Acta pharmacol. et toxicol. 1976, 38,289-298.
From the Institute of Occupational Health, Gydas vei 8, Oslo 3, Norway
The Importance of Organ Blood Mercury when Comparing Foetal and Maternal Rat Organ Distribution of Mercury after Methyl Mercury Exposure BY Axel Wannag (Received August 4, 1975; Accepted September 3, 1975)
Abstract: Foetal rat brain has previously been shown to contain twice as much mercury as maternal brain, after methyl mercury injection in the mother rat. However when brain mercury is corrected for the mercury which is present in the blood of the brain, foetal rat brain will contain 4 to 5 times as much mercury as maternal brain (depending on the stage of gestation), 24 hours after methyl mercuric chloride injection in the mother. Even when methyl mercuric chloride was injected in the mother about 14 days before term, near-term foetal brain contains 1.4 times as much mercury as the maternal brain. Likewise when corrected for mercury in the blood of the organ, foetal rat liver contains from 2.0 to 2.6 times more mercury than the maternal liver, and the foetal kidney contains from 13 to 23 times less mercury than the maternal kidney. The amount of mercury in foetal blood is about 65 % of the mercury in maternal blood 24 hours after methyl mercuric chloride injection in the mother, but maternal and foetal blood contain equal amounts 14 days after the injection. Except for the foetal membranes, no inorganic mercury released by biotransformation of methyl mercuric chloride was detected in the foetal-placental unit.
Key-words: Methyl mercuric compounds - rats - blood volume.
Methyl mercury exposure in the pregnant female has been found to involve greater risk of damage to the foetus than to the mother in humans (MATSUMOTO et al. 1965) mice (SPYKER& SMITHBERG 1972; SPYKERet al. 1972) and rats (MATSUMOTO et al. 1967). In rats, the mercury concentrations in the foetal brains have been found to be around twice that in the maternal brains (NULLet al. 1973; YANGet al. 1972). However, as the erythrocytes contain much of the total body burden of methyl mercury in the rat, variations in the blood content between foetal and maternal brains could account for the observed differences in mercury content. DEAVERS et al. (1971) and WINTROBE & SHUMACKER (1936) have shown that the blood volume changes continuously during foetal and neonatal life, and that this is reflected in the
290
AXEL WANNAG
blood contents of the organs. This study was undertaken in order to compare the mercury content in maternal and foetal brain, liver and kidney, taking the mercury in the blood of the organs into consideration. Materials and Methods Female inbred Wistar rats weighing from 200 to 250 g before pregnancy were used. They were observed when mating, and the pregnancy was recorded from that date. Initially, they were all kept in one cage, food (Rasmussens diet) and water being given ad libitum. Radioactive *OsHg-methylmercuric chloride was obtained from New England Nuclear. This compound, however, is fairly rapidly contaminated with inorganic zOSHg, due to radioactive auto-decomposition. To ensure a stable solution, the purchased isotope was purified before use. A sample was taken into 10 ml concentrated HCl, which was shaken for 10 minutes with 10 ml benzene. The benzene extract was separated from HCl, and evaporated to dryness at 40”. Finally the remaining methyl mercuric chloride was dissolved in 5 mM-Na,Co,. In this solution the ,OSHg-rnethyl mercuric chloride remained stable for months as tested with the Conway diffusion chamber technique (NORSETH & CLARKSON 1970a). At different stages of gestation the pregnant rats were injected with ZOSHg-methyl mercuric chloride 1 mg/kg into the tail vein. The injected animals were placed in metabolism cages with access to food and water ad libitum. At the end of the experiment an incision was made in the abdomen of the anaesthesized animal (nembutal@ Vet. (mebumalum NFN), Abbott), giving access to the uterus, which was partially opened exposing one foetal-placental unit at a time. The foetal membranes were opened and the foetal blood collected with a heparinized syringe from the umbilical blood vessels. Then the placenta was loosened from the uterus, the foetal-placental unit removed, and the umbilical cord cut a t the navel of the foetus. When all the foetal-placental units had been removed, 0.5 ml of heparin (loo0 unit/ml) was injected into the aorta of the mother and as much blood as possible drawn into the syringe. Generally 7-8 ml blood was collected. The maternal liver, kidneys and brain were then removed. The foetal liver, kidneys, brain, foetal membranes and placenta were collected from the foetal-placental units. All specimen were counted in a Packard Auto-gamma spectrometer 3001. 125I-human serum albumin was obtained from the Institute of Atomic Energy, Kjeller, Norway. This was injected intravenously into anaestesized animals (nembutal@ Vet. Abbott), using the tail vein in the mother rat, and the umbilical vein in the foetus. After allowing time for the “SI-serum albumine to be distributed evenly (10-20 min. in the adults, 4-5 min. in the foetus) as much blood as possible was drawn from the aorta in the adults and from the umbilical artery in the foetuses. The brain, kidneys and liver were then removed. Blood and organs were counted in a Packard Autogamma spectrometer 3001. The blood volume of an organ was calculated from the 1261-activity in the organ and the l2S1-activity in the arterial blood of the animal (maternal aorta, and foetal umbilical artery). The mercury in the blood of an organ was calculated from the median blood volume of the organ and the mercury content in the arterial blood of the animal. The amount of inorganic mercury in the organs was determined with the Conway diffusion chamber technique (NORSETH & CLARKSON 1970a). Unless stated otherwise, significance is referred t o the one sided descriptive significance level (a’) of the Wilcoxon-Mann-Whitney test. In some cases the Sign Test was used.
29 1
MERCURY ORGAN DISTRIBUTION IN RAT
Results
Blood volume measurements. The median blood volumes in the foetal organs on the last four days of pregnancy are presented in table 1. The largest amount of blood in the brain was found to be 24 mg/g on day 19 (a’, 0.002 comparing day 19 and 20), in the liver 127 mg/g and in the kidney 102 mglg both on day 20 (a’,0.047 and a’, 0.008, respectively, comparing day 20 and 21). There were no significant differences in blood content of the organs between litters on the same day of pregnancy. Nor were there any significant differences in the blood content of an organ between the mothers on different days of pregnancy. The values for blood content in maternal organs were therefore combined (table 1). Three non-pregnant rats had the same amount of blood in the organs as the pregnant rats. Mercury in organs. Twenty-four hours after methyl mercuric chloride injection the maternal liver contained 5.13 ymol Hg/kg; of this 2.48 pmol H o g was in the blood. Thus, the liver corrected for blood mercury, contained 2.65 pmol H o g . The maternal kidney contained 32.22 pmol Hg/kg, corrected for blood mercury 33.86 ymol Hg/kg, and the maternal brain 0.87 ymol Hg/kg, corrected for blood mercury 0.41 ymol H o g (table 2). As there were no significant differences in the mercury content of maternal livers, kidneys, or brains on the last four days of pregnancy, the values from all the mothers were combined. Three non-pregnant rats had similar mercury contents in the organs as the pregnant rats. The amount of mercury in foetal rat organs on each of the last four days of pregnancy, after methyl mercuric chloride injection in
Table 1 . Blood content in maternal and foetal organs on the last four days of pregnancy.
Foetus day 18 Foetus day 19 Foetus day 20 Foetus day 21 Mother rat
*
No. of litters
No. of animals
4 4 2 1
13 18 10 9 9
Liver 97* 108 127 99 123
116-83 125-100 158-98 130-76 128-101
Kidney 73 87-66 80 94-73 102 154-89 82 104-69 137 161-111
Brain 20 24 20 17 19
26-18 26-22 22-18 19-15 21-17
Values are given in mg/g organ. The median and the confidence interval of the median (0.95) are given.
7'60 6'83
7.19 6.36
6.52 5.66
6.64 5.87
5.13 2.65
Foetus organ day 19 organ cor.
Foetus organ day 20 organ cor.
Foetus organ day 21 organ cor.
Mother organ organ cor.
32.22 33.86
6'25 4.85 37'26 29.79
4.23 3.42
3.93 2S9
3'78 3'56
3'05
3'50
0.87 0.41
1.77 1.58
1.88 1.59
2.18 1.88
2.14 1.92
Brain
7.49
2.01 1.58 0.94 0.81
7.89
7.81
6.76
1'88 1.81
2.18 1.96
2'24 2.04 2.67 2.94
2.21
8.77 7.30 7.77 7.05
2.45
2.62 1.38
7.63 2.88
3.07 2.60
3.28 2.39
Foetal membranes
15.32 7.73
9.10 6.50
Placenta
23.80
13,64
15.90
14.71
14.04
25'34 22.46
14.71 12.23
17.57 12.59
15.33 14.18
15.39 12.00
Blood
Mercury is given in ymol Hgkg. For organs, median value* and range+ are given. Organ cor. is the median mercury in the organ corrected for mercury in the blood of the organ. The values are from 3 litters on day 19, 20 and 21 of pregnancy, 4 litters on day 18 and from 12 mothers.
4.11 3.39
3.76 2.19
3.75 2.77
3.40 2.58
Kidney
7.40 5.94
7'46 5*40
5*86'
7'06+
6.33' 5.62
Foetus organ day 18 organ cor.
Liver
Mercury in foetal and maternal organs 24 hours after methyl mercuric chloride injection into the mother.
Table 2.
5b
sz
Q
*X m r
293
MERCURY ORGAN DISTRIBUTION IN RAT
the mother 24 hours before each of these days, are presented in table 2. As can be seen 20-25 % of the mercury in a foetal liver is found in the blood, as against about 55 % of the mercury in the maternal liver. From 25-50 % of the mercury in foetal kidney is in the blood as compared to about 10 % in the blood of the maternal kidney. About 15 % of the mercury in foetal brain is found in the blood, as against around 50 % in maternal brain. The differences between foetal and maternal organs on the last 4 days of the pregnancy were all significant (a’, 0.000) (table 2). Foetal to maternal liver mercury ratio was 2/1 with some day to day variation. The foetallmaternal kidney mercury ratio varied from about 1/13 to 1/10. Foetal brain contained about 5 times as much mercury as maternal brain on day 18, while the ratio changed and reached about 4 on day 21. Maternal blood had about 1.6 times as much mercury as foetal blood (a’, 0.000). The ratios between mercury in organs, corrected for mercury in the blood of the organs, and mercury in blood on the last four days of pregnancy, are presented in table 3. Foetal liver and brain accumulate more of the available mercury than the maternal organs, while foetal kidney accumulates less than the maternal kidney. The mercury content of maternal and foetal organs were also studied at various time intervals after methyl mercuric chloride injection into the mother. After the injection, the pregnancy was allowed to proceed to day 19 or 20, before the animals were sacrificed. The mercury in organs, corrected for mercury in the blood of the organs, are presented in table 4. Maternal liver and kidney contain most mercury one day after injection. The mercury content then declines, and after 14 days the liver contains about 60 % and the kidney about 75 % of the mercury found one day after
Table 3 . Ratios in foetus and mother, between mercury content in organs, corrected for mercury in the blood of the organs, and mercury in the blood, 24 hours after methyl mercuric chloride injection in the mother. Foetus Day of pregnancy Liver Kidney Brain
18
0,40 0,18
0,14
19
20
21
0,43
0,36 0,14 0,lO
0,43 0,24 0,12
0,19 0,13
Mother 0,11 1,42 0,02
Values are calculated from the median organ and blood mercury of mothers and foetuses. From 3 litters on days 19, 20 and 21 of pregnancy, from 4 litters on day 18, and from 12 mothers.
294
AXEL WANNAG
Table 4. Mercury in foetal and maternal organs, corrected for mercury in the blood of the organs, and 20aHg++as % of the mercury in the organs, at various time intervals after methyl mercuric chloride injection in the mother. 3
1
Days after injection
Mother
Liver Kidney Brain Blood
Foetus
Liver Kidney Brain Blood
% Hg++ Hg
Hg
% Hgtt
Hg
2,65 33,86 0,41 23,80
8-10
2,36 31,56 0,97 21,95
2,08 27,77 1,26 13,70
6,Ol 2,48 1,72 15,31
From the Institute of Occupational Health, Gydas vei 8, Oslo 3, Norway
The Importance of Organ Blood Mercury when Comparing Foetal and Maternal Rat Organ Distribution of Mercury after Methyl Mercury Exposure BY Axel Wannag (Received August 4, 1975; Accepted September 3, 1975)
Abstract: Foetal rat brain has previously been shown to contain twice as much mercury as maternal brain, after methyl mercury injection in the mother rat. However when brain mercury is corrected for the mercury which is present in the blood of the brain, foetal rat brain will contain 4 to 5 times as much mercury as maternal brain (depending on the stage of gestation), 24 hours after methyl mercuric chloride injection in the mother. Even when methyl mercuric chloride was injected in the mother about 14 days before term, near-term foetal brain contains 1.4 times as much mercury as the maternal brain. Likewise when corrected for mercury in the blood of the organ, foetal rat liver contains from 2.0 to 2.6 times more mercury than the maternal liver, and the foetal kidney contains from 13 to 23 times less mercury than the maternal kidney. The amount of mercury in foetal blood is about 65 % of the mercury in maternal blood 24 hours after methyl mercuric chloride injection in the mother, but maternal and foetal blood contain equal amounts 14 days after the injection. Except for the foetal membranes, no inorganic mercury released by biotransformation of methyl mercuric chloride was detected in the foetal-placental unit.
Key-words: Methyl mercuric compounds - rats - blood volume.
Methyl mercury exposure in the pregnant female has been found to involve greater risk of damage to the foetus than to the mother in humans (MATSUMOTO et al. 1965) mice (SPYKER& SMITHBERG 1972; SPYKERet al. 1972) and rats (MATSUMOTO et al. 1967). In rats, the mercury concentrations in the foetal brains have been found to be around twice that in the maternal brains (NULLet al. 1973; YANGet al. 1972). However, as the erythrocytes contain much of the total body burden of methyl mercury in the rat, variations in the blood content between foetal and maternal brains could account for the observed differences in mercury content. DEAVERS et al. (1971) and WINTROBE & SHUMACKER (1936) have shown that the blood volume changes continuously during foetal and neonatal life, and that this is reflected in the
290
AXEL WANNAG
blood contents of the organs. This study was undertaken in order to compare the mercury content in maternal and foetal brain, liver and kidney, taking the mercury in the blood of the organs into consideration. Materials and Methods Female inbred Wistar rats weighing from 200 to 250 g before pregnancy were used. They were observed when mating, and the pregnancy was recorded from that date. Initially, they were all kept in one cage, food (Rasmussens diet) and water being given ad libitum. Radioactive *OsHg-methylmercuric chloride was obtained from New England Nuclear. This compound, however, is fairly rapidly contaminated with inorganic zOSHg, due to radioactive auto-decomposition. To ensure a stable solution, the purchased isotope was purified before use. A sample was taken into 10 ml concentrated HCl, which was shaken for 10 minutes with 10 ml benzene. The benzene extract was separated from HCl, and evaporated to dryness at 40”. Finally the remaining methyl mercuric chloride was dissolved in 5 mM-Na,Co,. In this solution the ,OSHg-rnethyl mercuric chloride remained stable for months as tested with the Conway diffusion chamber technique (NORSETH & CLARKSON 1970a). At different stages of gestation the pregnant rats were injected with ZOSHg-methyl mercuric chloride 1 mg/kg into the tail vein. The injected animals were placed in metabolism cages with access to food and water ad libitum. At the end of the experiment an incision was made in the abdomen of the anaesthesized animal (nembutal@ Vet. (mebumalum NFN), Abbott), giving access to the uterus, which was partially opened exposing one foetal-placental unit at a time. The foetal membranes were opened and the foetal blood collected with a heparinized syringe from the umbilical blood vessels. Then the placenta was loosened from the uterus, the foetal-placental unit removed, and the umbilical cord cut a t the navel of the foetus. When all the foetal-placental units had been removed, 0.5 ml of heparin (loo0 unit/ml) was injected into the aorta of the mother and as much blood as possible drawn into the syringe. Generally 7-8 ml blood was collected. The maternal liver, kidneys and brain were then removed. The foetal liver, kidneys, brain, foetal membranes and placenta were collected from the foetal-placental units. All specimen were counted in a Packard Auto-gamma spectrometer 3001. 125I-human serum albumin was obtained from the Institute of Atomic Energy, Kjeller, Norway. This was injected intravenously into anaestesized animals (nembutal@ Vet. Abbott), using the tail vein in the mother rat, and the umbilical vein in the foetus. After allowing time for the “SI-serum albumine to be distributed evenly (10-20 min. in the adults, 4-5 min. in the foetus) as much blood as possible was drawn from the aorta in the adults and from the umbilical artery in the foetuses. The brain, kidneys and liver were then removed. Blood and organs were counted in a Packard Autogamma spectrometer 3001. The blood volume of an organ was calculated from the 1261-activity in the organ and the l2S1-activity in the arterial blood of the animal (maternal aorta, and foetal umbilical artery). The mercury in the blood of an organ was calculated from the median blood volume of the organ and the mercury content in the arterial blood of the animal. The amount of inorganic mercury in the organs was determined with the Conway diffusion chamber technique (NORSETH & CLARKSON 1970a). Unless stated otherwise, significance is referred t o the one sided descriptive significance level (a’) of the Wilcoxon-Mann-Whitney test. In some cases the Sign Test was used.
29 1
MERCURY ORGAN DISTRIBUTION IN RAT
Results
Blood volume measurements. The median blood volumes in the foetal organs on the last four days of pregnancy are presented in table 1. The largest amount of blood in the brain was found to be 24 mg/g on day 19 (a’, 0.002 comparing day 19 and 20), in the liver 127 mg/g and in the kidney 102 mglg both on day 20 (a’,0.047 and a’, 0.008, respectively, comparing day 20 and 21). There were no significant differences in blood content of the organs between litters on the same day of pregnancy. Nor were there any significant differences in the blood content of an organ between the mothers on different days of pregnancy. The values for blood content in maternal organs were therefore combined (table 1). Three non-pregnant rats had the same amount of blood in the organs as the pregnant rats. Mercury in organs. Twenty-four hours after methyl mercuric chloride injection the maternal liver contained 5.13 ymol Hg/kg; of this 2.48 pmol H o g was in the blood. Thus, the liver corrected for blood mercury, contained 2.65 pmol H o g . The maternal kidney contained 32.22 pmol Hg/kg, corrected for blood mercury 33.86 ymol Hg/kg, and the maternal brain 0.87 ymol Hg/kg, corrected for blood mercury 0.41 ymol H o g (table 2). As there were no significant differences in the mercury content of maternal livers, kidneys, or brains on the last four days of pregnancy, the values from all the mothers were combined. Three non-pregnant rats had similar mercury contents in the organs as the pregnant rats. The amount of mercury in foetal rat organs on each of the last four days of pregnancy, after methyl mercuric chloride injection in
Table 1 . Blood content in maternal and foetal organs on the last four days of pregnancy.
Foetus day 18 Foetus day 19 Foetus day 20 Foetus day 21 Mother rat
*
No. of litters
No. of animals
4 4 2 1
13 18 10 9 9
Liver 97* 108 127 99 123
116-83 125-100 158-98 130-76 128-101
Kidney 73 87-66 80 94-73 102 154-89 82 104-69 137 161-111
Brain 20 24 20 17 19
26-18 26-22 22-18 19-15 21-17
Values are given in mg/g organ. The median and the confidence interval of the median (0.95) are given.
7'60 6'83
7.19 6.36
6.52 5.66
6.64 5.87
5.13 2.65
Foetus organ day 19 organ cor.
Foetus organ day 20 organ cor.
Foetus organ day 21 organ cor.
Mother organ organ cor.
32.22 33.86
6'25 4.85 37'26 29.79
4.23 3.42
3.93 2S9
3'78 3'56
3'05
3'50
0.87 0.41
1.77 1.58
1.88 1.59
2.18 1.88
2.14 1.92
Brain
7.49
2.01 1.58 0.94 0.81
7.89
7.81
6.76
1'88 1.81
2.18 1.96
2'24 2.04 2.67 2.94
2.21
8.77 7.30 7.77 7.05
2.45
2.62 1.38
7.63 2.88
3.07 2.60
3.28 2.39
Foetal membranes
15.32 7.73
9.10 6.50
Placenta
23.80
13,64
15.90
14.71
14.04
25'34 22.46
14.71 12.23
17.57 12.59
15.33 14.18
15.39 12.00
Blood
Mercury is given in ymol Hgkg. For organs, median value* and range+ are given. Organ cor. is the median mercury in the organ corrected for mercury in the blood of the organ. The values are from 3 litters on day 19, 20 and 21 of pregnancy, 4 litters on day 18 and from 12 mothers.
4.11 3.39
3.76 2.19
3.75 2.77
3.40 2.58
Kidney
7.40 5.94
7'46 5*40
5*86'
7'06+
6.33' 5.62
Foetus organ day 18 organ cor.
Liver
Mercury in foetal and maternal organs 24 hours after methyl mercuric chloride injection into the mother.
Table 2.
5b
sz
Q
*X m r
293
MERCURY ORGAN DISTRIBUTION IN RAT
the mother 24 hours before each of these days, are presented in table 2. As can be seen 20-25 % of the mercury in a foetal liver is found in the blood, as against about 55 % of the mercury in the maternal liver. From 25-50 % of the mercury in foetal kidney is in the blood as compared to about 10 % in the blood of the maternal kidney. About 15 % of the mercury in foetal brain is found in the blood, as against around 50 % in maternal brain. The differences between foetal and maternal organs on the last 4 days of the pregnancy were all significant (a’, 0.000) (table 2). Foetal to maternal liver mercury ratio was 2/1 with some day to day variation. The foetallmaternal kidney mercury ratio varied from about 1/13 to 1/10. Foetal brain contained about 5 times as much mercury as maternal brain on day 18, while the ratio changed and reached about 4 on day 21. Maternal blood had about 1.6 times as much mercury as foetal blood (a’, 0.000). The ratios between mercury in organs, corrected for mercury in the blood of the organs, and mercury in blood on the last four days of pregnancy, are presented in table 3. Foetal liver and brain accumulate more of the available mercury than the maternal organs, while foetal kidney accumulates less than the maternal kidney. The mercury content of maternal and foetal organs were also studied at various time intervals after methyl mercuric chloride injection into the mother. After the injection, the pregnancy was allowed to proceed to day 19 or 20, before the animals were sacrificed. The mercury in organs, corrected for mercury in the blood of the organs, are presented in table 4. Maternal liver and kidney contain most mercury one day after injection. The mercury content then declines, and after 14 days the liver contains about 60 % and the kidney about 75 % of the mercury found one day after
Table 3 . Ratios in foetus and mother, between mercury content in organs, corrected for mercury in the blood of the organs, and mercury in the blood, 24 hours after methyl mercuric chloride injection in the mother. Foetus Day of pregnancy Liver Kidney Brain
18
0,40 0,18
0,14
19
20
21
0,43
0,36 0,14 0,lO
0,43 0,24 0,12
0,19 0,13
Mother 0,11 1,42 0,02
Values are calculated from the median organ and blood mercury of mothers and foetuses. From 3 litters on days 19, 20 and 21 of pregnancy, from 4 litters on day 18, and from 12 mothers.
294
AXEL WANNAG
Table 4. Mercury in foetal and maternal organs, corrected for mercury in the blood of the organs, and 20aHg++as % of the mercury in the organs, at various time intervals after methyl mercuric chloride injection in the mother. 3
1
Days after injection
Mother
Liver Kidney Brain Blood
Foetus
Liver Kidney Brain Blood
% Hg++ Hg
Hg
% Hgtt
Hg
2,65 33,86 0,41 23,80
8-10
2,36 31,56 0,97 21,95
2,08 27,77 1,26 13,70
6,Ol 2,48 1,72 15,31
