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This article addresses the issue of hyperthermia-induced birth defects with an accompanying additional teratogen, be it a chemical or a physical agent (i.e., a simultaneous “combinational” exposure to two teratogens, one of which is hyperthermia). Hyperthermia per se is a recognized human and animal teratogen. An excellent example of such combinational exposures is an epileptic woman who becomes pregnant while taking valproic acid (VPA) to control seizures. VPA is a recognized chemical teratogen, and fever (hyperthermia) is not an uncommon event during pregnancy. While VPA also may occasionally induce fever as a side effect, we are concerned here with fevers arising from other, unrelated causes. There is a small but internally consistent literature on these combinational-teratogen exposures involving hyperthermia plus a chemical teratogen; in each instance, the effect level has been observed to be synergistically elevated above levels induced by the separate teratogenic components. The data were empirical. The observed synergy is, however, consistent with Arrhenius thermodynamics, a well-known chemical rate equation. The need for information about combinational teratogen exposures is acute; fever is a common occurrence during pregnancy; and there are many instances whereby there is also the simultaneous presence of some other teratogen(s). Given that the rate of autism spectrum disorders in the United States was recently presented as 1 in 88 births, it seems reasonable to suspect that such combinational regimens are much more prevalent than previously thought. Our hypothesis is that synergistic birth defect levels from combinational regimens are consistent with Arrhenius thermodynamics. Birth Defects Research (Part C) 99:50–60, 2013. © 2013 Wiley Periodicals, Inc.

There are a number of seemingly "usual" thermal episodes during pregnancy for which it is relatively easy to determine a rudimentary aspect of thermal dose; these episodes include fever, labor, labor plus epidural, and the normally-occurring 0.5 degrees C temperature elevation above maternal core temperature of the fetus during the entirety of the third trimester. Complications can involve, for instance, fever during the third trimester. We consider the thermal doses of five different but "usual" or "normal" hyperthermic episodes during human pregnancy and compare those doses with the thermal doses involved with both single and cohort exposures of pregnant guinea pigs throughout their gestational period. The end-point studied in the guinea pigs was microencephaly. In nine of the 10 comparisons (human fetal thermal dose vs. guinea pig fetal thermal dose) the human dose is substantially larger than that of the guinea pig thermal dose, which was substantially teratogenic. This situation is essentially the inverse of the type of information contained in the Physician's Desk Reference (PDR) on drugs, in which it is not unusual to discern that at high drug levels there may be teratogenic effects in laboratory animals, but such effects were not observed at "clinical" drug levels in animals or subsequent clinical trials. With hyperthermic events, however, it appears that the teratogenically-effective thermal dose levels associated with animal testing are quite low relative to those thermal doses associated with relatively "normal" obstetric observations during a pregnancy.

Biomedical ultrasound may induce adverse effects in patients by either thermal or non-thermal means. Temperatures above normal can adversely affect biological systems, but effects also may be produced without significant heating. Thermally induced teratogenesis has been demonstrated in many animal species as well as in a few controlled studies in humans. Various maximum ‘safe’ temperature elevations have been proposed, although the suggested values range from 0.0 to 2.5° C. Factors relevant to thermal effects are considered, including the nature of the acoustic field in situ, the state of perfusion of the embryo/fetus, and the variation of sensitivity to thermal insult with gestational stage of development. Non-thermal mechanisms of action considered include acoustic cavitation, radiation force, and acoustic streaming. While cavitation can be quite destructive, it is extremely unlikely in the absence of stabilized gas bodies, and although the remaining mechanisms may occur in utero, they have not been shown to induce adverse effects. For example, pulsed, diagnostic ultrasound can increase fetal activity during exposure, apparently due to stimulation of auditory perception by radiation forces on the fetal head or auditory structures. In contrast, pulsed ultrasound also produces vascular damage near developing bone in the late-gestation mouse, but by a unknown mechanism and at levels above current US FDA output limits. It is concluded that: (1) thermal rather than nonthermal mechanisms are more likely to induce adverse effects in utero, and (2) while the probability of an adverse thermal event is usually small, under some conditions it can be disturbingly high.

This paper concerns the rate of birth defects that is induced by hyperthermic conditions during gestation. These defects occur with low rates under apparently normal physiologic conditions, but with substantially higher rates with appropriate timing of a hyperthermic event during gestation. The concept of “free energy of activation” is invoked to explain not only the background rate but also the effect of a short-term, acute (additive) thermal dosing at a highly heat-sensitive stage of embryogenesis. In this respect, this paper is both retrospective and prospective in approach. It briefly and retrospectively addresses the historical data in this area and then, following a novel mathematical procedure whereby the activation energy for a specific type of hyperthermia-induced birth defect is determined, prospectively applies this information to predict what the outcomes would be in a clinical situation such as obstetric diagnostic ultrasound where it is known that the clinical procedure can involve a temperature increment for a specific duration. A major outcome of this review is that there appear to be no thresholds for hyperthermic events, that any temperature elevation for any duration during pregnancy has some potential for inducing a deleterious effect. The paper includes an analysis that for the first time allows a calculation of risk for such defined thermal insults, with broader implications for any natural or other clinical situation in which the temperature of the embryo or fetus is raised above the normal physiologic level. The topic of hyperthermia-induced birth defects and related activation energies needs thorough, long-term testing of this prospective position.

The results of this project provide proof of concept for an analysis of hyperthermia-induced in vitro cellular effects in relation to a thermal dose determination with a temperature-differential basis from each cell line's historical physiological temperature (HPT). Human ( Homo sapiens ) fetal cells, whose HPT was assumed to be 37°C, and guinea pig ( Cavia porcellus ) fetal cells, whose HPT was assumed to be 39.5°C, were used. It was hypothesized that in vitro cellular functionality (e.g. , fastest cell doubling time) would be maximal at each line's respective HPT and decrease with temperatures above and below each line's HPT. The hypothesis was supported.

This project was an initial test of the hypothesis that there would be a Δ T thermal dose relationship for birth defects in rats ( Rattus norvegicus ) during neural tube closure (NTC). Additionally, the same thermal dose as applied during NTC in utero in vivo, was also applied to exteriorized (i.e., ex vivo) in utero pregnant uterine horns at a comparable stage of organogenesis. Since the yields of the two regimens were comparable, the hyperthermia-induced teratogenic effects appear to result from the thermal dosing of the in utero embryos and not the elevated temperature of the mother. The hypothesis was supported.

This commentary addresses the matter of misinterpretation of thermal dose as discussed by Herman and Harris (2002), and shows that the thermal doses they would consider as ineffective ( i.e., “safe”) for producing a hyperthermia-induced teratologic effect, can be highly effective ones. The matter of whether or not thermal thresholds exist for teratologic effects is reviewed. There are only opinions about thresholds. The critical experiments for ascertaining whether or not a threshold exists have not been undertaken. A power computation illustrates the requisite sample sizes (litters, fetuses) for undertaking an experimental test of whether the hyperthermic teratologic response in rats is characterized by threshold or simple linearity kinetics. (E-mail: Morton_Miller@urmc.rochester.edu)

This project continued testing of the general working hypothesis that cell size is a physical determinant in extent of ultrasound (US)-induced hemolysis, the larger the cell the greater the lysis. For this project, the specific hypothesis tested was that human erythrocytes, being larger than rabbit erythrocytes, would be the more sensitive to sonolysis induced by inertial cavitation in the presence of Albunex, a US contrast agent. The rationale behind this hypothesis was 1. an earlier-published analytic construct indicating an inverse relation between particle size and the shear force required for deformation, and 2. a number of independent demonstrations that, among sized populations of erythrocytes, an inverse relation exists between erythrocyte volume and mechanically-induced shear forces in the cell-bathing medium; namely, the larger the cell, the less shear force required to rupture the cell's membrane. The present data support the hypothesis; over six independent trials, the mean corpuscular volumes of human (H) and rabbit (R) erythrocytes were 89.5 and 64.1 microm(3), respectively, H > R (p << 0.001), and the ratio of US-induced hemolysis in H to R blood in vitro was 1.12:1.0 (p < 0.004).

The data collected in this project supported the a priori hypothesis that the concentration of dissolved oxygen in whole human blood in vitro affected the extent of ultrasound (US)-induced hemolysis under conditions conducive to the occurrence of inertial cavitation. Aliquots of whole human blood in vitro with a relatively high O 2 level had statistically significantly more 1-MHz US-induced hemolysis than aliquots with a relatively low O 2 level in the presence of controlled gas nucleation (Albunex® or ALX, supplementation), with US-induced hemolytic yields being substantially less at 2.2- and 3.5-MHz exposures or in the absence of ALX-supplementation at otherwise comparable acoustic pressures, pulse lengths and duty factors. Passive cavitation detection (pcd) measures indicated a linear relationship for hemolysis up to about 70% and pcd values (R 2 = 0.99). (E-mail: Morton_Miller@urmc.rochester.edu)

This project tested the hypothesis that a "second-generation" ultrasound (US) contrast agent (Optison), offering extended echogenicity over that of its "first-generation" predecessor (Albunex), would have the greater potential for sonolysis of human erythrocytes in vitro. Whole human blood, obtained from apparently healthy donors, was anticoagulated and subsequently exposed in vitro to US in the presence of one of each or neither of the two US contrast agents. The US exposures were for 30 s and involved frequency (1.0, 2.2 and 3.4 MHz) and amplitude (approximately 2.8 to 0.38 MPa P(-)) regimens; pulse duration (200 micros) and interpulse interval (20 ms) were held constant. The data supported the hypothesis, with an overall ratio of approximately 2.5 for relative extent of background-corrected US-induced hemolysis of the Optison/Albunex regimens. Passive cavitation detection analyses corroborated the results obtained with hemolysis.

Human fetal and adult erythrocytes differ significantly in mean corpuscular volume (MCV), the fetal cells being larger than adult cells and diminishing in MCV as gestational age (GA) increases. Previous studies have shown that the sensitivity of erythrocytes from different species to lysis by mechanically applied shear stress increases as MCV increases. The tested hypotheses in the present project were: 1. fetal erythrocytes would be more sensitive to sonolysis than adult erythrocytes because of the former's larger size, and 2. erythrocyte sonolytic sensitivity would scale with MCV. Fetal and adult erythrocytes were resuspended to 40% hematocrit in oxygenated isotonic saline solution and 500 microL aliquots were exposed for 60 s to 200 micros bursts of 1-MHz ultrasound (US) (peak pressures: approximately 4.8 MPa positive, approximately 2.7 MPa negative; duty factor = 0.01), either with or without 3.6 volume % Albunex (ALX) present. Background-corrected hemolysis was indistinguishable from zero in sham-exposed fetal or adult erythrocyte suspensions. Without ALX, mean background-corrected US-induced hemolysis was significantly greater than zero for fetal and adult cells (0.42 +/- 0.15% vs. 0.62 +/- 0.15), but fetal cell lysis was not significantly greater than adult cell lysis. With ALX, US-induced hemolytic yields increased approximately 80-fold (fetal: 50.53 +/- 2.14; adult: 46.40 +/- 1.85%), and were significantly higher for fetal than for adult cells. There was also a statistically significant correlation between MCV and US-induced background-corrected hemolysis. Thus, the two hypotheses were supported.

This project tested the hypothesis that human erythrocytes, being larger than bovine erythrocytes, would be the more sensitive to sonolysis induced by inertial cavitation. The rationale behind this hypothesis was an earlier demonstration that, among sized populations of erythrocytes, an inverse relation existed between erythrocyte volume and mechanically-induced shear forces in the surrounding medium; viz, the larger the cell, the less shear force required to rupture the cell's membrane. At low erythrocyte densities (i.e., approximately 5% hematocrit) the hypothesis was supported; at high cell densities (i.e., approximately 35% hematocrit) it was not supported. The data are consistent with an ultrasound (US)-induced symmetric implosion of affected gas nuclei as causing the effect at low cell densities; under such conditions there is ample spacing among cells for US-induced symmetric growth and collapse of gas nuclei and the concomitant production of radially-expanding shock waves (which lyse the cells); at high cell densities there is not sufficient spacing among cells for US-induced symmetric growth and collapse of bubbles and an alternative mechanism, possibly asymmetric bubble collapse, becomes operational.

Phosphate buffered saline (PBS) was insonated or sham-insonated (1 MHz, 35 W/cm2, continuous wave, 30 min) in rotating (200 rpm) sterile polystyrene culture tubes. After treatment, the PBS was used immediately to suspend washed Chinese hamster V79 cells in vitro. Cells were incubated in the PBS at 37 degrees C for 15 min and then transferred to complete growth medium. Some insonation regimens also involved the inclusion of Albunex (ALX; an ultrasound microbubble contrast agent) to enhance ultrasound-induced inertial cavitation. Following exposure to the pretreated PBS and 6 d of subculture in complete medium, the cells were assayed for plating efficiencies and mutation frequencies (resistance to 6-thioguanine). X-rays (3 Gy) served as a positive control. Cells exposed to insonated PBS with or without ALX or x-rays had statistically significantly elevated mean mutation frequencies (4.37+/-0.97, 4.54+/-1.00, and 24.28+/-3.83 mutant colonies/10(6) viable cells, respectively) relative to corresponding control regimens (ultrasound sham, 2.44+/-0.56; x-ray sham, 2.96+/-0.88 mutant colonies/10(6) viable cells. The data supported the hypothesis that sonochemicals resulting from inertial cavitation have mutagenic potential.

We address the issue of health and safety in relation to exposure to diagnostic ultrasound, with particular emphasis given to obstetrics. In terms of fetal and maternal outcomes, the epidemiologic record of diagnostic ultrasound is exemplary but is primarily made on the basis of data derived from clinical devices whose outputs were relatively low compared with what is now allowable and available. The power outputs of clinical devices have been increasing over the past decade such that the potential for thermal and nonthermal insults is increased. For obstetric devices that use these higher outputs, the Food and Drug Administration now requires the presentation of 2 on-screen indexes, the thermal index and the mechanical index, in recognition of the 2 major mechanisms by which ultrasonography is known to affect cells and tissues. Greater responsibility for patient safety is now placed on the diagnostician; for the new indexes to be meaningful the diagnostician must be cognizant of the health and safety implications. The purpose of this article is to provide some guidance to the obstetrician in interpreting the indexes and to review the current status of ultrasonography biophysics in relation to the use of diagnostic ultrasound in obstetrics.

This brief review addresses the issue of health and safety from exposure to diagnostic ultrasound. The exemplary historical record of diagnostic ultrasound exposures is coupled with great patient benefit. However, the power outputs of clinical devices have been increasing over the past decade such that inertial cavitation seems reasonably likely to occur if appropriate gas nuclei are present. The use of microbubble contrast agents for certain diagnostic procedures ensures a well nucleated system. Under such conditions, the use of low signal output levels and short exam times will decrease the chance of cavitation related bioeffects.

Many studies of ultrasonic hemolysis have used erythrocytes; other blood cells are less well studied. The hypothesis tested was that human lymphocytes, being large and relatively fragile, are more sensitive to sonolysis than are erythrocytes at equivalent cell concentrations. Human lymphocytes (RPMI 1788) grown in vitro and erythrocytes obtained by venipuncture were used at a nominal cell concentration of 2.5 × 10 6 cells/mL. Cells were contained in rotated (200 rpm) exposure vessels and were exposed/sham-exposed to 1-MHz continuous-wave ultrasound for 60 s. Cell lysis was determined by hemacytometer counts of aliquots taken before/after treatment. The hypothesis was supported; the mean levels of lysis in insonated lymphocyte and erythrocyte preparations were 89.8% ± 0.6% and 78.9% ± 3.1%, respectively. This difference was significant at p

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.

Diminution of apparent ultrasonic cell lysis in vitro with increasing cell concentration/volume fraction has often been observed. A substantial fraction of the cells may be lysed by ultrasound at low cell concentrations, but only a few percent are lysed when the concentration is a fraction of that of whole blood. This suggests that sonolysis of cells in vitro is suppressed by high cell concentrations, and, therefore, that sonolysis of cells in vivo is unlikely. We present the results of a retrospective analysis of experimental data and a theoretical analysis; these indicate that while relative sonolytic yield declines with increasing cell concentration, the “absolute” extent of ultrasound-induced lysis remains large. We find evidence that in vitro sonolysis of cells is limited at high cell densities by the number of available microbubbles and/or the number of cells each bubble may encounter and lyse prior to bubble “inactivation.” Theory indicates the latter arises in consequence to the cell concentration-dependent formation of cell aggregates around pulsating bubbles.

To test the hypothesis that the full extent of in vitro cell lysis due to ultrasound becomes evident with time lapse following insonation, human erythrocytes (2% hematocrit) in autologous plasma were mixed with Albunex(R), a pulse echo contrast agent, and exposed to 1-MHz, continuous-wave ultrasound (US) (5 W/cm(2) SPTA intensity) for 60 seconds while in a rotating (200 rpm) dialysis membrane vessel. Exposed and sham-exposed samples were subsequently assayed for hemolysis colorimetrically, either immediately or after a delay of 3 hours. Hemolysis was dependent on the interval between US exposure and assay, with significantly greater lysis evident with delayed assay. There was also temporality in lytic yield with sample number, i.e., with time postpreparation of the blood sample, US-induced cell lysis decreased. The temporality of lytic yield was eliminated by maintenance at ice water temperatures, or by waiting about 1 hour before beginning treatments. The collective data indicate that the full extent of US-induced cell lysis is not evident upon assay immediately after insonation, and that with time postpreparation and preinsonation, erythrocytes may undergo changes in sensitivity to US. (ECHOCARDIOGRAPHY, Volume 13, January 1996)

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 valuesapproximately 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).

Human red blood cells (RBCs) in vitro at various fractional hematocrits (HCTs) were exposed for 60–120 seconds in a dialysis tubing vessel to 1 MHz continuous wave ultrasound (0–5 W/cm2 SPTA intensity); exposure vessels were either rotated at 200 rpm or stationary. Some RBC suspensions also contained Albunex® (ALX; a commercially-produced microbubble clinical ultrasound contrast agent) at final concentrations ranging from 0–41 μL/mL RBC suspension. Isonation was either by one transducer or by two opposing, continuously-gated, balanced transducers. For the vessel rotation / no rotation experiments, ultrasound-induced cell lysis was always increased with the ALX regimen relative to that of the no-ALX regimen at HCTs up to about 10%; at higher HCTs (including whole blood), ultrasound-induced hemol-ysis essectially ceased to occur. The data are consistent with reports of the ineffectiveness of continuous wave ultrasound at 3–5 W/cm2 to lyse cells in vitro at physiological cell densities with or without echocontrast medium supplemantation.

Microbubble contrast agents have been shown to enhance ultrasonic cell lysis in vitro when exposed to continuous-wave ultrasound having spatial peak temporal average (SPTA) intensities of a few W/cm2. The response is strongly dependent upon the hematocrit (HCT) of the cell sample; detectable cell lysis essentially disappears as the HCT approaches 5%-10%. This study was conducted to determine whether high intensity pulsedsound is an effective lytic agent in the presence of preexisting potential cavitation nuclei (Albunex® contrast agent). Human erythrocytes weresuspended in autologous plasma to HCTs ranging from 1%–40%. Suspensions were exposed or sham exposed for 60 seconds to focused, pulsed ultrasound. The pulse duration was 1 msec, and the pulse repetition frequency was 20 Hz. The pressure amplitudes, spatial peak pulse average (SPPA) intensity, and SPTA intensity were 4.7 MPa peak positive pressure, -2.7 MPa peak negative pressure, 420 W/cm2, and 8.5 /cm2, respectively. Samples were exposed to ultrasound in a dialysis membrane exposure vessel rotating at 200 rpm. When included in the erythrocyte samples, the Albunex concentration was 35 μL/mL suspension. Significant ultrasound-induced hemolysis in the absence of Albunex was observed only at the lowest HCT value tested (1%). In the presence of Albunex significant cell lysis was observed at all tested HCT values. The relative fraction of cells lysed by the combination of ultrasound exposure and Albunex diminished with increasing HCT, but the number of cells lysed per sample was nearly constant over the range of 5%–40% HCT. The ultrasound exposure parameters used in this study differ substantially from those associated with diagnostic imaging equipment; it is not valid to infer from the present results that the use of Albunex in diagnostic applications will induce or enhance hemolysis in vivo.

This article addresses the issue of hyperthermia-induced birth defects with an accompanying additional teratogen, be it a chemical or a physical agent (i.e., a simultaneous "combinational" exposure to two teratogens, one of which is hyperthermia). Hyperthermia per se is a recognized human and animal teratogen. An excellent example of such combinational exposures is an epileptic woman who becomes pregnant while taking valproic acid (VPA) to control seizures. VPA is a recognized chemical teratogen, and fever (hyperthermia) is not an uncommon event during pregnancy. While VPA also may occasionally induce fever as a side effect, we are concerned here with fevers arising from other, unrelated causes. There is a small but internally consistent literature on these combinational-teratogen exposures involving hyperthermia plus a chemical teratogen; in each instance, the effect level has been observed to be synergistically elevated above levels induced by the separate teratogenic components. The data were empirical. The observed synergy is, however, consistent with Arrhenius thermodynamics, a well-known chemical rate equation. The need for information about combinational teratogen exposures is acute; fever is a common occurrence during pregnancy; and there are many instances whereby there is also the simultaneous presence of some other teratogen(s). Given that the rate of autism spectrum disorders in the United States was recently presented as 1 in 88 births, it seems reasonable to suspect that such combinational regimens are much more prevalent than previously thought. Our hypothesis is that synergistic birth defect levels from combinational regimens are consistent with Arrhenius thermodynamics.

Neonatal rats were exposed or sham exposed for 30 min to pulsed ultrasound [2.25 MHz carrier frequency, 1 microsecond pulse length, 50 Hz pulse repetition frequency (PRF), 50 W/cm2 Imax, 2 mW/cm2 ITA], euthanised and prepared for electron microscopic analysis of the nodes of Ranvier of the dorsal and ventral roots of the spinal cord. There was also a cage control. All materials were processed and scored blindly, evaluating whether perinodal myelin was normal. Rats from all regimens had areas of disrupted myelination. There was no statistically significant difference among the regimens for absence of myelination. The results did not confirm an earlier report that diagnostic ultrasound disrupts myelination in neonatal rats.

Giant internodal cells (∼2–3 cm long) of the Characean alga Nitella clavata were exposed longitudinally to 60 Hz or direct current (d.c.) electric fields (EFs) ranging from 0 to 600 V/m or 0 to 200 V/m, respectively, and scored for membrane banding activity (i.e. the number and location of inhibited and active alkaline bands, which are macroscopically discernible by use of an indicator solution during exposure/sham exposure). Alkaline banding activity decreased with increasing 60 Hz EF strength, thus supporting the hypothesis that effects are proportional to an induced transmembrane potential. Application of d.c. fields likewise decreased alkaline banding activity with increasing EF strength, with the maximum effect near the ends of the cell. Because the d.c. fields were unidirectional, it was possible to analyze the results with respect to the polarity of the induced membrane potential changes. The decrease in banding activity was greater for d.c. EF-induced transmembrane potential hyperpolarizations than for similar transmembrane potential hypopolarizations. Additionally, bands which had been inactivated by the d.c. EF-induced hyperpolarizing membrane potentials could be reactivated by imposition of a d.c. EF-induced hypopolarizing membrane potential.

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.

Ultrasonic cell lysis in vitro shows a strong dependence on the cell density of the suspensions. The major determinant in this phenomenon appears to be the suspended particle density per se. Reported are the results of experiments designed to test the hypothesis that the majority of the cell density effect arises as a consequence of the cell density-dependent formation of cellular aggregates around oscillating bubbles in an ultrasound field (Nyborg and Miller 1982), which in turn diminishes the potential for the occurrence and sustenance of destructive cavitational events in the bulk suspension fluid. The hypothesis was tested indirectly using manometric methods to measure ultrasound-induced release of dissolved gas from saline solutions and fixed erythrocyte suspensions. Ultrasonic degassing of the fluids in excess of that attributable to thermal effects was observed, and was suppressed by the presence of fixed erythrocytes when the cell density was greater than 5 x 10(6) cells/mL. At higher cell densities, the inhibition of ultrasonic degassing by fixed cells increased monotonically with increasing cell density, attaining complete suppression at a cell density of 5 x 10(8) cells/mL. The data thus support the hypothesis.

Previous work in this laboratory has demonstrated that 60-Hz electric fields inhibit the growth of plant roots used as model eukaryotic cell systems. The inhibition of root growth is proportional to the 60-Hz membrane potential induced by the applied field and to field-induced inhibition of net, transmembrane H + flux ( Brayman and Miller , Radiat. Res. 123 , 22–31, 1990). We have proposed that the interference with transmembrane ion traffic is one of the coupling mechanisms by which field-exposed cells transduce the stimulus associated with field exposure ( viz. 60-Hz membrane potential induction) into gross effects on growth and cellular development in the root model system. We report here the results of experiments undertaken with the giant Characean alga Nitella clavata with which we demonstrate that 60-Hz electric field interference with electrogenic transmembrane ion transport processes is not unique to higher plant cells. Both the frequency of affected bands and the frequency of affected cells increased in response to increasing 60-Hz field strength. The sensitivity of acid/base banding to perturbation by 60-Hz electric fields is similar to that of higher plant root growth when compared on the basis of field strength, but qualitatively different when compared on the basis of induced membrane potentials.

Two quite different but potentially complementary hypothetical mechanisms have been proposed to explain the dependence of ultrasonic cell lysis in vitro on exposure vessel rotation. One mechanism proposes that resonant-size bubbles are trapped in planar arrays by standing waves in the sample and that exposure vessel rotation sweeps cells through the bubble arrays. The other mechanism, for which there is now considerable support, proposes that exposure vessel rotation simply allows bubbles to recycle after having been driven through the medium by the ultrasound. We report here the results of efforts to further discriminate between, and quantify, the relative contributions of these two mechanisms. Murine P388 cells were exposed to 1.07 MHz continuouswave (CW) ultrasound for 5 min at 5 W/cm2 (ISP) using various exposure vessel compositions and exposure configurations. Experimental treatments were designed either to minimize or maximize standing waves in the sample tube, or to minimize the potential for bubble recycling while maximizing standing wave formation. The results of these experiments indicate that bubble cycling is responsible for the majority of cell lysis occurring in the rotating exposure vessels, but that a significant contribution to total lysis is provided by trapped bubbles under some 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".

The postulate that 60-Hz electric field-induced bioeffect severity is proportional to induced transmembrane potential [\(V_{m^i } \)] magnitude was tested and supported using a plant root model cell system. Statistically significant correlations were obtained upon regression of the relative rates of exposedVicia faba andZea mays root segment growth on the average\(V_{m^i } \) (calculated) arising in those segments under specified 60 Hz field exposure conditions. The\(V_{m^i } \) associated with the apparent threshold for growth inhibition was similar inZea andVicia roots (2.5 vs 2.4 mV, respectively). At\(V_{m^i } \) greater than this threshold,Zea root growth declined by about 9% per mV, andVicia root growth by about 19% per mV induced potential.

V-79 cells were suspended in medium with 10 or 20% serum, and exposed to a continuous wave of 1 or 3.4 MHz ultrasound (0.7–1 W/cm2) for 0–90 min in polystyrene test tubes at 37 or 43°C, and subsequently assessed for cell lysis, plating efficiency and surviving fraction. The effectiveness of the ultrasound regimens was enhanced at 43°C. Thermocouple measurements of temperatures in the cell culture medium in the test-tube revealed an increase of about 0.5°C under insonation conditions, using the 1 MHz; 10% serum; 37°C regimen. The results were broadly consistent with an ultrasound-induced incremental theraml burden causing an increased effectiveness of the ultrasound exposures at hyperthermic temperatures.

A fibroblast cell line (Balb/c 3T3) was sham/exposed to ultrasound (2.2 MHz resonant frequency, 35.4 W/cm2 SPTP, 3 μs pulse duration, 200 Hz prf) for 30 min and subsequently scored for cell motility and alterations in cell morphology. Cell motility was assessed by computerized tracking of cells. Cell morphology (“normal” or “abnormal”) was determined blindly by three independent scorers. A positive control (1–4 Gy) yielded statistically significant alterations in cell motility and morphology; no such differences were observed for the ultrasound regimen. The results thus fail to confirm an earlier report by Liebeskind et al. (Brit. J. Cancer 45(Suppl. V):176–186; 1982) of ultrasound-induced alterations in cell motility and morphology.

This commentary addresses the matter of misinterpretation of thermal dose as discussed by Herman and Harris (2002), and shows that the thermal doses they would consider as ineffective (i.e., "safe") for producing a hyperthermia-induced teratologic effect, can be highly effective ones. The matter of whether or not thermal thresholds exist for teratologic effects is reviewed. There are only opinions about thresholds. The critical experiments for ascertaining whether or not a threshold exists have not been undertaken. A power computation illustrates the requisite sample sizes (litters, fetuses) for undertaking an experimental test of whether the hyperthermic teratologic response in rats is characterized by threshold or simple linearity kinetics.

This paper describes a further test of the hypothesis that cell size is an important physical parameter in ultrasound (US)-induced hemolysis, that is, the larger the cell the greater the potential for sonolysis by a cavitational mechanism. Mouse (M) and human (Hu) erythrocytes in vitro were used; their mean corpuscular volumes were 49.0 and 89.5 fL, respectively. At a US exposure in vitro in the presence of Albunex that yielded an average of 36.8% hemolysis for M blood, the Hu blood yielded an average of 54.0% hemolysis. The data supported the hypothesis. This paper also briefly discusses the difficulty of extrapolating sonolytic in vitro results to those derived in vivo.

Gas-based ultrasound (US) contrast agents increase erythrocyte sonolysis, presumably via enhancing inertial cavitation (IC) activity. The amount of IC activity (IC "dose") and hemolysis generated by exposure to 1.15 MHz US were examined with different US pulse lengths, but with the same delivered acoustic energy, for Optison and Albunex. The hypotheses were that 1. at longer pulse lengths, IC would generate more bubbles that could nucleate additional IC activity; 2. if the interval between pulse pairs were short enough for the next pulse to hit derivative bubbles before their dissolution, more IC could be induced; and 3. hemolysis would be proportional to IC activity. Two types of studies were performed. In the first, bubble generation after each burst of IC activity was quantified using an active cavitation detector (ACD), for different pulse lengths (5, 10, 20, 30, 50, 100 or 200 cycles), but the same pressure level (3 MPa) and total "on" time (173.16 ms). Low concentrations of either Optison or Albunex were added into the tank with high-intensity and interrogating transducers orthogonal to each other. For pulse lengths > 100 cycles, and pulse repetition intervals < 5 ms, a "cascade" effect (explosive bubble generation) was observed. In the second, IC was measured by passive detection methods. IC dose and hemolysis were determined in whole blood samples at a pressure level (3 MPa) and interpulse interval (5 ms) that induced the "cascade" effect. Each blood sample was mixed with the same number of contrast microbubbles (Optison approximately 0.3 v/v % and Albunex approximately 0.5 v/v %), but exposed to different pulse lengths (5, 10, 20, 30, 50, 100 or 200 cycles). With Optison, up to 60% hemolysis was produced with long pulses (100 and 200 cycles), compared with < 10% with short pulses (5 and 10 cycles). Albunex generated considerably less IC activity and hemolysis. The r(2) value was 0.99 for the correlation between hemolysis and IC dose. High pulse-repetition frequency (PRF) (500 Hz) generated more hemolysis than the low PRF (200 Hz) at 3 MPa. All experimental results could be explained by the dissolution times of IC-generated bubbles.

Whole human anticoagulated blood in vitro underwent controlled plasma replacement with either isotonic (0.9%) or hypotonic (0.5%) saline to 1. restore the blood to its original volume (which resulted in different hematocrits) or 2. bring the blood to a singular hematocrit (40%). The hypotonic cell MCVs were, on average, considerably larger than their isotonic counterpart by a ratio of 1.4:1. The blood samples were then subjected to two tests, one of mechanical fragility, the other to ultrasound (US)-induced hemolysis. The US exposure metrics were: 1.0-MHz center frequency, 200-micros pulse duration, 20-ms interpulse interval, exposure durations of 10 to 30 s in the presence of Albunex, as a control on blood gas nucleation, and exposure vessel rotation at 200 rpm. In all instances, the hypotonic blood displayed higher levels of hemolysis than the corresponding isotonic treatment. The highest ratio of US-induced hemolysis for the hypotonic:isotonic regimens was 2.2. In some instances, the ratio was somewhat less but appeared to be related to differences in whole blood viscosities among the regimens or other factors. The data supported the a priori hypothesis that hypotonicity will result in an increased tension on the cell membrane and render it more susceptible to shear-induced hemolysis, including exposure to US under conditions known to foster the occurrence of inertial cavitation. There was no temperature increase during the insonations of the blood.

This paper reports the results of a further test of the hypothesis that the extent of ultrasound (US)-induced cell lysis in the presence of a US contrast agent to enhance cavitational effects is a function of cell size. The present data support the hypothesis. Human adult erythrocytes in vitro derived from patients with HIV (n = 15) and apparently healthy individuals (n = 15) were compared for US-induced hemolysis in vitro. The anticoagulated whole blood from patients with HIV and macrocytic erythrocytes had significantly greater (p0.0001) mean corpuscular volume (MCV) and a significantly greater (p0.03) extent of US-induced hemolysis in vitro relative to blood from apparently normal, healthy individuals. As a control to determine if disease state (i.e., HIV infection per se) might be a contributing factor in US-induced hemolysis in vitro, the blood from patients with HIV and apparently normal MCVs (n = 15) was also tested against an additional population of apparently normal, healthy individuals (n = 15); there were no statistically significant differences in MCVs or US-induced hemolysis between the two groups (p0.05). There were also no statistically significant differences in viscosities or hematocrits of the whole blood or plasma in vitro from HIV-macrocytic or apparently healthy individuals but, for all blood types, a pooled correlation existed between hematocrit and whole blood viscosity.

This project tested the hypothesis that human erythrocytes pretreated with Trolox (a water-soluble analog of vitamin E) would be more susceptible to ultrasound (US)-induced hemolysis by a cavitational mechanism because of an increased fragility of the erythrocyte membrane over that without Trolox supplementation. Samples of whole human blood from apparently healthy donors (hematocrit approximately 40%) in vitro were supplemented or not supplemented with Trolox at various concentrations, ranging from 1.8 to 0.0018 mg/mL plasma. Mechanical fragility tests indicated the Trolox-treated blood in vitro exhibited greater hemolysis than untreated blood in vitro (p < 0.001). US exposures at comparable acoustic amplitude, pulse length and duty factor in the presence of the US contrast agent Albunex yielded differing results; at 1 MHz, the Trolox-supplemented blood had significantly greater hemolysis in vitro than non-Trolox-supplemented blood; at 3 MHz, there was a substantial reduction in hemolysis relative to that obtained at 1 MHz, and no statistically significant difference between the Trolox-supplemented and -unsupplemented blood. There was also essentially no support for an alternative hypothesis that the Trolox was functioning primarily as a pro-oxidant. These collective experimental results support the hypothesis and suggest duality in the functionality of membranous antioxidant inclusions or associations; they may foster protection against oxidative damage, yet render the cell less capable of withstanding mechanical stress.

This research project tested the hypothesis that cold-equilibrated (approximately 0 degrees C) human erythrocytes in vitro in the presence of an ultrasound contrast agent (Albunex) will undergo greater ultrasound-induced hemolysis than physiologically equilibrated (37 degrees C) human erythrocytes in vitro because of a temperature-related transition in membrane fluidity leading to increased fragility. First, it was shown that cold-equilibrated erythrocytes are more susceptible to mechanically induced hemolysis than physiologically equilibrated erythrocytes. Second, when adjustments were made for (1) temperature-dependent efficiencies of a 1-MHz transducer (200 micros pulse length, 20 ms interpulse interval, 30 s exposure duration) such that when cold or physiological temperatures were employed, there were equivalent acoustic outputs in terms of peak negative pressure (MPa P-) and (2) comparable viscosities of the 0 and 37 degrees C blood plasmas, the cold (approximately 0 degrees C) erythrocytes displayed substantially greater amounts of ultrasound-induced hemolysis than the physiological (37 degrees C) erythrocytes. The data supported the hypothesis.

In polystyrene exposure vessels, a substantial fraction of suspended P388 cells was lysed by exposure to cw, 1-MHz ultrasound (5 W cm-2 spatial peak intensity, 5 min) in stationary vessels or those subjected to a small number of half rotations. Lysis was increased significantly by two half-rotations during exposure relative to stationary vessels. Lysis in stationary exposure vessels is generally considered to be minimal; it is shown that approximately 40% cell lysis occurs in 15%-20% of the stationary tubes. This result appears to relate to the distribution of random defects on the exposure vessel walls.

Hyperthermia is a recognized teratogen in mammalian laboratory animals and is a suspected teratogen for humans. The purpose of this synopsis is to reanalyse existing data on hyperthermia-induced teratogenic effects in experimental mammalian systems in terms of a thermal dose (temperature:time) concept, and then to illustrate the utility of this concept to human situations involving potential thermal increments to post-implantation embryos and foetuses. For example, the threshold temperature elevation for hyperthermia-induced teratogenic effects in experimental mammals is estimated (but not rigorously tested) to be approximately 1.5 degrees C above core values for exposures of long duration, possibly with a thermal dose of approximately 5 min duration or more at 4 degrees C. This level of tissue temperature increment is within the capability of some modern diagnostic ultrasound (DUS) devices sold within the USA and abroad. Epidemiological studies have not indicated any hazard from the use of DUS, but such studies are limited in sensitivity and were conducted with DUS devices whose acoustic outputs were relatively low compared to those presently available. After a regulatory change that allowed for substantially increased acoustic outputs, modern DUS devices were mandated to provide the user with on-screen information (the Thermal Index, or 'TI') about ultrasound-induced temperature increments in the target tissue. The TI is generally accurate to within a factor of 2, but the factor may be as high as 6 in certain obstetric settings. Thus, informed use of and attention to the TI is strongly advised, with this admonition gaining increased emphasis if the present regulations regarding allowable acoustic outputs of DUS devices were to be further relaxed or eliminated.

The erosion of cells from fibroblast monolayers simulating the vascular endothelium by 20 μs pulses of ultrasound at 500 Hz PRF was studied in relation to the peak negative acoustic pressure (P−; 0.0–2.5 MPa), ultrasound (US) frequency (1.0, 2.1 or 3.5 MHz), orientation of the monolayer (i.e., simulating the sites of ultrasound entry/exit from a blood vessel) and the presence or absence of a microbubble contrast agent (3 Vol% Albunex®). The a priori hypotheses were that erosion of the monolayers would: 1. arise due to insonation treatment, 2. arise as a consequence of cavitation activity and, thus, increase with increasing P− at constant frequency, and decrease with increasing frequency at constant P−, 3. be significantly increased by the presence of a microbubble contrast agent, and 4. have a weak dependence on monolayer orientation. The data support these hypotheses. Under the most severe exposure conditions used, most of the affected cells appeared to have been lysed; however, a substantial number of viable cells were dislodged from the monolayer surface.

The hypotheses tested were that sonolysis of erythrocytes in the presence of a gas-based ultrasound contrast agent in vitro will be related quantitatively to the duration and number of ultrasound pulses applied using a constant pulse repetition period and, at least qualitatively, to the total exposure duration (i.e., the product of pulse number x pulse duration). An objective was to determine the influence of sample rotation during insonation on the amount of hemolysis produced under these conditions. Human erythrocytes, suspended to 40% hematocrit in autologous plasma containing 3.6% (V:V) Albunex, were exposed/sham-exposed to 1-100 pulses of 1-MHz ultrasound (6.2 MPa peak positive, 3.6 MPa peak negative acoustic pressures; I(SPTP) approximately 800 W/cm2) using a 1-s pulse repetition period. Pulse durations ranged from 20-20,000 micros; samples were either stationary or rotated (200 rpm) during insonation. Hemolysis was independent of vessel rotation treatment at all tested pulse durations and pulse numbers. Levels of hemolysis statistically greater than in sham-exposed samples were obtained withor = 50 pulses of 20 micros duration, andor = 1 pulse of 200, 2000 or 20,000 micros duration. Hemolysis increased with increasing pulse number and pulse duration. Approximately equivalent levels of hemolysis were produced by different pulse number x pulse duration combinations, yielding the same total exposure duration.

The hypothesis tested was that gas bodies capable of nucleating violent cavitation activity in vitro would survive the rapid disruption of Albunex ® microspheres by 1-MHz ultrasound. Human erythrocyte hemolysis was used as a proxy measure of cavitation. Fluid (5% human serum albumin [HSA]) with or without Albunex ® (ALX) was exposed or sham-exposed to 1-MHz ultrasound (P + = 1.25 ± 0.01 MPa, P − = 0.81 ± 0.01 MPa; I SPTP ∼ 35 W/cm 2 ) for 60 s using 10-μs pulses and a duty factor of 0.5. An equal volume of whole human blood was then added to the fluid, followed by a second 60-s treatment. Insonation of cell suspensions prepared in previously sham-exposed HSA + ALX fluid produced about 4% hemolysis, a level significantly greater than in the controls. Insonation of cell suspensions prepared in previously insonated HSA + ALX fluid produced about 0.4% hemolysis; this also differed significantly from the controls. The data thus support the hypothesis.

The dependence of hemolysis produced by pulsed ultrasound on ultrasound frequency, acoustic pressure and pulse length was explored. Human erythrocytes (40% hematocrit; in Albunex-supplemented autologous plasma) were exposed (60 s) to 20 or 200 microns pulses of ultrasound at frequencies of 1.02, 2.24 or 3.46 MHz and at peak negative pressures [P-] ranging from 0.0 to approximately 3.0 MPa in 0.5 MPa increments. The duty factor was 0.01. At each frequency, hemolysis increased with increasing acoustic pressure and depended weakly on pulse duration. At relatively high acoustic pressures, hemolysis depended strongly on ultrasound frequency; at lower pressures, the frequency dependence was weaker. The potential clinical significance of ultrasonic hemolysis is discussed.