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Heme synthesis in the lead-intoxicated mouse embryo
Toxicology, 9 (1978) 173--179 © Elsevier/North-Holland Scientific Publishers Ltd.
HEME S Y N T H E S I S IN THE L E A D - I N T O X I C A T E D M O U S E E M B R Y O *
GEORG B. GERBER and JOZEF MAES Euratom/C.E.N., Department of Radiobiology, B-2400 Mol (Belgium) (Received May 12th, 1977) (Revision received September 27th, 1977) (Accepted October 3rd, 1977)
SUMMARY
Incorporation of SSFe and of [14C] glycine was studied in control embryos and mothers and in those which had received lead in the diet from day 7 of pregnancy. Incorporation of Fe into heme of embryonic liver which increases markedly for controls on day 17 of pregnancy was depressed greatly and showed no such increase in lead-intoxicated embryos. These embryos were retarded in growth but had normal heme concentrations in body and liver. Incorporation of glycine into embryonic heme and proteins was n o t affected. Data on incorporation in the mothers are also presented. It is t h o u g h t t h a t the impaired synthesis of heme in lead-intoxicated embryos embryos limits their body growth during the late phase of pregnancy.
INTRODUCTION Exposure of pregnant mice to dietary lead causes retardation of growth of the embryo during the end of the pregnancy and can lead to prenatal death [7]. Earlier, we observed in these embryos a decrease in 5-aminolevulinic acid dehydratase to about one-third of the activity of control animals and a 2--3-fold increase in prophyrin content, but no changes in heme concentration [8]. Heme synthesis thus appears to be sufficient for the reduced b o d y mass of the lead-treated embryos, but this fact does n o t exclude the possibility that the a m o u n t of heme synthetized, nevertheless, limits the growth of these embryos, nor t h a t synthesis of certain types of heme proteins is affected by * Supported by Contract 036-74-7 ENV.B of the E.C. Environmental Research Program. Publication No. 1388 of the Euratom Biology Division.
173
lead more than that of others. In order to obtain more information on the action of lead on heme synthesis, we have now studied incorporation of SSFe and of [ 14C]glycine into heme isolated from the liver of embryos as well as from liver, bone marrow or blood from their mothers. In addition, incorporation of glycine into proteins was also followed. METHODS Mice of the C57B1 strain were mated and, when displaying a vaginal plug, were given a diet containing 0, 0.25, 0.5 or 1% of lead as lead acetate from the 7th day of pregnancy [7]. This procedure differs from our earlier one, where lead was given from the first day of pregnancy on. It was chosen since due to the impaired implantation the number of pregnant mice is very small when high doses of lead are given from the beginning of pregnancy. On the 15th, 16th or 17th day of pregnancy the mice received an intraperitoneal injection of either 10 t~Ci of [SSFe]citrate (from the I.R.E. (Fleurus, Belg., specific activity 10.6 mCi/mg) or of 10 pCi of [2-t4C]glycine (from the I.R.E., specific activity 48.9 mCi/mg). The animals were sacrificed 24 h later, the embryos were removed and their livers were dissected. Blood, femoral bone marrow and livers of mothers were also taken. Liver was homogenized in chloroform--methanol (2 : 1), the precipitate was washed with 50% ethanol and hemin was extracted with acid acetone. After spectrophotometric determination of the hemin c o n t e n t using Drabkin's solution, the extract was taken to dryness, redissolved in pyridine, and heroin was isolated by thin-layer chromatography in m e t h a n o l - a c e t i c acid--water (40 : 3 : 7). Hemin was eluted with acid acetone, the quantity of hemin was determined spectrophotometrically and, after decolorization with hydrogen peroxide, radioactivity of hemin was measured in a liquid scintillation counter using the appropriate conditions for counting the two isotopes. Protein from the residue of the homogenate after acid acetone extraction was washed, hydrolyzed in 6 N HC1, dried and counted for radioactivity. Bone marrow cells were washed with 50% ethanol and hemin was extracted with acid acetone; the blood samples were treated with 0.6 N perchloric acid (PCA). The washed precipitate was extracted with acid acetone and the hemin was purified as described above. RESULTS Incorporation of SSFe into hepatic heme of control embryos increases markedly on day 17 of pregnancy when the embryo grows from ~ 4 0 0 mg (day 16) to ~ 6 0 0 mg (day 17). (Fig. 1), whereas incorporation of [14C]glycine into heme does n o t change significantly with the age of the embryo. Incorporation of glycine into hepatic proteins increases only slightly during this time (Fig. 2). When the mothers were given 0.25% of lead in the diet incorporation of
174
SSFe
100-
ncorporation
x:
Controls
n :
0,5
e:
0,25
*/,
into
fetal
heme
Lead
A
*/,
Lead
80-
T
E (3. u
_L
60 >,
t t
> (J
40
20 ¸ {3. U3
0
i
0
i
400
I
I
800 Embryoweight
I
I
1200 (mg)
Fig. 1. Incorporation o f F6 into h e m e isolated from the liver o f control e m b r y o s and o f e m b r y o s w h o s e m o t h e r s had received 0 . 2 5 or 0.5% o f lead in the diet. The points represent the m e a n and standard errors from o n e litter ( 4 - - 8 animals). The weights w i t h i n a litter varied from 7--10%. The ages o f pregnancy corresponding to the weights are day 16, all animals b e l o w 4 0 0 mg, day 17 those b e l o w 7 0 0 mg and day 18 those above 8 0 0 mg e x c e p t for the 0.25% lead-treated litter weighing 6 8 0 mg w h i c h was obtained on day 18.
SSFe into heine is significantly reduced (Fig. 1), but an increase with age can still be discerned. I n c o r p o r a t i o n o f glycine into h e m e is n o t altered by lead t r e a t m e n t but t h a t i n t o hepatic proteins of the e m b r y o is increased (Fig. 2). N e x t , a s o m e w h a t higher dose (0.5%) w h i c h inhibits more strongly e m b r y o n i c g r o w t h was c h o s e n to investigate the effect on i n c o r p o r a t i o n of SSFe i n t o heme whereas the highest d o s e at w h i c h a few live e m b r y o s can still be o b t a i n e d was utilized to f o l l o w glycine incorporation. As Fig. 1 s h o w s the increase with age o f SSFe i n c o r p o r a t i o n is abolished at 0.5% of lead whereas even a dose of 1% of lead does n o t depress significantly i n c o r p o r a t i o n of glycine i n t o h e m e (specific activities o f all ages c o m b i n e d : means + S.E. controls 2 . 1 5 ± 0 . 1 7 , 0.25% lead 2 . 5 7 -+ 0 . 2 8 , 1% of lead 3 . 0 0 +_ 0 . 4 2 ) . On the other hand, the i n c o r p o r a t i o n o f glycine i n t o proteins w h i c h is elevated at 0.25% of lead is normal at 1% (specific activities of all ages c o m b i n e d : controls 8.31 -+ 0.58, 0.25% lead 2 6 . 5 3 + 4 . 2 6 , 1% lead 9 . 3 7 +- 1 . 2 6 ) . Data
175
l~C
40-
E
Glycine x:
Controls
D:
0,25
~:
I
"/,
Incorporat
on
into
Protein
~]
*/,= L e a d Lead
30
E u
~
20
°--
U
<
10
d U'I
0
I
0
I
400
I
I
I
800 Embryoweight
I
1200 (mg)
Fig. 2. Incorporation of [2-'4C]glycine into total liver proteins of control embryos and of embryos whose mothers had received 0.25 or 1% of lead in the diet. The points represent the mean and standard errors from one litter. The ages of pregnancy corresponding to the weights are day 16 below 420 mg,~lay 17 below 700 rag. The value for 1% of lead is from day 17. No significant difference was found for incorporation of [2-~4C]glycine into heine isolated from embryonic liver. on the i n c o r p o r a t i o n o f SSFe and [14C]glycine into organs o f the m o t h e r are p r e s e n t e d in Table I. Since n o significant differences in these values with r e s p e c t t o the age o f p r e g n a n c y was seen, all values have been p o o l e d . In liver and b l o o d , i n c o r p o r a t i o n o f SSFe in lead-treated animals is m a r k e d l y depressed b u t in b o n e m a r r o w it is e n h a n c e d c o m p a r e d to controls. I n c o r p o r a t i o n o f glycine into h e m e o f e r y t h r o c y t e s or liver is n o t affected b y lead but, in b o n e m a r r o w a slight depression o f i n c o r p o r a t i o n is n o t e d . The specific activity o f proteins is u n a l t e r e d in liver and elevated in b l o o d and b o n e m a r r o w a f t e r 0.25% o f lead. The value in b l o o d varies, however, greatly a m o n g the animals studied. DISCUSSION B l o o d f o r m a t i o n in the e m b r y o p r o c e e d s t h r o u g h several stages o f develo p m e n t . During the early p e r i o d up to d a y 12 the y o l k sack is the principal
176
b-a --O.
Liver Liver Liver Bone m a r r o w Bone m a r r o w Bone m a r r o w Blood Blood Blood
Incorporation Incorporation Incorporation Incorporation Incorporation Incorporation Incorporation Incorporation Incorporation
7.82 0.245 1.51 2.70 0.246 3.42 5.24 0.287 2.83
± 1.36 [12] ± 0.055 [6] ± 0.156 ± 0.86 ± 0.062 ± 0.33 ± 1.21 + 0.037 ± 0.55
Control
Specific activity
± ± ± ± ± ± ± +
5.46 0.382 1.66 4.56 12,15 2.47 0.221 6.97
0.25% Pb
2,62 0.167 0.037 2.25
1.35 [6] 0.092 [7] 0.67 1.25
± 0.49 [ 1 7 ]
0.41
± 0.06
13,3 ± 2.55 0.101 ± 0.0187
0.57
0.5% Pb
0.242 ± 0.04 2.25 ± 0.07
0.216 + 0.041 [4] 1.92 ± 0.52
1% Pb
This table w h i c h p r e s e n t m e a n ~ S.E. c o m b i n e s all data from m o t h e r s w h o had been e x p o s e d f r o m 9 t o 11 days, i.e., the 7th d a y o f p r e g n a n c y to lead in the diet. The n u m b e r o f animals used is indicated in b r a c k e t s for Fe and glycine i n c o r p o r a t i o n , respectively. Significant d i f f e r e n c e s from c o n t r o l s are u n d e r l i n e d o n c e (P < 0.05) or twice (P ~ 0.01). I n c o r p o r a t i o n i n t o heine is e x p r e s s e d as c p m / u g h e i n e , i n c o r p o r a t i o n into p r o t e i n s as c p m / m g tissue.
5SFe into heine glycine into h e m e glycine into p r o t e i n s 5SFe into heine glycine into h e m e glycine into p r o t e i n s o f SSFe into h e m e o f glycine into h e m e o f glycine into p r o t e i n s
Organ
S y s t e m studied
I N C O R P O R A T I O N OF SSFe and [14C]GLYCINE INTO D I F F E R E N T O R G A N S OF P R E G N A N T MICE
TABLE I
site of erythropoiesis, which then is superseded by liver and later on by spleen [1,10]. Blood formation in bone marrow takes over only during postnatal life. The relative activities of the various enzymes participating in heme biosynthesis also change during development [14]. In the adult mouse, 5-aminolevulinic acid synthetase is probably rate limiting for heme synthesis but in the fetal and newborn mouse the activity of 6 -aminolevulinic acid dehydratase (ALAD) and ferrochelatase are comparatively small and could become a critical factor when diminished by toxic effects. Lead is known to depress several reactions in the pathways leading to hemoglobin [11,12]. The depression in ALAD which in blood occurs after minimal exposure to lead has found much attention but its significance for actual synthesis of heme, at least in the adult, remains doubtful in view of the fact that simultaneously a successor product, porphyrin, may accumulate in excess and t h a t ALAD is much less affected in organs than it is in blood (Gerber, unpublished results). Another enzyme sensitive towards the toxic action of lead is ferrochelatase which couples iron to the porphyrin moiety [3,5,6,9]. Indeed, in vitro incorporation of Fe is decreased in bone marrow cells from workers intoxicated with lead [13] a n d this defect is probably responsible for the accumulation of porphyrins in erythrocytes and for its excess excretion into urine, as well as for the cellular retention of unused iron as ferritin [2]. Incorporation of glycine into heme of bone marrow cells reflecting the entire synthetic chain of heme formation also is diminished after lead exposure [11]. The data presented show a marked increase in Fe incorporation from day 16 to 18 of pregnancy. Most likely this rise reflects the fact that the stem cells which had attained maximal levels in liver at day 13 of pregnancy [10] had matured to synthesize hemoglobin 3--4 days later. Incorporation of Fe into heme is markedly depressed and the physiological increase in incorporation during the latter period of pregnancy is abolished in embryos whose mothers had been treated with 0.5% of lead. It is still markedly diminished in embryos treated with 0.25% of lead, no corresponding effect of lead on incorporation of glycine into fetal heme or fetal proteins is observed. Earlier we had reported [7,8] that ALAD is depressed and that growth is retarded seriously in the embryos when the mothers are given 0.25--0.5% of lead in the diet. On the other hand, heme concentration in the embryo was f o u n d unaltered. It is, however, quite possible that any deficit in heme at this time of rapid growth will immediately entrain a retardation in growth. Moreover, in the embryo the enzymes particularly sensible to the action of lead are also those which could become rate limiting [14]. The lack of change found in glycine incorporation does not constitute a proof against a decrease in heme synthesis since it must be kept in mind that it depends on several competing pathways. Indeed, uptake of an amino acid is enhanced in lead-treated embryos [4] and this may obscure any depression caused by lead. In the mothers, incorporation of Fe into heme of liver is depressed and incorporation of glycine is unaltered, an observation which concurs with that
178
o n the e m b r y o . O n the o t h e r h a n d , while r a d i o a c t i v i t y in t h e e r y t h r o c y t e o f l e a d - t r e a t e d m o t h e r s is r e d u c e d t h a t in b o n e m a r r o w is increased. This finding is, h o w e v e r , c o n t i n g e n t o f the 24 h interval b e t w e e n injection o f the i s o t o p e s and sacrifice. This p e r i o d was c h o s e n as an o p t i m a l t i m e f o r i n c o r p o r a t i o n i n t o t h e e m b r y o , b u t p a r t o f the n e w l y f o r m e d e r y t h r o c y t e s has t h e n already b e e n released f r o m the b o n e m a r r o w into the b l o o d o f the m o t h e r . When o n e follows i n c o r p o r a t i o n o f Fe into heine f r o m e r y t h r o c y t e s and b o n e m a r r o w as a f u n c t i o n o f time a f t e r i n j e c t i o n it b e c o m e s a p p a r e n t t h a t a f t e r 16 h i n c o r p o r a t i o n Fe in the b o n e m a r r o w is indeed depressed t o a b o u t 60% after 0.25% lead t r e a t m e n t and t h a t the release o f labeled cells is d e l a y e d c o m p a r e d t o c o n t r o l s . REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14
J.E. Barker, M.A. Keenan and L. Raphals, J. Cell Physiol., 74 (1969) 51. M.C. Bessis and W.N. Jensen, Brit. J. Hematol., 11 (1965) 49. E.I.B. Dressel and J.E. Falk, Biochem. J., 63 (1956) 80. G.B. Gerber, J. Maes and J. Deroo, in preparation. S.L.M. Gibson and A. Goldberg, Clin. Sci., 38 (1970) 63. A. Goldberg, H. Ashenbrucker, G.E. Cartwright and M.M. Wintrobe, Blood, 11 (1956) 821. P. Jacquet, A. L~onard and G.B. Gerber, Experientia, 31 (1975) 1312. P. Jacquet, G.B. Gerber and J. Maes, Bull. Environ. Contain. Toxicol., 18 (1977) 271. Kreimer, M. Birnbaum and M. Grinstein, Biochim. Biophys. Acta, 111 (1965) 110. D. Metcalf and M.A.S. Moore, Haeniopoietic Cells, North-Holland Publ., Amsterdam, 1971, p. 10. G.F. Nordberg, Effects and Dose-Relationships of Toxic Metals, Elsevier, Amsterdam, 1976, pp. 446--454. B.L. Vallee and D.D. Ulmer, Ann. Rev. Biochem., 41 (1972) 91. O. Wada, Y. Yano, K. Toyokawa, T. Suzuki, S. Suzuki and H. Katsanuma, Industr. Hlth, 10 (1972) 84. J.S. Woods, Biochem. Pharmacol., 25 (1976) 2147.
179
HEME S Y N T H E S I S IN THE L E A D - I N T O X I C A T E D M O U S E E M B R Y O *
GEORG B. GERBER and JOZEF MAES Euratom/C.E.N., Department of Radiobiology, B-2400 Mol (Belgium) (Received May 12th, 1977) (Revision received September 27th, 1977) (Accepted October 3rd, 1977)
SUMMARY
Incorporation of SSFe and of [14C] glycine was studied in control embryos and mothers and in those which had received lead in the diet from day 7 of pregnancy. Incorporation of Fe into heme of embryonic liver which increases markedly for controls on day 17 of pregnancy was depressed greatly and showed no such increase in lead-intoxicated embryos. These embryos were retarded in growth but had normal heme concentrations in body and liver. Incorporation of glycine into embryonic heme and proteins was n o t affected. Data on incorporation in the mothers are also presented. It is t h o u g h t t h a t the impaired synthesis of heme in lead-intoxicated embryos embryos limits their body growth during the late phase of pregnancy.
INTRODUCTION Exposure of pregnant mice to dietary lead causes retardation of growth of the embryo during the end of the pregnancy and can lead to prenatal death [7]. Earlier, we observed in these embryos a decrease in 5-aminolevulinic acid dehydratase to about one-third of the activity of control animals and a 2--3-fold increase in prophyrin content, but no changes in heme concentration [8]. Heme synthesis thus appears to be sufficient for the reduced b o d y mass of the lead-treated embryos, but this fact does n o t exclude the possibility that the a m o u n t of heme synthetized, nevertheless, limits the growth of these embryos, nor t h a t synthesis of certain types of heme proteins is affected by * Supported by Contract 036-74-7 ENV.B of the E.C. Environmental Research Program. Publication No. 1388 of the Euratom Biology Division.
173
lead more than that of others. In order to obtain more information on the action of lead on heme synthesis, we have now studied incorporation of SSFe and of [ 14C]glycine into heme isolated from the liver of embryos as well as from liver, bone marrow or blood from their mothers. In addition, incorporation of glycine into proteins was also followed. METHODS Mice of the C57B1 strain were mated and, when displaying a vaginal plug, were given a diet containing 0, 0.25, 0.5 or 1% of lead as lead acetate from the 7th day of pregnancy [7]. This procedure differs from our earlier one, where lead was given from the first day of pregnancy on. It was chosen since due to the impaired implantation the number of pregnant mice is very small when high doses of lead are given from the beginning of pregnancy. On the 15th, 16th or 17th day of pregnancy the mice received an intraperitoneal injection of either 10 t~Ci of [SSFe]citrate (from the I.R.E. (Fleurus, Belg., specific activity 10.6 mCi/mg) or of 10 pCi of [2-t4C]glycine (from the I.R.E., specific activity 48.9 mCi/mg). The animals were sacrificed 24 h later, the embryos were removed and their livers were dissected. Blood, femoral bone marrow and livers of mothers were also taken. Liver was homogenized in chloroform--methanol (2 : 1), the precipitate was washed with 50% ethanol and hemin was extracted with acid acetone. After spectrophotometric determination of the hemin c o n t e n t using Drabkin's solution, the extract was taken to dryness, redissolved in pyridine, and heroin was isolated by thin-layer chromatography in m e t h a n o l - a c e t i c acid--water (40 : 3 : 7). Hemin was eluted with acid acetone, the quantity of hemin was determined spectrophotometrically and, after decolorization with hydrogen peroxide, radioactivity of hemin was measured in a liquid scintillation counter using the appropriate conditions for counting the two isotopes. Protein from the residue of the homogenate after acid acetone extraction was washed, hydrolyzed in 6 N HC1, dried and counted for radioactivity. Bone marrow cells were washed with 50% ethanol and hemin was extracted with acid acetone; the blood samples were treated with 0.6 N perchloric acid (PCA). The washed precipitate was extracted with acid acetone and the hemin was purified as described above. RESULTS Incorporation of SSFe into hepatic heme of control embryos increases markedly on day 17 of pregnancy when the embryo grows from ~ 4 0 0 mg (day 16) to ~ 6 0 0 mg (day 17). (Fig. 1), whereas incorporation of [14C]glycine into heme does n o t change significantly with the age of the embryo. Incorporation of glycine into hepatic proteins increases only slightly during this time (Fig. 2). When the mothers were given 0.25% of lead in the diet incorporation of
174
SSFe
100-
ncorporation
x:
Controls
n :
0,5
e:
0,25
*/,
into
fetal
heme
Lead
A
*/,
Lead
80-
T
E (3. u
_L
60 >,
t t
> (J
40
20 ¸ {3. U3
0
i
0
i
400
I
I
800 Embryoweight
I
I
1200 (mg)
Fig. 1. Incorporation o f F6 into h e m e isolated from the liver o f control e m b r y o s and o f e m b r y o s w h o s e m o t h e r s had received 0 . 2 5 or 0.5% o f lead in the diet. The points represent the m e a n and standard errors from o n e litter ( 4 - - 8 animals). The weights w i t h i n a litter varied from 7--10%. The ages o f pregnancy corresponding to the weights are day 16, all animals b e l o w 4 0 0 mg, day 17 those b e l o w 7 0 0 mg and day 18 those above 8 0 0 mg e x c e p t for the 0.25% lead-treated litter weighing 6 8 0 mg w h i c h was obtained on day 18.
SSFe into heine is significantly reduced (Fig. 1), but an increase with age can still be discerned. I n c o r p o r a t i o n o f glycine into h e m e is n o t altered by lead t r e a t m e n t but t h a t i n t o hepatic proteins of the e m b r y o is increased (Fig. 2). N e x t , a s o m e w h a t higher dose (0.5%) w h i c h inhibits more strongly e m b r y o n i c g r o w t h was c h o s e n to investigate the effect on i n c o r p o r a t i o n of SSFe i n t o heme whereas the highest d o s e at w h i c h a few live e m b r y o s can still be o b t a i n e d was utilized to f o l l o w glycine incorporation. As Fig. 1 s h o w s the increase with age o f SSFe i n c o r p o r a t i o n is abolished at 0.5% of lead whereas even a dose of 1% of lead does n o t depress significantly i n c o r p o r a t i o n of glycine i n t o h e m e (specific activities o f all ages c o m b i n e d : means + S.E. controls 2 . 1 5 ± 0 . 1 7 , 0.25% lead 2 . 5 7 -+ 0 . 2 8 , 1% of lead 3 . 0 0 +_ 0 . 4 2 ) . On the other hand, the i n c o r p o r a t i o n o f glycine i n t o proteins w h i c h is elevated at 0.25% of lead is normal at 1% (specific activities of all ages c o m b i n e d : controls 8.31 -+ 0.58, 0.25% lead 2 6 . 5 3 + 4 . 2 6 , 1% lead 9 . 3 7 +- 1 . 2 6 ) . Data
175
l~C
40-
E
Glycine x:
Controls
D:
0,25
~:
I
"/,
Incorporat
on
into
Protein
~]
*/,= L e a d Lead
30
E u
~
20
°--
U
<
10
d U'I
0
I
0
I
400
I
I
I
800 Embryoweight
I
1200 (mg)
Fig. 2. Incorporation of [2-'4C]glycine into total liver proteins of control embryos and of embryos whose mothers had received 0.25 or 1% of lead in the diet. The points represent the mean and standard errors from one litter. The ages of pregnancy corresponding to the weights are day 16 below 420 mg,~lay 17 below 700 rag. The value for 1% of lead is from day 17. No significant difference was found for incorporation of [2-~4C]glycine into heine isolated from embryonic liver. on the i n c o r p o r a t i o n o f SSFe and [14C]glycine into organs o f the m o t h e r are p r e s e n t e d in Table I. Since n o significant differences in these values with r e s p e c t t o the age o f p r e g n a n c y was seen, all values have been p o o l e d . In liver and b l o o d , i n c o r p o r a t i o n o f SSFe in lead-treated animals is m a r k e d l y depressed b u t in b o n e m a r r o w it is e n h a n c e d c o m p a r e d to controls. I n c o r p o r a t i o n o f glycine into h e m e o f e r y t h r o c y t e s or liver is n o t affected b y lead but, in b o n e m a r r o w a slight depression o f i n c o r p o r a t i o n is n o t e d . The specific activity o f proteins is u n a l t e r e d in liver and elevated in b l o o d and b o n e m a r r o w a f t e r 0.25% o f lead. The value in b l o o d varies, however, greatly a m o n g the animals studied. DISCUSSION B l o o d f o r m a t i o n in the e m b r y o p r o c e e d s t h r o u g h several stages o f develo p m e n t . During the early p e r i o d up to d a y 12 the y o l k sack is the principal
176
b-a --O.
Liver Liver Liver Bone m a r r o w Bone m a r r o w Bone m a r r o w Blood Blood Blood
Incorporation Incorporation Incorporation Incorporation Incorporation Incorporation Incorporation Incorporation Incorporation
7.82 0.245 1.51 2.70 0.246 3.42 5.24 0.287 2.83
± 1.36 [12] ± 0.055 [6] ± 0.156 ± 0.86 ± 0.062 ± 0.33 ± 1.21 + 0.037 ± 0.55
Control
Specific activity
± ± ± ± ± ± ± +
5.46 0.382 1.66 4.56 12,15 2.47 0.221 6.97
0.25% Pb
2,62 0.167 0.037 2.25
1.35 [6] 0.092 [7] 0.67 1.25
± 0.49 [ 1 7 ]
0.41
± 0.06
13,3 ± 2.55 0.101 ± 0.0187
0.57
0.5% Pb
0.242 ± 0.04 2.25 ± 0.07
0.216 + 0.041 [4] 1.92 ± 0.52
1% Pb
This table w h i c h p r e s e n t m e a n ~ S.E. c o m b i n e s all data from m o t h e r s w h o had been e x p o s e d f r o m 9 t o 11 days, i.e., the 7th d a y o f p r e g n a n c y to lead in the diet. The n u m b e r o f animals used is indicated in b r a c k e t s for Fe and glycine i n c o r p o r a t i o n , respectively. Significant d i f f e r e n c e s from c o n t r o l s are u n d e r l i n e d o n c e (P < 0.05) or twice (P ~ 0.01). I n c o r p o r a t i o n i n t o heine is e x p r e s s e d as c p m / u g h e i n e , i n c o r p o r a t i o n into p r o t e i n s as c p m / m g tissue.
5SFe into heine glycine into h e m e glycine into p r o t e i n s 5SFe into heine glycine into h e m e glycine into p r o t e i n s o f SSFe into h e m e o f glycine into h e m e o f glycine into p r o t e i n s
Organ
S y s t e m studied
I N C O R P O R A T I O N OF SSFe and [14C]GLYCINE INTO D I F F E R E N T O R G A N S OF P R E G N A N T MICE
TABLE I
site of erythropoiesis, which then is superseded by liver and later on by spleen [1,10]. Blood formation in bone marrow takes over only during postnatal life. The relative activities of the various enzymes participating in heme biosynthesis also change during development [14]. In the adult mouse, 5-aminolevulinic acid synthetase is probably rate limiting for heme synthesis but in the fetal and newborn mouse the activity of 6 -aminolevulinic acid dehydratase (ALAD) and ferrochelatase are comparatively small and could become a critical factor when diminished by toxic effects. Lead is known to depress several reactions in the pathways leading to hemoglobin [11,12]. The depression in ALAD which in blood occurs after minimal exposure to lead has found much attention but its significance for actual synthesis of heme, at least in the adult, remains doubtful in view of the fact that simultaneously a successor product, porphyrin, may accumulate in excess and t h a t ALAD is much less affected in organs than it is in blood (Gerber, unpublished results). Another enzyme sensitive towards the toxic action of lead is ferrochelatase which couples iron to the porphyrin moiety [3,5,6,9]. Indeed, in vitro incorporation of Fe is decreased in bone marrow cells from workers intoxicated with lead [13] a n d this defect is probably responsible for the accumulation of porphyrins in erythrocytes and for its excess excretion into urine, as well as for the cellular retention of unused iron as ferritin [2]. Incorporation of glycine into heme of bone marrow cells reflecting the entire synthetic chain of heme formation also is diminished after lead exposure [11]. The data presented show a marked increase in Fe incorporation from day 16 to 18 of pregnancy. Most likely this rise reflects the fact that the stem cells which had attained maximal levels in liver at day 13 of pregnancy [10] had matured to synthesize hemoglobin 3--4 days later. Incorporation of Fe into heme is markedly depressed and the physiological increase in incorporation during the latter period of pregnancy is abolished in embryos whose mothers had been treated with 0.5% of lead. It is still markedly diminished in embryos treated with 0.25% of lead, no corresponding effect of lead on incorporation of glycine into fetal heme or fetal proteins is observed. Earlier we had reported [7,8] that ALAD is depressed and that growth is retarded seriously in the embryos when the mothers are given 0.25--0.5% of lead in the diet. On the other hand, heme concentration in the embryo was f o u n d unaltered. It is, however, quite possible that any deficit in heme at this time of rapid growth will immediately entrain a retardation in growth. Moreover, in the embryo the enzymes particularly sensible to the action of lead are also those which could become rate limiting [14]. The lack of change found in glycine incorporation does not constitute a proof against a decrease in heme synthesis since it must be kept in mind that it depends on several competing pathways. Indeed, uptake of an amino acid is enhanced in lead-treated embryos [4] and this may obscure any depression caused by lead. In the mothers, incorporation of Fe into heme of liver is depressed and incorporation of glycine is unaltered, an observation which concurs with that
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o n the e m b r y o . O n the o t h e r h a n d , while r a d i o a c t i v i t y in t h e e r y t h r o c y t e o f l e a d - t r e a t e d m o t h e r s is r e d u c e d t h a t in b o n e m a r r o w is increased. This finding is, h o w e v e r , c o n t i n g e n t o f the 24 h interval b e t w e e n injection o f the i s o t o p e s and sacrifice. This p e r i o d was c h o s e n as an o p t i m a l t i m e f o r i n c o r p o r a t i o n i n t o t h e e m b r y o , b u t p a r t o f the n e w l y f o r m e d e r y t h r o c y t e s has t h e n already b e e n released f r o m the b o n e m a r r o w into the b l o o d o f the m o t h e r . When o n e follows i n c o r p o r a t i o n o f Fe into heine f r o m e r y t h r o c y t e s and b o n e m a r r o w as a f u n c t i o n o f time a f t e r i n j e c t i o n it b e c o m e s a p p a r e n t t h a t a f t e r 16 h i n c o r p o r a t i o n Fe in the b o n e m a r r o w is indeed depressed t o a b o u t 60% after 0.25% lead t r e a t m e n t and t h a t the release o f labeled cells is d e l a y e d c o m p a r e d t o c o n t r o l s . REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14
J.E. Barker, M.A. Keenan and L. Raphals, J. Cell Physiol., 74 (1969) 51. M.C. Bessis and W.N. Jensen, Brit. J. Hematol., 11 (1965) 49. E.I.B. Dressel and J.E. Falk, Biochem. J., 63 (1956) 80. G.B. Gerber, J. Maes and J. Deroo, in preparation. S.L.M. Gibson and A. Goldberg, Clin. Sci., 38 (1970) 63. A. Goldberg, H. Ashenbrucker, G.E. Cartwright and M.M. Wintrobe, Blood, 11 (1956) 821. P. Jacquet, A. L~onard and G.B. Gerber, Experientia, 31 (1975) 1312. P. Jacquet, G.B. Gerber and J. Maes, Bull. Environ. Contain. Toxicol., 18 (1977) 271. Kreimer, M. Birnbaum and M. Grinstein, Biochim. Biophys. Acta, 111 (1965) 110. D. Metcalf and M.A.S. Moore, Haeniopoietic Cells, North-Holland Publ., Amsterdam, 1971, p. 10. G.F. Nordberg, Effects and Dose-Relationships of Toxic Metals, Elsevier, Amsterdam, 1976, pp. 446--454. B.L. Vallee and D.D. Ulmer, Ann. Rev. Biochem., 41 (1972) 91. O. Wada, Y. Yano, K. Toyokawa, T. Suzuki, S. Suzuki and H. Katsanuma, Industr. Hlth, 10 (1972) 84. J.S. Woods, Biochem. Pharmacol., 25 (1976) 2147.
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