ToxicologyLetters, 64/65 (1992) 773477 0 1992 Elsevier Science Publishers B.V., All rights reserved 03784274192/$5.00
773
Behavioral toxicity of guanidinosuccinic acid in adult and young mice Rudi D’Hooge, Yin-Quan Pei, Bart Marescau and Peter P. De Deyn Laboratory ofNeurochemistry and Behauior, Born-Bunge Foundation, University of Antwerp (WA), and Department OfNeurology, Middelheim General Hospital, Antwerp (Belgium) Key words: Guanidinosuccinic acid; Guanidino compounds; Uremia; Epilepsy SUMMARY Guanidinosuccinic acid (GSA), a guanidino compound found to be greatly increased in uremia, was administered by intraperitoneal (i.p.1 injection to adult albino mice and to young mice 7, 14 and 21 days old. Epileptogenic and toxic properties were assessed and GSA brain levels following i.p. injection were determined. In adult mice, GSA induced long-lasting generalized clonic and clonic-tonic convulsions in a dose-dependent manner with a CDm (and 95% confidence interval) of 363 (287-458) mg/kg (n=35), and an LDm of 579 (445-756) mg/kg. The CDm of GSA corresponded with a brain concentration of 56 nmol/g tissue. Electrocorticographic recording in five adult mice revealed epileptiform discharges (spikes, spike-waves, and polyspike-waves) which appeared concomitant with the convulsions, When young mice were i.p. injected with a (for adults) subconvulsive dose of GSA (250 mg/kg), an age-dependent decrease was noted in GSA-induced convulsions and in the resulting brain concentration. The presented findings suggest that GSA could be an important uremic toxin which could contribute to the epileptic symptomatology in uremia.
INTRODUCTION
Guanidino compounds are generated as a result of protein and amino acid metabolism. Endogenous guanidino compounds have been frequently proposed to play a role in behavioral toxicity and epileptic symptomatology accompanying many acquired and inborn errors of metabolism where guanidino compounds are increased in patients suffering from these disorders D-31. Renal failure resulting in uremia is one of these disorders, and Correspondence to: P.P. De Deyn, Laboratory of Neurochemistry and Behavior, Born-Bunge Foundation, University of Antwerp, Universiteitsplein I,2610 Antwerp, Belgium.
774
work by our group has shown that a number of guanidino compounds are greatly increased in serum, cerebrospinal fluid and brain tissue of uremic patients [Z&4,51.Guanidinosuccinic acid (GSA) is the most increased of these uremic guanidino compounds. For example, GSA levels in cerebrospinal fluid of uremics are up to 100 times higher than in controls 141.However, although De Deyn and Macdonald [61 showed glycine and GABA antagonism by GSA on cultured mouse neurons, no in vivo convulsive properties of GSA have been reported as yet. Nevertheless, many other guanidino compounds have been shown to induce behavioral convulsions as well as epileptiform electrographic discharges [Il. This study was designed to deal with the convulsive properties of GSA in adult and young mice. MATERIALS AND METHODS
Randomly bred Swiss mice (male and female, body weight 13-25 g for the first series of experiments) were housed under standard environmentally controlled conditions. For another series of experiments, young Swiss mice 7, 14 and 21 days old were used. Guanidinosuccinic acid (MW 175.2) was purchased from Sigma (St. Louis, USA) and GSA suspensions were made in 30% polyethylene glycol solution. Injections of GSA suspensions were delivered in volumes of 0.1 ml per 10 g body weight, i.p. and in doses between 250 and 1000 mg/kg (5 mice per dose, in duplicate). After injection, the mice were put in individual cylindrical transparent cages and observed for 1 h (for 2 h in the case of the young mice). Both CD50and LDso (convulsive and lethal dose in 50% of the animals) were calculated by probit analysis or moving average interpolation. Mice with guanidino compounds administered i.p. were decapitated, immediately following the observation period (or earlier if death occurred within this period). Whole brains were quickly removed, rinsed in ice cold saline and stored at -75°C until analysis of GSA brain concentration. Before analysis, brain tissue was homogenized at 0°C with a Potter homogenizer in 1 ml water and the obtained homogenate was preprocessed according to Marescau et al. 171.Concentrations of GSA were determined according to an earlier described method 181,modified from Hiraga and Kinoshita 191. For the study of the electrographic effects of GSA, mice (body weight 30*2 g) were anesthetized with Nembutal@ (30 mg/kg i.p.1 and four cortical electrodes were implanted. Left and right from the sagittal suture, holes were drilled in the skull with a dental drill without damaging the dura mater. Two holes anterior, left and right, and two holes posterior, left and right, were made and four stainless steel electrodes provided with plastic stoppers were slid through these holes to touch the dura mater and fixed with Cyanolit? rapid tissue glue. Four small additional holes were drilled,
775
two rostra1 and two caudal, to fix two stainless steel hooks. A cap was made with dental cement that contained the four recording electrodes and the hooks, thus keeping the electrodes rigid and tightly fixed. Two weeks later, electrocorticographic (ECoG) recordings were made with a Siemens Mingograf EEG recorder (70 Hz filter, time constant 0.03 s) in non-anesthetized animals. Four tightly fitting sockets were attached to the cortical electrodes, a reference electrode was clipped to the right ear and a ground electrode to the left ear. A control ECoG recording was made prior to the drug injection to check for any post-operative ECoG abnormalities. Normal mouse ECoG showed basic activity of 7-3 Hz and about 100 PV in amplitude. RESULTS AND DISCUSSION
Convulsive response after i.p. injection of doses of GSA of 500 n&kg usually developed slowly with a median latency of about 25 min, and began with hyperventilation, leading to hyperactivity and rearing. Next followed wild running and bouncing which was followed by jerking and twitching, eventually leading to generalized clonic seizures comprising automatic crawling or grasping movements with head jerked backwards and tail raised. Some of the animals kept on convulsing severely, and lost their balance. They rolled around and vigorously convulsed, foamed at the mouth and micturated. In some of the animals these severe clonic convulsions led to tonic extension of the four limbs, often lethal. As shown in Figure 1, the occurrence of clonic convulsions as well as of the tonic extension phase were related to the dose of GSA injected (dose range between 250 and 1000 mg/kg). A CD50 (and 95% confidence interval) of 363 (287-458) mg/kg (n=35) was calculated, and an LDr,oof 579 (445-756) mg/kg. The CD97 for clonic seizures was 700 mg/kg; CD97 for tonic extension was about 1000 mg/kg. GSA brain levels were determined to find out how much GSA actually entered the brain of the animals, and whether this brain level was comparable with those previously reported in uremic patients. It was found that GSA brain concentration increased in a linear fashion with the dose injected within the dose range studied. It was calculated that the CD50 of GSA corresponded with a brain concentration of 56 nmol/g tissue. In cerebral cortex of uremic patients, comparable levels of up to 21 nmol/g tissue were demonstrated 121,and in other brain regions even higher levels were found (control levels of GSA are similar in mice and men i.e. 10.4 nmol/g tissue). To assess the occurrence of abnormal ECoG discharges, ECoG recordings were made in 5 non-anesthetized mice following the injection of the CD97 dose of GSA, 700 mg/kg. Spike-wave and polyspike-wave discharges appeared 10 to 15 min following GSA injection. These appeared concomitant
776
loo-
5-
I
8
250
500 IF DOSE(mg/
1000 kg)
Fig. 1. Dose-dependent increase in the number of mice displaying convulsions, and in GSA brain concentration following i.p. injection of GSA suspensions. Abscissa shows the dose of GSA injected i.p., left ordinate, the proportion of the animals (in percent of total number) displaying generalized clonic convulsions (white area) and tonic extension of the limbs (grciv area), and right ordinate, the brain concentration of GSA (in nmol/g tissue), 1 h following the injection (black circles depict the mean of five determinations with SEM indicated).
f-150
t
14
_
21
AGE (days) Fig. 2. Age-related decrease in GSA-induced convulsions and in GSA brain concentration following i.p. injection of a 250 mg/kg GSA suspension. Abscissa shows the age of the mice (in days after birth), ordinates and figure construction are analogous to Fig. 1.
with clonic convulsions. After 30 min, regular spike-waves appeared, larger in amplitude. As the severity of the convulsions increased, ECoG further deteriorated, and around 50 min following injection, irritative spiking and muscle artifacts appeared concomitant with severe clonic convulsions. A dose of 250 mg/kg was injected i.p. in mice 7, 14 and 21 days old, and brain levels were determined 2 h following injection. As Figure 2 demonstrates, brain levels reached higher values in young mice than in older ones, although the dose injected was the same (250 mg/kg, i.p.). This difference
777
could be due to differences in systemic absorption and/or blood-brain barrier permeability. Interestingly, the behavioral response of the animals paralleled the differences in brain levels. Whereas in 7-day-old mice, all 5 injected mice displayed clonic convulsions (comprising wild crawling or running), leading to tonic extension and death in three of them, the reaction in 14 and 2l-day-old mice was much less severe (none of them died). In conclusion, GSA induces behavioral convulsions as well as epileptiform ECoG discharges. The convulsive properties of GSA could at least partially be explained by its antagonistic action on GABA and glycine neurotransmission reported earlier. Indeed, antagonism of inhibitory amino acid neurotransmission has been proposed to underlie the pathogenesis of epilepsy [3,101. Following i.p. injection, GSA reaches higher brain levels in young mice than in older ones. Behavioral toxicity of GSA parallels these different brain levels. Furthermore, our findings suggest that GSA could be an important uremic toxin, contributing to the epileptic symptomatology associated with renal failure. REFERENCES 1 Mori, A. (1987) Biochemistry and neurotoxicology of guanidino compounds: history and recent advances. Pav. J. Biol. Sci. 22,85-94. 2 De Deyn, P.P. (1989) Analytical studies and pathophysiological importance of guanidino compounds in uremia and hyperargininemia. Thesis submitted to obtain the degree of Geaggregeerde voor het Hoger Onderwijs. University of Antwerp &X4), Belgium. 3 De Deyn, P.P., Marescau, B. and Macdonald, R.L. (1990) Epilepsy and the GABA hypothesis: a brief review and some examples. Acta Neurol. Belg. 90,65-81. 4 De Deyn, P.P., Marescau, B., Cuykens, J.J., Van Gorp, L., Lowenthal, A. and De Potter, W.P. (1987) Guanidino compounds in serum and cerebrospinal fluid of non-dialysed patients with renal insufficiency. Clin. Chim. Ada 167,81-88. 5 Marescau, B., De Deyn, P.P., Wiechert, P., Hiramatsu, M., Van Gorp, L., De Potter, W.P. and Lowenthal, A. (1989) Guanidino compounds in serum and cerebrospinal fluid of epileptic and some other neurological patients. In: A. Mori, B.D. Cohen and H. Koide (Eds.), Guanidines 2, Plenum Press, New York and London, pp. 203-212. 6 De Deyn, P.P. and Macdonald, R.L. (1990) Guanidino compounds that are increased in cerebrospinal fluid and brain of uremic patients inhibit GABA and glycine responses on mouse neurons in cell culture. Ann. Neurol. 28,627-633. 7 Marescau, B., De Deyn, P.P., Wiechert, P., Van Gorp, L. and Lowenthal, A. (1986) Comparative study of guanidino compounds in serum and brain of mouse, rat, rabbit, and man. J. Neurochem. 46,717-720. 8 Marescau, B., Deshmukh, D.R., Kockx, M., Possemiers, I., Qureshi, I.A., Wiechert, P. and De Deyn, P.P. (1992) Guanidino compounds in serum, urine, liver, kidney and brain of man and some ureotelic animals. Metabolism 41, l-6. 9 Hiraga, Y. and Kinoshita, T. (1981) Post-column derivatization of guanidino compounds in high-performance liquid chromatography using ninhydrin. J. Chromatogr. 226,43+X. 10 Meldrum, B.S. (1989) GABAergic mechanisms in the pathogenesis and treatment of epilepsy. Br. J. Clin. Pharmac. 27,3S-11s.
773
Behavioral toxicity of guanidinosuccinic acid in adult and young mice Rudi D’Hooge, Yin-Quan Pei, Bart Marescau and Peter P. De Deyn Laboratory ofNeurochemistry and Behauior, Born-Bunge Foundation, University of Antwerp (WA), and Department OfNeurology, Middelheim General Hospital, Antwerp (Belgium) Key words: Guanidinosuccinic acid; Guanidino compounds; Uremia; Epilepsy SUMMARY Guanidinosuccinic acid (GSA), a guanidino compound found to be greatly increased in uremia, was administered by intraperitoneal (i.p.1 injection to adult albino mice and to young mice 7, 14 and 21 days old. Epileptogenic and toxic properties were assessed and GSA brain levels following i.p. injection were determined. In adult mice, GSA induced long-lasting generalized clonic and clonic-tonic convulsions in a dose-dependent manner with a CDm (and 95% confidence interval) of 363 (287-458) mg/kg (n=35), and an LDm of 579 (445-756) mg/kg. The CDm of GSA corresponded with a brain concentration of 56 nmol/g tissue. Electrocorticographic recording in five adult mice revealed epileptiform discharges (spikes, spike-waves, and polyspike-waves) which appeared concomitant with the convulsions, When young mice were i.p. injected with a (for adults) subconvulsive dose of GSA (250 mg/kg), an age-dependent decrease was noted in GSA-induced convulsions and in the resulting brain concentration. The presented findings suggest that GSA could be an important uremic toxin which could contribute to the epileptic symptomatology in uremia.
INTRODUCTION
Guanidino compounds are generated as a result of protein and amino acid metabolism. Endogenous guanidino compounds have been frequently proposed to play a role in behavioral toxicity and epileptic symptomatology accompanying many acquired and inborn errors of metabolism where guanidino compounds are increased in patients suffering from these disorders D-31. Renal failure resulting in uremia is one of these disorders, and Correspondence to: P.P. De Deyn, Laboratory of Neurochemistry and Behavior, Born-Bunge Foundation, University of Antwerp, Universiteitsplein I,2610 Antwerp, Belgium.
774
work by our group has shown that a number of guanidino compounds are greatly increased in serum, cerebrospinal fluid and brain tissue of uremic patients [Z&4,51.Guanidinosuccinic acid (GSA) is the most increased of these uremic guanidino compounds. For example, GSA levels in cerebrospinal fluid of uremics are up to 100 times higher than in controls 141.However, although De Deyn and Macdonald [61 showed glycine and GABA antagonism by GSA on cultured mouse neurons, no in vivo convulsive properties of GSA have been reported as yet. Nevertheless, many other guanidino compounds have been shown to induce behavioral convulsions as well as epileptiform electrographic discharges [Il. This study was designed to deal with the convulsive properties of GSA in adult and young mice. MATERIALS AND METHODS
Randomly bred Swiss mice (male and female, body weight 13-25 g for the first series of experiments) were housed under standard environmentally controlled conditions. For another series of experiments, young Swiss mice 7, 14 and 21 days old were used. Guanidinosuccinic acid (MW 175.2) was purchased from Sigma (St. Louis, USA) and GSA suspensions were made in 30% polyethylene glycol solution. Injections of GSA suspensions were delivered in volumes of 0.1 ml per 10 g body weight, i.p. and in doses between 250 and 1000 mg/kg (5 mice per dose, in duplicate). After injection, the mice were put in individual cylindrical transparent cages and observed for 1 h (for 2 h in the case of the young mice). Both CD50and LDso (convulsive and lethal dose in 50% of the animals) were calculated by probit analysis or moving average interpolation. Mice with guanidino compounds administered i.p. were decapitated, immediately following the observation period (or earlier if death occurred within this period). Whole brains were quickly removed, rinsed in ice cold saline and stored at -75°C until analysis of GSA brain concentration. Before analysis, brain tissue was homogenized at 0°C with a Potter homogenizer in 1 ml water and the obtained homogenate was preprocessed according to Marescau et al. 171.Concentrations of GSA were determined according to an earlier described method 181,modified from Hiraga and Kinoshita 191. For the study of the electrographic effects of GSA, mice (body weight 30*2 g) were anesthetized with Nembutal@ (30 mg/kg i.p.1 and four cortical electrodes were implanted. Left and right from the sagittal suture, holes were drilled in the skull with a dental drill without damaging the dura mater. Two holes anterior, left and right, and two holes posterior, left and right, were made and four stainless steel electrodes provided with plastic stoppers were slid through these holes to touch the dura mater and fixed with Cyanolit? rapid tissue glue. Four small additional holes were drilled,
775
two rostra1 and two caudal, to fix two stainless steel hooks. A cap was made with dental cement that contained the four recording electrodes and the hooks, thus keeping the electrodes rigid and tightly fixed. Two weeks later, electrocorticographic (ECoG) recordings were made with a Siemens Mingograf EEG recorder (70 Hz filter, time constant 0.03 s) in non-anesthetized animals. Four tightly fitting sockets were attached to the cortical electrodes, a reference electrode was clipped to the right ear and a ground electrode to the left ear. A control ECoG recording was made prior to the drug injection to check for any post-operative ECoG abnormalities. Normal mouse ECoG showed basic activity of 7-3 Hz and about 100 PV in amplitude. RESULTS AND DISCUSSION
Convulsive response after i.p. injection of doses of GSA of 500 n&kg usually developed slowly with a median latency of about 25 min, and began with hyperventilation, leading to hyperactivity and rearing. Next followed wild running and bouncing which was followed by jerking and twitching, eventually leading to generalized clonic seizures comprising automatic crawling or grasping movements with head jerked backwards and tail raised. Some of the animals kept on convulsing severely, and lost their balance. They rolled around and vigorously convulsed, foamed at the mouth and micturated. In some of the animals these severe clonic convulsions led to tonic extension of the four limbs, often lethal. As shown in Figure 1, the occurrence of clonic convulsions as well as of the tonic extension phase were related to the dose of GSA injected (dose range between 250 and 1000 mg/kg). A CD50 (and 95% confidence interval) of 363 (287-458) mg/kg (n=35) was calculated, and an LDr,oof 579 (445-756) mg/kg. The CD97 for clonic seizures was 700 mg/kg; CD97 for tonic extension was about 1000 mg/kg. GSA brain levels were determined to find out how much GSA actually entered the brain of the animals, and whether this brain level was comparable with those previously reported in uremic patients. It was found that GSA brain concentration increased in a linear fashion with the dose injected within the dose range studied. It was calculated that the CD50 of GSA corresponded with a brain concentration of 56 nmol/g tissue. In cerebral cortex of uremic patients, comparable levels of up to 21 nmol/g tissue were demonstrated 121,and in other brain regions even higher levels were found (control levels of GSA are similar in mice and men i.e. 10.4 nmol/g tissue). To assess the occurrence of abnormal ECoG discharges, ECoG recordings were made in 5 non-anesthetized mice following the injection of the CD97 dose of GSA, 700 mg/kg. Spike-wave and polyspike-wave discharges appeared 10 to 15 min following GSA injection. These appeared concomitant
776
loo-
5-
I
8
250
500 IF DOSE(mg/
1000 kg)
Fig. 1. Dose-dependent increase in the number of mice displaying convulsions, and in GSA brain concentration following i.p. injection of GSA suspensions. Abscissa shows the dose of GSA injected i.p., left ordinate, the proportion of the animals (in percent of total number) displaying generalized clonic convulsions (white area) and tonic extension of the limbs (grciv area), and right ordinate, the brain concentration of GSA (in nmol/g tissue), 1 h following the injection (black circles depict the mean of five determinations with SEM indicated).
f-150
t
14
_
21
AGE (days) Fig. 2. Age-related decrease in GSA-induced convulsions and in GSA brain concentration following i.p. injection of a 250 mg/kg GSA suspension. Abscissa shows the age of the mice (in days after birth), ordinates and figure construction are analogous to Fig. 1.
with clonic convulsions. After 30 min, regular spike-waves appeared, larger in amplitude. As the severity of the convulsions increased, ECoG further deteriorated, and around 50 min following injection, irritative spiking and muscle artifacts appeared concomitant with severe clonic convulsions. A dose of 250 mg/kg was injected i.p. in mice 7, 14 and 21 days old, and brain levels were determined 2 h following injection. As Figure 2 demonstrates, brain levels reached higher values in young mice than in older ones, although the dose injected was the same (250 mg/kg, i.p.). This difference
777
could be due to differences in systemic absorption and/or blood-brain barrier permeability. Interestingly, the behavioral response of the animals paralleled the differences in brain levels. Whereas in 7-day-old mice, all 5 injected mice displayed clonic convulsions (comprising wild crawling or running), leading to tonic extension and death in three of them, the reaction in 14 and 2l-day-old mice was much less severe (none of them died). In conclusion, GSA induces behavioral convulsions as well as epileptiform ECoG discharges. The convulsive properties of GSA could at least partially be explained by its antagonistic action on GABA and glycine neurotransmission reported earlier. Indeed, antagonism of inhibitory amino acid neurotransmission has been proposed to underlie the pathogenesis of epilepsy [3,101. Following i.p. injection, GSA reaches higher brain levels in young mice than in older ones. Behavioral toxicity of GSA parallels these different brain levels. Furthermore, our findings suggest that GSA could be an important uremic toxin, contributing to the epileptic symptomatology associated with renal failure. REFERENCES 1 Mori, A. (1987) Biochemistry and neurotoxicology of guanidino compounds: history and recent advances. Pav. J. Biol. Sci. 22,85-94. 2 De Deyn, P.P. (1989) Analytical studies and pathophysiological importance of guanidino compounds in uremia and hyperargininemia. Thesis submitted to obtain the degree of Geaggregeerde voor het Hoger Onderwijs. University of Antwerp &X4), Belgium. 3 De Deyn, P.P., Marescau, B. and Macdonald, R.L. (1990) Epilepsy and the GABA hypothesis: a brief review and some examples. Acta Neurol. Belg. 90,65-81. 4 De Deyn, P.P., Marescau, B., Cuykens, J.J., Van Gorp, L., Lowenthal, A. and De Potter, W.P. (1987) Guanidino compounds in serum and cerebrospinal fluid of non-dialysed patients with renal insufficiency. Clin. Chim. Ada 167,81-88. 5 Marescau, B., De Deyn, P.P., Wiechert, P., Hiramatsu, M., Van Gorp, L., De Potter, W.P. and Lowenthal, A. (1989) Guanidino compounds in serum and cerebrospinal fluid of epileptic and some other neurological patients. In: A. Mori, B.D. Cohen and H. Koide (Eds.), Guanidines 2, Plenum Press, New York and London, pp. 203-212. 6 De Deyn, P.P. and Macdonald, R.L. (1990) Guanidino compounds that are increased in cerebrospinal fluid and brain of uremic patients inhibit GABA and glycine responses on mouse neurons in cell culture. Ann. Neurol. 28,627-633. 7 Marescau, B., De Deyn, P.P., Wiechert, P., Van Gorp, L. and Lowenthal, A. (1986) Comparative study of guanidino compounds in serum and brain of mouse, rat, rabbit, and man. J. Neurochem. 46,717-720. 8 Marescau, B., Deshmukh, D.R., Kockx, M., Possemiers, I., Qureshi, I.A., Wiechert, P. and De Deyn, P.P. (1992) Guanidino compounds in serum, urine, liver, kidney and brain of man and some ureotelic animals. Metabolism 41, l-6. 9 Hiraga, Y. and Kinoshita, T. (1981) Post-column derivatization of guanidino compounds in high-performance liquid chromatography using ninhydrin. J. Chromatogr. 226,43+X. 10 Meldrum, B.S. (1989) GABAergic mechanisms in the pathogenesis and treatment of epilepsy. Br. J. Clin. Pharmac. 27,3S-11s.
