Arrhenius thermodynamics and birth defects: chemical teratogen synergy. Untested, testable, and projected relevance.

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.

Fetal thermal dose considerations during the obstetrician's watch: Implications for the pediatrician's observations.

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.

Quantification of risk from fetal exposure to diagnostic ultrasound.

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 degrees 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.

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 5. Temperature.

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.

Cell size relations for sonolysis.

The occurrence of cell lysis following exposure to ultrasound (US) has been well documented; the specifics of the mechanistic process(es) involved have proven to be difficult to characterize. There appear to be two major mechanisms of US-induced cell lysis in vitro, acoustic cavitation and bubble transport. Both involve shear forces. Earlier research showed that the size of oil droplets was a crucial factor in their rupture by shear forces; the greater the size the greater the rupture per unit shear force. Here we find further support (Miller et al. 2000, 2001a, 2003a; Miller and Battaglia 2003; Abramwocz et al. 2003) that, under a number of experimental US conditions causing hemolysis in vitro, cell volume (i.e., size) is an apparently critical factor.

An extended commentary on "Models and regulatory considerations for transient temperature rise during diagnostic ultrasound pulses" by Herman and Harris (2002).

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.

The relevance of cell size on ultrasound-induced hemolysis in mouse and human blood in vitro.

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.

Comparative hemolytic effectiveness of 1 MHz ultrasound on human and rabbit blood in vitro.

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).

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: medium tonicity.

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.

The pulse length-dependence of inertial cavitation dose and hemolysis.

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.

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 2. Medium dissolved gas (pO2) content.

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).

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 1. HIV macrocytosis (cell size).

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 (p <0.0001) mean corpuscular volume (MCV) and a significantly greater (p <0.03) 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 (p >> 0.05). 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.

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 3. Antioxidant (Trolox) inclusion.

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.

Biological Effects of Ultrasound. The Perceived Safety of Diagnostic Ultrasound Within the Context of Ultrasound Biophysics: A Personal Perspective.

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.

Temporality in Ultrasound-Induced Cell Lysis In Vitro.

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)