European Journal of Pharmacology - Encironmental Toxicology and Pharmacology Section, 228 (1992) 37-44 © 1992 Elsevier Science Publishers B.V. All rights reserved 0926-6917/92/$05.00
37
EJPTOX 40005
Effects of N-ethyl,N-nitrosourea on m o n o a m i n e concentrations and metabolizing enzymes in m o u s e brain regions 1 Sunil J a y a s e k a r a
2, R a g h u b i r
P. S h a r m a a n d D a v i d B. D r o w n
Toxicology Program, Departments of Biology, and Animal, Dairy, and Veterinary Sciences, Utah State Unit,ersity, Logan, UT84322, USA Received 21 January 1992, accepted 28 January 1992
N-ethyl,N-nitrosourea is a well known alkylating agent and produces central nervous system-specific tumors in several laboratory animal species. In the present study, young male CD-I mice were treated by i.p. injections of 0, 2, 8, or 32 mg/kg body weight N-ethyl,N-nitrosourea, twice a week for 3 weeks. Endogenous levels of brain monoamine neurotransmitters and their selected metabolites; norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), vanillylmandelic acid (VMA), dihydroxyphenyl acetic acid (dopac), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and dihydroxyphenylalanine (dopa) were measured using HPLC with electrochemical detection. N-ethyl,N-nitrosourea treatment caused an increase of NE and 5-HT in the hypothalamus and striatum. Increased levels of 5-HIAA were noticed in the same brain regions. Elevated levels of NE were also observed in the cerebral cortex, medulla oblongata and the cerebellum. The major metabolite of NE, VMA, was decreased in several brain regions to non-detectable levels. Histopathological examination of brain tissue did not reveal any pathologic lesions. The increases in brain amines were associated with increased activity of tryptophan hydroxylase in the hypothalamus and corpus striatum. Dopa-decarboxylase was elevated in the cerebral cortex at a low dose of N-ethyl,Nnitrosourca only, whereas the monoamine oxidase activity was unaltered. Results indicated that N-ethyl,N-nitrosourea exposure may cause an elevation of steady state levels of various biogenic amines in brain areas and these changes to some extent are consistent with the altered activity of metabolizing enzymes. N-Ethyl,N-nitrosourea; Neurotransmitters; Brain enzymes; Biogenic amine metabolism; (CD1 mouse)
1. Introduction The nitrosamides and nitrosamines are synthethic or naturally occurring chemicals that have potent biological activities. They produce carcinogenic and mutagenic lesions in a variety of tissues of different species of laboratory animals (Margison and O'Connor, 1979). A m o n g many such chemicals, N-ethyl,N-nitrosourea is a well known alkylating agent and produces central nervous system-specific neuroectodermal carcinomas in rats and gliomas in Mongolian gerbils (Goth and Rajewsky, 1974; Naito et al., 1985; Nehls and Rajewsky, 1985). N-ethyl,N-nitrosourea has been shown to be a transplacental carcinogen that is capable of inducing neurogenic tumors in prenatally exposed rats (Stoica
Correspondence to: R.P. Sharma, Utah State University, UMC 5600, Logan, UT 84322-5600, USA. 1 Published as paper no. 3881 of Utah State University Agricultural Experiment Station. 2 Current address: Faculty of Veterinary Medicine, University of Paradeniya, Sri-Lanka.
and Koestner, 1984). Large doses of N-ethyl,Nnitrosourea were capable of inducing a variety of tumors in young male rats and the tumor incidence in selected organs was correlated with molecular tissue damage. In addition, a high incidence of neurogenic tumors was produced by repetitive injections of methyl-nitrosourea in young adult rats (Druckrey et al., 1965). Although it is not known if N-nitroso compounds are carcinogenic in man, they have been found to be so in many susceptible animal species and thus, are potentially hazardous. When animals were exposed prenatally or postnatally in single or multiple doses of N-ethyl,N-nitrosourea, binding of the reactive moieties of N-ethyl,N-nitrosourea with the D N A and subsequent cellular transformations were reported in both fetal and adult rat brain (Goth and Rajewsky, 1972, 1974). Whether these biochemical alterations correlated with cellular changes or the changes in neurotransmitter concentrations in the nervous system has not been investigated. Significant alterations of the concentrations of specific brain catecholamines, indoleamines and their major metabolites are of impor-
38 tance, since they are known to play an essential role in brain function. Catecholamines, indoleamines, and their metabolizing enzymes were investigated, as these are easily measured and would indicate any effects on cellular metabolism in the nervous system. We recently reported (Jayasekara et al., 19891 that aflatoxin B1, a well known carcinogen via activation to alkylating metabolites, caused alterations in the regional biogenic amine concentrations and their metabolites in mouse brain. The concentrations of biogenic amines were generally increased whereas the levels of their metabolites decreased. The changes were associated with corresponding changes in the activity of various metabolizing enzymes. In the present study we have examined the dose-related changes in concentrations of brain amines and their metabolizing enzymes after repeated exposures to N-ethyl,N-nitrosourea.
2. Materials and methods
2.1. Chemicals
N-ethyl,N-nitrosourea was obtained from Sigma Chemical Co. (St. Louis, MO). Because of the high rate of spontaneous decomposition of N-ethyl,N-nitrosourea at pH values above 7 in aqueous medium, the chemical was dissolved in citric acid-disodium phosphate buffer of pH 6.0 to give desired concentrations (Goth and Rajewsky, 1974). Care was taken to prevent decomposition from light during preparation. The Nethyl,N-nitrosourea solution was then stored frozen until used. The neurotransmitter standards dopamine (DA), norepinephrine (NE), serotonin (5-HT), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5HIAA), dihydroxyphenyl acetic acid (dopac), vanillylmandelic acid (VMA), and L-3,4-dihydroxyphenylalanine (dopa), were purchased from Sigma Chemical Co. (St. Louis, MO). L-[3,5-3H]tyrosine (specific activity, 40 C i / m m o l ) , L-[5- 3H]tryptophan (specific activity, 20 Ci/mmol), 5-hydroxy[G-3H]tryptamine creatinine sulfate (specific activity, 20 Ci/mmol), and L-3,4[2,5,63H]dihydroxyphenylalanine (specific activity, 40 C i / m m o l ) were purchased from New England Nuclear Co. (Boston, MA). Catalase, Pipes (piperazine-N,N'bis,2-ethanesulfonic acid) buffer, 6-methyl-5,6,7,8-tetrahydropterin (6MPH 4) and dithiothreitol were obtained from Sigma. 2.2. Animals and treatment
Young adult male CD-1 mice (Charles River, Wilmington, MA) of 18-20 g initial body weight were randomly housed, five animals in a group. They were
acclimated to the animal facility (accredited by American Association for the Accreditation of Laboratory Animal Care) environment for 1 week before treatment. Animals were maintained on rodent chow (Wayne Blox, Wayne Laboratories, Chicago, ILl and drinking water ad libitum during thc study. Room temperature as maintained at 22 +_ I°C and 50_+ 10% humidity with a 12 h light cycle. Mice were injected i.p. with 0, 2, 8, or 32 m g / k g N-ethyl,N-nitrosourea twice a week for 3 weeks. 2 days after the last injection animals were decapitated and the following brain regions were sampled: hypothalamus, cortex, corpus striatum, midbrain, medulla oblongata, and cerebellum as described earlier (Glowinski and lversen, 1966). Weights of the whole brain were also recorded. Tissues for neurochemical evaluation were placed immediately in tared vials containing several volumes of ice cold 0.05 M HCIO 4 (containing (I.5% cysteine) in relation to the tissue weight and homogenized. The samples were frozen at - 8 0 ° C until analysis. For enzyme analyses brain regions were homogenized in known volumes of 50 mM Tris-HC1, 2 mM dithiothreitol, pH 7.4 at 4°C and stored at -80°C. The tissues were sampled between 9:00 am. and 12:(10 noon to avoid diurnal variations. 2.3. Histopatholog3,
The whole brain specimens (from separate experiments) for histopathological evaluation were immediately fixed in buffered-formalin solution and were later processed by sectioning at three levels and stained with hematoxylin and eosin. They were examined by a veterinary pathologist for any cellular alterations. 2.4. Neurotransmitter analysis
Brain homogenates were centrifuged for 30 min at 10,000 × g and supernatants prepared for high performance liquid chromatography (HPLC) analysis by filtration (MF-1 filters, Bioanalytical Syst., West Lafayette, IN) for 5 min at 2000 × g. The filtrates were directly injected into the HPLC for the analysis of catecholamines, indoleamines and their selected metabolites, according to the method described earlier (Mayer and Shoup, 19831. The electrochemical multiple electrode HPLC system consisted of a Bioanalytical System model LC-150 EC analyzer equipped with a model LC-22A temperature controller and BiophaseODS column. The pH of the mobile phase was 2.75 with 1.3% tetrahydrofuran. The column temperature was maintained at 30°C. The standard amines were dissolved in 0.05 M HCIO 4 solution containing 0.5% cysteine.
39
NE
5-HT
DA
2.8-
0.7-
2.4-
0.6-
HIAA 0,70.6-
0.82
0.5.
1.6
0.4-
1.2-
0.3-
0.8-
0.2-
0.4 +
0.1-
Cn
0.5 ~
I
0.60.4-
.c
E "< C
0.3-
0.4,
en
Dose(mg/kg)
0.20.2"
1"-1 CONTROL I~
0.1.
2.0
I-~ 8.0 32.0
0
,/,
+
0.0
0
0.0
Fig. 1. Effect of N-ethyl,N-nitrosourea treatment on concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-HT), and 5-hydroxyindoleacetic acid (HIAA) in the hypothalamus. The doses indicated are m g / k g injected i.p. twice weekly for 3 weeks. Asterisks denote a significant effect relative to the control group (P _< 0.05). Values represent the mean _+S.E.M. for five animals.
2.5. Enzyme analyses
2.6. Statistical
The activities of tyrosine hydroxylase, tryptophan hydroxylase, dopa-decarboxylase and monoamine oxidase were determined in selected brain regions as indicated earlier (Jayasekara et al., 1989). Tyrosine hydroxylase was measured by the tritium release method (Nagatsu et al., 1964) using [3,5-3H]tyrosine; tryptophan hydroxylase assay utilized [5-3H]tryptophan as a substrate (Beevers et al., 1983). A similar method was developed for measuring dopa-decarboxylase in our laboratory (Jayasekara et al., 1989). The assay for monoamine oxidase used [3H]5-hydroxytryptamine as a substrate; the labeled product was separated and measured (Jarrot, 1971).
The statistical significance between the control and treatment groups was determined by using one-way A N O V A procedures. Significant differences between means in the experimental groups were determined by Duncan's multiple mean comparison test.
DA
DOPA 12-
IO@,
3. Results
The doses of N-ethyl,N-nitrosourea administered in the present study had no significant effect on body weight gain (data not presented). There were no treatment related changes in food or water intake in Nethyl,N-nitrosourea treated animals (data not shown).
5-HT
HIAA
0.8 -
0.4 -
0.6 -
0.3
0.4 -
0.2
0.2 -
0.1
IOlO'
.S.c ,o.
37
EJPTOX 40005
Effects of N-ethyl,N-nitrosourea on m o n o a m i n e concentrations and metabolizing enzymes in m o u s e brain regions 1 Sunil J a y a s e k a r a
2, R a g h u b i r
P. S h a r m a a n d D a v i d B. D r o w n
Toxicology Program, Departments of Biology, and Animal, Dairy, and Veterinary Sciences, Utah State Unit,ersity, Logan, UT84322, USA Received 21 January 1992, accepted 28 January 1992
N-ethyl,N-nitrosourea is a well known alkylating agent and produces central nervous system-specific tumors in several laboratory animal species. In the present study, young male CD-I mice were treated by i.p. injections of 0, 2, 8, or 32 mg/kg body weight N-ethyl,N-nitrosourea, twice a week for 3 weeks. Endogenous levels of brain monoamine neurotransmitters and their selected metabolites; norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), vanillylmandelic acid (VMA), dihydroxyphenyl acetic acid (dopac), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and dihydroxyphenylalanine (dopa) were measured using HPLC with electrochemical detection. N-ethyl,N-nitrosourea treatment caused an increase of NE and 5-HT in the hypothalamus and striatum. Increased levels of 5-HIAA were noticed in the same brain regions. Elevated levels of NE were also observed in the cerebral cortex, medulla oblongata and the cerebellum. The major metabolite of NE, VMA, was decreased in several brain regions to non-detectable levels. Histopathological examination of brain tissue did not reveal any pathologic lesions. The increases in brain amines were associated with increased activity of tryptophan hydroxylase in the hypothalamus and corpus striatum. Dopa-decarboxylase was elevated in the cerebral cortex at a low dose of N-ethyl,Nnitrosourca only, whereas the monoamine oxidase activity was unaltered. Results indicated that N-ethyl,N-nitrosourea exposure may cause an elevation of steady state levels of various biogenic amines in brain areas and these changes to some extent are consistent with the altered activity of metabolizing enzymes. N-Ethyl,N-nitrosourea; Neurotransmitters; Brain enzymes; Biogenic amine metabolism; (CD1 mouse)
1. Introduction The nitrosamides and nitrosamines are synthethic or naturally occurring chemicals that have potent biological activities. They produce carcinogenic and mutagenic lesions in a variety of tissues of different species of laboratory animals (Margison and O'Connor, 1979). A m o n g many such chemicals, N-ethyl,N-nitrosourea is a well known alkylating agent and produces central nervous system-specific neuroectodermal carcinomas in rats and gliomas in Mongolian gerbils (Goth and Rajewsky, 1974; Naito et al., 1985; Nehls and Rajewsky, 1985). N-ethyl,N-nitrosourea has been shown to be a transplacental carcinogen that is capable of inducing neurogenic tumors in prenatally exposed rats (Stoica
Correspondence to: R.P. Sharma, Utah State University, UMC 5600, Logan, UT 84322-5600, USA. 1 Published as paper no. 3881 of Utah State University Agricultural Experiment Station. 2 Current address: Faculty of Veterinary Medicine, University of Paradeniya, Sri-Lanka.
and Koestner, 1984). Large doses of N-ethyl,Nnitrosourea were capable of inducing a variety of tumors in young male rats and the tumor incidence in selected organs was correlated with molecular tissue damage. In addition, a high incidence of neurogenic tumors was produced by repetitive injections of methyl-nitrosourea in young adult rats (Druckrey et al., 1965). Although it is not known if N-nitroso compounds are carcinogenic in man, they have been found to be so in many susceptible animal species and thus, are potentially hazardous. When animals were exposed prenatally or postnatally in single or multiple doses of N-ethyl,N-nitrosourea, binding of the reactive moieties of N-ethyl,N-nitrosourea with the D N A and subsequent cellular transformations were reported in both fetal and adult rat brain (Goth and Rajewsky, 1972, 1974). Whether these biochemical alterations correlated with cellular changes or the changes in neurotransmitter concentrations in the nervous system has not been investigated. Significant alterations of the concentrations of specific brain catecholamines, indoleamines and their major metabolites are of impor-
38 tance, since they are known to play an essential role in brain function. Catecholamines, indoleamines, and their metabolizing enzymes were investigated, as these are easily measured and would indicate any effects on cellular metabolism in the nervous system. We recently reported (Jayasekara et al., 19891 that aflatoxin B1, a well known carcinogen via activation to alkylating metabolites, caused alterations in the regional biogenic amine concentrations and their metabolites in mouse brain. The concentrations of biogenic amines were generally increased whereas the levels of their metabolites decreased. The changes were associated with corresponding changes in the activity of various metabolizing enzymes. In the present study we have examined the dose-related changes in concentrations of brain amines and their metabolizing enzymes after repeated exposures to N-ethyl,N-nitrosourea.
2. Materials and methods
2.1. Chemicals
N-ethyl,N-nitrosourea was obtained from Sigma Chemical Co. (St. Louis, MO). Because of the high rate of spontaneous decomposition of N-ethyl,N-nitrosourea at pH values above 7 in aqueous medium, the chemical was dissolved in citric acid-disodium phosphate buffer of pH 6.0 to give desired concentrations (Goth and Rajewsky, 1974). Care was taken to prevent decomposition from light during preparation. The Nethyl,N-nitrosourea solution was then stored frozen until used. The neurotransmitter standards dopamine (DA), norepinephrine (NE), serotonin (5-HT), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5HIAA), dihydroxyphenyl acetic acid (dopac), vanillylmandelic acid (VMA), and L-3,4-dihydroxyphenylalanine (dopa), were purchased from Sigma Chemical Co. (St. Louis, MO). L-[3,5-3H]tyrosine (specific activity, 40 C i / m m o l ) , L-[5- 3H]tryptophan (specific activity, 20 Ci/mmol), 5-hydroxy[G-3H]tryptamine creatinine sulfate (specific activity, 20 Ci/mmol), and L-3,4[2,5,63H]dihydroxyphenylalanine (specific activity, 40 C i / m m o l ) were purchased from New England Nuclear Co. (Boston, MA). Catalase, Pipes (piperazine-N,N'bis,2-ethanesulfonic acid) buffer, 6-methyl-5,6,7,8-tetrahydropterin (6MPH 4) and dithiothreitol were obtained from Sigma. 2.2. Animals and treatment
Young adult male CD-1 mice (Charles River, Wilmington, MA) of 18-20 g initial body weight were randomly housed, five animals in a group. They were
acclimated to the animal facility (accredited by American Association for the Accreditation of Laboratory Animal Care) environment for 1 week before treatment. Animals were maintained on rodent chow (Wayne Blox, Wayne Laboratories, Chicago, ILl and drinking water ad libitum during thc study. Room temperature as maintained at 22 +_ I°C and 50_+ 10% humidity with a 12 h light cycle. Mice were injected i.p. with 0, 2, 8, or 32 m g / k g N-ethyl,N-nitrosourea twice a week for 3 weeks. 2 days after the last injection animals were decapitated and the following brain regions were sampled: hypothalamus, cortex, corpus striatum, midbrain, medulla oblongata, and cerebellum as described earlier (Glowinski and lversen, 1966). Weights of the whole brain were also recorded. Tissues for neurochemical evaluation were placed immediately in tared vials containing several volumes of ice cold 0.05 M HCIO 4 (containing (I.5% cysteine) in relation to the tissue weight and homogenized. The samples were frozen at - 8 0 ° C until analysis. For enzyme analyses brain regions were homogenized in known volumes of 50 mM Tris-HC1, 2 mM dithiothreitol, pH 7.4 at 4°C and stored at -80°C. The tissues were sampled between 9:00 am. and 12:(10 noon to avoid diurnal variations. 2.3. Histopatholog3,
The whole brain specimens (from separate experiments) for histopathological evaluation were immediately fixed in buffered-formalin solution and were later processed by sectioning at three levels and stained with hematoxylin and eosin. They were examined by a veterinary pathologist for any cellular alterations. 2.4. Neurotransmitter analysis
Brain homogenates were centrifuged for 30 min at 10,000 × g and supernatants prepared for high performance liquid chromatography (HPLC) analysis by filtration (MF-1 filters, Bioanalytical Syst., West Lafayette, IN) for 5 min at 2000 × g. The filtrates were directly injected into the HPLC for the analysis of catecholamines, indoleamines and their selected metabolites, according to the method described earlier (Mayer and Shoup, 19831. The electrochemical multiple electrode HPLC system consisted of a Bioanalytical System model LC-150 EC analyzer equipped with a model LC-22A temperature controller and BiophaseODS column. The pH of the mobile phase was 2.75 with 1.3% tetrahydrofuran. The column temperature was maintained at 30°C. The standard amines were dissolved in 0.05 M HCIO 4 solution containing 0.5% cysteine.
39
NE
5-HT
DA
2.8-
0.7-
2.4-
0.6-
HIAA 0,70.6-
0.82
0.5.
1.6
0.4-
1.2-
0.3-
0.8-
0.2-
0.4 +
0.1-
Cn
0.5 ~
I
0.60.4-
.c
E "< C
0.3-
0.4,
en
Dose(mg/kg)
0.20.2"
1"-1 CONTROL I~
0.1.
2.0
I-~ 8.0 32.0
0
,/,
+
0.0
0
0.0
Fig. 1. Effect of N-ethyl,N-nitrosourea treatment on concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-HT), and 5-hydroxyindoleacetic acid (HIAA) in the hypothalamus. The doses indicated are m g / k g injected i.p. twice weekly for 3 weeks. Asterisks denote a significant effect relative to the control group (P _< 0.05). Values represent the mean _+S.E.M. for five animals.
2.5. Enzyme analyses
2.6. Statistical
The activities of tyrosine hydroxylase, tryptophan hydroxylase, dopa-decarboxylase and monoamine oxidase were determined in selected brain regions as indicated earlier (Jayasekara et al., 1989). Tyrosine hydroxylase was measured by the tritium release method (Nagatsu et al., 1964) using [3,5-3H]tyrosine; tryptophan hydroxylase assay utilized [5-3H]tryptophan as a substrate (Beevers et al., 1983). A similar method was developed for measuring dopa-decarboxylase in our laboratory (Jayasekara et al., 1989). The assay for monoamine oxidase used [3H]5-hydroxytryptamine as a substrate; the labeled product was separated and measured (Jarrot, 1971).
The statistical significance between the control and treatment groups was determined by using one-way A N O V A procedures. Significant differences between means in the experimental groups were determined by Duncan's multiple mean comparison test.
DA
DOPA 12-
IO@,
3. Results
The doses of N-ethyl,N-nitrosourea administered in the present study had no significant effect on body weight gain (data not presented). There were no treatment related changes in food or water intake in Nethyl,N-nitrosourea treated animals (data not shown).
5-HT
HIAA
0.8 -
0.4 -
0.6 -
0.3
0.4 -
0.2
0.2 -
0.1
IOlO'
.S.c ,o.
