Department of Medicine, Hematology/Oncology Section, Baylor College of Medicine, BCMD 525D, One Baylor Plaza, Houston, Texas 77030
Received January 15, 2002; accepted May 31, 2002
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ABSTRACT |
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Key Words: 2-butoxyethanol; ethylene glycol monobutyl ether; butoxyacetic acid; glycol ethers; hemolysis; rat erythrocytes; human erythrocytes.
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INTRODUCTION |
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The mechanism of rat red-cell damage is not known. Similarly, there is no understanding of human resistance to BAA in vitro. Bartnik et al. (1987) found no evidence for hemolysis of human RBCs incubated with concentrations of 115 mM BAA for up to 3 h. Ghanayem (1989) noted a slight degree of hemolysis of human RBCs incubated in 8.0 mM BAA for 4 h. 2-BE can cause hemolysis of human RBCs, but only when cells are exposed to 125200 mM for 2 or 3 h (Bartnik et al., 1987). At such high levels of 2-BE, rat erythrocytes are only marginally more sensitive to the solvent than humans. Non-specific detergent effects of 2-BE probably might account for the red-cell damage observed at such high concentrations of 2-BE.
In the rat, an increase in mean cellular volume (MCV) accompanies hemolysis during in vivo exposure to 2-BE (Ghanayem et al., 1990). A similar degree of cell swelling occurs in vitro with exposure of red cells to BAA. Measurements of MCV were used by Ghanayem to identify susceptible species for 2-BE toxicity by demonstrating an increase in MCV after in vitro incubation of RBCs with 2.0 mM BAA (Ghanayem et al., 1993). Rat erythrocyte deformability, as assessed by a polycarbonate filtration method, also decreases after exposure to BAA in vitro. Human erythrocyte deformability was unaffected by incubation with 2.0 mM BAA for 4 h, while rat erythrocytes were considerably more rigid after exposure to 0.2 mM BAA, which caused no in vitro hemolysis (Udden and Patton, 1994
). Rat erythrocyte morphology undergoes a transformation from a normal discocytic shape to spherocytic and stomatocytic, or cup-shaped, red blood cell when exposed to BAA in vitro or after 2-BE administered by gavage (Udden, 2000
). Human erythrocyte morphology was not affected by exposure to 2.0 mM BAA (Udden and Patton, 1994
).
In the present study, changes in human RBC, deformability along with changes in other functional aspects of the red cell, are described after exposure to high concentrations (up to 10 mM) of BAA in vitro. Blood samples from healthy donors and hospitalized patients including children were screened for evidence of increased erythrocyte susceptibility to hemolysis or cell swelling. A comparison is also presented of similar pre-hemolytic damage to rat RBCs after exposure to low concentrations of BAA (up to 0.1 mM). The absence of an in vitro threshold for hemolysis of human RBCs has made it difficult to infer from animal studies an estimate of relative toxicity of BAA, and therefore of 2-BE for man. The experiments presented here offer a new approach to making this comparison by focusing instead on pre-hemolytic RBC changes observed at high concentrations of BAA for human RBCs and similar changes in rat RBCs exposed to 100-fold lower concentrations of BAA.
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MATERIALS AND METHODS |
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Butoxyacetic acid.
A 99% pure reagent was obtained from the Shell Development Company, Westhollow Technical Center, Houston, Texas.
Red blood cell indices: Morphology and hemolysis:
Erythrocyte suspensions were sampled at intervals for determination of microhematocrit, hemoglobin, RBC count, and percent hemolysis, utilizing standard methods. The mean cellular volume (MCV) for blood samples obtained in heparin from healthy donors and for rat RBCs was determined from the microhematocrit and the red-cell count. The distribution of RBC size was determined using a Coulter model Z2 cell-size analyzer. For blood samples obtained from the clinical laboratory and stored in EDTA, the MCV and size distribution were determined using the Z2 analyzer.
Deformability.
Erythrocytes were diluted with buffer to 2.5 x 108 cells/ml and then infused via a Harvard syringe pump at 2.0 ml/min through a 3.0-micron polycarbonate filter (lot number 8084011, Nuclepore Filter Corporation, Pleasanton, CA). During filtration at a constant rate, pressure increases were measured by a Statham P23Db pressure transducer with graphic output, on a Gould Chart recorder. The typical pressure-time curve and a mathematical expression describing the curve as a function of initial filtration pressure (P0), parameters determined by the electronic filter in the chart recorder, and filling of the filter chamber are described in detail elsewhere (O`Rear et al., 1979). The initial filtration pressure, or P0, is a function of RBC deformability and was determined by a software program that analyzed digitized curves using a Marquardt non-linear regression numerical method (Biological Research Systems, Houston TX).
Erythrocyte osmotic fragility, density, and cations.
Osmotic fragility was determined on washed RBCs as described by Beutler (1995)). Density distribution of red cells was determined on mixtures of phthalate esters (Danon and Marikovsky, 1964). Erythrocyte sodium and potassium concentrations were determined using an atomic absorption spectrophotometer (Perkin-Elmer, Norwalk, CT) in the emission mode. Measurements were made on erythrocytes washed 4 times with isotonic MgCl2 and then lysed in 0.1% LiCl.
Microscopy.
Red blood cells were prepared for light-phase microscopy (1000 x magnification) by addition of 20 µl of cell suspension to 1.0 ml of buffer containing 1.0% glutaraldehyde.
Statistics.
Mean and SE for the number of different subjects tested, n, were used to characterize the effects of BAA at varying concentrations when compared to a control (0 mM BAA) group. Significance was determined by one-way analysis of variance, with additional use of an all pair-wise multiple comparison procedure (Tukey) test to determine significance of difference in effect due to different concentrations of BAA, compared to control (no BAA) incubation. Paired t-tests were used for normally distributed data, and the Wilcoxon Rank Sum test was used to characterize the differences in effect of BAA on non-normally distributed results of studies on patients` blood samples. Calculations were made using a Sigmastat software program.
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RESULTS |
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DISCUSSION |
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Normal RBC deformability depends upon an increased surface area to volume ratio of the cell, intrinsic membrane deformability, and viscosity of the internal hemoglobin milieu (Chien, 1977). The polycarbonate sieve method used in these experiments is very sensitive to the ratio of surface area to volume (Chien, 1977
; O`Rear et al., 1979
). This is because a relatively under-filled, or collapsed balloon-like object is easier to push through a small pore than a filled, spherical object. Thus, the RBC deformability changes observed here could be related to cell swelling and greater sphericity. An effect on membrane structure causing a change in deformability is also possible since the increase in cell size was relatively modest. In these measurements, filters with the same pore size (3.0 micron) were used for rat and human RBCs. The human red blood cell is larger than the rat red cell, accounting for the higher P0 shown in the human control erythrocytes.
The changes in rat erythrocyte deformability are very consistent with cell swelling and increased water content. During exposure to sub-hemolytic concentrations of BAA, the MCV increased, as did the osmotic fragility. Lysis occurs in the osmotic fragility test because red cells behave as osmometerstaking on water as the external environmental osmolality decreases. When the cell reaches a size in which the surface area is fully expanded, the critical hemolytic volume is attained, after which further uptake of water results in lysis of the cell (Beutler, 1995). The rat erythrocyte osmotic fragility was consistent with cells being closer to their critical hemolytic volume after exposure to BAA. A remarkable shift towards lower erythrocyte density shown in the phthalate ester density distribution of the rat RBCs is consistent with cell swelling and dilution of hemoglobin, hemoglobin concentration being the most important determinant of red cell density (Mohandas et al., 1980
).
Although the change in MCV in the human and rat red cells was of about the same magnitude (3.7 µm3 for human, 5.7 µm3 for the rat), the relative increase in the human red cell, which starts out as a larger cell (98 µm3 versus 56 µm3 in rat), was smaller. The size distribution as determined by impedance cell sizing is demonstrably changed after exposure to BAA in vitro. In these studies the coincidence of cells makes it difficult to detect a sub-population of enlarged cells, although a loss of the smallest cells and a shift of the main peak to a higher cell volume is apparent. Certain size analyzers are unable to detect such changes in rat MCV after BAA exposure (Ghanayem et al., 1990). There was a significant increase in osmotic fragility for human RBCs, indicating a greater surface area to volume ratio. However, the absence of a significant shift towards lower density, demonstrated by the phthalate ester density distribution, suggests that water content did not greatly increase. A possible explanation is that the changes occurred in a sub-population of human erythrocytes, or that exposure to BAA renders the human red blood cell more vulnerable to osmotic stress. However, most of the rat erythrocytes subjected to a lower concentration of BAA had an increased osmotic fragility and decreased cell density. Spherocytosis was evident in the rat red blood cells exposed to BAA, but human red blood cell morphology was not affected by BAA at much higher concentrations.
Both human and rat RBCs showed a clear increase in cell sodium as a result of exposure to BAA. Rat erythrocytes appeared to immediately respond with a small potassium loss that failed to compensate for the net increase in sodium. Because water follows cations into the red cell, the net increase in total cation content of the rat erythrocytes exposed to 0.1 mM BAA for 4 h could have contributed to increased cell size and decreased density. The redistribution of cell density to lower values and the increased osmotic fragility are consistent with increased cell volume due to increased water.
Although similar (quantitatively and qualitatively) changes occurred for human and rat RBC deformability, osmotic fragility, cell sodium levels, and perhaps MCV, it is not certain that these changes happened for the same reasons. The absence of a major decrease in cell density and the absence of morphologic changes in the human red blood cell suggests that the mechanism for pre-hemolytic changes are different in the two species. However, the clear increase in sodium content of human and rat erythrocytes suggests that the effect of BAA is to disturb cation transport. Even so, it is now possible to assess the concentration-dependent effects of BAA on human RBCs.
These studies are consistent with a 100-fold difference between concentrations at which pre-hemolytic effects of BAA are observed on rat and human red blood cells. Although there was a very small (but statistically significant) increase in hemolysis of red blood cells exposed to BAA from hospitalized adults, none of the blood samples from 97 adults and children demonstrated an increase in hemolysis of a degree that would predict an increased susceptibility to BAA accumulating in the blood from an exposure to 2-BE. There was an increase in the MCV, as expected. There was no evidence of differences due to age or sex. A human pharmacokinetic model (Corely et al., 1994) indicated that the predicted level of BAA in humans exposed continuously by inhalation to a saturated vapor of 2-BE or by dermal exposure to a 40% 2-BE solution was less than 2 mM. Thus, the pre-hemolytic changes described here are unlikely to occur in humans under normal exposure conditions. These results are particularly important when uncertainty factors used in risk assessment are based on inter- or intra-species differences in hemolytic effects.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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