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Metal induced changes in the erythrocyte membrane of rats
Toxicology Letters ELSEVIER
Toxicology Letters 78 (1995) 127-133
Metal induced changes in the erythrocyte membrane of rats Z. Sadia Jehan*, D.B. Motlag Department
of Biochemistry,
University of Madras, Guindy Campus, Madras, 600 025. India
Received 4 January 1994; revision received 23 October 1994; accepted 30 November 1994
Abstract Lead, zinc and copper were administered i.p singly or in combination as acetate salts to rats for 14 consecutive days. It was observed that lead induced drastic changes, copper induced moderate changes but zinc did not cause any significant change in the cholesterol and phospholipid content, hexose, hexosamine and sialic acid levels and activities of the erythrocyte membrane enzymes - acetylcholinesterase (AChE), NADH dehydrogenase and Na+-K’ ATPase. In the combined metal treatment the presence of zinc considerably reduced the changes induced by lead and copper. Keywork
Lead; Zinc; Copper; Erythrocyte membrane; Rats
1. Introduction Exposure to more than one metal is prevalent in view of the presence of several other metals in the environment. The combination of certain metals when administered to animals can influence their distribution in the tissues [l]. A decrease in the absorption of lead was reported when rats were preinjected with zinc which leads to a decrease in the total body burden of lead [2]. That zinc moderates the toxic effects of lead on the hemoglobin concentration, erythrocyte number and hematocrit values has been reported by Kielan-Bak et al. [3]. The adverse effects of lead on growth and hematopoietic parameters are minim&d when dietary copper, iron and zinc are adequate [4]. The interaction of lead with negatively charged liposomes in the erythrocyte membrane has been reported by Fiorini et al. [5]. l
Corresponding author.
0378-4274/95609.50 0 1995 Ekvier SSDI 0378-4274(94)03245-3
The present study was undertaken to examine the effect of lead, zinc and copper singly and in combination on the erythrocyte membrane of rats. 2. Materials and methods Male albino rats weighing about 100 f 10 g were obtained from the Fredrick Institute of Plant Protection and Toxicology (FIPPAT), Padappai, Madras. They were divided into 5 groups, housed in well-ventilated cages and given a normal laboratory animal diet (M/S. Hindustan Lever Ltd, Bangalore, India) and distilled water ad libitum. Group 1 were controls, given i.p. 0.2 ml physiological saline for 14 consecutive days. Group 2 received i.p. 20 mg PbJkg body weight as lead acetate in 0.2 ml physiological saline for 14 consecutive days. Group 3 were given i.p. 5 mg ZnJkg body weight as zinc acetate in 0.2 ml physiological saline for 14 consecutive days.
Science Ireland Ltd. All rights reserved
Z.S. Jehan, D.B. Motlag/ Toxicology Letters 78 (1995) 127-133
128
Table 1 Effect of lead, zinc and copper individually and in combination on cholesterol and phospholipid content of erythrocyte membrane Group
Group Group Group Group Group
1 2 3 4 5
Cholesterol (pmol/mg protein)
Phospholipid (~mopmg protein)
CholesteroVphospholipid ratio
10.410 l 0.521 l 0.419 l 0.498 zt 0.445 f
0.476 0.698 0.484 0.588 0.522
0.861 0.746**** 0.86!F 0.846*** 0.852**
0.037 0.014**** 0.008NS 0.041*** 0.018*
f zt f f f
0.038 0.012**** 0.027NS 0.052*** 0.016**
Values represent the mean f S.D. of 6 animals in each group. l***P < 0.001, ***P < 0.01, **k’ < 0.02, +P c 0.05. NS, not significant.
Group 4 received i.p. 2 mg Cu/kg body weight as copper acetate in 0.2 ml physiological saline for 14 consecutive days. Group 5 were given i.p. 20 mg Pb/kg body weight, 5 mg Zn/kg body weight and 2 mg Cu/kg body weight as acetate salts in 0.2 ml physiological saline for 14 consecutive days. The dose was fixed based on the reports of Hasan and Seth [6] and Malhotra et al. [7]. At the end of the experimental period the animals were killed by stunning and decapitation. Blood was collected with heparin as anticoagulant and plasma separated. The red cells were washed 3 times with ice-cold saline. An aliquot of red blood cells was taken and the membrane was isolated according to the method of Dodge et al. [8]. The pellet containing the membrane was suspended in Tris-HCl buffer to carry out the following estimations. Membrane protein was estimated by the method of Lowry et al. [9]. Cholesterol content was estimated by the method of Parekh and Jung
[lo] and phospholiid content by the Fiske and Subba Row method [ 1I]. Hexose was estimated by the method of Niebes [12], while Wagner’s procedure was adopted for the estimation of hexosamine [13]. Sialic acid was estimated by the method of Warren [ 141. Acetylcholinesterase (AChE) was determined spectrophotometrically by the method of Ellman et al. [15]. NADH dehydrogenase activity was assayed according to the method of Minakami et al. [la] and Na+-K+ ATPase was assayed by the method of Bonting u71. The results were analysed statistically by Student’s I-test. A P value less than 0.01 was considered significant. The experimental values in the metal-injected groups were compared with values in the control group. 3. Results Table 1 shows the levels of cholesterol, phos-
Table 2 Effect of lead, zinc and copper individually and in combination on the giycoprotein content of erythrocyte membrane Group
Group Group Group Group Group
1 2 3 4 5
Hexose (pg/mg protein)
Hexosamine (&mg protein)
Sialic acid (&mg protein)
516.96 f 388.47 f 523.02 f 463.79 zt 498.83 zt
433.62 f 27.97 330.42 zt 8.74-** 424.59 t 26.18NS 363.18 f 15.27*** 395.80 zk 37.87+
72.84 f 53.88 f 75.90 + 59.13 t 65.12 *
50.12 9.81**** 19.28NS 13.85** 2.59NS
Values represent the mean f S.D. of 6 animals in each group. ****P < 0.001, l**P < 0.01, l*P < 0.02, lP < 0.05. NS, not significant.
6.68 2.17**** 3.42NS 5.70*** 1.96.
Z.S. Jehan, D.B. Motlag / Toxicology Letters 78 (1995) 127-133
pholipid and the cholesterol/phospholipid ratio in the erythrocyte membrane of control and experimental animals. A statistically significant (P < 0.001) elevation in the phospholipid content with
129
a concomittant increase in cholesterol level was observed in the lead-injected group. The copperinjected group also indicated a significant (P < 0.01) increase in cholesterol and phospholipid con-
0.12
0.11
N.S.
* *
0.10
0.00
0.06
0.04
0.03
0.02
0.01
0.00 Control
Lead
Zinc
Copper
Lead
+ Zinc
Copper
l
***
P < 0.001
l
** 1’ < 0.01
Fig. 1. Activity of AChE in the erythrocyte
l
* P < 0.02
N.S.
-
Not
significant
membrane of control and experimental
animals.
+
Z.S. Jehan. D.B. Mohg/
130
ToxicologyLetters 78 (1995) 127-133
tent. The zinc-injected group and the combined metal-injected group did not show any significant change. The cholesterol/phospholipid ratio show-. ed a&nilar pattern as above.
Membrane glycoprotein levels indicated by the levels of sialic acid, hexose and hexosamine in the erythrocyte membrane of control and experimental animals are presented in Table 2. The lead- and
N.S.
0.12
0.11 0.10
* * *
0.09
T
0.06
0.07
*
0.06
c * *
0.06
0.04
0.03
0.02
0.01
1
0.00
Lead
Control
Zinc
I
Copper
Lead
;
Zin
Copper
++**
p < 0.001
l
** P c 0.01
* P < 0.05
N.S.
- Not significant
Fig. 2. Activity of NADH dehydrogenase in the erythrocyte membrane of control and experimental animals.
Z.S. Jehan, D.B. Motlag/ Toxicology
copper-injected groups indicated a decrease in the glycoprotein content, the decrease being more pronounced in the lead-injected group. There were no significant changes in the zinc-injected group. The
0.6
7
8.4
-
t.l
-
1.0
-
1.8
-
Letters 78 (1995) 127-133
131
combined metal-injected group indicated a marginal decrease in the glycoprotein content when compared to the control. The activities of erythrocyte membrane enzymes
0
: * T
N.S. T
Control
l
*** P < 0.001
Zinc
Lead
l
** P < 0.01
Fig. 3. Activity of Na+-K+ ATPase in the erythrocyte
l
P < 0.05
Copper
Lead + Zinc + Copper
N.S. - Not significant
membrane of control and experimental
animals.
132
Z.S. Jehan. D. B. Motlag/ Toxicology Letters 78 (1995) 127-133
- AChE, NADH dehydrogenase and Na+-K+ ATPase - in the experimental animals as compared with that of the control are presented in Figs. 1, 2 and 3. A significant variation (P < 0.001) in the activities of AChE and NADH dehydrogenase was observed in the lead-injected group, while in the copper-injected group the decrease was less significant (P < 0.01). The zincinjected group did not indicate any significant change in the activities of these 2 enzymes. In the combined metal-injected group the activities were near normal. A statistically significant (P < 0.001) increase in the activity of Na+-K+ ATPase of the erythrocyte membrane was observed in the lead-injected group with the copper-injected group indicating a less significant (P < 0.01) increase. 4. Discussion Cholesterol and phospholipid form an integral part of the erythrocyte membrane. The area of red cell surface is altered by changes in the cholesterol content in the membrane, which in turn alters the shape of the cells [18]. Enrichment of cholesterol in red cell membrane causes profound and reversible changes in lipid composition and membrane fluidity [19]. From the results it can be observed that lead and copper when injected individually resulted in an increase in cholesterol content thereby altering the membrane composition while the zinc and the combined metals when injected did not induce much change in the erythrocyte membrane. Ladbrooke et al. [20] have proposed that the function of cholesterol in the erythrocyte membrane is to maintain the membrane in its original form. Apart from the specific role of certain phospholipids for the activity of membrane-located enzymes [21], the phospholipids play an important role in maintaining the fluidity of the membrane [20]. Cooper et al. [22] have reported that the cho1esteroYphospholipid ratio is of great importance and it is carefully regulated in order to prevent abrupt changes in fluidity. An increase in the phospholipid content of rat erythrocyte on treatment with lead and chromium has been reported by Kuktha et al. [23]. It is observed from the re-
sults that though the lead- and copper-injected groups alter the cholesterol-phospholipid levels, the presence of zinc in the combined metal-injected group minim& the changes caused by lead and copper so that the integrity of the erythrocyte membrane is not lost completely. Glycoproteins of erythrocyte membrane are involved in many biological phenomena concerned with specificity at the molecular level [24]. Terayama and Muratsugu [25] have reported a decrease in the sialic acid content of the rat erythrocyte membrane after i.p. injection of lead. The decrease in the sialic acid level of the erythrocyte membrane in the lead- and copperinjected groups may be related to (1) a selfdigestion process by the action of sialidase which can release sialic acid, and (2) the release of sialoglycopeptide by proteinase linked to the membrane [25]. As a result of this self-digestion process accelerated by injected metals, the decrease in sialic acid content of the rat erythrocyte membrane may be observed. The activity of membrane-bound enzymes of erythrocyte is maintained by the fluidity of the membrane. Cholesterol appears to maintain the bilayer matrix in an intermediate fluid state by regulating the mobility of the phospholipid fatty acyl chain [26). The decrease in the activity of AChE and NADH dehydrogenase observed in the lead- and copper-injected groups may be due to the impairment of red cell membrane as reported by Broody et al. [27]. A significant increase in the activity of erythrocyte Na+-K+ ATPase in workers occupationally exposed to lead was reported by Karai [28]. It has been reported that Na+-K+ ATPase activity in the lead- and copperinjected groups may be due to alteration in cholesterol and phospholipid levels as reported in Table 1. The increase in Na+-K+ ATPase activity due to alteration in lipid content was reported by Fukomoto et al. [30]. The results observed indicate that the increase in Na+-K+ ATPase activity in the erythrocyte membrane when injected with lead or copper individually may be due to alteration in the lipid content, which was not significant in the zinc- or combined metal-injected groups. Rats injected with lead and copper individually had profound influence on the erythrocyte mem-
Z.S. Jehan. D. B. Motlag / Toxicology Letters 78 (1995) 127- I33
brane leading to adverse effects, while zinc did not bring about any significant alterations. When zinc was given along with lead and copper, it moderated the changes caused by lead and copper. References 111Webb, M. (1972) Protection by zinc against cadmium toxicity. Biochem. Pharmacol. 21, 2767-2772.
PI Iseki, S. (1989) Effects of zinc on absorption, distribution and excretion of lead, correlating with body copper distribution. Gifu. Daigaku. Igakubu. Kiyo. 37, 192-222. [31 Kielan-Bak, Z., Tara, B. and Strzelec, M. (1984) Lead and zinc toxicity on the blood profile of rats. Pr. Nauk. Uniw. Slask. Katowicach. 642, 132-140. 141 Klauder, D.S. and Petering, H.G. (1975) Protective value of dietary copper and iron against some toxic effects of lead in rats. Environ. Health Perspect 12, 77-80. 151 Fiorini, R.M., Curatola, G., Valentino, M. and Govema, M. (1982) Effects of lead on the physical state of erythrocyte and model membranes. Boll&c. Ital. Biol. Sper. 58, 871-875. PI Hasan, M.Z. and Seth, T.D. (1981) Effect of lead and zinc administration on liver, kidney and brain levels of copper, lead, manganese and zinc and on erythrocyte ALA-D activity in rats. Toxicol. Lett. 7, 353-358. 171 Malhotra, K.M., Shukla, G.S. and Chandra, S.V. (1982) Neurochemical changes in rats co-exposed to lead and copper. Arch. Toxicol. 49, 331-336. 181 Dodge, J.T., Mitchell, C. and Hanahan, D.J. (1963) The preparation and chemical characteristics of hemoglobin free ghosts of human erythrocytes. Arch. Biochem. Biophys. 100, 131-139. 191 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. HOI Parekh, AC. and Jung, D.H. (1970) Cholesterol determination with ferric acetate, uranium acetate and sulphuric acid - ferrous sulphate reagents. Anal. Chem. 42, 1423-1427. u11 Fiske, C.H. and Subba Row, Y. (1925) The calorimetric determination of phosphorous. J. Biol. Chem. 66, 375-400. WI Niebes, P. (1972) Determination of enzymes and degradation products of glycosaminoglycan metabolism in the serum of healthy and varicose subjects. Clin. Chim. Acta. 42, 399-408. [I31 Wagner, W.D. (1979) A more sensitive assay discriminating galactosamine and glucosamine in mixtures. Anal. B&hem. 94, 394-398. I141 Warren, L. (1959) The thiobarbituric acid assay of sialic acid. J. B&hem. 234, 1971-1975.
133
I151 Ellman, G.L., Courtney, K.D., Andres, V. Jr. and Featherstone, R.M. (1961) A new rapid calorimetric determination of acetylcholine esterase activity. B&hem. Pharmacol. 7, 88-94. I161 Minakami, S., Rengter, R.C.F. and Singer, P. (1962) The respiratory chain linked reduced nicotinamide adenine dinucleotide dehydrogenase. J. Biol. Chem. 237, 569-576. 1171 Bonting, S.L. (1970) Membrane and ion transport. In: E.Z. Bittar, (Ed.), Wiley, Interscience, London, Vol. I, p. 257. 1181 Murphy, J.R. (1965) Erythrocyte metabolism. 11. Cell shape and location of cholesterol in RBC membrane. J. Lab. Clin. Med. 65, 756-774. I191 Cooper, R.A. (1970) Lipids of human red cell membranes: normal composition and variability in disease. Semin. Haematol. 7, 296-322. [201 Ladbrooke, B.D., William, R.M. and Chapman, D. (1968) Studies on lecithin-cholesterol water interactions by differential scanning calorimetry and X-ray diffraction. B&hem. Biophys. Acta. 150, 333-340. 1211 Friedeman, H. and Rapoport, SM. (1974) Enzymes of red cell: a critical catalogue. In: H. Yoshikawa and S.M. Rapoport (Eds.), Cellular and Molecular Biology of Erythrocytes, Univ. Pack. Press, Baltimore, pp. 181-259. WI Cooper, R.A., Durocher, J.R. and Leslie, M.H. (1977) Decreased fluidity of red cell membrane lipids in abetahpoproteinemia. J. Clin. Invest. 60, 1I5- 121. v31 Kuktha, V.K., Shkrebneva, 1.1. and Nurman, T.N. (1983) Effect of heavy metals (lead and chromium) on the composition of blood plasma phosphohpids in rats. Vesti. Akad. Navuk. BSSR, Ser. Biyal. Navuk 4, 69-71. [241 Ashwell, G. and Morell, A.G. (1977) Membrane glycoproteins and recognition phenomena. Trends Biochem. Sci. 2, 76-78. v51 Terayama, K. and Muratsugu, M. (1988) Effects of lead on sialic acid content and survival of rat erythrocytes. Toxicology 53, 269-276. 1261 Chapman, D. (1968) Biological Membrane Physical Fact and Function. Academic Press, New York. [271 Broody, A.R., Gerwyn, G. and Lila, H.H. (1983) Interactions of chrysolite and crocidolite asbestos with red blood cell membrane: chrysolite binds to sialic acid. Lab. Invest. 49, 468-475. P81 Karai, I. (1982) Studies on the effect of lead on Na+-K+ ATPase activity of erythrocyte membranes Osaka Shiritsu. Daigaku. Igaku. Zassh. 31, 15-27. v91 Miner, P.B. Jr., Sneller, M. and Crawford, S.S. (1983) Spironolactone and canrenone induced changes in cholesterol and phospholipid, and fluorescence polarization in rats. Hepatology 3, 481-488. 1301 Fukumoto, K., Karai, I. and Horiguchi, S. (1983) Effect of lead on erythrocyte membranes. Br. J. Ind. Med. 40, 220-223.
Toxicology Letters 78 (1995) 127-133
Metal induced changes in the erythrocyte membrane of rats Z. Sadia Jehan*, D.B. Motlag Department
of Biochemistry,
University of Madras, Guindy Campus, Madras, 600 025. India
Received 4 January 1994; revision received 23 October 1994; accepted 30 November 1994
Abstract Lead, zinc and copper were administered i.p singly or in combination as acetate salts to rats for 14 consecutive days. It was observed that lead induced drastic changes, copper induced moderate changes but zinc did not cause any significant change in the cholesterol and phospholipid content, hexose, hexosamine and sialic acid levels and activities of the erythrocyte membrane enzymes - acetylcholinesterase (AChE), NADH dehydrogenase and Na+-K’ ATPase. In the combined metal treatment the presence of zinc considerably reduced the changes induced by lead and copper. Keywork
Lead; Zinc; Copper; Erythrocyte membrane; Rats
1. Introduction Exposure to more than one metal is prevalent in view of the presence of several other metals in the environment. The combination of certain metals when administered to animals can influence their distribution in the tissues [l]. A decrease in the absorption of lead was reported when rats were preinjected with zinc which leads to a decrease in the total body burden of lead [2]. That zinc moderates the toxic effects of lead on the hemoglobin concentration, erythrocyte number and hematocrit values has been reported by Kielan-Bak et al. [3]. The adverse effects of lead on growth and hematopoietic parameters are minim&d when dietary copper, iron and zinc are adequate [4]. The interaction of lead with negatively charged liposomes in the erythrocyte membrane has been reported by Fiorini et al. [5]. l
Corresponding author.
0378-4274/95609.50 0 1995 Ekvier SSDI 0378-4274(94)03245-3
The present study was undertaken to examine the effect of lead, zinc and copper singly and in combination on the erythrocyte membrane of rats. 2. Materials and methods Male albino rats weighing about 100 f 10 g were obtained from the Fredrick Institute of Plant Protection and Toxicology (FIPPAT), Padappai, Madras. They were divided into 5 groups, housed in well-ventilated cages and given a normal laboratory animal diet (M/S. Hindustan Lever Ltd, Bangalore, India) and distilled water ad libitum. Group 1 were controls, given i.p. 0.2 ml physiological saline for 14 consecutive days. Group 2 received i.p. 20 mg PbJkg body weight as lead acetate in 0.2 ml physiological saline for 14 consecutive days. Group 3 were given i.p. 5 mg ZnJkg body weight as zinc acetate in 0.2 ml physiological saline for 14 consecutive days.
Science Ireland Ltd. All rights reserved
Z.S. Jehan, D.B. Motlag/ Toxicology Letters 78 (1995) 127-133
128
Table 1 Effect of lead, zinc and copper individually and in combination on cholesterol and phospholipid content of erythrocyte membrane Group
Group Group Group Group Group
1 2 3 4 5
Cholesterol (pmol/mg protein)
Phospholipid (~mopmg protein)
CholesteroVphospholipid ratio
10.410 l 0.521 l 0.419 l 0.498 zt 0.445 f
0.476 0.698 0.484 0.588 0.522
0.861 0.746**** 0.86!F 0.846*** 0.852**
0.037 0.014**** 0.008NS 0.041*** 0.018*
f zt f f f
0.038 0.012**** 0.027NS 0.052*** 0.016**
Values represent the mean f S.D. of 6 animals in each group. l***P < 0.001, ***P < 0.01, **k’ < 0.02, +P c 0.05. NS, not significant.
Group 4 received i.p. 2 mg Cu/kg body weight as copper acetate in 0.2 ml physiological saline for 14 consecutive days. Group 5 were given i.p. 20 mg Pb/kg body weight, 5 mg Zn/kg body weight and 2 mg Cu/kg body weight as acetate salts in 0.2 ml physiological saline for 14 consecutive days. The dose was fixed based on the reports of Hasan and Seth [6] and Malhotra et al. [7]. At the end of the experimental period the animals were killed by stunning and decapitation. Blood was collected with heparin as anticoagulant and plasma separated. The red cells were washed 3 times with ice-cold saline. An aliquot of red blood cells was taken and the membrane was isolated according to the method of Dodge et al. [8]. The pellet containing the membrane was suspended in Tris-HCl buffer to carry out the following estimations. Membrane protein was estimated by the method of Lowry et al. [9]. Cholesterol content was estimated by the method of Parekh and Jung
[lo] and phospholiid content by the Fiske and Subba Row method [ 1I]. Hexose was estimated by the method of Niebes [12], while Wagner’s procedure was adopted for the estimation of hexosamine [13]. Sialic acid was estimated by the method of Warren [ 141. Acetylcholinesterase (AChE) was determined spectrophotometrically by the method of Ellman et al. [15]. NADH dehydrogenase activity was assayed according to the method of Minakami et al. [la] and Na+-K+ ATPase was assayed by the method of Bonting u71. The results were analysed statistically by Student’s I-test. A P value less than 0.01 was considered significant. The experimental values in the metal-injected groups were compared with values in the control group. 3. Results Table 1 shows the levels of cholesterol, phos-
Table 2 Effect of lead, zinc and copper individually and in combination on the giycoprotein content of erythrocyte membrane Group
Group Group Group Group Group
1 2 3 4 5
Hexose (pg/mg protein)
Hexosamine (&mg protein)
Sialic acid (&mg protein)
516.96 f 388.47 f 523.02 f 463.79 zt 498.83 zt
433.62 f 27.97 330.42 zt 8.74-** 424.59 t 26.18NS 363.18 f 15.27*** 395.80 zk 37.87+
72.84 f 53.88 f 75.90 + 59.13 t 65.12 *
50.12 9.81**** 19.28NS 13.85** 2.59NS
Values represent the mean f S.D. of 6 animals in each group. ****P < 0.001, l**P < 0.01, l*P < 0.02, lP < 0.05. NS, not significant.
6.68 2.17**** 3.42NS 5.70*** 1.96.
Z.S. Jehan, D.B. Motlag / Toxicology Letters 78 (1995) 127-133
pholipid and the cholesterol/phospholipid ratio in the erythrocyte membrane of control and experimental animals. A statistically significant (P < 0.001) elevation in the phospholipid content with
129
a concomittant increase in cholesterol level was observed in the lead-injected group. The copperinjected group also indicated a significant (P < 0.01) increase in cholesterol and phospholipid con-
0.12
0.11
N.S.
* *
0.10
0.00
0.06
0.04
0.03
0.02
0.01
0.00 Control
Lead
Zinc
Copper
Lead
+ Zinc
Copper
l
***
P < 0.001
l
** 1’ < 0.01
Fig. 1. Activity of AChE in the erythrocyte
l
* P < 0.02
N.S.
-
Not
significant
membrane of control and experimental
animals.
+
Z.S. Jehan. D.B. Mohg/
130
ToxicologyLetters 78 (1995) 127-133
tent. The zinc-injected group and the combined metal-injected group did not show any significant change. The cholesterol/phospholipid ratio show-. ed a&nilar pattern as above.
Membrane glycoprotein levels indicated by the levels of sialic acid, hexose and hexosamine in the erythrocyte membrane of control and experimental animals are presented in Table 2. The lead- and
N.S.
0.12
0.11 0.10
* * *
0.09
T
0.06
0.07
*
0.06
c * *
0.06
0.04
0.03
0.02
0.01
1
0.00
Lead
Control
Zinc
I
Copper
Lead
;
Zin
Copper
++**
p < 0.001
l
** P c 0.01
* P < 0.05
N.S.
- Not significant
Fig. 2. Activity of NADH dehydrogenase in the erythrocyte membrane of control and experimental animals.
Z.S. Jehan, D.B. Motlag/ Toxicology
copper-injected groups indicated a decrease in the glycoprotein content, the decrease being more pronounced in the lead-injected group. There were no significant changes in the zinc-injected group. The
0.6
7
8.4
-
t.l
-
1.0
-
1.8
-
Letters 78 (1995) 127-133
131
combined metal-injected group indicated a marginal decrease in the glycoprotein content when compared to the control. The activities of erythrocyte membrane enzymes
0
: * T
N.S. T
Control
l
*** P < 0.001
Zinc
Lead
l
** P < 0.01
Fig. 3. Activity of Na+-K+ ATPase in the erythrocyte
l
P < 0.05
Copper
Lead + Zinc + Copper
N.S. - Not significant
membrane of control and experimental
animals.
132
Z.S. Jehan. D. B. Motlag/ Toxicology Letters 78 (1995) 127-133
- AChE, NADH dehydrogenase and Na+-K+ ATPase - in the experimental animals as compared with that of the control are presented in Figs. 1, 2 and 3. A significant variation (P < 0.001) in the activities of AChE and NADH dehydrogenase was observed in the lead-injected group, while in the copper-injected group the decrease was less significant (P < 0.01). The zincinjected group did not indicate any significant change in the activities of these 2 enzymes. In the combined metal-injected group the activities were near normal. A statistically significant (P < 0.001) increase in the activity of Na+-K+ ATPase of the erythrocyte membrane was observed in the lead-injected group with the copper-injected group indicating a less significant (P < 0.01) increase. 4. Discussion Cholesterol and phospholipid form an integral part of the erythrocyte membrane. The area of red cell surface is altered by changes in the cholesterol content in the membrane, which in turn alters the shape of the cells [18]. Enrichment of cholesterol in red cell membrane causes profound and reversible changes in lipid composition and membrane fluidity [19]. From the results it can be observed that lead and copper when injected individually resulted in an increase in cholesterol content thereby altering the membrane composition while the zinc and the combined metals when injected did not induce much change in the erythrocyte membrane. Ladbrooke et al. [20] have proposed that the function of cholesterol in the erythrocyte membrane is to maintain the membrane in its original form. Apart from the specific role of certain phospholipids for the activity of membrane-located enzymes [21], the phospholipids play an important role in maintaining the fluidity of the membrane [20]. Cooper et al. [22] have reported that the cho1esteroYphospholipid ratio is of great importance and it is carefully regulated in order to prevent abrupt changes in fluidity. An increase in the phospholipid content of rat erythrocyte on treatment with lead and chromium has been reported by Kuktha et al. [23]. It is observed from the re-
sults that though the lead- and copper-injected groups alter the cholesterol-phospholipid levels, the presence of zinc in the combined metal-injected group minim& the changes caused by lead and copper so that the integrity of the erythrocyte membrane is not lost completely. Glycoproteins of erythrocyte membrane are involved in many biological phenomena concerned with specificity at the molecular level [24]. Terayama and Muratsugu [25] have reported a decrease in the sialic acid content of the rat erythrocyte membrane after i.p. injection of lead. The decrease in the sialic acid level of the erythrocyte membrane in the lead- and copperinjected groups may be related to (1) a selfdigestion process by the action of sialidase which can release sialic acid, and (2) the release of sialoglycopeptide by proteinase linked to the membrane [25]. As a result of this self-digestion process accelerated by injected metals, the decrease in sialic acid content of the rat erythrocyte membrane may be observed. The activity of membrane-bound enzymes of erythrocyte is maintained by the fluidity of the membrane. Cholesterol appears to maintain the bilayer matrix in an intermediate fluid state by regulating the mobility of the phospholipid fatty acyl chain [26). The decrease in the activity of AChE and NADH dehydrogenase observed in the lead- and copper-injected groups may be due to the impairment of red cell membrane as reported by Broody et al. [27]. A significant increase in the activity of erythrocyte Na+-K+ ATPase in workers occupationally exposed to lead was reported by Karai [28]. It has been reported that Na+-K+ ATPase activity in the lead- and copperinjected groups may be due to alteration in cholesterol and phospholipid levels as reported in Table 1. The increase in Na+-K+ ATPase activity due to alteration in lipid content was reported by Fukomoto et al. [30]. The results observed indicate that the increase in Na+-K+ ATPase activity in the erythrocyte membrane when injected with lead or copper individually may be due to alteration in the lipid content, which was not significant in the zinc- or combined metal-injected groups. Rats injected with lead and copper individually had profound influence on the erythrocyte mem-
Z.S. Jehan. D. B. Motlag / Toxicology Letters 78 (1995) 127- I33
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