Neutrophils in lung inflammation: Which reactive oxygen species are being measured?

The oxidative burst in circulating polymorphonuclear leukocytes (PMN) plays a fundamental role in pulmonary defense and injury. Flow cytometric techniques have been developed for quantitation of oxidative burst activity at the single cell level using 2',7'-dichlorofluorescin (DCFH). However, the specific reactive oxidant species being measured using this method are not clearly defined. Isolated human PMN were loaded with DCFH diacetate, stimulated with phorbol myristate acetate (PMA) in the presence or absence of specific reagents, and analyzed using flow cytometry. Addition of PMA resulted in a 90-fold increase in the fluorescence intensity of DCFH-loaded neutrophils (p <.01). Inhibition of NADPH oxidase activity using a calmodulin antagonist (W-13) decreased PMA-induced DCFH oxidation by 70% (p <.05). Inhibition of nitric oxide synthase using N(G)-monomethyl-L-arginine (NMMA) did not significantly reduce DCFH oxidation, and did not alter the action of W-13. Addition of superoxide dismutase (SOD) had no effect, but catalase, with or without SOD, suppressed DCFH oxidation by 90% (p <.01). These data suggest that hydrogen peroxide, and not NO, is primarily responsible for the PMA-induced oxidation of DCFH in human PMN under these conditions.

Correlation of ultrasound-induced hemolysis with cavitation detector output in vitro.

A 20-MHz passive acoustic detector was used to quantify the amount of transient acoustic cavitation occurring in a sample exposed to intense pulsed ultrasound. A dilute suspension of human erythrocytes with and without a microbubble echo-contrast agent was exposed in vitro to 500 W/cm2 (SPPA) ultrasound of center frequency 1 MHz and tone burst duration 20, 100, 200, 500 and 1000 microseconds at a pulse repetition frequency of 20 Hz. Inertial cavitation occurring within the sample, as measured by the temporal average of the detector output, correlated well with hemolysis, suggesting that violent bubble collapse is responsible for cell damage. The result also raises the prospect of cavitation monitoring as a possible predictor of adverse bioeffects when echo-contrast agents are used clinically.

Effect of static pressure on acoustic transmittance of Albunex microbubble suspensions.

Albunex (ALX), an albumin-stabilized microbubble echo contrast agent, is sensitive to pressures similar to those produced by the heart. The tested hypothesis was that the acoustic transmittance of ALX suspensions will increase with increasing hydrostatic pressure (Ps). The test involved an acoustic setup analogous to a spectrophotometer. The acoustic transmittance of microbubble suspensions was strongly Ps dependent. Transmittance at 1 MHz was essentially zero at ambient pressure, increasing to approximately 50%, approximately 63%, and nearly 100% at Ps of 80, 120, and 400 mm Hg, respectively. The ultrasound pulses used to interrogate samples were without measurable effect on the acoustic transmittance of suspensions maintained at ambient pressure during experimental measurements. The data indicate that many of the microbubbles are destroyed at Ps comparable to those produced by the heart.

Hemolysis of albunex-supplemented, 40% hematocrit human erythrocytes in vitro by 1-MHz pulsed ultrasound: acoustic pressure and pulse length dependence.

The tested hypothesis was that ultrasound-induced hemolysis in blood supplemented with a microbubble contrast agent varies with ultrasound intensity and pulse duration. Human erythrocytes in autologous plasma containing 3.6% v:v Albunex microspheres were exposed to 1.07-MHz ultrasound pulses of 5 to 1000 mus at SPTP intensities of 0 to 1100 W/cm2. The dependence of hemolysis on the mechanical index (MI) value of the exposures was also examined. Ultrasound-induced hemolysis: (1) was evident at all pulse/intensity combinations; (2) increased generally with increasing pulse duration at constant intensity; and (3) increased with increasing MI at constant pulse duration. For pulses of 10 to 30 mus, ultrasound-induced hemolysis remained low (< or = 2%) at MI values < approximately 2 and increased sharply with further increase in MI; for 5-mus pulses, this abrupt increase in hemolysis was associated with a larger MI (approximately 3).

A test of the hypothesis that a 60-Hz magnetic field affects ornithine decarboxylase activity in mouse L929 cells in vitro.

Four replicate experiments were performed to test the hypothesis that a 4-hr exposure of L929 cells in vitro to a 60 Hz, 10 microT magnetic field results in a large increase in ornithine decarboxylase (ODC) enzyme activity (1-2). A positive control yielded a highly statistically significant increase in ODC activity. However, magnetic field exposure had no statistically significant effect on extractable ODC activity of L929 cells relative to that of sham-exposed cells.

Bubble recycling and ultrasonic cell lysis in a stationary exposure vessel.

An exposure system producing interleafed pulses of 1-MHz ultrasound from balanced, opposing transducers was used to test the bubble recycling hypothesis (as formulated with respect to rotating exposure vessels) in a stationary exposure vessel containing P388 cell suspensions. Cell lysis was measured in cell suspensions which had been exposed or sham-exposed to 1-MHz ultrasound (5 W/cm2 spatial peak pulse average intensity) delivered in continuously alternating, opposing pulses. The opposing pulse cycle frequency (i.e., gating frequency) varied from 0.1 Hz to 100 kHz, corresponding to single pulse durations ranging from 5 microseconds to 5 s. Unequivocal support for the bubble recycling hypothesis was obtained. Lytic yield increased as the gating frequency increased, passing through a maximum at gating frequencies of 100 Hz-1 kHz. As the gating frequency increased further, lytic yield declined rapidly. The results are discussed in relation to recent theoretical estimates of bubble translation speeds in an ultrasound field, and are found to be consistent with those estimates.

On the mechanism of a 60-Hz electric field induced growth reduction of mammalian cells in vitro.

Data on 60-Hz electric field (EF) induced reduction in growth rate of plant roots have strongly supported the hypothesis that the effect is related to an EF-induced transmembrane potential (Vim). An investigation was undertaken to determine if this hypothesis is also applicable to 60-Hz EF-induced reductions in growth rate of mammalian cells in vitro. Human lymphoblastic (RPMI 1788) and human carcinoma (HeLa) cells were selected for study, the former having a relatively small diameter (11.2 microns), and the latter having a relatively large diameter (15.4 microns). The 60-Hz EFs ranged from 430-1200 V/m in the culture medium. The growth rate of RPMI 1788 cells after 4-days was depressed by about 42% at a 60-Hz EF of 1000-1200 V/m with a response threshold occurring at 950 V/m; the Vim at the response threshold was 8 mV. There was no 60-Hz EF-induced effect on HeLa cell growth rate of a Vim of 8 mV (60-Hz EF = 700 V/m); a statistically significant effect was achieved at Vim of 11 mV (950 V/m). The data support the hypothesis that above a threshold 60-Hz EF, Vim acts as the initial signal leading to growth rate reductions.

3H-uridine uptake in human leukemia HL-60 cells exposed to extremely low frequency electromagnetic fields.

Human leukemia HL-60 cells in vitro were exposed or sham-exposed to a 60 Hz electric (3.40 mV/m) or magnetic 10 G (1 muT) field for 5 hr in an attempt to replicate the results of a previously published report (1) of increased uptake of [3H] uridine in response to similar exposure conditions. The results of the present experiments indicated no treatment effect of the fields on uridine incorporation. The ability to detect differences in [3H] uridine uptake were confirmed in a negative control experiment. A 'negative control' experiment demonstrated a statistically significant increase in 3H-uridine uptake for cells at 37 degrees C relative to those at 4 degrees C.

Failure to reproduce increased calcium uptake in human lymphocytes at purported cyclotron resonance exposure conditions.

An attempt was made to verify a report (Liboff et al. 1987) that a unique combination of DC and AC magnetic field exposures at room temperature results in a 3-fold increase in 45Ca2+ uptake by human lymphocytes in vitro. Exposures at "resonance condition", as well as at frequencies and amplitudes above and below the reported effective exposure conditions, were without effect, as were exposures at 37 degrees C. Treatment with ionomycin (0.25 microM), a positive control, resulted in a highly significant increase 45Ca2+ uptake. Some experiments were performed simultaneously by different investigators. Their results did not differ significantly. All experiments were conducted "double blindly".

Lack of induced increase in sister chromatid exchanges in human lymphocytes exposed to in vivo therapeutic ultrasound.

In 1984 Stella et al. reported that each of 10 patients exposed to therapeutic ultrasound had a statistically significant increase in sister chromatid exchanges (SCEs) at mid- and end of therapy; there was a wide range of clinical symptoms (e.g., elbow epicondylitis to knee arthrosis) in this set of patients. In the present study, a set of 4 patients each with some diagnosis recommending therapeutic ultrasound was similarly tested for SCE induction; an additional set of 4 healthy persons underwent sham-therapeutic ultrasound exposures. For both sets of subjects (patients, nonpatients) there was no increase in the frequency of SCEs.

Repetitive pulsed-train "off" duration mitigates reductions in root growth rates of Pisum sativum L. induced by 60-Hz electric field.

An investigation was undertaken to define a 60-Hz electric field exposure system which would affect a eukaryotic cell system while mitigating a potential thermal rise. The biological effectiveness of pulsed 60-Hz electric fields on a cell system of defined sensitivity to continuous-wave 60-Hz electric fields was sought. Roots of garden pea (Pisum sativum L.) were exposed to pulsed trains of 60-Hz, 430 V/m electric fields. The "on" time was constant at 1 s and the "off" time varied. The repetitive on:off regimens used were 1:20, 1:50, 1:100, 1:130, 1:200, and 1:300. With continuous or 1:20 pulsed fields the growth response was equivalent (representing a 60% depression in root growth rate). The severity of the growth effect diminished as the off time increased; for the 1:100 regimen, the relative growth rate was depressed by about 30%; for the 1:300 regimen, the relative growth rate was equal to that of the controls.

Sister chromatid exchanges in Chinese hamster ovary cells exposed to high intensity pulsed ultrasound: inability to confirm previous positive results.

This study was undertaken in an attempt to determine a physical mechanism of action for a recently published report of a small but statistically significant increase in sister chromatid exchanges (SCEs) in Chinese hamster ovary cells exposed to high-intensity pulsed ultrasound. The "positive" report's protocol involved a sizeable chance of ultrasound beam impingement on the side wall of the cell exposure chamber. Ten experiments per regimen were conducted; the regimens included exposures of (a) chamber center, (b) chamber wall, (c) nine grid sites, 0.5 mm between sites, and (d) nine grid sites, 1.5 mm between sites. The last was an exact replication of the conditions previously reported to induce the small SCE effect. The results did not support the postulate of an increase in SCEs with the ultrasound exposures.