- Email: [email protected]
Effects of Na2CaEDTA on lead deposits in rabbit osseous tissue
Toxicology Letters, 19 (1983) l-5 Elsevier
EFFECTS OF NazCaEDTA ON LEAD DEPOSITS IN RABBIT OSSEOUS TISSUE (Lead; chelation; bone; rabbits)
JACEK DONIEC, BARBARA TROJANOWSKA, IRENA GARLICKA
MALGORZATA
Institute of Occupational Medicine, P.O. Box 199, 90-950 tddi
TRZCINKA-OCHOCKA
and
(Poland)
(Received September 1lth, 1982) (Revision received May 6th, 1983) (Accepted May 27th, 1983)
SUMMARY Radiochemical and autoradiographic methods were used for *iOPb determination after NasCaEDTA administration to rabbits. ‘t”Pb was determined in soft tissues, compact and trabecular bones and growing microareas on the endosteum of the long bone. After NazCaEDTA injection lead was depleted from ‘new’ deposits mainly on the growing surface of both trabecular and compact bones.
INTRODUCTION
Investigations of the toxic effects of lead (Pb) in man have established the mechanisms responsible for hematotoxicity. In addition, methods have been developed for examining the clinical effects of Pb intoxication [ 11. Osseous tissue is the principal site of Pb deposition [2]. The purpose of this paper is to collect data on the distribution of Pb in different types of osseous tissue, and data on the effects of Na&aEDTA in decreasing the Pb content in the deposits. MATERIALS AND METHODS
Male rabbits (Chinchilla strain) were given feed pellets (19.6% protein, 11.7% carbohydrate, 2.3% fat) and water ad lib. Group 1:ll rabbits, 386 + 24 days old, 4.28 f 0.96 kg. Group 2 (control): 19 rabbits 397 f 49 days old, 4.09 f 0.32 kg. Groups 1 and 2 were administered “‘Pb(NO& (The Radiochemical Centre, Amersham, U.K.) in solution pH =4, containing 260 &i “‘Pb/ml and non0378-4274/83/$ 03.00 0 Elsevier Science Publishers B.V.
2
TABLE I ‘i”Pb CONTENT (EXPRESSED AS FRACTION OF DOSE) IN RABBIT SOFT TISSUES Hours after ‘iOPb Hours after injection NaKaEDTA injection
Average content of 210Pb in the soft tissues
Standard deviation
Number of measurements
240 240 288 288 408 408 1200 1200
0.074 0.109 0.084 0.136 0.061 0.065 0.074 0.100
0.033 0.011 0.038 0.052 0.018 0.037 0.017 0.020
3 3 2 2 3 3 3 3
168 120 _ 168 _ 216
radioactive Pb (as nitrate) in concentration 35 pg/ml. Daughter products of ‘lOPb were removed [3]. 26 &i of 210Pb and 3.5 pg of stable Pb/kg were injected i.v. Tetracycline hydrochloride (Tetracyclinum 0.25, Polfa, Tarchomin, Poland) was injected i.v. into groups 1 and 2 at 20 mg/kg body weight, 3 days before and 3 days following “‘Pb injection [4]. Group 1 was injected i.v. 14 mg/kg of NazCaEDTA (‘Chelaton’, Polfa, Warsaw, Poland) on 3, 7, 10 and 41 days following “‘Pb injection. Rabbit tissues were ashed at 800°C [5] and the dry residue was dissolved in a solution of HCl. The activity of “‘Pb was measured by liquid scintillation method after radioactive equilibrium between ‘*‘Pb and ‘loBi had been reached (about 1 month) [6]. It was taken as a criterion, that the proximal end, shaft, and distal end of the femur are 0.3, 0.5 and 0.2 of the bone length, respectively [7]. The shafts of the left long bones were ashed and the activity of 210Pb was measured [5,6]. The shafts of the right long bones were embedded in polymethylmetacrylate [8] and cut [9] into
TABLE II 2’oPb CONTENT (EXPRESSED AS FRACTION OF DOSE) IN RABBIT OSSEOUS TISSUE Hours after ‘i”Pb injection 240 240 288 288 408 408 1200 1200
Hours after NazCaEDTA injection
168 120 _ 168 _ 216
Average content of ‘i”Pb in the osseous tissue
Standard deviation
0.51ga 0.186” 0.356 0.357 0.403 0.348 0.158 0.162
0.054 0.014 0.048 0.033 0.082 0.045 0.059 0.068
“Statistically significant difference at P
Number of measurements
3
TABLE “‘Pb
III
CONTENT
Hours
after “‘Pb
(EXPRESSED Hours
after
AS FRACTION Average
OF DOSE) content
IN RABBIT
of
FEMUR
Standard
Number
NazCaEDTA
*i’Pb in the femur
injection
ends
240
-
8.5”
0.45
240
168
0.418”
0.080
3
288
-
9.85
0.88
2
288
120
9.2
2.8
2
408
-
6.7
1.5
3
408
168
7.2
1.8
3
1200
_
4.56
0.88
3
6.2
1.4
3
injection
1200
216
“Statistically
significant
difference
deviation
ENDS
(. IO’)
of
measurements
(. 10’)
at P< 0.002 (Student’s
3
r-test).
slices of 120 f 32 pm. Bone slices with tetracycline bands (green-yellow fluorescence in UV light) on the endosteum were used for autoradiography [lo] and Pb concentration was measured on the endosteal surface and in compact bone as a ‘diffuse’ activity. RESULTS
AND DISCUSSION
The results (Tables I and II) suggest that injection of NaKaEDTA changes the distribution of Pb in the organism, that chelation liberates deposits of Pb from osseous tissue and that plasma and intercellular fluid transfer these deposits to soft tissues. Table II shows the Pb content in whole osseous tissue of rabbits. Effects of NaKaEDTA are evident in the first group only (i.e. when Na2CaEDTA is injected
TABLE “‘Pb
IV
CONTENT
Hours injection
after “‘Pb
(EXPRESSED Hours
after
AS FRACTION Average
OF DOSE) content
of
NazCaEDTA
“‘Pb
in the femur
(. 10’)
IN RABBIT
deviation
injection
shaft
240
_
14.5
2.3
240 288
168 -
11.5
1.0
288 408
120 _
15.0 11.7
1.8
11.2
2.9
408 1200
168 _
15.0
3.9 2.1
1200
216
6.5 9.1
FEMUR
Standard
1 .o
3.4
SHAFT Number
(. 103)
of
measurements
TABLE “‘Pb
V
CONCENTRATION
FACES
ON
(EXPRESSED
ENDOSTEUM
OF
AS FRACTION RABBIT
OF DOSE/cm’)
LONG
BONE
IN THE GROWING
SHAFT
SUR-
MEASURED
BY
AUTORADIOGRAPH after
Average
content
Standard
“‘Pb
Hours
after
NazCaEDTA
Hours
of “‘Pb
in the
deviation
iniection
iniection
endosteum
Number
(. 10e4)
(. lo-?
240
_
22.3”
5.5
28
240
168
4.3”
1.7
26
288
_
6.5
2.5
31
288
120
1.9
2.5
33
408
_
3.2
1.5
37
5.6
2.3
20
3.7
1.2
21
4.7
1.8
29
408
168
1200
_
1200
216
“Statistically
significant
difference
of
measurements
at P< 0.002 (Student’s
t-test).
3 days after *l’Pb injection). In other groups (i.e. with a longer time between *“Pb and Na2CaEDTA injection) chelation did not remove Pb deposits from the osseous tissue. The decrease of *l’Pb content in the rabbit body after chelation is a result of Pb removal mainly from osseous tissue but not from soft tissues (Table I) and acceleration of this process is effective shortly after Pb injection only. Similar results were obtained by Schworer and Kaul [ 1l] in experiments on rats. They suggested that NazCaEDTA removes Pb from the bone and that prolonged chelation does not accelerate the process. Additional confirmation of this hypothesis (Tables III and IV) is shown by the
TABLE “‘Pb
VI
CONCENTRATION
BONE
OF THE
Hours “‘Pb
after
injection
(EXPRESSED
LONG
BONE
AS FRACTION MEASURED
OF DOSE g Ca) IN RABBIT
content
Standard
of *i’Pb of the
deviation
injection
compact
_
240 288
168 _
288 408
120 _
408 1200 1200
after
Average
COMPACT
BY AUTORADIOGRAPH
NazCaEDTA
Hours
240
%tatistically
SHAFT
Number (. lo- ‘)
bone (. 10e3) 0.28
1.43a 1.01=
13 21
1.33
0.23 0.28
1.53
0.34
6
1.41
0.20
6
168 -
2.20 1.02
0.48 0.27
216
1.41
0.37
significant
difference
at P
f-test).
of
measurements
19
18 28 9
5
amount of Pb in different types of bone. A large amount of Pb is removed from trabecular bone (ends of femur), but it is not removed from the compact bone (shaft of femur). Elimination of lead by chelation from the trabecular bone does not act effectively each time following 210Pb injection. Most Pb is removed shortly after Pb absorption, when Pb is not bound firmly to the bone and is in a form which easily yields a stable compound with Na2CaEDTA. On day 7 following the injection of Pb, chelation does not liberate Pb from the bone. Autoradiographic studies of the shaft of the long bone confirmed that only ‘new’ deposits are removed from bone and demonstrated that chelation markedly decreases Pb concentration in ‘growing microareas’ on the endosteum (Table V). Chelation has little effect on deposits in compact bone (Table VI). This conclusion is in agreement with the data found for trabecular bone. Acceleration of Pb removal from the organism after NaKaEDTA injection occurs in ‘new’ deposits of osseous tissue, mainly on growing surfaces of both trabecular and compact bones. ACKNOWLEDGEMENT
This investigation was supported by contract MZ-IX. 1.03 from the Ministry of Health and Social Welfare, Poland. REFERENCES 1 L.J. Casarett and J. Doull (Eds.), Toxicology, The Basic Science of Poisons, Macmillan, New York, 1975. 2 S.R. Bernard, Dosimetric data and metabolic model for lead, Health Phys., (1977) 44-46. 3 N. Cohen and T.J. Kneip, A method for the analysis of ‘i”Pb in the urines of uranium miners, Health Phys., 17 (1969) 125-130. 4 H.M. Frost, Tetracycline bone labelling in anatomy. Am. J. Phys. Anthropol., 29 (1968) 183-196. 5 D. Kello, K. Kostial and G.E. Harrison, Influence of age and temperature of incineration on the loss of lead from rat bone, Health Phys., 28 (1975) 169-170. 6 B.W. Fox, Techniques of Sample Preparation for Liquid Scintillation Counting, North-Holland, Amsterdam, 1976. 7 D. Witkowska, The effect of stable strontium on the incorporation and metabolism of radioactive strontium in young and adult rabbit bones, Ph.D. Thesis, Academy of Medicine, todi, 1973. 8 T. Domanski, Investigation of penetration processes of calcium, strontium, barium, into bone tissue, Studies and Monographic Materials, Institute of Occupational Medicine, tddi, Vol. 3(7), 1981. 9 T. Domanski, A. Tomczak, J. Krzywariski and A. KoIodziejczak, Thin sectioning saw for bone samples, Int. Lab., 10(3) (1980) 33-37. 10 J. Doniec, Determination of “‘Pb in microareas of bone by quantitative autoradiographic method, submitted to J. Biochem. Biophys. Methods. 11 I. Schworer and A. Kaul, Tierexperimentelle Untersuchungen ilber die Elimination von inkorporiertern Blei unter biologischen Stress-situationen und Applikation von NazCaEDTA, J. Clin. Chem. Clin. Biochem., 18 (1980) 163-168.
EFFECTS OF NazCaEDTA ON LEAD DEPOSITS IN RABBIT OSSEOUS TISSUE (Lead; chelation; bone; rabbits)
JACEK DONIEC, BARBARA TROJANOWSKA, IRENA GARLICKA
MALGORZATA
Institute of Occupational Medicine, P.O. Box 199, 90-950 tddi
TRZCINKA-OCHOCKA
and
(Poland)
(Received September 1lth, 1982) (Revision received May 6th, 1983) (Accepted May 27th, 1983)
SUMMARY Radiochemical and autoradiographic methods were used for *iOPb determination after NasCaEDTA administration to rabbits. ‘t”Pb was determined in soft tissues, compact and trabecular bones and growing microareas on the endosteum of the long bone. After NazCaEDTA injection lead was depleted from ‘new’ deposits mainly on the growing surface of both trabecular and compact bones.
INTRODUCTION
Investigations of the toxic effects of lead (Pb) in man have established the mechanisms responsible for hematotoxicity. In addition, methods have been developed for examining the clinical effects of Pb intoxication [ 11. Osseous tissue is the principal site of Pb deposition [2]. The purpose of this paper is to collect data on the distribution of Pb in different types of osseous tissue, and data on the effects of Na&aEDTA in decreasing the Pb content in the deposits. MATERIALS AND METHODS
Male rabbits (Chinchilla strain) were given feed pellets (19.6% protein, 11.7% carbohydrate, 2.3% fat) and water ad lib. Group 1:ll rabbits, 386 + 24 days old, 4.28 f 0.96 kg. Group 2 (control): 19 rabbits 397 f 49 days old, 4.09 f 0.32 kg. Groups 1 and 2 were administered “‘Pb(NO& (The Radiochemical Centre, Amersham, U.K.) in solution pH =4, containing 260 &i “‘Pb/ml and non0378-4274/83/$ 03.00 0 Elsevier Science Publishers B.V.
2
TABLE I ‘i”Pb CONTENT (EXPRESSED AS FRACTION OF DOSE) IN RABBIT SOFT TISSUES Hours after ‘iOPb Hours after injection NaKaEDTA injection
Average content of 210Pb in the soft tissues
Standard deviation
Number of measurements
240 240 288 288 408 408 1200 1200
0.074 0.109 0.084 0.136 0.061 0.065 0.074 0.100
0.033 0.011 0.038 0.052 0.018 0.037 0.017 0.020
3 3 2 2 3 3 3 3
168 120 _ 168 _ 216
radioactive Pb (as nitrate) in concentration 35 pg/ml. Daughter products of ‘lOPb were removed [3]. 26 &i of 210Pb and 3.5 pg of stable Pb/kg were injected i.v. Tetracycline hydrochloride (Tetracyclinum 0.25, Polfa, Tarchomin, Poland) was injected i.v. into groups 1 and 2 at 20 mg/kg body weight, 3 days before and 3 days following “‘Pb injection [4]. Group 1 was injected i.v. 14 mg/kg of NazCaEDTA (‘Chelaton’, Polfa, Warsaw, Poland) on 3, 7, 10 and 41 days following “‘Pb injection. Rabbit tissues were ashed at 800°C [5] and the dry residue was dissolved in a solution of HCl. The activity of “‘Pb was measured by liquid scintillation method after radioactive equilibrium between ‘*‘Pb and ‘loBi had been reached (about 1 month) [6]. It was taken as a criterion, that the proximal end, shaft, and distal end of the femur are 0.3, 0.5 and 0.2 of the bone length, respectively [7]. The shafts of the left long bones were ashed and the activity of 210Pb was measured [5,6]. The shafts of the right long bones were embedded in polymethylmetacrylate [8] and cut [9] into
TABLE II 2’oPb CONTENT (EXPRESSED AS FRACTION OF DOSE) IN RABBIT OSSEOUS TISSUE Hours after ‘i”Pb injection 240 240 288 288 408 408 1200 1200
Hours after NazCaEDTA injection
168 120 _ 168 _ 216
Average content of ‘i”Pb in the osseous tissue
Standard deviation
0.51ga 0.186” 0.356 0.357 0.403 0.348 0.158 0.162
0.054 0.014 0.048 0.033 0.082 0.045 0.059 0.068
“Statistically significant difference at P
Number of measurements
3
TABLE “‘Pb
III
CONTENT
Hours
after “‘Pb
(EXPRESSED Hours
after
AS FRACTION Average
OF DOSE) content
IN RABBIT
of
FEMUR
Standard
Number
NazCaEDTA
*i’Pb in the femur
injection
ends
240
-
8.5”
0.45
240
168
0.418”
0.080
3
288
-
9.85
0.88
2
288
120
9.2
2.8
2
408
-
6.7
1.5
3
408
168
7.2
1.8
3
1200
_
4.56
0.88
3
6.2
1.4
3
injection
1200
216
“Statistically
significant
difference
deviation
ENDS
(. IO’)
of
measurements
(. 10’)
at P< 0.002 (Student’s
3
r-test).
slices of 120 f 32 pm. Bone slices with tetracycline bands (green-yellow fluorescence in UV light) on the endosteum were used for autoradiography [lo] and Pb concentration was measured on the endosteal surface and in compact bone as a ‘diffuse’ activity. RESULTS
AND DISCUSSION
The results (Tables I and II) suggest that injection of NaKaEDTA changes the distribution of Pb in the organism, that chelation liberates deposits of Pb from osseous tissue and that plasma and intercellular fluid transfer these deposits to soft tissues. Table II shows the Pb content in whole osseous tissue of rabbits. Effects of NaKaEDTA are evident in the first group only (i.e. when Na2CaEDTA is injected
TABLE “‘Pb
IV
CONTENT
Hours injection
after “‘Pb
(EXPRESSED Hours
after
AS FRACTION Average
OF DOSE) content
of
NazCaEDTA
“‘Pb
in the femur
(. 10’)
IN RABBIT
deviation
injection
shaft
240
_
14.5
2.3
240 288
168 -
11.5
1.0
288 408
120 _
15.0 11.7
1.8
11.2
2.9
408 1200
168 _
15.0
3.9 2.1
1200
216
6.5 9.1
FEMUR
Standard
1 .o
3.4
SHAFT Number
(. 103)
of
measurements
TABLE “‘Pb
V
CONCENTRATION
FACES
ON
(EXPRESSED
ENDOSTEUM
OF
AS FRACTION RABBIT
OF DOSE/cm’)
LONG
BONE
IN THE GROWING
SHAFT
SUR-
MEASURED
BY
AUTORADIOGRAPH after
Average
content
Standard
“‘Pb
Hours
after
NazCaEDTA
Hours
of “‘Pb
in the
deviation
iniection
iniection
endosteum
Number
(. 10e4)
(. lo-?
240
_
22.3”
5.5
28
240
168
4.3”
1.7
26
288
_
6.5
2.5
31
288
120
1.9
2.5
33
408
_
3.2
1.5
37
5.6
2.3
20
3.7
1.2
21
4.7
1.8
29
408
168
1200
_
1200
216
“Statistically
significant
difference
of
measurements
at P< 0.002 (Student’s
t-test).
3 days after *l’Pb injection). In other groups (i.e. with a longer time between *“Pb and Na2CaEDTA injection) chelation did not remove Pb deposits from the osseous tissue. The decrease of *l’Pb content in the rabbit body after chelation is a result of Pb removal mainly from osseous tissue but not from soft tissues (Table I) and acceleration of this process is effective shortly after Pb injection only. Similar results were obtained by Schworer and Kaul [ 1l] in experiments on rats. They suggested that NazCaEDTA removes Pb from the bone and that prolonged chelation does not accelerate the process. Additional confirmation of this hypothesis (Tables III and IV) is shown by the
TABLE “‘Pb
VI
CONCENTRATION
BONE
OF THE
Hours “‘Pb
after
injection
(EXPRESSED
LONG
BONE
AS FRACTION MEASURED
OF DOSE g Ca) IN RABBIT
content
Standard
of *i’Pb of the
deviation
injection
compact
_
240 288
168 _
288 408
120 _
408 1200 1200
after
Average
COMPACT
BY AUTORADIOGRAPH
NazCaEDTA
Hours
240
%tatistically
SHAFT
Number (. lo- ‘)
bone (. 10e3) 0.28
1.43a 1.01=
13 21
1.33
0.23 0.28
1.53
0.34
6
1.41
0.20
6
168 -
2.20 1.02
0.48 0.27
216
1.41
0.37
significant
difference
at P
f-test).
of
measurements
19
18 28 9
5
amount of Pb in different types of bone. A large amount of Pb is removed from trabecular bone (ends of femur), but it is not removed from the compact bone (shaft of femur). Elimination of lead by chelation from the trabecular bone does not act effectively each time following 210Pb injection. Most Pb is removed shortly after Pb absorption, when Pb is not bound firmly to the bone and is in a form which easily yields a stable compound with Na2CaEDTA. On day 7 following the injection of Pb, chelation does not liberate Pb from the bone. Autoradiographic studies of the shaft of the long bone confirmed that only ‘new’ deposits are removed from bone and demonstrated that chelation markedly decreases Pb concentration in ‘growing microareas’ on the endosteum (Table V). Chelation has little effect on deposits in compact bone (Table VI). This conclusion is in agreement with the data found for trabecular bone. Acceleration of Pb removal from the organism after NaKaEDTA injection occurs in ‘new’ deposits of osseous tissue, mainly on growing surfaces of both trabecular and compact bones. ACKNOWLEDGEMENT
This investigation was supported by contract MZ-IX. 1.03 from the Ministry of Health and Social Welfare, Poland. REFERENCES 1 L.J. Casarett and J. Doull (Eds.), Toxicology, The Basic Science of Poisons, Macmillan, New York, 1975. 2 S.R. Bernard, Dosimetric data and metabolic model for lead, Health Phys., (1977) 44-46. 3 N. Cohen and T.J. Kneip, A method for the analysis of ‘i”Pb in the urines of uranium miners, Health Phys., 17 (1969) 125-130. 4 H.M. Frost, Tetracycline bone labelling in anatomy. Am. J. Phys. Anthropol., 29 (1968) 183-196. 5 D. Kello, K. Kostial and G.E. Harrison, Influence of age and temperature of incineration on the loss of lead from rat bone, Health Phys., 28 (1975) 169-170. 6 B.W. Fox, Techniques of Sample Preparation for Liquid Scintillation Counting, North-Holland, Amsterdam, 1976. 7 D. Witkowska, The effect of stable strontium on the incorporation and metabolism of radioactive strontium in young and adult rabbit bones, Ph.D. Thesis, Academy of Medicine, todi, 1973. 8 T. Domanski, Investigation of penetration processes of calcium, strontium, barium, into bone tissue, Studies and Monographic Materials, Institute of Occupational Medicine, tddi, Vol. 3(7), 1981. 9 T. Domanski, A. Tomczak, J. Krzywariski and A. KoIodziejczak, Thin sectioning saw for bone samples, Int. Lab., 10(3) (1980) 33-37. 10 J. Doniec, Determination of “‘Pb in microareas of bone by quantitative autoradiographic method, submitted to J. Biochem. Biophys. Methods. 11 I. Schworer and A. Kaul, Tierexperimentelle Untersuchungen ilber die Elimination von inkorporiertern Blei unter biologischen Stress-situationen und Applikation von NazCaEDTA, J. Clin. Chem. Clin. Biochem., 18 (1980) 163-168.