Assessment of sublethal endpoints for toxicity testing with the nematode Caenorhabditis elegans.

Toxicity tests in invertebrates often use sublethal endpoints, which may exhibit different sensitivity for various toxicants. Our objective was to characterize the sensitivity of movement, feeding, growth, and reproduction as endpoints for heavy metal toxicity testing with Caenorhabditis elegans. Growth and feeding were assessed in the same nematode samples used to assess movement and reproduction. Median effective concentrations (EC50s) for 24-h exposures to Pb, Cu, and Cd were determined for movement, feeding, and growth and a 72-h EC50 was derived for reproduction. The order of toxicity was Cu > Pb > Cd for each endpoint, including lethality and movement. There were no differences in sensitivity among endpoints for any metal. When exposed for 4 h at (sublethal) concentrations that were 14 times the 24-h EC50 value, Pb and Cu reduced feeding to the same extent while movement was reduced significantly more by Pb than by Cu. Thus, a difference in sensitivity of endpoints was apparent at 4 h, which was not evident at 24 h. These observations suggest potentially different mechanisms of toxicity for 24- and 4-h tests.

Effects of dextran sulfate on tracheal mucociliary velocity in dogs.

We have shown that low molecular weight dextran, as a potential mucolytic agent, reduced the viscoelasticity and spinnability of cystic fibrosis (CF) sputum and improved its ciliary transportability in vitro; it also reduced viscoelasticity of healthy dog mucus in in vitro testing. In anesthetized dogs, dextran administered by aerosol at 65 mg/mL increased tracheal mucus velocity, but this increase was not sustained for higher concentrations. The purpose of the present study is to evaluate whether low mol. wt. dextran sulfate, a charged oligosaccharide, exhibits similar effects to previously tested neutral dextran when administered by aerosol to anesthetized dogs in terms of mucus rheology and mucociliary clearance rate. Healthy mongrel dogs were anesthetized with pentobarbital and intubated. Aerosols of Ringer's solution or dextran sulfate (m.w. 5000) dissolved in Ringer's were generated by Pari LC STAR nebulizer, and delivered during 30-min periods of spontaneous breathing. Tracheal transepithelial potential difference (PD, using agar filled electrodes) and tracheal mucociliary velocity (TMV, by charcoal marker particle transport) were measured under bronchoscopic control, and mucus for viscoelasticity analysis by magnetic rheometry was collected by the endotracheal tube method. We performed experiments in seven dogs, involving 30-min administrations of aerosol, separated by 30-min periods of no aerosol. All dogs received inhalations of 6.5 mg/mL, 20 mg/mL, and 65 mg/mL dextran sulfate. Tracheal mucus viscoelasticity (average log G* over 1-100 rad/s) decreased progressively with increasing dose of dextran sulfate; for the highest concentration (65 mg/mL), log G* decreased by a factor of 2.61 (p = 0.021). A modest increase in the TMV was observed for the first dose of dextran sulfate (128% of baseline at 6.5 mg/mL, p = 0.066); thereafter TMV was stable. PD increased significantly at each concentration of dextran sulfate compared with Ringer control; however, there was no additional change between the three groups. The solids content of collected airway fluid (%SC) was gradually increased during successive 30-min dextran sulfate aerosols, indicating a significant residence time for the dextran in the mucus, and correlating with the decrease in viscoelasticity. These results suggest that dextran sulfate may be potentially of therapeutic value as a mucolytic agent, assisting mucus clearance by cough and physiotherapy, although whether it stimulates mucociliary clearance remains to be proven.

Effects of methacholine and uridine 5'-triphosphate on tracheal mucus rheology in mice.

We compared the action of methacholine (MCh) and uridine 5'-triphosphate (UTP) with and without pretreatment with the chloride channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) on the transepithelial potential difference (PD), the mucus collection rate (MCR), and tracheal mucus rheology using anesthetized C57BL/6 mice. The cystic fibrosis transmembrane conductance regulator (CFTR) blocker 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) was also used as a pretreatment for MCh. After collecting baseline mucus for 1.5 h, mucus secretion was stimulated by instilling 5 microl of 10(-2) M MCh or UTP around the upper trachea. There was a significant increase in PD after MCh or UTP stimulation (-21.3+/-2.0 mV MCh versus -14.1+/-1.6 mV control; -25.4+/-2.5 mV UTP versus -19.2+/-1.9 mV control). When UTP administration was preceded by DIDS, PD shifted from -15.2+/-2.9 to -12.0+/-2.2 mV. When MCh was preceded by DIDS or by NPPB, there was no change in PD. There was a significant decrease in mucus rigidity index, logG*, with MCh (2.54+/-0.09 versus 2.99+/-0.14 for control), similar to that previously reported in other species. With UTP, 14 of 16 mice responded in terms of PD becoming more negative, and of these, there was a significant difference in logG* after UTP administration (2.29 +/-0.10 versus 2.57+/-0.10 for control), whereas there was no change in logG* with DIDS administration before UTP. When DIDS administration preceded MCh, there was a diminished but still significant decrease in logG* from control, whereas there was no change in logG* when NPPB was preadministered. The control mucus collection rate was 0.19+/-0.09 mg/h, whereas after MCh stimulation, it increased to 2.83+/-0.78 mg/h. No significant difference was measured in the MCR after either UTP or DIDS+UTP stimulation. DIDS+MCh and NPPB+MCh both resulted in significant increases in MCR, but of a much smaller magnitude than that for MCh alone. We conclude that hypersecretion owing to UTP in C57BL/6 mice is less vigorous than with MCh, reflecting the limited population of Ca(2+)-dependent Cl(-) channels stimulated by UTP P(2) receptors. The action of MCh on tracheal mucus secretion in mice appears to involve both CFTR- and non-CFTR-dependent chloride channels.

Effects of combined treatment with rhDNase and airflow oscillations on spinnability of cystic fibrosis sputum in vitro.

Treatment with either rhDNase or high-frequency oscillation has been shown to be effective in improving the physical and transport properties of airway secretions in cystic fibrosis (CF). The objects of this in vitro study was to examine whether combined treatment with oscillation and rhDNase results in greater change of CF sputum spinnability than either treatment by itself. Aliquots of sputum (0.4 g) from eight CF patients were subjected to the following protocols for 15 minutes and then followed for a total of 30 minutes: 1) incubation with 0.04 ml DNase 50 micrograms rhDNase/normal saline (10% dilution) at 37 degrees C to achieve 5 micrograms DNase/g of sputum final concentration; 2) airflow oscillation at 27 Hz similar to the airflow magnitude produced by a commercial high-frequency chest compression (HFCC) device; 3) negative control with no treatment; 4) positive (dilution) control, incubating with 10% saline by volume; 5) combination of DNase and oscillation, and 6) combination of saline and oscillation. For each protocol, sputum spinnability (in mm, mean +/- SD) was measured by means of a filancemeter at baseline, 15, and 30 minutes. Treatment with DNase decreased spinnability significantly more than either saline or oscillation at 15 and 30 minutes (P < 0.02 and P < 0.04, respectively). Incubation with saline or oscillation of CF sputum for 15 and 30 minutes decreased spinnability significantly compared with control. The combination of DNase and oscillation decreased spinnability significantly more than treatment with DNase alone (3.74 +/- 0.45 vs. 6.54 +/- 0.73 at 15 minutes, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The Denver Sexually Transmitted Diseases Prevention/Training Center: a two-year performance evaluation.

The Venereal Disease Control Division of the Centers for Disease Control (Atlanta, Ga.) has funded the development of nine regional Sexually Transmitted Diseases Prevention/Training Centers. During its first two years of operation, the Denver center offered 17 courses of three types to 158 students. The performance of the Denver Center as to appropriateness of student selection and outcome measures of training was evaluated. The evaluation concentrated on students from the more extensive, two-week Level I course. Comparison of students' written examinations before and after the course demonstrated an average improvement in correct responses of 24.9%. Students rated overall course quality at 3.66 on a four-point scale. Post-course surveys 9.5 months later were completed by 74% of students and 72% of their supervisors. All student respondents take sexual and sexually transmitted disease histories, and 82% routinely prepare specimens from men and women for culture of Neisseria gonorrhoeae. Nine percent of students were no longer employed at STD clinics, but supervisors agreed that for 92% of students, training benefits outweighed lost work time. It is concluded that the perceived needs of Level I students and their supervisors have been met and that data from the survey infer satisfactory patient care in their clinics.

A nuclear magnetic double resonance study of N-beta-bis-(beta'-chloroethyl) phosphonylethyl-DL-phenylalanine.

Studies were carried out on the effect of decoupling, deuterium labeling, concentration, temperature, and solvent media on the NMR parameters of the vinyl phosphonate adduct of phenylalanine, C6H5CH2CH(COO-)NH2+CH2CH2PO(OCH2CH2C1)2. The results permit assignments of chemical shifts and coupling constants to the various protons of this molecule which contains unique structural features. The NH2+-CH2-protons are deshielded by more than 1 ppm than the CH2-PO-protons. The -OCH2-protons are nonequivalent exhibiting a fine split. Possible sources of the fine split include NH...O=P hydrogen bonding. The deuterium-labeling method should be applicable for synthesizing deuterium-and tritium-labeled crosslinked amino acids such as lysinoalanine and lanthionine and demonstrating analgous dehydroalanine-alpha-amino group-crosslinking.