tuberculosis organisms. A positive threshold (PT) value, above which 95% of the SDA results were judged to be M. tuberculosis positive (sensitivity = 95%), was found to be 2.4 M. tuberculosis organisms per SDA reaction. The analogous PT value for 95% sensitivity on smear-positive specimens was 3.6 M. tuberculosis organisms per reaction. The PT of 2.4 M. tuberculosis organisms per reaction detected 100% of culture-positive, smear-positive specimens (sensitivity = 100%), while 95% sensitivity was achieved with a PT of 15.5 M. tuberculosis organisms per reaction. Specificities, which were calculated with respect to culture- and smear-negative specimens, ranged from 96% at a PT of 15.5 M. tuberculosis organisms to 84% at a PT of 2.4 M. tuberculosis organisms per reaction. The M. tuberculosis-negative specimens were also segregated according to whether the patients received antituberculosis chemotherapy. SDA specificity ranged from 90% (PT = 2.4 M. tuberculosis organisms) to 98% (PT = 15.5 M. tuberculosis organisms) for the M. tuberculosis-negative specimens from patients who had not received chemotherapy. SDA specificity in the M. tuberculosis-negative specimens from patients who received chemotherapy was lower (85 to 94%). This study represents the first large-scale demonstration of M. tuberculosis detection in clinical sputum specimens by isothermal DNA amplification with SDA.

Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR.

Two criteria must be met before mycobacterial specimens can be tested by DNA amplification methods: (i) the sample must be rendered noninfectious, and (ii) the organisms must be lysed to free the DNA. Previous publications reporting DNA amplification of mycobacteria have concentrated on lysis and amplification procedures and have not addressed the issue of sample safety. We have shown that heating of samples below 100 degrees C may not consistently kill mycobacteria; however, heating at 100 degrees C in a boiling-water bath or a forced-air oven for a minimum of 5 min kills mycobacteria, including Mycobacterium thermoresistibile. Furthermore, heating at 100 degrees C for 30 min consistently lyses mycobacteria to produce short fragments of DNA that are suitable for amplification by PCR and strand displacement amplification. This procedure works with clinical samples digested by the n-acetyl cysteine-NaOH method as well as with suspensions of organisms in phosphate buffer. This paper also demonstrates the feasibility of using strand displacement amplification with clinical specimens.

Paraquat pharmacokinetics using a subcutaneous toxic low dose in the rat.

The pharmacokinetics of paraquat were examined at a dose which produced lung disease but avoided renal damage. Following single sc injections of 14CH3-paraquat (72 mumols/kg) in male Sprague-Dawley rats, blood was sampled via indwelling jugular cannulas. Noncannulated rats were exsanguinated by cardiac puncture during a 7-day test period. Blood, liver, kidney, lung, brain, heart, spleen, gi tract, injection site, adrenals, body, urine, and feces were analyzed for total radioactivity. Histology of lung after 7 days revealed (+1) paraquat lung disease. No evidence of renal damage was observed. Paraquat was rapidly absorbed. Peak blood concentrations of 58 nmol/ml were measured at 20 min. Peak lung and kidney paraquat concentrations at 40 min were 65 and 359 nmol/g, respectively. Paraquat pharmacokinetics (NONLIN) were best described by a two-compartment open model; the mean biological half-life was 40.9 hr. Eighty-five percent of the dose was eliminated in urine by 7 days. The body contained 79% of the remaining radioactivity. The residual radioactivity is associated with prolonged paraquat excretion and, perhaps, progressive lung disease.

Paraquat-induced, dose-dependent conditioned taste aversions and weight loss mediated by the area postrema.

Paraquat's (PQ) effect on feeding behavior in the rat was examined using a conditioned taste aversion (CTA) paradigm. CTA is a learned avoidance of tastes closely associated with prior illness. Male Sprague-Dawley rats trained to drink an instant breakfast solution were subsequently offered a novel-flavored solution and consumption was measured over 30 min. Following consumption of the novel solution, PQ (0.48-48.0 mumol/kg) was injected subcutaneously. Peak blood PQ concentrations were measured by serially sampling blood (0.15 ml) from an indwelling jugular cannula between 10 and 35 min after injection. Two days later, the rats were offered the same novel-flavored solution. Paraquat produced dose-dependent avoidance of the novel solution when injected subcutaneously. A PQ dosage of 2.7 mumol/kg or less did not alter consumption. The ED50 for CTA production of 13.0 mumol/kg was determined by log-probit analysis. The minimum effective dosage was 4.2 micron/kg. The doses examined did not produce overt clinical or histological signs of toxicity. Peak blood paraquat concentration was linearly related (r = 0.995) to dosage. Additionally when administered by gavage CTAs occurred only with a much larger PQ dosage (480 mumol/kg). Thermal lesions of a hindbrain circumventricular organ, the area postrema (AP), prevented PQ-induced CTAs despite repeated PQ injections. Additionally, weight loss following PQ exposure was also attenuated by AP lesions. CTAs were induced in these same AP-lesioned rats by oral administration of copper sulfate. This substance conditions taste aversions by activating vagal afferent neurons. The fact that copper sulfate-induced aversions were not blocked by lesions of the area posterema indicates that the lesioned rats are capable of forming CTAs when treated with a toxicant which does not act via the AP. These data indicate that PQ produces CTAs in a dose-dependent manner. Furthermore, PQ-induced CTAs and weight loss are mediated by the AP. The AP may contain receptors which detect xenobiotics, enabling animals to avoid future contact with these compounds.

Carbofuran metabolism and toxicity in the rat.

The influence of carbofuran metabolism on acetylcholinesterase inhibition has been defined after low dose (50 micrograms/kg, iv and oral) [carbonyl-14C]carbofuran exposures to male Sprague-Dawley rats. Red blood cell acetylcholinesterase (RBC AchE) inhibition (83% at 2 min, 37% at 15 min for iv and oral, respectively, with recovery by 3 hr), was correlated with carbofuran plasma concentrations (r = 0.97). Eight-hour sample collection indicated that ultimate carbofuran fate (41-47% 14CO2, 14-15% urine, less than 1% feces, and 30-31% carcass) was independent of exposure route. Carbofuran absorption (peak plasma levels less than 7 min), distribution, and elimination (t1/2 = 29 +/- 5 min) occurred rapidly. 3-Hydroxycarbofuran, a significant oxidative metabolite of carbofuran with anticholinesterase activity, was rapidly formed and subject to enterohepatic circulation (plasma t1/2 = 64 +/- 5 min). Results indicated that rapid RBC AchE recovery closely paralleled carbofuran metabolism and the primary in vivo disposition of 3-hydroxycarbofuran was metabolic conjugation.