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Surface charge and hemolytic activity of asbestos
BNVIKONMENTAL RESEARCH13, 13.5-145 (1977)
Surface
Charge
and Hemolytic W. G.
Department
LIGHT
of Asbestos
E. T. WEI
AND
of Biomedical and Environmental Health. University of California,
Activity
Health Berkeley,
Sciences, California
School 94720
of Public
Received March 20, 1976 Surface charge. represented by zeta potential, and hemolytic activity were measured for the five asbestos reference samples of the International Union against Cancer (UICC). Zeta potentials in distilled water at pH 7.4 for chrysotiles A and B were +40.5 and +52.5 mV. and for amphiboles crocidolite, amosite, and anthophyllite were -50.4, -58.5, and -54.0 mV, respectively. For fiber samples of equal surface area, chrysotiles had a greater hemolytic activity than amphiboles: 60.5 and 81.9% for chrysotiles A and B compared to 7.7. 9.8, and 15.7% for crocidolite, amosite, and anthophyllite, respectively. A lysing mechanism based on charge polarity is proposed to account for this difference in activity. Zeta potential and hemolytic activity were reduced by adding the main component of pulmonary surfactant. dipalmitoyl phosphatidylcholine (DPPC), to fiber suspensions. DPPC was more effective in decreasing both zeta potential and hemolytic activity for amphiboles than for chrysotiles. On the basis of these reductions, a significant correlation (r = 0.84) was found between zeta potential and hemolytic activity for all five UICC samples. This correlation suggests that surface charge is the principal factor in hemolysis by asbestos. The relevance of surface charge to asbestos toxicity is discussed. INTRODUCTION
The physical and structural properties of fibers are generally considered to be the primary determinants of their biological effects. For asbestos fibers, it has been suggested that the most important properties are surface charge, solubility, surface area, and dimensions of the fibers (Morgan, 1975). The relative significance of solubility, surface area. and dimensions in determining cytotoxic, tibrogenic, and carcinogenic activity of asbestos has been discussed elsewhere (Harington, Allison, and Badami, 1975; Morgan, 1975). In the present investigation, the relationship between surface charge and hemolytic activity was examined for the five UICC reference samples of asbestos. Asbestos fibers, when added to macrophages itz vitro, damage plasma and lysosomal membranes and cause cell death (Allison, 1971; Miller and Harington, 1972; Harington, Allison, and Badami, 1975). This cytotoxicity is more readily obtained with chrysotiles than amphiboles, and it appears that the in viva correlate of this cytotoxic effect is fibrosis (Klosterkotter and Robock, 1975; Morgan, 1975). To study the disruptive effects of asbestos on membranes, a simple in vitro technique based on rupture of the erythrocyte membrane (hemolysis) has been developed (Desai, Hext, and Richards, 1975; Harington, Miller, and Macnab, 1971; Schnitzer and Pundsack, 1970). Using this technique, it has been demonstrated that chrysotiles also have a greater hemolytic activity than amphiboles. It is suspected that the hemolytic activity of asbestos is principally determined by the surface charge the fibers acquire in solution. However, there have been virtually no studies in which surface charge was measured directly and related to biological activity. In the present study, surface charge was represented by zeta 135 Copyrtghl Q 1977 by Academic Press. inc. All rights of reproduction in any form reserved.
ISSN 0013-9351
136
LIGHT
AND
WEI
potential which is a measure of the potential energy difference between the bulk solution and the boundary separating solvent adhering to a fiber and free solvent and, although it is smaller in magnitude, approximates total surface potential. The effect of dipalmitoyl phosphatidylcholine (DPPC), the main component of pulmonary surfactant, on zeta potential and hemolytic activity was also examined since it has been shown that pulmonary surfactant (Desai, Hext, and Richards, 1975) and bronchial secretions (Allison, 1971) can reduce the hemolytic activity of asbestos. The adsorption of DPPC by fibers may have significance in viva since DPPC is present at the alveolar air-liquid interface (King, 1974) and would adsorb onto surfaces of inhaled fibers. MATERIALS
AND METHODS
Reagents The five UICC standard reference samples of asbestos, kindly supplied by the MRC Pneumoconiosis Unit, Penarth, South Wales, and the Johns-Manville Research and Development Center, Denver, were used in all experiments. The preparation of these samples has been described by Timbre11 and Rendall (1971- 1972). Synthetic dipalmitoyl L-a-phosphatidylcholine was obtained from the Sigma Chemical Company, St. Louis. Sterile sheep erythrocytes in the form of equal parts of whole blood and modified Alsever’s solution were acquired from Microbiological Media, Concord, California. Prior to use, erythrocytes were suspended in sterile Tyrode’s solution (prepared with sodium replacing calcium and adjusted to pH 7.4) and washed three times. After each washing, the suspensions were centrifuged for 10 minutes at 1500 rpm. Standard 1.5% erythrocyte suspensions were prepared by combining 1.5 ml of packed erythrocytes with 100 ml of sterile Tyrode’s solution. Zeta-Potential Studies Ten-milligram samples of asbestos were uniformly dispersed in bottles containing 100 ml of triple-distilled water. DPPC was added and the suspensions were adjusted to appropriate pH values using 1O-2 M HCl or 1O-2 M NaOH. Samples were then incubated for 1 day at between 41” (the melting point of DPPC) and 43°C in a shaker bath. Following this equilibration period, the samples were cooled to room temperature (23°C) and the pH was measured. Zeta potentials were determined using a microelectrophoresis instrument commercially known as a Zeta Meter (Zeta-Meter, Inc.). A Zeta Meter cell (#G-T248) was filled with 35-ml aliquots of the fiber suspensions and an electric field was applied. The movement of fibers at least 0.1 pm in size was then observed through a stereoscopic microscope. The time required for 15 to 25 fibers each to traverse 1 ocular-micrometer division was recorded and the average time calculated. Fiber velocity (prn/sec) in a given electric field (volt/cm), the electrophoretic mobility, was determined. Zeta potential was calculated from electrophoretic mobility using the Helmholtz-Smoluchowski equation (Riddick, 1961). Zeta potential was not converted to surface charge because the term zeta potential readily conveys the concept of charge. The decrease in zeta potential for fibers
ASBESTOS
SCRFACE
CHARGE,
HEMOLYTIC
ACTIVITY
treated with DPPC was expressed as a percentage of the zeta potential obtained when no DPPC was added to the fiber suspensions: % decrease in ZP =
137 (ZP)
x 100. -ZP fibers treated with DPPC ZP untreated fibers >
In the zeta-potential experiments, distilled water was used as the suspending liquid rather than Tyrode’s solution because inorganic solutions of high molarity and liquid conductance generate excessive heat. Heat generation at concentrations above 0.01 M can result in convection currents which cause charged particles to deviate from the linear path of an electric field. Hemolysis Studies The experimental procedures used here were similar to those described by others (Desai, Hext, and Richards, 1975: Harington, Miller, and Macnab, 1971; Schnitzer and Pundsack, 1970). Asbestos fibers in pre-weighed amounts were placed in lo-ml tubes. To each tube. 2 ml of suspending liquid (Tyrode’s solution or Tyrode’s solution containing DPPC) were added. A vortex mixer was used to ensure uniform distribution of fibers in the suspending liquid. Following a l-hour incubation period at 41-43”C, the tubes were cooled to room temperature (23°C) and 2 ml of 1.5% erythrocyte suspension were added to each tube. The tubes were sealed with Parafilm-covered corks and inverted twice every 10 minutes for 1 hour. The samples were then centrifuged at 2000 rpm for 20 minutes. Supernatant solutions were transferred from the tubes using a 5-ml syringe. The tilled syringe was attached to a Millipore Swinnex-25 filter holder containing a filter of 0.22~pm pore size, and the supematant was filtered to remove any fibers that may have been taken up by the syringe. Filters were prewashed with the supernatant of a centrifuged erythrocyte suspension to avoid excessive adsorption of hemoglobin onto clean filters. The optical densities of the filtered supematant solutions and controls were read at 541 nm in a Perkin-Elmer. Model 54, Coleman Digital Spectrophotometer. UICC chrysotiles have a specific surface area approximately two times greater than that of anthophyllite and four times greater than that of other amphiboles (Morgan, 1975). To adjust for these differences in surface area as well as obtain measurable hemolytic activities, fibers were used in the following quantities: 5 mg of chrysotiles, 10 mg of anthophyllite, and 20 mg of crocidolite or amosite. Tubes not containing fibers functioned as the three types of controls used in the experiments: totally lysed control (2 ml erythrocyte suspension plus 2 ml triple-distilled water), fragility control (2 ml of erythrocyte suspension plus 2 ml of Tyrode’s solution or Tyrode’s solution containing DPPC), and Tyrode’s solution control. Six replicate tubes were used for each suspending liquid and fiber type. The hemolytic activity of asbestos was calculated from the average optical density (OD) of the six replicates and was expressed as a percentage of the totally lysed control OD: Hemolytic
activity
=
average OD - fragility control OD x 100. lysed control OD - Tyrode’s solution OD >
138
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AND
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The decrease in hemolytic activity of the fibers treated with DPPC was expressed as a percentage of the hemolytic activity (HA) obtained when no DPPC was added to the fiber suspensions: % decrease in HA =
-HA
fibers treated with HA untreated fibers RESULTS
Zeta potentials for the five UICC samples were plotted as a function of pH in Figs. 1 and 2. At pH 7.4, zeta potentials in distilled water for chrysotiles A and B were +40.5 and +52.5 mV, and for amphiboles crocidolite, amosite, and anthophyllite were -50.5, -58.5, and -54.0 mV, respectively. These results clearly illustrate that zeta potentials for the two types of asbestos were similar in absolute magnitude, but opposite in polarity. The data plotted in Fig. 2 were indicative of the two types of asbestos and show that for both types the addition of DPPC to fiber suspensions produced a dose-dependent decrease in the magnitude of zeta potential. To compare the relative effects of the surfactant DPPC on the zeta potentials for the five different fibers, a dimensionless variable, mg DPPC/mg fibers, was defined and plotted against the decrement in zeta potential produced by DPPC (Fig. 3). The data show that the relative efficacy of DPPC in reducing zeta potential can
-30
ANTHOPnYLLlTE
t
FIG. function
1. Zeta potentials of pH.
for UICC
chrysotile
B, amosite,
and anthophyllite
in distilled
water
as a
ASBESTOS
SlJRFACE
FIN;. 2. Zeta potentials for IO mg of UICC of pH and in the presence of the surfactant
CHARCE,
chrysotile dipalmitoyl
HEMOLYTIC
AC’I‘IVI
I‘Y
A and crocidolite in distilled water phosphatidylcholine (DPPC).
139
as a function
be ranked as crocidolite > amosite > anthophyllite > chrysotile A > chrysotile B. At a ratio of 0.1, for example, zeta potentials for amosite and crocidolite were decreased by about 74%, while zeta potentials for chrysotiles A and B were only decreased by about 11%. After taking into account differences in specific surface area, it was found that zeta potentials for amphiboles were still more readily reduced by DPPC than those for chrysotiles. DPPC was more effective in reducing the zeta potential for amosite and crocidolite than for anthophyllite because anthophyllite has a surface area approximately twice as great as the other amphiboles. For fiber samples of equal surface area, chrysotiles had a greater hemolytic activity than amphiboles. The relative activities in Tyrode’s solution were the following: chrysotile B (81.9%) > chrysotile A (60.5%) > anthophyllite (15.7%) > amosite (9.8%) > crocidolite (7.7%). When DPPC was added to fiber suspensions, hemolytic activity decreased for all five UICC samples (Fig. 4). At a ratio of 0.1 for example, hemolytic activities for amosite and crocidolite were decreased by about 87% while hemolytic activities for chrysotiles A and B were decreased by about 25%. On the basis of fiber samples of either equal mass or equal surface area, DPPC was more antihemolytic for amphiboles than for chrysotiles. Thus, the relative efficacy of DPPC in decreasing hemolytic activity paralleled its relative efficacy in reducing zeta potential.
140
LIGHT I
AND
WEI
I
I
I
0 IJ n B 0
I
’
CHRYSOTILE A CHRYSOTILE B AMOSITE CROCIDOLITE ANTHOPHYLLITE
s
4:
0.1
0.2 SURFACTANT/
FIG. 3. The effect of the surfactantifiber UICC samples at pH 7.4.
ratio
Fl&?S
I
3 ? 0
I
0.1
FIG. 4. The effect ofthe UICC samdes.
surfactantkber
1
0.2
I.0
h&i4
on the percentage
1
I
decrease
in zeta potential
for the five
AMOSITE CROClOOLlTE ANTHOPHYLLITE
L
0.3 0.4 0.5 SURFACTANT / FBERS
ratio on the percentage
I
0.6 (mglmg)
decrease
I
I
0.7
0.8
in hemolytic
activity
for the five
ASBESTOS
SURFACE
CHARGE,
HEMOLYTIC
141
ACTIVITY
When either chrysotiles or amphiboles were added to neutral solutions, an increase in pH occurred. To demonstrate sufficiently whether surface charge is the principal factor in asbestos hemolysis, it was necessary to determine what effect these shifts in pH had on hemolytic activity. Increases in pH for 0.01% neutral suspensions of chrysotile B were measured after 1 day of aging and used as a measure of all fibers since chrysotile B resulted in the largest shifts. While an increase of 2.5 pH units was obtained in distilled water, an increase of only 0.2 pH units was measured in Tyrode’s solution (due to the action of effective bicarbonate and phosphate buffers), and in Tyrode’s solution diluted to 10% of normal concentration (comparable to the ratio of buffer/fibers used in hemolysis experiments) an increase of 0.5 pH units was observed. To test the effect this 0.5 pH unit increase has on hemolytic activity, erythrocyte fragility was measured in Tyrode’s solution adjusted to pH 7.4 and to pH 7.9. No significant difference in hemolysis was observed between these two pH values. An antihemolytic action of surfactant could be exerted directly on the erythrocyte membrane rather than on the surface of the asbestos fiber. To examine this possibility, erythrocyte fragility was measured in Tyrode’s solutions diluted to 10, 50, and 90% of normal concentration and containing DPPC. The surfactant did not protect cells from osmotic rupture; in fact, its presence may have slightly enhanced hemolysis. These results further indicate that the antihemolytic effect of surfactant is secondary to its interaction with the asbestos fiber.
90 > 5
3
80 : V 0 0
F ::
CHRYSOTILE A CHRYSOTILE B AMOSITE CROCIOOLITE ANTHOPHYLLITE GLASS FIBERS
0
70 2 I> 6
60 I-
/
-I
E I z
50
1
1 GO
PERCENTAGE
FIG. 5. The correlation between in zeta potential for the five UICC fiber ratios (P < 0.01, Y = 0.84).
DECREASE
IN
ZETA
POTENTIAL
percentage decrease in hemolytic activity and percentage dec :rease samples and glass fibers at pH 7.4 on the basis of equal surfactanti
142
LIGHT
AND
WEI
To summarize the relationship between zeta potential and hemolytic activity, the data in Figs. 3 and 4 were combined to yield Fig. 5. A least-squares regression analysis showed that the two variables were significantly correlated (P < 0.01, r = 0.84) for all five UICC samples. Thus, even though zeta potentials for amphiboles and chrysotiles were opposite in polarity, the net effect of DPPC was the same for both types: a decrease in hemolytic activity proportional to a decrease in zeta potential. It was of interest to determine whether other fibers had hemolytic characteristics similar to those of asbestos fibers. In preliminary experiments, the zeta potential (-53.5 mV) and hemolytic activity (28.3% for lo-mg fibers) for “code 100 microfiber, 475 glass” (Johns-Manville Research and Development Center) were measured. The data suggest that the hemolytic activity of glass fibers, like that of asbestos fibers, may be strongly dependent on the zeta potential.
DISCUSSION
Zeta potentials calculated for the five UICC reference samples in distilled water at pH 7.4 are consistent with previously published results. Correcting the original value of Martinez and Zucker (1960), recognized as being high by a factor of 2 (Harington, Allison, and Badami, 1975), gave a value of +46.5 mV for the equilibrium zeta potential of chrysotile. For MgO, the oxide on the surface of chrysotile, Lai (1970) measured an electrophoretic mobility corresponding to a zeta potential of +50.9 mV. These two values are comparable to the present results of +40.5 and +52.5 mV for chrysotiles A and B, respectively. Zeta potentials for amphiboles (-50.5, -54.0, and -58.5 mV for crocidolite, anthophyllite, and amosite, respectively) are in accord with those calculated by Prasad and Pooley (1973) for amosite and crocidolite of approximately -50 mV for both fibers in 1O-6 N Ca(NO,), and -45 mV for both fibers in lo-* N NaCl. Although the values they obtained in distilled water were lower in magnitude, it would be expected that in distilled water the zeta potentials would be greater since the lower the ionic strength of an indifferent electrolyte, the higher the zeta potential. For silica, Lai (1970) obtained an electrophoretic mobility corresponding to a zeta potential of -63.6 mV. This zeta potential probably is greater in magnitude than that for amphiboles because the negative surface-charge density of amphiboles is less than if the entire fiber surface were silica. How pH controls surface potential has been the subject of considerable research (Fuerstenau, 1970), and for solid oxides, there is substantial evidence that hydrogen ions determine the potential (Lai, 1970). This is explained by the theory that in air there is a layer of hydroxide on a solid-oxide surface which, on immersion into an aqueous solution, can either give off a proton to form a negative site, MO-, as occurs for amphiboles at neutral pH, or it can adsorb a proton to form a substituted-hydronium ion, MOH, +, as occurs for chrysotiles at neutral pH, where M is a surface cation. By this mechanism, chrysotiles acquire a positive charge not because of the release of surface-hydroxyl ions as has been previously suggested, but because chrysotiles adsorb hydrogen ions. The reduction in zeta potential obtained by adding the amphoteric surfactant
ASBESTOS
SURFACE
CHARGE,
HEMOLYTIC
ACTIVI’I‘\
143
DPPC to suspensions of asbestos is in accord with previous findings that anionic (Harington, Allison, and Badami, 1975; Otouma and Take, 197.5; Gracheva, 1973) and cationic (Prasad and Pooley, 1973) surfactants are capable of reducing the surface charge on chrysotiles and amphiboles, respectively, in some cases to the extent of reversing polarity. The adsorption of DPPC is probably a two-step process consisting of an initial adsorption layer due to coulombic interactions between amphoteric heads of DPPC and charged fibers, and a second layer due to physical adsorption of nonpolar palmitic acid groups of free DPPC to identical groups located on the first layer with the ionic heads directed outward (Otouma and Take, 1975; Fuerstenau, 1971). DPPC was found to be more effective in reducing the zeta potential for amphiboles than for chrysotiles. This suggests that amphiboles could have a greater affinity for other similar phospholipids, possibly those in the cell membrane lipid bilayer, and could be more susceptible to potential reduction in I,~\v than chrysotiles. The present hemolysis experiments confirm previous observations that chrysotiles are strongly hemolytic, B being more active than A, and that amphiboles are less hemolytic, anthophyllite being the most active amphibole. These studies also show that hemolytic activity of amphiboles was more susceptible to reduction by DPPC than that of chrysotiles. This could indicate that amphiboles have fewer hemolytic sites which DPPC must block in order to obtain a reduction in activity. On the other hand, there could be a comparable number of sites for both amphiboles and chrysotiles, but negative-amphibole sites are less effective in inducing hemolysis and have a greater affinity for DPPC and, consequently, are more easily blocked. Results of the zeta-potential studies support the latter explanation since amphiboles and chrysotiles have comparable magnitudes of zeta potential and, thus, a similar number of charge sites. There is an apparent correlation between the magnesium concentration in fibers and hemolytic activity (Harington, Miller, and Macnab, 1971; Harington, Allison, and Badami, 1975). However, strict adherence to this correlation is questionable. While Morgan (1975) found that hemolytic activity of chrysotiles was reduced progressively as magnesium was removed, Desai, Hext, and Richards (1975) found that 20 and 80% magnesium-depleted chrysotiles were still potently hemolytic. Moreover, magnesium is not required for hemolysis by silica and glass fibers. Although magnesium may be involved in determining the ion-adsorption characteristics of fibers, particularly chrysotiles, it is the adsorption or desorption of hydrogen ions which probably gives the fibers their surface potential which seems to be the critical factor in hemolysis by asbestos. Harington, Allison, and Badami (1975) have proposed the following mechanism for hemolysis by chrysotile. In a normal erythrocyte membrane, glycoproteins extend through the entire lipid bilayer and are arranged at random due to mutually repulsive forces of ionized sialic acid groups they contain. However, in the vicinity of chrysotile fibers, glycoproteins are trapped and form clusters by the coulombic interaction of their negatively charged sialic acid groups with positively charged fibers. Although ion permeability through lipid bilayers is very low, ions can readily penetrate regions of the membrane where there are clusters of glycoproteins. The formation of clusters increases passive permeability of small ions
144
LIGHT
AND
WEI
causing a Donnan redistribution of ions and an accumulation of sodium ions and water inside the cell. Eventually this accumulation results in osmotic rupture of the cell. The reverse of this mechanism could be applied to the negatively charged amphiboles. Lipid-bilayer clusters would be formed near fibers due to the coulombic repulsion of negatively charged sialic acid groups with negatively charged amphiboles. This would result in extremely low ion permeability in the vincinity of fibers and a net increase in overall permeability of the membrane distant from fibers. In addition, fiber interactions with membrane phospholipids may be important, possibly those involving lipid peroxidation of fatty acids (Gabor and Anca, 1975). Such fiber-lipid interactions in conjunction with an increase in overall permeability could explain the hemolytic activity of amphiboles as well as other negatively charged particles. If, for both amphiboles and chrysotiles, the crucial interactions with erythrocytes primarily depend on the presence of a fiber-surface potential, then the relative efficacy of a particular mechanism in inducing hemolysis should directly depend on the magnitude of the potential. That is, if the magnitude of the surface potential is decreased, there should be a corresponding proportional decrease in hemolytic activity regardless of the mechanisms. Figure 5 shows that the experimental data are in agreement with this hypothesis and that there is a significant correlation between zeta potential and hemolytic activity for all five UICC reference samples. Compared to chrysotiles. amphiboles had a less effective lysing mechanism and a zeta potential that was more readily reduced by DPPC. This difference in activities and in adsorption properties could explain why amphiboles are less acutely cytotoxic than chrysotiles. However, with carcinogenicity the activity of chrysotiles is reduced relative to that of amphiboles, possibly due to the effect fiber solubility has on surface charge (Morgan, 1975). While amphiboles are stable in V&O over a long periods of time. chrysotiles break down, and there may be leaching of as much as 35% of the structural-magnesium atoms in 1 month (Morgan, Holmes, and Gold. 1971) which consequently reduces surface charge. The observation that amphiboles may be more carcinogenic that chrysotiles could be a result of the predominance of this dissolution effect in chrysotiles. In either acute or chronic toxicity, charge polarity could determine the mechanism of action, and adsorption and dissolution properties could determine the magnitude of charge and, thus, the effectiveness of the mechanism. ACKNOWLEDGMENTS This paper is dedicated to the memory of Professor B. D. Tebbens. Fuerstenau for advice and use of the Zeta Meter and Dr. W. C. Cooper topic. This work was funded by NIOSH Training Grant 5T01-OH-00020.
We thank Professor D. W. for suggesting the research
REFERENCES Allison. A. C. (1971). Effects of silica and asbestos on cells in culture. In “Inhaled Particles, (W. H. Walton, Ed.), Vol. I, pp. 437-445. Unwin. Surrey, England. Desai, R., Hext, P., and Richards, R. (1975). The prevention of asbestos-induced hemolysis. Liff 16, 1931- 1938.
III” Sci.
ASBESTOS
SURFACE
CHANGE,
HEMOLYTIC
ACTIVITI
14.5
Fuerstenau. D. W. (1970). Interfacial processes in mineral/water systems. Pure Appl. Chem. 24, 135- 164. Fuerstenau. D. W. (1971). The adsorption of surfactants at solid-water interfaces. In “The Chemistry of Biosurfaces” (M. L. Hair. Ed.), Vol. 1, pp. 143- 176. Dekker, New York. Gabor. S.. and Anca. Z. (1975). Effect of asbestos on lipid peroxidation in the red cells. Brit. J. Ind. Med. 32, 39-41. Gracheva, 0. I. (1973). Investigation of the effect of surfactants on the properties of chrysotile asbestos. h’olloid Zh. 35, 748-751. Harington. J. S.. Allison, A. C., and Badami, D. V. (1975). Mineral fibers: chemical, physicochemical. and biological properties. Adtwn. Pharmacol. Chemother. 12, 191-402. Harington, J. S., Miller, K., and Macnab. G. (1971). Hemolysis by asbestos. Eul~iron. Rcs. 4, 95-l 17. King, R. J. (1974). The surfactant system of the lung. Fed. Proc. 33, 2238-2246. Klosterkotter, W., and Robock, K. (1975). New aspects of dust and pneumoconiosis research. Amer. Ind. Hyg. As. J. 36, 659-668. Lai, R. W. (1970). Surface charge, adsorption of ionic surfactants, and the wettability of oxide minerals. Ph.D. Thesis. University of California. Berkeley. Martinez. E., and Zucker, G. L. (1960). Asbestos ore body minerals studied by zeta potential measurements. J. Phys. Chrm. 64, 924-926. Miller, K., and Harington, J. S. (1972). Some biochemical effects of asbestos on macrophages. Brit. J. E.rp. Pathol. 53, 397-405. Morgan. A. (1975). The physical and biological properties of the asbestos standard reference samples prepared under the auspices of the UICC. In “Third International Conference on the Physics and Chemistry of Asbestos Minerals.” Abstract Paper 8.36. Lava1 University, Quebec City, Canada. Morgan, A.. Holmes. A., and Gold. C. (1971). Studies of the solubility of constituents of chrysotile asbestos in tsivo using radioactive tracer techniques. Environ. Res. 4, 558-570. Gtouma. T.. and Take. S. (1975). Effect of anionic surface active agents on chrysotile. In “Third International Conference on the Physics and Chemistry of Asbestos Minerals.” Abstract Paper 5.21. Lava1 University, Quebec City, Canada. Prasad, N. A., and Pooley, F. D. (1973). Characteristics of amphibole asbestos dust surfaces in aqueous media with reference to quartz. J. Appl. Chern. Biorechnol. 23. 675-687. Riddick, T. M. (1961). Notes and comment on the zeta potential and ZP techniques. 112 *‘Zeta-Meter Manual,” pp. 43373. Zeta-Meter, Inc., New York. Schnitzer, R. J.. and Pundsack. F. L. (1970). Asbestos hemolysis. E~lviron. Re.\. 3, 1-13. Timbrell, V.. and Rendall. R. E. G. ( 1971- 1972). Preparation of the UICC standard reference samples of asbestos. Ponader Technol. 5, 279-287.
Surface
Charge
and Hemolytic W. G.
Department
LIGHT
of Asbestos
E. T. WEI
AND
of Biomedical and Environmental Health. University of California,
Activity
Health Berkeley,
Sciences, California
School 94720
of Public
Received March 20, 1976 Surface charge. represented by zeta potential, and hemolytic activity were measured for the five asbestos reference samples of the International Union against Cancer (UICC). Zeta potentials in distilled water at pH 7.4 for chrysotiles A and B were +40.5 and +52.5 mV. and for amphiboles crocidolite, amosite, and anthophyllite were -50.4, -58.5, and -54.0 mV, respectively. For fiber samples of equal surface area, chrysotiles had a greater hemolytic activity than amphiboles: 60.5 and 81.9% for chrysotiles A and B compared to 7.7. 9.8, and 15.7% for crocidolite, amosite, and anthophyllite, respectively. A lysing mechanism based on charge polarity is proposed to account for this difference in activity. Zeta potential and hemolytic activity were reduced by adding the main component of pulmonary surfactant. dipalmitoyl phosphatidylcholine (DPPC), to fiber suspensions. DPPC was more effective in decreasing both zeta potential and hemolytic activity for amphiboles than for chrysotiles. On the basis of these reductions, a significant correlation (r = 0.84) was found between zeta potential and hemolytic activity for all five UICC samples. This correlation suggests that surface charge is the principal factor in hemolysis by asbestos. The relevance of surface charge to asbestos toxicity is discussed. INTRODUCTION
The physical and structural properties of fibers are generally considered to be the primary determinants of their biological effects. For asbestos fibers, it has been suggested that the most important properties are surface charge, solubility, surface area, and dimensions of the fibers (Morgan, 1975). The relative significance of solubility, surface area. and dimensions in determining cytotoxic, tibrogenic, and carcinogenic activity of asbestos has been discussed elsewhere (Harington, Allison, and Badami, 1975; Morgan, 1975). In the present investigation, the relationship between surface charge and hemolytic activity was examined for the five UICC reference samples of asbestos. Asbestos fibers, when added to macrophages itz vitro, damage plasma and lysosomal membranes and cause cell death (Allison, 1971; Miller and Harington, 1972; Harington, Allison, and Badami, 1975). This cytotoxicity is more readily obtained with chrysotiles than amphiboles, and it appears that the in viva correlate of this cytotoxic effect is fibrosis (Klosterkotter and Robock, 1975; Morgan, 1975). To study the disruptive effects of asbestos on membranes, a simple in vitro technique based on rupture of the erythrocyte membrane (hemolysis) has been developed (Desai, Hext, and Richards, 1975; Harington, Miller, and Macnab, 1971; Schnitzer and Pundsack, 1970). Using this technique, it has been demonstrated that chrysotiles also have a greater hemolytic activity than amphiboles. It is suspected that the hemolytic activity of asbestos is principally determined by the surface charge the fibers acquire in solution. However, there have been virtually no studies in which surface charge was measured directly and related to biological activity. In the present study, surface charge was represented by zeta 135 Copyrtghl Q 1977 by Academic Press. inc. All rights of reproduction in any form reserved.
ISSN 0013-9351
136
LIGHT
AND
WEI
potential which is a measure of the potential energy difference between the bulk solution and the boundary separating solvent adhering to a fiber and free solvent and, although it is smaller in magnitude, approximates total surface potential. The effect of dipalmitoyl phosphatidylcholine (DPPC), the main component of pulmonary surfactant, on zeta potential and hemolytic activity was also examined since it has been shown that pulmonary surfactant (Desai, Hext, and Richards, 1975) and bronchial secretions (Allison, 1971) can reduce the hemolytic activity of asbestos. The adsorption of DPPC by fibers may have significance in viva since DPPC is present at the alveolar air-liquid interface (King, 1974) and would adsorb onto surfaces of inhaled fibers. MATERIALS
AND METHODS
Reagents The five UICC standard reference samples of asbestos, kindly supplied by the MRC Pneumoconiosis Unit, Penarth, South Wales, and the Johns-Manville Research and Development Center, Denver, were used in all experiments. The preparation of these samples has been described by Timbre11 and Rendall (1971- 1972). Synthetic dipalmitoyl L-a-phosphatidylcholine was obtained from the Sigma Chemical Company, St. Louis. Sterile sheep erythrocytes in the form of equal parts of whole blood and modified Alsever’s solution were acquired from Microbiological Media, Concord, California. Prior to use, erythrocytes were suspended in sterile Tyrode’s solution (prepared with sodium replacing calcium and adjusted to pH 7.4) and washed three times. After each washing, the suspensions were centrifuged for 10 minutes at 1500 rpm. Standard 1.5% erythrocyte suspensions were prepared by combining 1.5 ml of packed erythrocytes with 100 ml of sterile Tyrode’s solution. Zeta-Potential Studies Ten-milligram samples of asbestos were uniformly dispersed in bottles containing 100 ml of triple-distilled water. DPPC was added and the suspensions were adjusted to appropriate pH values using 1O-2 M HCl or 1O-2 M NaOH. Samples were then incubated for 1 day at between 41” (the melting point of DPPC) and 43°C in a shaker bath. Following this equilibration period, the samples were cooled to room temperature (23°C) and the pH was measured. Zeta potentials were determined using a microelectrophoresis instrument commercially known as a Zeta Meter (Zeta-Meter, Inc.). A Zeta Meter cell (#G-T248) was filled with 35-ml aliquots of the fiber suspensions and an electric field was applied. The movement of fibers at least 0.1 pm in size was then observed through a stereoscopic microscope. The time required for 15 to 25 fibers each to traverse 1 ocular-micrometer division was recorded and the average time calculated. Fiber velocity (prn/sec) in a given electric field (volt/cm), the electrophoretic mobility, was determined. Zeta potential was calculated from electrophoretic mobility using the Helmholtz-Smoluchowski equation (Riddick, 1961). Zeta potential was not converted to surface charge because the term zeta potential readily conveys the concept of charge. The decrease in zeta potential for fibers
ASBESTOS
SCRFACE
CHARGE,
HEMOLYTIC
ACTIVITY
treated with DPPC was expressed as a percentage of the zeta potential obtained when no DPPC was added to the fiber suspensions: % decrease in ZP =
137 (ZP)
x 100. -ZP fibers treated with DPPC ZP untreated fibers >
In the zeta-potential experiments, distilled water was used as the suspending liquid rather than Tyrode’s solution because inorganic solutions of high molarity and liquid conductance generate excessive heat. Heat generation at concentrations above 0.01 M can result in convection currents which cause charged particles to deviate from the linear path of an electric field. Hemolysis Studies The experimental procedures used here were similar to those described by others (Desai, Hext, and Richards, 1975: Harington, Miller, and Macnab, 1971; Schnitzer and Pundsack, 1970). Asbestos fibers in pre-weighed amounts were placed in lo-ml tubes. To each tube. 2 ml of suspending liquid (Tyrode’s solution or Tyrode’s solution containing DPPC) were added. A vortex mixer was used to ensure uniform distribution of fibers in the suspending liquid. Following a l-hour incubation period at 41-43”C, the tubes were cooled to room temperature (23°C) and 2 ml of 1.5% erythrocyte suspension were added to each tube. The tubes were sealed with Parafilm-covered corks and inverted twice every 10 minutes for 1 hour. The samples were then centrifuged at 2000 rpm for 20 minutes. Supernatant solutions were transferred from the tubes using a 5-ml syringe. The tilled syringe was attached to a Millipore Swinnex-25 filter holder containing a filter of 0.22~pm pore size, and the supematant was filtered to remove any fibers that may have been taken up by the syringe. Filters were prewashed with the supernatant of a centrifuged erythrocyte suspension to avoid excessive adsorption of hemoglobin onto clean filters. The optical densities of the filtered supematant solutions and controls were read at 541 nm in a Perkin-Elmer. Model 54, Coleman Digital Spectrophotometer. UICC chrysotiles have a specific surface area approximately two times greater than that of anthophyllite and four times greater than that of other amphiboles (Morgan, 1975). To adjust for these differences in surface area as well as obtain measurable hemolytic activities, fibers were used in the following quantities: 5 mg of chrysotiles, 10 mg of anthophyllite, and 20 mg of crocidolite or amosite. Tubes not containing fibers functioned as the three types of controls used in the experiments: totally lysed control (2 ml erythrocyte suspension plus 2 ml triple-distilled water), fragility control (2 ml of erythrocyte suspension plus 2 ml of Tyrode’s solution or Tyrode’s solution containing DPPC), and Tyrode’s solution control. Six replicate tubes were used for each suspending liquid and fiber type. The hemolytic activity of asbestos was calculated from the average optical density (OD) of the six replicates and was expressed as a percentage of the totally lysed control OD: Hemolytic
activity
=
average OD - fragility control OD x 100. lysed control OD - Tyrode’s solution OD >
138
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The decrease in hemolytic activity of the fibers treated with DPPC was expressed as a percentage of the hemolytic activity (HA) obtained when no DPPC was added to the fiber suspensions: % decrease in HA =
-HA
fibers treated with HA untreated fibers RESULTS
Zeta potentials for the five UICC samples were plotted as a function of pH in Figs. 1 and 2. At pH 7.4, zeta potentials in distilled water for chrysotiles A and B were +40.5 and +52.5 mV, and for amphiboles crocidolite, amosite, and anthophyllite were -50.5, -58.5, and -54.0 mV, respectively. These results clearly illustrate that zeta potentials for the two types of asbestos were similar in absolute magnitude, but opposite in polarity. The data plotted in Fig. 2 were indicative of the two types of asbestos and show that for both types the addition of DPPC to fiber suspensions produced a dose-dependent decrease in the magnitude of zeta potential. To compare the relative effects of the surfactant DPPC on the zeta potentials for the five different fibers, a dimensionless variable, mg DPPC/mg fibers, was defined and plotted against the decrement in zeta potential produced by DPPC (Fig. 3). The data show that the relative efficacy of DPPC in reducing zeta potential can
-30
ANTHOPnYLLlTE
t
FIG. function
1. Zeta potentials of pH.
for UICC
chrysotile
B, amosite,
and anthophyllite
in distilled
water
as a
ASBESTOS
SlJRFACE
FIN;. 2. Zeta potentials for IO mg of UICC of pH and in the presence of the surfactant
CHARCE,
chrysotile dipalmitoyl
HEMOLYTIC
AC’I‘IVI
I‘Y
A and crocidolite in distilled water phosphatidylcholine (DPPC).
139
as a function
be ranked as crocidolite > amosite > anthophyllite > chrysotile A > chrysotile B. At a ratio of 0.1, for example, zeta potentials for amosite and crocidolite were decreased by about 74%, while zeta potentials for chrysotiles A and B were only decreased by about 11%. After taking into account differences in specific surface area, it was found that zeta potentials for amphiboles were still more readily reduced by DPPC than those for chrysotiles. DPPC was more effective in reducing the zeta potential for amosite and crocidolite than for anthophyllite because anthophyllite has a surface area approximately twice as great as the other amphiboles. For fiber samples of equal surface area, chrysotiles had a greater hemolytic activity than amphiboles. The relative activities in Tyrode’s solution were the following: chrysotile B (81.9%) > chrysotile A (60.5%) > anthophyllite (15.7%) > amosite (9.8%) > crocidolite (7.7%). When DPPC was added to fiber suspensions, hemolytic activity decreased for all five UICC samples (Fig. 4). At a ratio of 0.1 for example, hemolytic activities for amosite and crocidolite were decreased by about 87% while hemolytic activities for chrysotiles A and B were decreased by about 25%. On the basis of fiber samples of either equal mass or equal surface area, DPPC was more antihemolytic for amphiboles than for chrysotiles. Thus, the relative efficacy of DPPC in decreasing hemolytic activity paralleled its relative efficacy in reducing zeta potential.
140
LIGHT I
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I
I
I
0 IJ n B 0
I
’
CHRYSOTILE A CHRYSOTILE B AMOSITE CROCIDOLITE ANTHOPHYLLITE
s
4:
0.1
0.2 SURFACTANT/
FIG. 3. The effect of the surfactantifiber UICC samples at pH 7.4.
ratio
Fl&?S
I
3 ? 0
I
0.1
FIG. 4. The effect ofthe UICC samdes.
surfactantkber
1
0.2
I.0
h&i4
on the percentage
1
I
decrease
in zeta potential
for the five
AMOSITE CROClOOLlTE ANTHOPHYLLITE
L
0.3 0.4 0.5 SURFACTANT / FBERS
ratio on the percentage
I
0.6 (mglmg)
decrease
I
I
0.7
0.8
in hemolytic
activity
for the five
ASBESTOS
SURFACE
CHARGE,
HEMOLYTIC
141
ACTIVITY
When either chrysotiles or amphiboles were added to neutral solutions, an increase in pH occurred. To demonstrate sufficiently whether surface charge is the principal factor in asbestos hemolysis, it was necessary to determine what effect these shifts in pH had on hemolytic activity. Increases in pH for 0.01% neutral suspensions of chrysotile B were measured after 1 day of aging and used as a measure of all fibers since chrysotile B resulted in the largest shifts. While an increase of 2.5 pH units was obtained in distilled water, an increase of only 0.2 pH units was measured in Tyrode’s solution (due to the action of effective bicarbonate and phosphate buffers), and in Tyrode’s solution diluted to 10% of normal concentration (comparable to the ratio of buffer/fibers used in hemolysis experiments) an increase of 0.5 pH units was observed. To test the effect this 0.5 pH unit increase has on hemolytic activity, erythrocyte fragility was measured in Tyrode’s solution adjusted to pH 7.4 and to pH 7.9. No significant difference in hemolysis was observed between these two pH values. An antihemolytic action of surfactant could be exerted directly on the erythrocyte membrane rather than on the surface of the asbestos fiber. To examine this possibility, erythrocyte fragility was measured in Tyrode’s solutions diluted to 10, 50, and 90% of normal concentration and containing DPPC. The surfactant did not protect cells from osmotic rupture; in fact, its presence may have slightly enhanced hemolysis. These results further indicate that the antihemolytic effect of surfactant is secondary to its interaction with the asbestos fiber.
90 > 5
3
80 : V 0 0
F ::
CHRYSOTILE A CHRYSOTILE B AMOSITE CROCIOOLITE ANTHOPHYLLITE GLASS FIBERS
0
70 2 I> 6
60 I-
/
-I
E I z
50
1
1 GO
PERCENTAGE
FIG. 5. The correlation between in zeta potential for the five UICC fiber ratios (P < 0.01, Y = 0.84).
DECREASE
IN
ZETA
POTENTIAL
percentage decrease in hemolytic activity and percentage dec :rease samples and glass fibers at pH 7.4 on the basis of equal surfactanti
142
LIGHT
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To summarize the relationship between zeta potential and hemolytic activity, the data in Figs. 3 and 4 were combined to yield Fig. 5. A least-squares regression analysis showed that the two variables were significantly correlated (P < 0.01, r = 0.84) for all five UICC samples. Thus, even though zeta potentials for amphiboles and chrysotiles were opposite in polarity, the net effect of DPPC was the same for both types: a decrease in hemolytic activity proportional to a decrease in zeta potential. It was of interest to determine whether other fibers had hemolytic characteristics similar to those of asbestos fibers. In preliminary experiments, the zeta potential (-53.5 mV) and hemolytic activity (28.3% for lo-mg fibers) for “code 100 microfiber, 475 glass” (Johns-Manville Research and Development Center) were measured. The data suggest that the hemolytic activity of glass fibers, like that of asbestos fibers, may be strongly dependent on the zeta potential.
DISCUSSION
Zeta potentials calculated for the five UICC reference samples in distilled water at pH 7.4 are consistent with previously published results. Correcting the original value of Martinez and Zucker (1960), recognized as being high by a factor of 2 (Harington, Allison, and Badami, 1975), gave a value of +46.5 mV for the equilibrium zeta potential of chrysotile. For MgO, the oxide on the surface of chrysotile, Lai (1970) measured an electrophoretic mobility corresponding to a zeta potential of +50.9 mV. These two values are comparable to the present results of +40.5 and +52.5 mV for chrysotiles A and B, respectively. Zeta potentials for amphiboles (-50.5, -54.0, and -58.5 mV for crocidolite, anthophyllite, and amosite, respectively) are in accord with those calculated by Prasad and Pooley (1973) for amosite and crocidolite of approximately -50 mV for both fibers in 1O-6 N Ca(NO,), and -45 mV for both fibers in lo-* N NaCl. Although the values they obtained in distilled water were lower in magnitude, it would be expected that in distilled water the zeta potentials would be greater since the lower the ionic strength of an indifferent electrolyte, the higher the zeta potential. For silica, Lai (1970) obtained an electrophoretic mobility corresponding to a zeta potential of -63.6 mV. This zeta potential probably is greater in magnitude than that for amphiboles because the negative surface-charge density of amphiboles is less than if the entire fiber surface were silica. How pH controls surface potential has been the subject of considerable research (Fuerstenau, 1970), and for solid oxides, there is substantial evidence that hydrogen ions determine the potential (Lai, 1970). This is explained by the theory that in air there is a layer of hydroxide on a solid-oxide surface which, on immersion into an aqueous solution, can either give off a proton to form a negative site, MO-, as occurs for amphiboles at neutral pH, or it can adsorb a proton to form a substituted-hydronium ion, MOH, +, as occurs for chrysotiles at neutral pH, where M is a surface cation. By this mechanism, chrysotiles acquire a positive charge not because of the release of surface-hydroxyl ions as has been previously suggested, but because chrysotiles adsorb hydrogen ions. The reduction in zeta potential obtained by adding the amphoteric surfactant
ASBESTOS
SURFACE
CHARGE,
HEMOLYTIC
ACTIVI’I‘\
143
DPPC to suspensions of asbestos is in accord with previous findings that anionic (Harington, Allison, and Badami, 1975; Otouma and Take, 197.5; Gracheva, 1973) and cationic (Prasad and Pooley, 1973) surfactants are capable of reducing the surface charge on chrysotiles and amphiboles, respectively, in some cases to the extent of reversing polarity. The adsorption of DPPC is probably a two-step process consisting of an initial adsorption layer due to coulombic interactions between amphoteric heads of DPPC and charged fibers, and a second layer due to physical adsorption of nonpolar palmitic acid groups of free DPPC to identical groups located on the first layer with the ionic heads directed outward (Otouma and Take, 1975; Fuerstenau, 1971). DPPC was found to be more effective in reducing the zeta potential for amphiboles than for chrysotiles. This suggests that amphiboles could have a greater affinity for other similar phospholipids, possibly those in the cell membrane lipid bilayer, and could be more susceptible to potential reduction in I,~\v than chrysotiles. The present hemolysis experiments confirm previous observations that chrysotiles are strongly hemolytic, B being more active than A, and that amphiboles are less hemolytic, anthophyllite being the most active amphibole. These studies also show that hemolytic activity of amphiboles was more susceptible to reduction by DPPC than that of chrysotiles. This could indicate that amphiboles have fewer hemolytic sites which DPPC must block in order to obtain a reduction in activity. On the other hand, there could be a comparable number of sites for both amphiboles and chrysotiles, but negative-amphibole sites are less effective in inducing hemolysis and have a greater affinity for DPPC and, consequently, are more easily blocked. Results of the zeta-potential studies support the latter explanation since amphiboles and chrysotiles have comparable magnitudes of zeta potential and, thus, a similar number of charge sites. There is an apparent correlation between the magnesium concentration in fibers and hemolytic activity (Harington, Miller, and Macnab, 1971; Harington, Allison, and Badami, 1975). However, strict adherence to this correlation is questionable. While Morgan (1975) found that hemolytic activity of chrysotiles was reduced progressively as magnesium was removed, Desai, Hext, and Richards (1975) found that 20 and 80% magnesium-depleted chrysotiles were still potently hemolytic. Moreover, magnesium is not required for hemolysis by silica and glass fibers. Although magnesium may be involved in determining the ion-adsorption characteristics of fibers, particularly chrysotiles, it is the adsorption or desorption of hydrogen ions which probably gives the fibers their surface potential which seems to be the critical factor in hemolysis by asbestos. Harington, Allison, and Badami (1975) have proposed the following mechanism for hemolysis by chrysotile. In a normal erythrocyte membrane, glycoproteins extend through the entire lipid bilayer and are arranged at random due to mutually repulsive forces of ionized sialic acid groups they contain. However, in the vicinity of chrysotile fibers, glycoproteins are trapped and form clusters by the coulombic interaction of their negatively charged sialic acid groups with positively charged fibers. Although ion permeability through lipid bilayers is very low, ions can readily penetrate regions of the membrane where there are clusters of glycoproteins. The formation of clusters increases passive permeability of small ions
144
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causing a Donnan redistribution of ions and an accumulation of sodium ions and water inside the cell. Eventually this accumulation results in osmotic rupture of the cell. The reverse of this mechanism could be applied to the negatively charged amphiboles. Lipid-bilayer clusters would be formed near fibers due to the coulombic repulsion of negatively charged sialic acid groups with negatively charged amphiboles. This would result in extremely low ion permeability in the vincinity of fibers and a net increase in overall permeability of the membrane distant from fibers. In addition, fiber interactions with membrane phospholipids may be important, possibly those involving lipid peroxidation of fatty acids (Gabor and Anca, 1975). Such fiber-lipid interactions in conjunction with an increase in overall permeability could explain the hemolytic activity of amphiboles as well as other negatively charged particles. If, for both amphiboles and chrysotiles, the crucial interactions with erythrocytes primarily depend on the presence of a fiber-surface potential, then the relative efficacy of a particular mechanism in inducing hemolysis should directly depend on the magnitude of the potential. That is, if the magnitude of the surface potential is decreased, there should be a corresponding proportional decrease in hemolytic activity regardless of the mechanisms. Figure 5 shows that the experimental data are in agreement with this hypothesis and that there is a significant correlation between zeta potential and hemolytic activity for all five UICC reference samples. Compared to chrysotiles. amphiboles had a less effective lysing mechanism and a zeta potential that was more readily reduced by DPPC. This difference in activities and in adsorption properties could explain why amphiboles are less acutely cytotoxic than chrysotiles. However, with carcinogenicity the activity of chrysotiles is reduced relative to that of amphiboles, possibly due to the effect fiber solubility has on surface charge (Morgan, 1975). While amphiboles are stable in V&O over a long periods of time. chrysotiles break down, and there may be leaching of as much as 35% of the structural-magnesium atoms in 1 month (Morgan, Holmes, and Gold. 1971) which consequently reduces surface charge. The observation that amphiboles may be more carcinogenic that chrysotiles could be a result of the predominance of this dissolution effect in chrysotiles. In either acute or chronic toxicity, charge polarity could determine the mechanism of action, and adsorption and dissolution properties could determine the magnitude of charge and, thus, the effectiveness of the mechanism. ACKNOWLEDGMENTS This paper is dedicated to the memory of Professor B. D. Tebbens. Fuerstenau for advice and use of the Zeta Meter and Dr. W. C. Cooper topic. This work was funded by NIOSH Training Grant 5T01-OH-00020.
We thank Professor D. W. for suggesting the research
REFERENCES Allison. A. C. (1971). Effects of silica and asbestos on cells in culture. In “Inhaled Particles, (W. H. Walton, Ed.), Vol. I, pp. 437-445. Unwin. Surrey, England. Desai, R., Hext, P., and Richards, R. (1975). The prevention of asbestos-induced hemolysis. Liff 16, 1931- 1938.
III” Sci.
ASBESTOS
SURFACE
CHANGE,
HEMOLYTIC
ACTIVITI
14.5
Fuerstenau. D. W. (1970). Interfacial processes in mineral/water systems. Pure Appl. Chem. 24, 135- 164. Fuerstenau. D. W. (1971). The adsorption of surfactants at solid-water interfaces. In “The Chemistry of Biosurfaces” (M. L. Hair. Ed.), Vol. 1, pp. 143- 176. Dekker, New York. Gabor. S.. and Anca. Z. (1975). Effect of asbestos on lipid peroxidation in the red cells. Brit. J. Ind. Med. 32, 39-41. Gracheva, 0. I. (1973). Investigation of the effect of surfactants on the properties of chrysotile asbestos. h’olloid Zh. 35, 748-751. Harington. J. S.. Allison, A. C., and Badami, D. V. (1975). Mineral fibers: chemical, physicochemical. and biological properties. Adtwn. Pharmacol. Chemother. 12, 191-402. Harington, J. S., Miller, K., and Macnab. G. (1971). Hemolysis by asbestos. Eul~iron. Rcs. 4, 95-l 17. King, R. J. (1974). The surfactant system of the lung. Fed. Proc. 33, 2238-2246. Klosterkotter, W., and Robock, K. (1975). New aspects of dust and pneumoconiosis research. Amer. Ind. Hyg. As. J. 36, 659-668. Lai, R. W. (1970). Surface charge, adsorption of ionic surfactants, and the wettability of oxide minerals. Ph.D. Thesis. University of California. Berkeley. Martinez. E., and Zucker, G. L. (1960). Asbestos ore body minerals studied by zeta potential measurements. J. Phys. Chrm. 64, 924-926. Miller, K., and Harington, J. S. (1972). Some biochemical effects of asbestos on macrophages. Brit. J. E.rp. Pathol. 53, 397-405. Morgan. A. (1975). The physical and biological properties of the asbestos standard reference samples prepared under the auspices of the UICC. In “Third International Conference on the Physics and Chemistry of Asbestos Minerals.” Abstract Paper 8.36. Lava1 University, Quebec City, Canada. Morgan, A.. Holmes. A., and Gold. C. (1971). Studies of the solubility of constituents of chrysotile asbestos in tsivo using radioactive tracer techniques. Environ. Res. 4, 558-570. Gtouma. T.. and Take. S. (1975). Effect of anionic surface active agents on chrysotile. In “Third International Conference on the Physics and Chemistry of Asbestos Minerals.” Abstract Paper 5.21. Lava1 University, Quebec City, Canada. Prasad, N. A., and Pooley, F. D. (1973). Characteristics of amphibole asbestos dust surfaces in aqueous media with reference to quartz. J. Appl. Chern. Biorechnol. 23. 675-687. Riddick, T. M. (1961). Notes and comment on the zeta potential and ZP techniques. 112 *‘Zeta-Meter Manual,” pp. 43373. Zeta-Meter, Inc., New York. Schnitzer, R. J.. and Pundsack. F. L. (1970). Asbestos hemolysis. E~lviron. Re.\. 3, 1-13. Timbrell, V.. and Rendall. R. E. G. ( 1971- 1972). Preparation of the UICC standard reference samples of asbestos. Ponader Technol. 5, 279-287.