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Effect of lead on electrophoretic mobility of rat erythrocytes
Toxicology, 40 (1986) 259--265 Elsevier Scientific Publishers Ireland Ltd.
E F F E C T OF L E A D ON E L E C T R O P H O R E T I C M O B I L I T Y O F R A T ERYTHROCYTES
KAZUYUKI TERAYAMA a'* , NAOKI MAEHARA a, MAKOTO MURATSUGU b, MIKIO MAKINO b and KOHTAROH YAMAMURA a aDepartment of Hygiene, Asahikawa Medical College and bClinical Laboratory, Asahikawa Medical College Hospital, 3-11, 4-5 Nishikagura, Asahikawa, Hokkaido, 078-11 (Japan) (Received November 1st, 1985) (Accepted March 1st, 1986)
SUMMARY Lead o f t e n affects the e r y t h r o c y t e m e m b r a n e . T h e relationship b e t w e e n the changes in e r y t h r o c y t e m e m b r a n e and t h e a n e m i a caused b y lead is still unclear. Initially, the e f f e c t o f lead injected i n t r a p e r i t o n e a l l y o n the electrop h o r e t i c m o b i l i t y o f rat e r y t h r o c y t e s was investigated in o r d e r to s t u d y the relationship b e t w e e n t h e m . As indices o f lead e x p o s u r e , h e m o g l o b i n (Hb) levels, h e m a t o c r i t s (Ht), 5 -aminolevulinic acid d e h y d r a t a s e (ALA-D) activities and b l o o d lead ( b l o o d Pb) levels in the injected rats were also e x a m i n e d . E x p o s u r e t o lead significantly d e c r e a s e d the m o b i l i t y o f rat e r y t h r o c y t e s . The changes in m o b i l i t y s e e m e d t o be less sensitive t h a n those in ALA-D activity, h o w e v e r , t h e decreases in m o b i l i t y were simultaneous with or prior to t h o s e in Hb level and Ht. T h e decreases in m o b i l i t y were evident to some e x t e n t b e l o w a b l o o d Pb level o f 100 t~g/100 ml and generally present at a level o f 100 t~g/100 ml and over. In t h e rats e x p o s e d t o lead a significant negative c o r r e l a t i o n was f o u n d b e t w e e n the mobilities and the logarithms o f b l o o d Pb level.
Key
words," Lead; Blood; E r y t h r o c y t e ; E l e c t r o p h o r e t i c m o b i l i t y ; A n e m i a
INTRODUCTION
The e l e c t r o p h o r e t i c m o b i l i t y o f m a m m a l i a n red b l o o d cells is d e t e r m i n e d b y certain surface characteristics o f the cells and h e n c e is o f value in s t u d y i n g the c o n s t i t u t i o n o f t h e cell surface [1]. Providing t h a t the m e a s u r e m e n t is
*To whom correspondence should be addressed. 0300-483x/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
259
c o n d u c t e d under certain conditions (medium, temperature, pH, ionic strength, salt species and isotonicity), the mobility of normal human red cells is indep en d en t of race, sex, age and blood group [2,3]. For a given species, the mobilities of e r y t h r o c y t e s from respective animals seem to show little variation among individuals [1,4,5]. It is well known that lead exerts influences on the e r y t h r o c y t e membrane. For example, the osmotic fragility of a leaded red cell is decreased; by contrast, the mechanical fragility is much increased [6]. The e n h a n c e m e n t of potassium loss from red cells [ 7 ] and the reduction of Na*/K*-ATPase activity in the e r y t h r o c y t e membrane [8] were observed in comparative studies of lead-exposed and non-exposed workers. Some of these e r y t h r o c y t e m em brane p h e n o m e n a caused by lead may be concerned with shortening of e r y t h r o c y t e survival times [9]. The anemia frequently observed in lead poisoning is t h o u g h t to result from a shortening of e r y t h r o c y t e survival times in combination with disturbed hemoglobin for m at i on [6,9 11]. However, the exact mechanism whereby lead causes the shortening of e r y t h r o c y t e survival times is not fully understood [9]. Results presented by Durocher et al. [5] suggest that shortening of e r y t h r o c y t e survival times is related to a decrease in the electrophoretic mobility of erythrocytes. In the present study, we investigated the effects of lead administration on the electrophoretic mobility of rat er yt h rocyt es, and also examined the relationship between the changes in mobility and blood lead levels as well as other toxicological indices of lead exposure. MATERIALS AND METHODS Animal treatment and sample collection T w e n t y - f o u r male albino rats (Wistar strain) 6 weeks of age were housed in groups of 3 or 4 in a r oom maintained at a t em perat ure of 24 +- I°C and a relative humidity of 55 -+ 5% with a 12 h light (0700--1900 h)/dark {1900-0700 h) cycle. T hey were fed a commercial laboratory pelleted diet (CE-2, Clea Japan Inc., Japan) and tap water ad libitum. At 10 weeks of age, the rats were divided into a control group (Group C, n = 6) and 2 dosed groups (Group A, n = 8; Group B, n = 10) based on b o d y weight, hematocrit (Ht), hemoglobin (Hb) level, 5-aminolevulinic acid dehydratase (ALA-D) activity and electrophoretic mobility of e r y t h r o c y t e s measured prior to the administration of lead. The administration of lead was started when the rats were 11 weeks old. Either a 10 or 20 mM lead acetate (Kishida Chemicals, Japan) solution was injected intraperitoneally once a week for 5 weeks; the volume of each injection was I ml/100 g of b o d y weight. Namely, 100 and 200 t~mol/kg of lead were administered to the rats in Group A and B, respectively. The solution was freshly prepared with distilled, de-ionized boiled water for each injection. The control rats were injected with 1 ml/100 g of 145 mM sodium chloride solution. All rats were injected at 1000 -1100 h.
260
Blood samples were obtained from the rat tail vein by tip incision without anesthetic. About 24 h after each injection, 500 pl of blood was collected in a lead-free heparinized glass tube and placed in an iced water bath. Fifty microliters of this blood was centrifuged at 1500 g for 10 min immediately after collection and the erythrocytes were washed 3 times in 10 ml of cold isotonic buffer, made up of 145 mM sodium chloride and 0.3 mM sodium bicarbonate (approx. pH 7.2) [3]. The washed erythrocytes were suspended in 100 t~l of the isotonic buffer, kept at 4°C and used for electrophoretic measurement within 24 h after collection. The measurements of Ht, Hb level and ALA-D activity were performed within 3 h after blood collection. The remaining blood samples (approx. 200 pl each) were frozen at - 2 0 ° C for subsequent blood lead {blood Pb) measurement. Ht and Hb level Ht was measured by the microcapillary tube method, and the Hb level was measured by the cyanmethemoglobin m e t h o d [12]. A L A - D activity As an indicator of lead exposure, ALA-D activity in blood was assayed using the European standardized m e t h o d [13] with slight modification, i.e. duplicate 50-pl blood aliquots of each blood sample were used for the determination of ALA-D activity and another 50-~1 aliquots for the blank. Electrophoretic mobility Electrophoretic mobility of erythrocytes was measured by a microelectrophoretic apparatus (Apparatus Mark II, Rank Brothers, England). Five microliters of the erythrocyte suspension was diluted with a 10-ml solution containing 4 volumes of 5% sorbitol and 1 volume of 67 mM Sorensen's buffer (pH 7.2) [5] and transferred to an electrophoretic rectangular cuvette kept at 25 -+ 0.1°C. The mobilities of 10 cells per experimental point were recorded; the erythrocytes were timed over 171.4 t~m (85.7 pm in each direction) at the front stationary layer under 6.4 V/cm of potential gradient. The mobilities obtained were corrected for the viscosity of the medium which was determined by an Ostwald viscometer. Blood Pb level Blood Pb analysis was performed by using a Zeeman-effect atomic absorption spectrophotometer equipped with a graphite furnace and a deuterium background corrector (Model 180-80, Hitachi Co., Japan). A mixture of nitric acid/perchloric acid was used for the wet digestion of the conserved blood samples, and a standard addition m e t h o d was applied for the determination to eliminate matrix interferences [ 14,15 ]. Statistics Student's t-test and linear regression analysis were used.
261
RESULTS
Injected lead significantly reduced the body weights of rats in Group A and B after the 2nd and 1st injections, respectively. Four rats died during this experiment: 1 in Group A and 3 in Group B after the 1st or 2nd injection. The death seemd to be induced not by the injection per se but by the lead effect, as the injection of sodium chloride had no appreciable effect on
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Fig. 1. Changes in mean e l e c t r o p h o r e t i c m o b i l i t y of e r y t h r o c y t e s , b l o o d Pb level, Hb level, Ht and ALA-D activity of rats in G r o u p B (o). Each vertical bar r e p r e s e n t s s t a n d a r d deviation. The rats were injected i n t r a p e r i t o n e a l l y with 200 p m o l / k g o f lead at the 0th, 7th, 14th, 21st and 28th days, respectively. The c o n t r o l rats were injected with 1 ml/ 100 g of 145 mM s o d i u m chloride s o l u t i o n in the same manner. The data ( × ) in none x p o s e d ( c o n t r o l and p r e - e x p o s e d ) rats are also indicated in the figure. The symbols, * and ** indicate significant d i f f e r e n c e s f r o m c o n t r o l at the levels of P < 0.05 and P < 0.01, respectively.
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Fig. 2. R e l a t i o n s h i p b e t w e e n t h e e l e c t r o p h o r e t i c mobilities o f rat e r y t h r o e y t e s and the b l o o d Pb level. The regression line o f m o b i l i t y o n logarithms o f b l o o d Pb o f rats e x p o s e d to 100 ( ) and 200 (o) u m o l / k g o f lead at each injection is s h o w n as a solid line. The data ( × ) in n o n - e x p o s e d ( c o n t r o l and p r e - e x p o s e d ) rats are also i n d i c a t e d in the figure.
the control rats except for a slight loss of body weights at the 1st day after the 1st injection. Figure 1 shows ~he effects of lead on the electrophoretic mobility of rat erythrocytes and other parameters (blood Pb, Hb level, Ht and ALA-D activities). The values of 200 pmol/kg dose and the control are shown in the Fig. 1. The results of 100 pmol/kg dose were similar to those of 200/~mol/kg dose but of lower magnitude. The mobility of erythrocytes in Group B significantly decreased after the 1st injection compared with the control (P < 0.01). The mean blood Pb level in Group B showed a marked increase in proportion to the a m o u n t of injected lead, which was nearly 2-fold the level in Group A at each stage after injection except the 1st one. The Hb level and Ht in Group B significantly decreased after the 2nd and 3rd injections, respectively. The injected lead significantly inhibited the ALA-D activities in Group B after the 1st injection, and inhibition after the 2nd and 5th injections was almost complete. In Group A, significant decreases in the mobility, Hb level and Ht were observed after the 3rd injection, whereas decreases in the ALAD activities were found even after the 1st and 2nd injections. Figure 2 shows the relationship between the electrophoretic mobilities of rat erythrocytes and the blood Pb level. The mobilities in non-exposed {control and pre-exposed) rats (X) showed an almost constant value (2.52 +0.02 pm/s per V per cm) corresponding to blood Pb levels of I -12 pg/100 ml. In the rats exposed to 100 (o) and 200 (o) pmol/kg of lead, decreases in mobility were evident to some extent below a blo~)d Pb level of 100 pg/ 100 ml and generally present at a level of 100 pg/100 ml and over. A signifi-
263
cant negative correlation between the mobilities in the rats (o and . ) exposed to lead and the logarithms of blood Pb level was also f o u n d (r = - 0 . 6 0 0 , P < 0.01). DISCUSSION Our results clearly show that lead exposure decreased the mobility of rat erythrocytes. The decreases in mobility were simultaneous with or prior to those in Hb level and Ht. However, the changes in mobility seemed to be a less sensitive indicator than those in ALA-D activity, which is one of the major indicators of lead exposure [16]. We also investigated mobility in relation to blood Pb levels. Our blood Pb levels after the 1st to 3rd injections of 200 pm ol / kg of lead were similar to a recent report [17] of experiments c o n d u c t e d with almost the same conditions as used in this study. The decreases in mobility were evident to some e x t e n t below a blood Pb level of 100 pg/100 ml and generally present at a level of 100 pg/100 ml and over. F u r t h e r m o r e , a significant negative correlation between the mobilities in rats exposed to lead and the logarithms of blood Pb level was observed. The electrophoretic mobility of e r y t h r o c y t e s is determined by the negative surface charge density of the e r y t h r o c y t e s [18], and the carboxyl groups of sialic acid in the m e m br a ne are mainly responsible for the surface charge [4]. Some investigators have r e por t ed that old e r y t h r o c y t e s showed a decreased mobility compared with young cells [19 -21]. According to Durocher et al. [5], desialylated rat e r y t h r o c y t e s treated with neuraminidase showed a markedly shortened survival time as well as a decrease in cell mobility. This result suggests t hat a decrease in mobility of e r y t h r o c y t e s is related to a shortening of e r y t h r o c y t e survival times [5]. Furt herm ore, others have observed a shortening of e r y t h r o c y t e survival times in lead poisoning [6,10,11 ]. The anemia frequently observed in lead poisoning is t hought to result from shortening of e r y t h r o c y t e survival times in combination with inhibition of hemoglobin synthesis [6,9 -11]. In the present study, decreases in electrophoretic mobility of e r y t h r o c y t e s were observed in the rats exposed to lead. Therefore, one may speculate that a decrease in mobility is related to a shortening of e r y t h r o c y t e survival time and a subsequent decrease in the num be r of red cells even in anemia caused by lead. ACKNOWLEDGEMENTS The authors wish to thank Dr. K. Ichihara (Dept. of Pharmacology, Asahikawa Medical College) for his technical help. We are also indebted to Dr. N. Sugawara (Dept. of Public Health, Sapporo Medical College) and to Mr. E. Uchino (Hokkaido Institute of Public Health) for their useful advice o n measurement of blood lead. We are also obliged to Mr. J.A. Sunley for correcting the manuscript.
264
REFERENCES 1 H.A. Abramson, The cataphoretic velocity of mammalian red blood cells. J. Gen. Physiol., 12 (1929) 711. 2 A.D. Bangham, R. Flemans, D.H. Heard and G.V.F. Seaman, An apparatus for microelectrophoresis of small particles. Nature, 182 (1958) 642. 3 D.H. Heard and G.V.F. Seaman, The influence of pH and ionic strength on the electrokinetic stability of the human erythrocyte membrane. J. Gen. Physiol., 43 (1960) 635. 4 E.H. Eylar, M.A. Madoff, O.V. Brody and J.L. Oncley, The contribution of sialic acid to the surface charge of the erythrocytes. J. Biol. Chem., 237 (1962) 1992. 5 J.R. Durocher, R.C. Payne and M.E. Conrad, Role of sialic acid in erythrocyte survival. Blood, 45 (1975) 11. 6 H.A. Waldron, The anemia of lead poisoning: A review, Br. J. Ind. Med., 23 (1966)83. 7 J. Hasan, S. Hernberg, P. Mets~l~ and V. Vihko, Enhanced potassium loss in blood cells from men exposed to lead. Arch. Environ. Health, 14 (1967) 309. 8 J. Hasan, V. Vihko and S. Hernberg, Deficient red cell membrane (Na÷ + K÷)-ATPase in lead poisoning. Arch. Environ. Health, 14 (1967) 313. 9 J.M. Ratcliffe, Lead in Man and the Environment, Ellis Horwood Ltd., Chichester, 1981, p. 33. 10 R.C. Griggs, Lead poisoning: Hematologic aspects. Prog. Hematol., 4 (1964) 117. 11 S. Hernberg, M. Nurminen and J. Hasan, Nonrandom shortening of red cell survival times in men exposed to lead. Environ. Res., 1 (1967) 247. 12 E.J. Van Kampen and W.G. Zijlstra, Standardization of hemoglobinometry. II. The hemoglobincyanide method. Clin. Chim. Acta, 6 (1961) 538. 13 A. Berlin and K.H. Schaller, European standardized method for the determination of -aminolevulinic acid dehydratase activity in blood. Z. Kiln. Chem. Kiln. Biochem., 12 (1974) 389. 14 K. Yamamura, R. Kishi, N. Maehara, T. Sadamoto and E. Uchino, An experimental study of the effects of lead acetate on hearing, Cochlear microphonics and action potential of the guinea pig. Toxicol. Lett., 21 (1984) 41. 15 N. Maehara, K. Terayama, E. Uchino, H. Hirata, N. Ueno, H. Ohno and K. Yamamura, Effects of repeated reversal of the light-dark cycle on the peripheral nervous system, blood and spontaneous activity of lead-exposed rats. Int. J. Biometeorol., 29 (1985) 335. 16 S. Hernberg, J. Nikkanen, G. Mellin and H. Lilius, 6-Aminolevulinic acid dehydratase as a measure of lead exposure. Arch. Environ. Health, 21 (1970) 140. 17 N. Mobarak and A.Y.S. P'an, Lead distribution in the saliva and blood fractions of rats after intraperitoneal injections. Toxicology, 32 (1984) 67. 18 H.A. Abramson and L.S. Moyer, The electrical charge of mammalian red blood cells. J. Gen. Physiol., 19 (1936) 601. 19 D. Danon and Y. Marikovsky, Difference de charge ~lectrique de surface entre ~rythrocytes jeunes et fig~s. C.R. Acad. Sci. Ser. D, 253 (1961) 1271. 20 A. Yaari, Mobility of human red blood cells of different age groups in an electric field. Blood, 33 (1969) 159. 21 G. Bartosz, E. Grzelinska and A. Bartkowiak, Aging of the erythrocyte. XIX. Decrease in surface charge density of bovine erythrocytes. Mech. Ageing Dev., 24 (1984) 1.
265
E F F E C T OF L E A D ON E L E C T R O P H O R E T I C M O B I L I T Y O F R A T ERYTHROCYTES
KAZUYUKI TERAYAMA a'* , NAOKI MAEHARA a, MAKOTO MURATSUGU b, MIKIO MAKINO b and KOHTAROH YAMAMURA a aDepartment of Hygiene, Asahikawa Medical College and bClinical Laboratory, Asahikawa Medical College Hospital, 3-11, 4-5 Nishikagura, Asahikawa, Hokkaido, 078-11 (Japan) (Received November 1st, 1985) (Accepted March 1st, 1986)
SUMMARY Lead o f t e n affects the e r y t h r o c y t e m e m b r a n e . T h e relationship b e t w e e n the changes in e r y t h r o c y t e m e m b r a n e and t h e a n e m i a caused b y lead is still unclear. Initially, the e f f e c t o f lead injected i n t r a p e r i t o n e a l l y o n the electrop h o r e t i c m o b i l i t y o f rat e r y t h r o c y t e s was investigated in o r d e r to s t u d y the relationship b e t w e e n t h e m . As indices o f lead e x p o s u r e , h e m o g l o b i n (Hb) levels, h e m a t o c r i t s (Ht), 5 -aminolevulinic acid d e h y d r a t a s e (ALA-D) activities and b l o o d lead ( b l o o d Pb) levels in the injected rats were also e x a m i n e d . E x p o s u r e t o lead significantly d e c r e a s e d the m o b i l i t y o f rat e r y t h r o c y t e s . The changes in m o b i l i t y s e e m e d t o be less sensitive t h a n those in ALA-D activity, h o w e v e r , t h e decreases in m o b i l i t y were simultaneous with or prior to t h o s e in Hb level and Ht. T h e decreases in m o b i l i t y were evident to some e x t e n t b e l o w a b l o o d Pb level o f 100 t~g/100 ml and generally present at a level o f 100 t~g/100 ml and over. In t h e rats e x p o s e d t o lead a significant negative c o r r e l a t i o n was f o u n d b e t w e e n the mobilities and the logarithms o f b l o o d Pb level.
Key
words," Lead; Blood; E r y t h r o c y t e ; E l e c t r o p h o r e t i c m o b i l i t y ; A n e m i a
INTRODUCTION
The e l e c t r o p h o r e t i c m o b i l i t y o f m a m m a l i a n red b l o o d cells is d e t e r m i n e d b y certain surface characteristics o f the cells and h e n c e is o f value in s t u d y i n g the c o n s t i t u t i o n o f t h e cell surface [1]. Providing t h a t the m e a s u r e m e n t is
*To whom correspondence should be addressed. 0300-483x/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
259
c o n d u c t e d under certain conditions (medium, temperature, pH, ionic strength, salt species and isotonicity), the mobility of normal human red cells is indep en d en t of race, sex, age and blood group [2,3]. For a given species, the mobilities of e r y t h r o c y t e s from respective animals seem to show little variation among individuals [1,4,5]. It is well known that lead exerts influences on the e r y t h r o c y t e membrane. For example, the osmotic fragility of a leaded red cell is decreased; by contrast, the mechanical fragility is much increased [6]. The e n h a n c e m e n t of potassium loss from red cells [ 7 ] and the reduction of Na*/K*-ATPase activity in the e r y t h r o c y t e membrane [8] were observed in comparative studies of lead-exposed and non-exposed workers. Some of these e r y t h r o c y t e m em brane p h e n o m e n a caused by lead may be concerned with shortening of e r y t h r o c y t e survival times [9]. The anemia frequently observed in lead poisoning is t h o u g h t to result from a shortening of e r y t h r o c y t e survival times in combination with disturbed hemoglobin for m at i on [6,9 11]. However, the exact mechanism whereby lead causes the shortening of e r y t h r o c y t e survival times is not fully understood [9]. Results presented by Durocher et al. [5] suggest that shortening of e r y t h r o c y t e survival times is related to a decrease in the electrophoretic mobility of erythrocytes. In the present study, we investigated the effects of lead administration on the electrophoretic mobility of rat er yt h rocyt es, and also examined the relationship between the changes in mobility and blood lead levels as well as other toxicological indices of lead exposure. MATERIALS AND METHODS Animal treatment and sample collection T w e n t y - f o u r male albino rats (Wistar strain) 6 weeks of age were housed in groups of 3 or 4 in a r oom maintained at a t em perat ure of 24 +- I°C and a relative humidity of 55 -+ 5% with a 12 h light (0700--1900 h)/dark {1900-0700 h) cycle. T hey were fed a commercial laboratory pelleted diet (CE-2, Clea Japan Inc., Japan) and tap water ad libitum. At 10 weeks of age, the rats were divided into a control group (Group C, n = 6) and 2 dosed groups (Group A, n = 8; Group B, n = 10) based on b o d y weight, hematocrit (Ht), hemoglobin (Hb) level, 5-aminolevulinic acid dehydratase (ALA-D) activity and electrophoretic mobility of e r y t h r o c y t e s measured prior to the administration of lead. The administration of lead was started when the rats were 11 weeks old. Either a 10 or 20 mM lead acetate (Kishida Chemicals, Japan) solution was injected intraperitoneally once a week for 5 weeks; the volume of each injection was I ml/100 g of b o d y weight. Namely, 100 and 200 t~mol/kg of lead were administered to the rats in Group A and B, respectively. The solution was freshly prepared with distilled, de-ionized boiled water for each injection. The control rats were injected with 1 ml/100 g of 145 mM sodium chloride solution. All rats were injected at 1000 -1100 h.
260
Blood samples were obtained from the rat tail vein by tip incision without anesthetic. About 24 h after each injection, 500 pl of blood was collected in a lead-free heparinized glass tube and placed in an iced water bath. Fifty microliters of this blood was centrifuged at 1500 g for 10 min immediately after collection and the erythrocytes were washed 3 times in 10 ml of cold isotonic buffer, made up of 145 mM sodium chloride and 0.3 mM sodium bicarbonate (approx. pH 7.2) [3]. The washed erythrocytes were suspended in 100 t~l of the isotonic buffer, kept at 4°C and used for electrophoretic measurement within 24 h after collection. The measurements of Ht, Hb level and ALA-D activity were performed within 3 h after blood collection. The remaining blood samples (approx. 200 pl each) were frozen at - 2 0 ° C for subsequent blood lead {blood Pb) measurement. Ht and Hb level Ht was measured by the microcapillary tube method, and the Hb level was measured by the cyanmethemoglobin m e t h o d [12]. A L A - D activity As an indicator of lead exposure, ALA-D activity in blood was assayed using the European standardized m e t h o d [13] with slight modification, i.e. duplicate 50-pl blood aliquots of each blood sample were used for the determination of ALA-D activity and another 50-~1 aliquots for the blank. Electrophoretic mobility Electrophoretic mobility of erythrocytes was measured by a microelectrophoretic apparatus (Apparatus Mark II, Rank Brothers, England). Five microliters of the erythrocyte suspension was diluted with a 10-ml solution containing 4 volumes of 5% sorbitol and 1 volume of 67 mM Sorensen's buffer (pH 7.2) [5] and transferred to an electrophoretic rectangular cuvette kept at 25 -+ 0.1°C. The mobilities of 10 cells per experimental point were recorded; the erythrocytes were timed over 171.4 t~m (85.7 pm in each direction) at the front stationary layer under 6.4 V/cm of potential gradient. The mobilities obtained were corrected for the viscosity of the medium which was determined by an Ostwald viscometer. Blood Pb level Blood Pb analysis was performed by using a Zeeman-effect atomic absorption spectrophotometer equipped with a graphite furnace and a deuterium background corrector (Model 180-80, Hitachi Co., Japan). A mixture of nitric acid/perchloric acid was used for the wet digestion of the conserved blood samples, and a standard addition m e t h o d was applied for the determination to eliminate matrix interferences [ 14,15 ]. Statistics Student's t-test and linear regression analysis were used.
261
RESULTS
Injected lead significantly reduced the body weights of rats in Group A and B after the 2nd and 1st injections, respectively. Four rats died during this experiment: 1 in Group A and 3 in Group B after the 1st or 2nd injection. The death seemd to be induced not by the injection per se but by the lead effect, as the injection of sodium chloride had no appreciable effect on
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Fig. 1. Changes in mean e l e c t r o p h o r e t i c m o b i l i t y of e r y t h r o c y t e s , b l o o d Pb level, Hb level, Ht and ALA-D activity of rats in G r o u p B (o). Each vertical bar r e p r e s e n t s s t a n d a r d deviation. The rats were injected i n t r a p e r i t o n e a l l y with 200 p m o l / k g o f lead at the 0th, 7th, 14th, 21st and 28th days, respectively. The c o n t r o l rats were injected with 1 ml/ 100 g of 145 mM s o d i u m chloride s o l u t i o n in the same manner. The data ( × ) in none x p o s e d ( c o n t r o l and p r e - e x p o s e d ) rats are also indicated in the figure. The symbols, * and ** indicate significant d i f f e r e n c e s f r o m c o n t r o l at the levels of P < 0.05 and P < 0.01, respectively.
262
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Fig. 2. R e l a t i o n s h i p b e t w e e n t h e e l e c t r o p h o r e t i c mobilities o f rat e r y t h r o e y t e s and the b l o o d Pb level. The regression line o f m o b i l i t y o n logarithms o f b l o o d Pb o f rats e x p o s e d to 100 ( ) and 200 (o) u m o l / k g o f lead at each injection is s h o w n as a solid line. The data ( × ) in n o n - e x p o s e d ( c o n t r o l and p r e - e x p o s e d ) rats are also i n d i c a t e d in the figure.
the control rats except for a slight loss of body weights at the 1st day after the 1st injection. Figure 1 shows ~he effects of lead on the electrophoretic mobility of rat erythrocytes and other parameters (blood Pb, Hb level, Ht and ALA-D activities). The values of 200 pmol/kg dose and the control are shown in the Fig. 1. The results of 100 pmol/kg dose were similar to those of 200/~mol/kg dose but of lower magnitude. The mobility of erythrocytes in Group B significantly decreased after the 1st injection compared with the control (P < 0.01). The mean blood Pb level in Group B showed a marked increase in proportion to the a m o u n t of injected lead, which was nearly 2-fold the level in Group A at each stage after injection except the 1st one. The Hb level and Ht in Group B significantly decreased after the 2nd and 3rd injections, respectively. The injected lead significantly inhibited the ALA-D activities in Group B after the 1st injection, and inhibition after the 2nd and 5th injections was almost complete. In Group A, significant decreases in the mobility, Hb level and Ht were observed after the 3rd injection, whereas decreases in the ALAD activities were found even after the 1st and 2nd injections. Figure 2 shows the relationship between the electrophoretic mobilities of rat erythrocytes and the blood Pb level. The mobilities in non-exposed {control and pre-exposed) rats (X) showed an almost constant value (2.52 +0.02 pm/s per V per cm) corresponding to blood Pb levels of I -12 pg/100 ml. In the rats exposed to 100 (o) and 200 (o) pmol/kg of lead, decreases in mobility were evident to some extent below a blo~)d Pb level of 100 pg/ 100 ml and generally present at a level of 100 pg/100 ml and over. A signifi-
263
cant negative correlation between the mobilities in the rats (o and . ) exposed to lead and the logarithms of blood Pb level was also f o u n d (r = - 0 . 6 0 0 , P < 0.01). DISCUSSION Our results clearly show that lead exposure decreased the mobility of rat erythrocytes. The decreases in mobility were simultaneous with or prior to those in Hb level and Ht. However, the changes in mobility seemed to be a less sensitive indicator than those in ALA-D activity, which is one of the major indicators of lead exposure [16]. We also investigated mobility in relation to blood Pb levels. Our blood Pb levels after the 1st to 3rd injections of 200 pm ol / kg of lead were similar to a recent report [17] of experiments c o n d u c t e d with almost the same conditions as used in this study. The decreases in mobility were evident to some e x t e n t below a blood Pb level of 100 pg/100 ml and generally present at a level of 100 pg/100 ml and over. F u r t h e r m o r e , a significant negative correlation between the mobilities in rats exposed to lead and the logarithms of blood Pb level was observed. The electrophoretic mobility of e r y t h r o c y t e s is determined by the negative surface charge density of the e r y t h r o c y t e s [18], and the carboxyl groups of sialic acid in the m e m br a ne are mainly responsible for the surface charge [4]. Some investigators have r e por t ed that old e r y t h r o c y t e s showed a decreased mobility compared with young cells [19 -21]. According to Durocher et al. [5], desialylated rat e r y t h r o c y t e s treated with neuraminidase showed a markedly shortened survival time as well as a decrease in cell mobility. This result suggests t hat a decrease in mobility of e r y t h r o c y t e s is related to a shortening of e r y t h r o c y t e survival times [5]. Furt herm ore, others have observed a shortening of e r y t h r o c y t e survival times in lead poisoning [6,10,11 ]. The anemia frequently observed in lead poisoning is t hought to result from shortening of e r y t h r o c y t e survival times in combination with inhibition of hemoglobin synthesis [6,9 -11]. In the present study, decreases in electrophoretic mobility of e r y t h r o c y t e s were observed in the rats exposed to lead. Therefore, one may speculate that a decrease in mobility is related to a shortening of e r y t h r o c y t e survival time and a subsequent decrease in the num be r of red cells even in anemia caused by lead. ACKNOWLEDGEMENTS The authors wish to thank Dr. K. Ichihara (Dept. of Pharmacology, Asahikawa Medical College) for his technical help. We are also indebted to Dr. N. Sugawara (Dept. of Public Health, Sapporo Medical College) and to Mr. E. Uchino (Hokkaido Institute of Public Health) for their useful advice o n measurement of blood lead. We are also obliged to Mr. J.A. Sunley for correcting the manuscript.
264
REFERENCES 1 H.A. Abramson, The cataphoretic velocity of mammalian red blood cells. J. Gen. Physiol., 12 (1929) 711. 2 A.D. Bangham, R. Flemans, D.H. Heard and G.V.F. Seaman, An apparatus for microelectrophoresis of small particles. Nature, 182 (1958) 642. 3 D.H. Heard and G.V.F. Seaman, The influence of pH and ionic strength on the electrokinetic stability of the human erythrocyte membrane. J. Gen. Physiol., 43 (1960) 635. 4 E.H. Eylar, M.A. Madoff, O.V. Brody and J.L. Oncley, The contribution of sialic acid to the surface charge of the erythrocytes. J. Biol. Chem., 237 (1962) 1992. 5 J.R. Durocher, R.C. Payne and M.E. Conrad, Role of sialic acid in erythrocyte survival. Blood, 45 (1975) 11. 6 H.A. Waldron, The anemia of lead poisoning: A review, Br. J. Ind. Med., 23 (1966)83. 7 J. Hasan, S. Hernberg, P. Mets~l~ and V. Vihko, Enhanced potassium loss in blood cells from men exposed to lead. Arch. Environ. Health, 14 (1967) 309. 8 J. Hasan, V. Vihko and S. Hernberg, Deficient red cell membrane (Na÷ + K÷)-ATPase in lead poisoning. Arch. Environ. Health, 14 (1967) 313. 9 J.M. Ratcliffe, Lead in Man and the Environment, Ellis Horwood Ltd., Chichester, 1981, p. 33. 10 R.C. Griggs, Lead poisoning: Hematologic aspects. Prog. Hematol., 4 (1964) 117. 11 S. Hernberg, M. Nurminen and J. Hasan, Nonrandom shortening of red cell survival times in men exposed to lead. Environ. Res., 1 (1967) 247. 12 E.J. Van Kampen and W.G. Zijlstra, Standardization of hemoglobinometry. II. The hemoglobincyanide method. Clin. Chim. Acta, 6 (1961) 538. 13 A. Berlin and K.H. Schaller, European standardized method for the determination of -aminolevulinic acid dehydratase activity in blood. Z. Kiln. Chem. Kiln. Biochem., 12 (1974) 389. 14 K. Yamamura, R. Kishi, N. Maehara, T. Sadamoto and E. Uchino, An experimental study of the effects of lead acetate on hearing, Cochlear microphonics and action potential of the guinea pig. Toxicol. Lett., 21 (1984) 41. 15 N. Maehara, K. Terayama, E. Uchino, H. Hirata, N. Ueno, H. Ohno and K. Yamamura, Effects of repeated reversal of the light-dark cycle on the peripheral nervous system, blood and spontaneous activity of lead-exposed rats. Int. J. Biometeorol., 29 (1985) 335. 16 S. Hernberg, J. Nikkanen, G. Mellin and H. Lilius, 6-Aminolevulinic acid dehydratase as a measure of lead exposure. Arch. Environ. Health, 21 (1970) 140. 17 N. Mobarak and A.Y.S. P'an, Lead distribution in the saliva and blood fractions of rats after intraperitoneal injections. Toxicology, 32 (1984) 67. 18 H.A. Abramson and L.S. Moyer, The electrical charge of mammalian red blood cells. J. Gen. Physiol., 19 (1936) 601. 19 D. Danon and Y. Marikovsky, Difference de charge ~lectrique de surface entre ~rythrocytes jeunes et fig~s. C.R. Acad. Sci. Ser. D, 253 (1961) 1271. 20 A. Yaari, Mobility of human red blood cells of different age groups in an electric field. Blood, 33 (1969) 159. 21 G. Bartosz, E. Grzelinska and A. Bartkowiak, Aging of the erythrocyte. XIX. Decrease in surface charge density of bovine erythrocytes. Mech. Ageing Dev., 24 (1984) 1.
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