Toxicology, 11 (1978} 203--206 © Elsevier/North-Holland Scientific Publishers Ltd.
EFFECT OF M E T H Y L M E R C U R Y ON SOME CONSTITUENTS OF SERUM AND URINE FELICITAS P L A N A S - B O H N E * Kernforschungszentrum Karlsruhe, Institut fiirGenetik und filrToxikologie yon Spaltstoffen, Postfach 3640, D 7500 Karlsruhe 1 (G.F.R.)
(Received July 17th, 1978) (Accepted July 20th, 1978)
SUMMARY
To evaluate some of the early effects of methylmercury chloride (MMC) male rats were given 10, 20 or 30 mg MMC/kg intraperitoneaUy. Urine was analysed for vanilmandelic acid (VMA), leucine aminopeptidase (LAP), alkaline Dhosphatase (AP), and creatinine, blood for glucose-6-phosphatase (G-6-P) and glucose, serum for glutamate-oxalate-transaminase (GOT) and urea. Except for LAP and AP excretion there is no effect of MMC on the parameters investigated. However, the effects on these 2 renal enzymes are too variable to permit their use as a test for MMC toxicity.
INTRODUCTION
The toxicity of methylmercury compounds is well established since the severe outbreaks of poisoning in Japan in the 1950's and in Iraq in the 1970's. The most striking clinical symptoms are neurological disturbances, which however, are seen only some time after the beginning of methylmercury exposure [1,2]. By this time the toxic effects are in many cases irreversible. It seemed desirable, therefore, to find some parameter, by which the degree of a suspected methylmercury intoxication could be assessed at a stage when its effects were not yet manifest. During methylmercury exposure there is (besides the uptake by the brain) a rapid accumulation in liver and kidney [3--5], which leads to changes of * This is an abridged paper. Copies o f the full paper are available from the Editor on request, which should be accompanied by $5.00, or equivalent, to cover reproduction
and postage. Abbreviations: AP, alkaline phosphatase; GOT, glutamate-oxalate-transaminase; G-6-P, glucose-6-phosphatase ; LAP, leucine aminopeptidase ; M M C , methylmercury chloride; NE, norepinephrine; V M A , vanilmandelic acid.
203
structure and function [ 6 - - 9 ] . We, therefore, investigated some c o m p o n e n t s of blood and urine which reflect the function of these organs under the influence of MMC. In addition, as there is an increase of norepinephrine (NE) under the effect of this mercury c o m p o u n d in the brain, we studied the excretion o f the catecholamine metabolite vanilmandelic acid. MATERIALS AND METHODS
Group 1 Pretreatment
Parameters measured Treatment Parameters measured
Group 2
Group 3
Housed in metabolic cages, daily injection with 0.9% NaC1 (1 ml/100 g b o d y wt.) VMA in urine 30 mg/kg MMC VMA in urine 5 days after MMC [10]
0.9% NaC1, 10 or 20 mg/kg MMC Alkaline phosphatase, LAP, and creatinine in urine 7 days after MMC [11--13]
0.9% NaC1, 30 mg/kg MMC E r y t h r o c y t e glucose-6-phosphatase, blood glucose, serum urea and glutamate-oxalate transaminase 5 days after MMC [13,14]
(6 animals per group) RESULTS (1) The chromatographic procedure showed for control urine a spot o f similar R F value (0.87) and colour as the VMA-standards with an intensity between 8 and 16 gg/ml. Spots of the same intensity and R F were found in the urine collected after MMC-injection, i.e., there is no rise of VMA excretion after 30 mg/kg MMC. (2) Alkaline phosphatase and LAP excretion by the animals o f group 2 are shown in Table I. There is a depression of LAP in the urine from day 3--5 after 10 mg MMC/kg, b u t an elevation on days 4 and 5 after 20 mg/kg. The AP excretion shows a rise on the 3rd day after both doses. However, the excretion of this enzyme, is very inconstant and the increased levels are n o t statistically significant. Creatinine excretion in these animals was n o t different from the control values on any day. (3) None of the measured parameters showed any significant difference to the control values.
204
TABLE I I n f l u e n c e o f CH3HgCI o n L A P ( l e u c i n e a m i n o p e p t i d a s e ) a n d A P ( a l k a l i n e p h o s p h a t a s e ) e x c r e t i o n in rats e x p r e s s e d as % o f c o n t r o l a n i m a l s ( m e a n values _+ s t a n d a r d e r r o r , 6 animals/group).
CH3HgCI dose mg/kg LAP 10
1
2
3
4
5
6
7
103.2 +-4.8
95.2 ±15.9
76.0 a ±4.1 158 -+25
64.2 a ±9.0 214 a ±18
64.2 a ±6.2 178 a ±25
152 ±25
170 _+23
157.8 a ±13.9 213 b ±47
117.5 ±17.8 154 ±42
79.0 ±11.8 218 ±62
104 ±17
127 ±39
20
AP 10 20
70.7 ±17.8
75.0 ±17.0
a S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l (P < 0.05). bp = 0.05.
DISCUSSION
The build up of neurotoxic effects by MMC is accompanied by higher levels o f NE in the brain stem [ 1 5 ] . The results of e x p e r i m e n t 1 s h o w that this is n o t reflected by a higher excretion of the NE metabolite VMA. Methylmercury intoxication has been shown to affect renal ultrastructure [7,16] and to bring a b o u t e n z y m e changes [ 1 7 ] . Our experiment 2 shows that there is also a change in enzyme excretion, which, however, shows a large variability. As the effect b e c o m e s manifest only 3 days after MMC-injection we think that it m a y be d u e to HgC12 rather than to MMC itself because at this time in the kidney m o s t o f the MMC has been transformed to HgC12 [ 1 8 , 1 9 ] . HgC12 was shown to have an influence on the renal excretion of AP and LAP [ 2 0 ] . Apart from the changes in renal e n z y m e excretion there is no effect on renal function as far as this can be judged b y urine volume, urinary protein, osmolality and serum urea. This is in agreement with the observations o f Verschuuren [21] and Stroo and H o o k [ 6 ] . Liver function, also, is affected by MMC poisoning [ 1 7 , 2 1 - - 2 3 ] . There is for example a decrease o f G-6-P in adult and foetal rat liver [17,21] and a rise in foetal serum glucose [ 2 1 ] . In our experiments this was n o t f o u n d nor the rise of serum GOT which was reported by Snell [ 2 2 ] . This discrepancy m a y be due to the different t r e a t m e n t schedules.
205
ACKNOWLEDGEMENT
The excellent technical assistance of Mrs. G. Regula and Miss H. Olinger is gratefully acknowledged. REFERENCES 1 S.F. A1-Damluji and the Clinical Committee on Mercury Poisoning, Bull. WHO, 53 (1976) 65. 2 H. A1-Sharistani, K. Shihab and J.K. AI-Haddad, Bull. WHO, 53 (1976) 105. 3 G.G. Somjen, S.P. Herman, R. Klein, P.E. Brubaker, W.H. Briner, J.K. Goodrich, M.R. Krigman and J.K. Haseman, J. Pharmacol. Exp. Ther., 187 (1973) 602. 4 R. Klein, S.P. Herman, P.E. Brubaker, G.W. Lucier and M.R. Krigman, Arch. Pathol., 93 (1972} 408. 5 M. Berlin, J. Carlson and T. Norseth, Arch. Environ. Health, 30 (1975) 307. 6 W.E. Stroo and J.B. Hook, Toxicol. Appl. Pharmacol., 42 (1977) 399. 7 L.W. Chang and J.A. Sprecher, Environ. Res., 12 (1976) 218. 8 P.A. Desnoyers and L.W. Chang, Environ. Res., 9 (1975) 224. 9 P.A. Desnoyers and L.W. Chang, Environ. Res., 10 (1975) 59. 10 M. Badella, M.W. Routh, B.H. Gump and H.J. Gigliotti, Clin. Chem. 22 (1976) 2046. 11 T.-U. Hausamen, R. Helger, W. Rick and W. Gross, Clin. Chim. Acta, 15 (1967) 241. 12 W. J~sch and U.C. Dubach, Z. Klin. Chem. Kiln. Biochem., 5 (1967) 59. 13 H. Mattenheimer, Mikromethoden f'tlr das klinisch~chemische und biochemische Laboratorium, Walter de Gruyter & Co, Berlin, 1966. 14 M.A. Swanson, J. Biol. Chem., 184 (1950) 647. 15 P.D. Hrdina, D.A.V. Peters and R.L. Singhal, Res. Commun. Chem., Pathol. Pharmacol., 15 (1976) 483. 16 B.A. Fowler and J.S. Woods, Exp. Mol. Pathol., 27 (1977) 403. 17 L.W. Chang, R.A. Ware and P.A. Desnoyers, Food. Cosmet. Toxicol., 11 (1973) 283. 18 T. Norseth and T.W. Clarkson, Biochem. Pharmacol., 19 (1970) 2775. 19 T. Norseth and T.W. Clarkson, Arch. Environ. Health, 21 (1970) 717. 20 F. Planas-Bohne, Arch. Toxicol., 37 (1977) 219. 21 H.G. Verschuuren, R. Kroes, E.M. den Tonkelaar, J.M. Berkvens, P.W. Helleman, A.G. Rauws, P.L. Schuler and G.J. van Esch, Toxicology, 6 (1976) 85. 22 K. Snell, S.L. Ashby and S.J. Barton, Toxicology, 8 (1977) 277. 23 S.O. Welsh, J.H. Soares Jr., B.R. Stilling and H. Lagally, Nutr. Rep. Int., 8 (1973) 419.
206
EFFECT OF M E T H Y L M E R C U R Y ON SOME CONSTITUENTS OF SERUM AND URINE FELICITAS P L A N A S - B O H N E * Kernforschungszentrum Karlsruhe, Institut fiirGenetik und filrToxikologie yon Spaltstoffen, Postfach 3640, D 7500 Karlsruhe 1 (G.F.R.)
(Received July 17th, 1978) (Accepted July 20th, 1978)
SUMMARY
To evaluate some of the early effects of methylmercury chloride (MMC) male rats were given 10, 20 or 30 mg MMC/kg intraperitoneaUy. Urine was analysed for vanilmandelic acid (VMA), leucine aminopeptidase (LAP), alkaline Dhosphatase (AP), and creatinine, blood for glucose-6-phosphatase (G-6-P) and glucose, serum for glutamate-oxalate-transaminase (GOT) and urea. Except for LAP and AP excretion there is no effect of MMC on the parameters investigated. However, the effects on these 2 renal enzymes are too variable to permit their use as a test for MMC toxicity.
INTRODUCTION
The toxicity of methylmercury compounds is well established since the severe outbreaks of poisoning in Japan in the 1950's and in Iraq in the 1970's. The most striking clinical symptoms are neurological disturbances, which however, are seen only some time after the beginning of methylmercury exposure [1,2]. By this time the toxic effects are in many cases irreversible. It seemed desirable, therefore, to find some parameter, by which the degree of a suspected methylmercury intoxication could be assessed at a stage when its effects were not yet manifest. During methylmercury exposure there is (besides the uptake by the brain) a rapid accumulation in liver and kidney [3--5], which leads to changes of * This is an abridged paper. Copies o f the full paper are available from the Editor on request, which should be accompanied by $5.00, or equivalent, to cover reproduction
and postage. Abbreviations: AP, alkaline phosphatase; GOT, glutamate-oxalate-transaminase; G-6-P, glucose-6-phosphatase ; LAP, leucine aminopeptidase ; M M C , methylmercury chloride; NE, norepinephrine; V M A , vanilmandelic acid.
203
structure and function [ 6 - - 9 ] . We, therefore, investigated some c o m p o n e n t s of blood and urine which reflect the function of these organs under the influence of MMC. In addition, as there is an increase of norepinephrine (NE) under the effect of this mercury c o m p o u n d in the brain, we studied the excretion o f the catecholamine metabolite vanilmandelic acid. MATERIALS AND METHODS
Group 1 Pretreatment
Parameters measured Treatment Parameters measured
Group 2
Group 3
Housed in metabolic cages, daily injection with 0.9% NaC1 (1 ml/100 g b o d y wt.) VMA in urine 30 mg/kg MMC VMA in urine 5 days after MMC [10]
0.9% NaC1, 10 or 20 mg/kg MMC Alkaline phosphatase, LAP, and creatinine in urine 7 days after MMC [11--13]
0.9% NaC1, 30 mg/kg MMC E r y t h r o c y t e glucose-6-phosphatase, blood glucose, serum urea and glutamate-oxalate transaminase 5 days after MMC [13,14]
(6 animals per group) RESULTS (1) The chromatographic procedure showed for control urine a spot o f similar R F value (0.87) and colour as the VMA-standards with an intensity between 8 and 16 gg/ml. Spots of the same intensity and R F were found in the urine collected after MMC-injection, i.e., there is no rise of VMA excretion after 30 mg/kg MMC. (2) Alkaline phosphatase and LAP excretion by the animals o f group 2 are shown in Table I. There is a depression of LAP in the urine from day 3--5 after 10 mg MMC/kg, b u t an elevation on days 4 and 5 after 20 mg/kg. The AP excretion shows a rise on the 3rd day after both doses. However, the excretion of this enzyme, is very inconstant and the increased levels are n o t statistically significant. Creatinine excretion in these animals was n o t different from the control values on any day. (3) None of the measured parameters showed any significant difference to the control values.
204
TABLE I I n f l u e n c e o f CH3HgCI o n L A P ( l e u c i n e a m i n o p e p t i d a s e ) a n d A P ( a l k a l i n e p h o s p h a t a s e ) e x c r e t i o n in rats e x p r e s s e d as % o f c o n t r o l a n i m a l s ( m e a n values _+ s t a n d a r d e r r o r , 6 animals/group).
CH3HgCI dose mg/kg LAP 10
1
2
3
4
5
6
7
103.2 +-4.8
95.2 ±15.9
76.0 a ±4.1 158 -+25
64.2 a ±9.0 214 a ±18
64.2 a ±6.2 178 a ±25
152 ±25
170 _+23
157.8 a ±13.9 213 b ±47
117.5 ±17.8 154 ±42
79.0 ±11.8 218 ±62
104 ±17
127 ±39
20
AP 10 20
70.7 ±17.8
75.0 ±17.0
a S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l (P < 0.05). bp = 0.05.
DISCUSSION
The build up of neurotoxic effects by MMC is accompanied by higher levels o f NE in the brain stem [ 1 5 ] . The results of e x p e r i m e n t 1 s h o w that this is n o t reflected by a higher excretion of the NE metabolite VMA. Methylmercury intoxication has been shown to affect renal ultrastructure [7,16] and to bring a b o u t e n z y m e changes [ 1 7 ] . Our experiment 2 shows that there is also a change in enzyme excretion, which, however, shows a large variability. As the effect b e c o m e s manifest only 3 days after MMC-injection we think that it m a y be d u e to HgC12 rather than to MMC itself because at this time in the kidney m o s t o f the MMC has been transformed to HgC12 [ 1 8 , 1 9 ] . HgC12 was shown to have an influence on the renal excretion of AP and LAP [ 2 0 ] . Apart from the changes in renal e n z y m e excretion there is no effect on renal function as far as this can be judged b y urine volume, urinary protein, osmolality and serum urea. This is in agreement with the observations o f Verschuuren [21] and Stroo and H o o k [ 6 ] . Liver function, also, is affected by MMC poisoning [ 1 7 , 2 1 - - 2 3 ] . There is for example a decrease o f G-6-P in adult and foetal rat liver [17,21] and a rise in foetal serum glucose [ 2 1 ] . In our experiments this was n o t f o u n d nor the rise of serum GOT which was reported by Snell [ 2 2 ] . This discrepancy m a y be due to the different t r e a t m e n t schedules.
205
ACKNOWLEDGEMENT
The excellent technical assistance of Mrs. G. Regula and Miss H. Olinger is gratefully acknowledged. REFERENCES 1 S.F. A1-Damluji and the Clinical Committee on Mercury Poisoning, Bull. WHO, 53 (1976) 65. 2 H. A1-Sharistani, K. Shihab and J.K. AI-Haddad, Bull. WHO, 53 (1976) 105. 3 G.G. Somjen, S.P. Herman, R. Klein, P.E. Brubaker, W.H. Briner, J.K. Goodrich, M.R. Krigman and J.K. Haseman, J. Pharmacol. Exp. Ther., 187 (1973) 602. 4 R. Klein, S.P. Herman, P.E. Brubaker, G.W. Lucier and M.R. Krigman, Arch. Pathol., 93 (1972} 408. 5 M. Berlin, J. Carlson and T. Norseth, Arch. Environ. Health, 30 (1975) 307. 6 W.E. Stroo and J.B. Hook, Toxicol. Appl. Pharmacol., 42 (1977) 399. 7 L.W. Chang and J.A. Sprecher, Environ. Res., 12 (1976) 218. 8 P.A. Desnoyers and L.W. Chang, Environ. Res., 9 (1975) 224. 9 P.A. Desnoyers and L.W. Chang, Environ. Res., 10 (1975) 59. 10 M. Badella, M.W. Routh, B.H. Gump and H.J. Gigliotti, Clin. Chem. 22 (1976) 2046. 11 T.-U. Hausamen, R. Helger, W. Rick and W. Gross, Clin. Chim. Acta, 15 (1967) 241. 12 W. J~sch and U.C. Dubach, Z. Klin. Chem. Kiln. Biochem., 5 (1967) 59. 13 H. Mattenheimer, Mikromethoden f'tlr das klinisch~chemische und biochemische Laboratorium, Walter de Gruyter & Co, Berlin, 1966. 14 M.A. Swanson, J. Biol. Chem., 184 (1950) 647. 15 P.D. Hrdina, D.A.V. Peters and R.L. Singhal, Res. Commun. Chem., Pathol. Pharmacol., 15 (1976) 483. 16 B.A. Fowler and J.S. Woods, Exp. Mol. Pathol., 27 (1977) 403. 17 L.W. Chang, R.A. Ware and P.A. Desnoyers, Food. Cosmet. Toxicol., 11 (1973) 283. 18 T. Norseth and T.W. Clarkson, Biochem. Pharmacol., 19 (1970) 2775. 19 T. Norseth and T.W. Clarkson, Arch. Environ. Health, 21 (1970) 717. 20 F. Planas-Bohne, Arch. Toxicol., 37 (1977) 219. 21 H.G. Verschuuren, R. Kroes, E.M. den Tonkelaar, J.M. Berkvens, P.W. Helleman, A.G. Rauws, P.L. Schuler and G.J. van Esch, Toxicology, 6 (1976) 85. 22 K. Snell, S.L. Ashby and S.J. Barton, Toxicology, 8 (1977) 277. 23 S.O. Welsh, J.H. Soares Jr., B.R. Stilling and H. Lagally, Nutr. Rep. Int., 8 (1973) 419.
206
