JOURNAL OF APPLIED TOXICOLOGY, VOL. 12(6), 421-426 (1992)
Chick Embryo Exposure to Carbamates Alters
Neurochemical Parameters and Behavior M. Farage-Elawar" and W. D. BlakerS VAiMD Regional College of Veterinary Medicine, Department of Biomedical Sciences, Blacksburg, VA 24061, USA
Key words: carbamates; locomotion; dopamine; DOPAC; HVA; 5-HT, S-HIAA; chicks; in o v o .
Recent evidence has shown that exposure to pesticides can lead to long-term neurophysiological and functional deficits. We have demonstrated previously that locomotion in chicks exposed to some organophosphates and carbamates could be altered persistently without concomitant central or peripheral esterase inhibition. Furthermore, histopathology of the ataxic chickens showed no lesions in either the central or peripheral systems. In this study, we examined whether locomotion alterations seen in chicks exposed in ovo to carbaryl and aldicarb are accompanied by perturbations in particular central neurotransmitter systems. Carbaryl and aldicarb were injected in ovo on day 15 of incubation at 6, 16 and 65 mg kg-' egg weight and at 0.2, 0.4 and 3.5 mg kg-' egg weight, respectively. Neurotransmitter levels (assayed by high performance liquid chromatography with electrochemical detection) and locomotion were measured at various times ( 1 4 3 days) after dosing. At the lower doses of both carbaryl and aldicarb, a trend towards prolonged decreases in cerebral dopamine and homovanillic acid was seen. The high dose of carbaryl significantly reduced dopamine and the high dose of both compounds significantly decreased homovanillic acid and 5-hydroxyindoleacetic acid. Persistent locomotion alterations were observed only at the higher doses of both carbaryl and aldicarb when the specific neurotransmitters measured returned to normal levels.
0-(methylcarbamoyl)oxime), or Temiks, is a systemic oxime carbamate that is used on a variety of crops, INTRODUCTION including cotton, sugar cane, potatoes, peanuts and ornamental plants. Carbaryl (1-naphthyl-Nmethylcarbamate), or Sevins, is used extensively for Organophosphorus esters (OPs) and carbamate (CA) the control of ectoparasites on poultry, including compounds are used extensively as insecticides owing control of chicken mites and lice. Carbaryl has been to their acute neurotoxicity. These two classes of assumed to be a safe pesticide owing to the easy insecticides inhibit not only the acetylcholinesterase diagnosis of the acute toxicity and the rapid reversibility (AChE) in the nervous system of insects but also in of acute symptoms once the exposure is removed. brain, red blood cells, plasma, liver, pancreas, intestine and skeletal muscles of mammals. This inhibition Thus, although it has been assumed that carbaryl has results in an accumulation of acetylcholine at the no long-term toxic effects in humans, carbamates as post-synaptic membranes, mimicking the muscarinic, well as OPs have been shown to cause brain alterations.x,lo.lI nicotinic and central nervous system actions of acetylcholine. These symptoms in animals include Most studies of the effects of carbaryl on neurochemvomiting, diarrhoea, sweating, cyanosis, twitching of ical parameters have been performed on adult mamthe muscles and hypera~tivity."~ mals, Almost no studies have been done on chickens The acute toxicity of both the OPs and CA have been or chick embryos and none have investigated the effect studied extensively. Recent evidence from humans of aldicarb. We have demonstrated that aldicarb and and animals exposed to these insecticides shows that carbaryl cause alterations in the gait of young chicks additional effects, including behavioral and functional without affecting several esterases in the central nerdeficits, do occur. Exposure to OPs and CA have been vous system (CNS) and the peripheral nervous system reported to cause a number of neurophysiological (PNS).'*.''." Since no direct correspondence has been problems in humans, such as difficulty in cognition, found between the inhibition of esterases and effects on memory and communication."" Additionally. alterthe gait of chicks, we investigated possible correlations ations in the locomotion were shown to persist in between brain neurotransmitter levels and altered chicks exposed to some OPs and carbamates. These locomotion. alterations occur even after the recovery of the neuroIt is well accepted that the action of CNS neurotoxic esterase (NTE), AChE and other e s t e r a ~ e s . ' ~ - ~ ' transmitter systems, particularly the dopaminergic sysAldicarb (2-methyl-2-(methylthio)propionaldehyde tem, contributes to the control of motor behavior in a variety of species. Since our previous work showed that gait alteration is not associated with esterase * Present address: The Procter and Gamble Company. Ivorydale Technical Center, 5299 Spring Grove Avenue. Cincinnati.OH 45217. perturbation, we were interested in investigating IJSA. whether locomotion alterations seen in the chicks that ?Present address: Department of Biology. Furman University, were exposed to carbaryl and aldicarb will be associated Greenville, SC 29613. USA. 026(L437X/92/060421-06$08.00 (Q 1992 by John Wiley & Sons. Ltd.
Received 2 July 1991 Accepfed (revised) 14 February 1992
422
M. FARAGE-ELAWAR AND W. D. BLAKER
with perturbations of specific neurotransmitters. The neurotransmitters that were measured in this study were chosen to correlate with other studies that were performed on mammals and as representative examples of the many CNS neurotransmitter systems. The two chemicals carbaryl and aldicarb were of interest in this study because: (i)
they are the two most popular carbamate insecticides; (ii) carbaryl has been implicated in behavioral and functional deficits in humans;&'" (iii) aldicarb is shown to have the lowest L D ~ "of any pesticide registered in the USA; and (iv) they have been tested extensively in our previous studies. 12-"
EXPERIMENTAL Animals
Thirty to fifty fertile chicken eggs per dose group, hybrids between Peterson strain roosters and Hubbard hens, were obtained from Rocco hatchery, Harrisonburg, VA. The eggs were incubated at 38°C and candled on days 7 and 14 of incubation to discard infertile and dead eggs. After hatching, chicks were fed a corn-soybean diet, without antibiotics or other additives, designed for chicks at Virginia Tech. Both food and water were provided ad libitum. They were identified individually by wing bands. Chicks were kept in thermostatically controlled starter batteries for 2.5 weeks and then moved to cages containing 4-6 birds each. Chemicals and treatment
Aldicarb (2-methyl-2-(methylthio)propionaldehydeU (methylcarbamoyl)oxime), or Temiko (990/, pure, purchased from Chem Serv., PA), or carbaryl (1naphthyl-N-methylcarbamate), or SevinB (99% pure, a gift from Rhone-Poulonc, Research Triangle Park, NC), were dissolved in acetone, diluted in corn oil and then the acetone was evaporated by constant stirring at room temperature. The control solution of the corn oil was prepared similarly but without the carbamates. On day 15 of incubation, eggs were injected with either corn oil, aldicarb or carbaryl. Day 15 of incubation was chosen because the chick embryo is well developed at this period of time and thus chemicals would not interfere with organogenesis. The doses of aldicarb were 0.2, 0.4 and 3.5 mg kg-' egg weight (ew) and for carbaryl 6, 16 and 65 mg kg-' ew. These doses were chosen to correspond to our previous studies. The injection volume was 0.1 ml per egg and all doses were injected at the same time into the albumin through the small end of the egg. The injection site was first swabbed with 70% ethanol and, immediately prior to dosing, a small hole sufficient in size to insert a 22-g needle was made in the shell. After injection, the holes were sealed with melted paraffin. The chicks were allowed 7 days after hatching in order to acclimatize before initiation of gait analysis.
Biochemical assays
At a different time period after hatching, the brain was removed and the cerebrum and subcortical-telencephalon were rapidly dissected on ice. They were stored at -70°C and the levels of dopamine, its two metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), serotonin (5-HT) and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ED).'* Protein measurements of the acid precipitates of the individual brain samples were done according to the method of Lowry et al.'" Behavioral assays
Locomotion was evaluated for eight chicks per group at different time intervals after 7 days of acclimatization. The method of Newby-Schmidt and Norton,2" with some modifications, was used for evaluation of locomotion. The feet of each chick were dipped into water-soluble, non-toxic paint, using different colors to distinguish the left and right feet. A pathway 15-20 inches wide and 3-4 feet long, closed on one side by a wood board and on the other by a wall, was designed so that chicks could only see ahead of them. White paper lined the pathway, and a cage at the end containing other chicks provided impetus for the chick to walk towards the cage. Measurements made for each stride were: distance between consecutive placements of the same foot (stride length); the distance between one foot and the preceding and succeeding placements of the opposite foot (hypotenuse); and the stride width. The sine of the angle of placement was then derived by dividing the stride width by the hypotenuse. Statistics
Outlier values in each data group were eliminated by using the criteria of the Dixon gap test.21 Typically, < 2% of the values are eliminated. Either Student's t-test (for comparing two groups) or the Dunnett multiple comparison test (for comparing several treatment groups to a control g r o ~ p ) was ~ ~ ,used ~ ~ to compare effects of treatment on the length, width and sine of the angle of stride and on the brain monamine
Table 1. Effect of aldicarb and carbaryl treatment on per cent hatchability"
Group Control Aldicarb
Ca rbaryl
Dose (mg kg-' ew)
0.2 0.4 3.5 6 16
65 a
YO Hatchability 84 91 87 57 97 97 77.5
Thirty to fifty eggs per dose group.
423
CHICK EMBRYO EXPOSURE TO CARBAMATES
1500
I
b
levels. A P value of < 0.05 (two-sided) was considered to be statistically significant. controls
3.5 mg kg-'
Aldicarb
RESULTS
mg kg-'
Carbaryl
Per cent hatchability
65
A dose-response relationship was found with both carbaryl and aldicarb (Table 1). The lower dose of carbaryl was accompanied by 97% hatchability, and 77.5% hatchability was found at the highest dose. Lower doses of aldicarb did not affect the per cent hatchability, whereas the highest dose caused a reduction (Table 1).
1000
c,
L: -4 P
$ >.
V
m 5W
Growth
0 . 0
10
20
In ovo injection of aldicarb and carbaryl on day 15 of incubation to chick embryos did not affect significantly the body weight of the chicks after hatching. Figure 1 shows the body weight from days 7 to 43 after hatching of chicks that received 3.5 mg kgg' ew aldicarb and 65 mg kg-' ew carbaryl as embryos.
40
30
Days after hatching
Biochemical results
Figure 1. Body weights of control and treated chick embryos after hatching. Treated chick embryos received either 3.5 mg kg - l egg weight aldicarb or 65 mg kg-' egg weight carbaryl in ovo on day 15 of incubation. Controls were injected with equivalent volumes of corn oil.
The results shown in Table 2 show a decrease in HVA with 0.4 mg kg-l ew aldicarb treatment and a trend for such a decrease with carbaryl treatment. Importantly, in the same samples there were no effects on the levels of 5-HT or its metabolite 5-HIAA. Thus, with the
Table 2. Effect of in ovo injection of aldicarb and carbaryl on day 15 of incubation on monoamine levels in the cerebrum of chicks 1 day after hatching" Group
Dose (mg kg-' ew) Dopamine
Control Aldicarb Carbaryl
13.8 2 2.2 12.6 0.7 9 . 6 1.5 ~ 9 . 4 f 0.5 13.5f 1.6
*
0.2 0.4 6 16
DOPAC
(7) (7) (7) (6) (7)
1.2 ? 0.1 1.0 f 0.1 0.9 t 0.1 1.Of 0.1 1.1 0.1
*
5-HT
HVA
(7) (7) (6) (6) (6)
4.1 f 0.1 4.0 ? 0.4 2.8 f 0.2 3.3 ? 0.1 3.6* 0.3
(7) (7) (6Ib (5) (7)
*
25 2 24 2 2 23 1 24 ? 1 23 f 2
*
5-HIAA (7) (7) (6) (7) (7)
*
2.7 0.4 2.1 t 0.1 2.7 2 0.4 2.4 t 0.2 3 . 0 ? 0.4
(5) (5) (4) (6) (5)
a Results expressed as prnol rng-' protein ? S E M . The numbers in parentheses represent sample size. Significantly different from control; P < 0.05.
Table 3. Effect of in ovo injection of aldicarb and carbaryl on day 15 of incubation on monoamine levels in the cerebrum of chicks 2 days after hatching' Group
n
Dose (mg kg-' ew) Dopamine
Control Aldicarb Carbaryl
6 7 5
3.5 65
a
11.6 f 0.9 11.7 5 1.0 8.5 f 0.8
DOPAC
HVA
5-HT
5-HIM
8.8 ? 1.3 6.5 2 0.8 7.4 f 0.8
4.5 3.7 2.5
42.8 39.4 32.7
5.7 0.3 4 . 8 2 0.4 3.8f 0.7"
Results expressed as pmol mg-' protein 2 SEM Significantly different from control; P < 0.05. Significantly different from control; P < 0.01.
f 0.4
f 0.3b 5
0.2'
2.6 3.5 f 4.0
f ?
*
424
M. FARAGE-ELAWAR A N D W. D . BLAKER
Table 4. Effect of in ovo injection of aldicarb and carbaryl on day 15 of incubation on monoamine levels in the subcortical-telencephalon of chicks 2 days after hatching" Group
n
Dose (rng kg-l ew)
Dopamine
HVA
5-HT
5-HIAA
Control Aldicarb Carbaryl
6 7 5
3.5 65
93212 9955 9425
14.752.1 16.220.8 13.0k0.9
5628 5423 5323
5.1 f0.6 5.0f0.3 5.020.5
a
Results expressed as pmol m g - l protein
SEM.
5
Table 5. Effect of in ovo injection of aldicarb and carbaryl on day 15 of incubation on monoamine levels 43 days after hatching Group
Dose (mgikg ew)
Subcortical telencephalon Dopamine HVA
Doparnine
Control Aldicarb Carbaryl
3.5 65
72 84 70
30 43 37
f f
2
6 (7) 9 (5) 10 (5)
Results expressed as pmol per mgsize.
a
18
-
16
-
20
37 47 39
protein
2
6 (6) 8 (4) ? 7 (5)
5
?
f
f
5
Cerebrum HVA
3 (7) 7 (5) 4 (6)
7.1 7.0 5.7
f
2
2
1.1 (4) 0.6 (4) 0.4 (6)
SEM. Numbers in parentheses represent sample
T
T.
7 6 1
14
12 10
PI
2 +I
-
5-
4-
3-
8-
-
6-
0
3.5 mg kg-'
3.5 mg kg-l A l d i c a r b
65
I
Controls
Controls
-
2-
~-
PI
2
mg kg-'
'
Carbaryl
1
65
i "I-,
0
10
.
I
20
.
I
30
Aldicarb
mg k g - l C a r b a r y l
.
I
4 0
-
I
5 0
Days a f t e r h a t c h i n g Days a f t e r h a t c h i n g
Figure2. Stride length versus days after hatching in chicks treated in ovo on day 15 of incubation with either 3.5 rng kg-' egg weight aldicarb or 65 mg kg-l egg weight carbaryl. Results expressed as means f SEM. The points significantly different from controls are denoted by an asterisk. Locomotion was evaluated for eight chicks per group.
Figure3. Stride width versus days after hatching in chicks treated in ovo on day 15 of incubation with either 3.5 mg kg-' egg weight aldicarb or 65 mg kg-' egg weight carbaryl. Results expressed as means f SEM. The points significantly different from controls are denoted by an asterisk. Locomotion was evaluated for eight chicks per group.
425
CHICK EMBRYO EXPOSURE TO CARBAMATES
lower ranges of aldicarb and carbaryl administration there are indications of a perturbation of the dopamine system but not of the 5-HT system. When aldicarb and carbaryl were given at the higher doses of 3.5 and 65 mg k g ' ew, respectively, a significant reduction in HVA and 5-HIAA (with carbaryl only) was seen 2 days after the chicks have hatched (Table 3). No effect was seen in any of the monoamine levels in the subcortical-telencephalon of 2-day-old chicks (Table 4). When dopamine and HVA were measured in the subcortical-telencephalon and cerebrum 43 days after hatching. no significant differences in the levels of neurotransmitters were seen between treated and control groups (Table 5 ) . Behavioral results
The locomotion of the low-treated and control chicks were measured from days 7 to 22 after hatching. The lower doses of both carbaryl and aldicarb did not affect the stride length, stride width or the sine of the angle of placement (data not shown). The higher doses of both compounds caused an alteration in the gait of the chicks. The stride length of the treated chicks was significantly shorter than that of the controls and this effect lasted until the end of the experiment (Fig. 2).
u a
I
0.8
-
0.7
-
0.6
-
0.5
-
0.4
-
0.3
-
0.2
-
A'
- '
rd r(
a 0 al
4
tn
2
L
W
0 P)
2
rn
0.1
-
-
controls
3.5 mg kg 65
mg kg
-1
-1
Aldicarb Carbaryl
A significant effect on the stride width was seen also with both treatments (Fig. 3). Similarly, the sine of the angle of the treated chicks significantly increased from days 7 to 43 after hatching (Fig. 4).
DISCUSSION
Most studies have focused on the acute toxicity of these pesticides, measuring AChE levels in the central and peripheral nervous systems. When blood ChE is 95% inhibited, ACh in the brain rises to 2-3-fold in acute carbaryl poisoning. Multiple oral dosages of > 1 mg carbaryl kg-' day-' causes an increase in the levels of 5-HT in the blood and brain of rats for 2-4 months. followed by a decrease after 6 months of dosing.'" Changes in catecholamines also have been associated with sublethal single and repeated doses of carbaryl. When carbaryl is administered as a single oral dose of 60 mg kg-' to rats, an increase in the brain levels of 5-HT and its primary metabolite 5-HIAA is observed.'-5 Bursian and EdensZ6 demonstrated an increase in brain dopamine concentrations and an increase in norepinephrine (NE) in Japanese quail given single intramuscular injections of 30 mg carbaryl kg-'. Studies conducted with mammalian species indicate that carbaryl affects ChE activity, catecholamine and indolamine metabolism and plasma glucose level^.'^ Our study demonstrated that in ovo administration of aldicarb and carbaryl at certain doses causes longterm alterations in locomotion of the chicks with reductions in certain neurotransmitter metabolites in the CNS early after hatching. Other studies show that the same behavioral effects can be seen without having alterations in some esterase activities. ':-I7 In our study, even when the levels of neurotransmitters returned to normal, locomotion alterations persisted. Whether the initial alterations in certain neurotransmitters lead to such long-term locomotion alterations remains unknown. More studies should be conducted to investigate further the possible correlations between toxicantinduced behavioral effects seen in animals and humans and alterations in neurotransmitter systems. Certain CNS disorders, such as Alzheimer's disease and Parkinson's disease. are known to be associated with sDecific neurotransmitter alterations. Since xenobiotics such as pesticides are being implicated in long-term neurophysiological behavioral changes and neurotransmitter alterations. further investigation is needed to seek out and substantiate the exact nature of such correlations of pesticide exposure and CNS deficits. u
0.0 0
10
20
30
40
50
Days a f t e r h a t c h i n g
Figure4. Sine of the angle of placement versus days after hatching in chicks treated in ovo on day 15 of incubation with either 3.5 rng kg -' egg weight aldicarb or 65 m g kg -' egg weight carbaryl. Results expressed as means ? SEM. The points significantly different from controls are denoted by an asterisk. Locomotion was evaluated for eight chicks per group.
Acknowledgements The authors would like to thank Mr Gregory Thornwall for his technical assistance.
426
M. FARAGE-ELAWAR AND W. D . BLAKER
REFERENCES
1. R. L. Metcalf, Organic Insecticides. Interscience Publishers, New York (1955). 2. T. R. Fukuto, M. A. H. Fahmy and R. L. Metcalf, Alkaline hydrolysis, anticholinesterase and insecticidal properties of some nitro-substituted phenyl carbamates. J. Agric. food Chem. 15, 273-281 (1967). 3. T. R. Fukuto, R. L. Metcalf, R. L. Jones and R. 0. Myers, Structure reactivity and biological activity of Q (diethylphosphory1)oximes and Q(methylcarbamoyl) oximes of substituted acetophenones and substituted benzaldehydes. J. Agric. Food Chem., 17, 923-930 (1969). 4. F. Matsamara, Toxicology of Insecticides, 2nd Edn. Plenum Press, New York (1985). 5. R. D. O'Brien, Insecticides: Action and Metabolism. Academic Press. New York (1967). 6. D. R. Metcalf and J. H. Holmes, EEG, psychological and neurological alterations in humans with organophosphorus exposure. Ann. NY Acad. Sci. 160 (11, 357-365 (1969). 7. F. H. Duffy, J. L. Burchfiel, P. H. Bartels, M. Gaon and V. M. Sim, Long term effects of an organophosphate upon human electroencephalogram. Toxicol. Appl. Pharmacol. 47, 161-176, (1979). 8. F. H. Duffy and J. L. Burchfiel, Long term effects of the organophosphate sarin on EEG's in monkeys and humans. Neurotoxicology 1, 667-689 (1980). 9. E. P. Savage, Pesticides may alter brain function. Sci. News 129(6), 88 (1986). 10. R. A. Branch and E. Jacqz, Subacute neurotoxicity following long-term exposure to carbaryl. Am. J. Med. 80, 741-745 (1986). 11. R. A. Branch and E. Jacqz, Is carbaryl as safe as its reputation? Does it have a potential for causing chronic neurotoxicity in humans? Am. J. Med. 80, 659-664 (1986). 12. M. Farage-Elawar,Enzyme and behavioral changes in young chicks as a result of carbaryl treatment. J. Toxicol. Environ. Health 26, 119-131 (1989). 13. M. Farage-Elawar, Toxicity of aldicarb in young chicks. Neurotoxicol. Teratol. 10(6), 549-554 (1989). 14. M. Farage-Elawar, Effects of in ovo injection of carbamates on chick embryo hatchability, esterase enzyme activity and locomotion of chicks. J. Appl. Toxicol. 10, 197-201 (1990). 15. M. Farage-Elawar and B. Magnus Francis, Effect of multiple dosing of fenthion. fenitrothion, and desbromoleptophos
16.
17.
18.
19. 20. 21. 22. 23. 24.
25. 26.
27.
in young chicks. J. Toxicol. Environ. Health 23, 217-228 (1988). M. Farage-Elawar and B. Magnus Francis, Effects of fenthion, fenitrothion and desbromoleptophos on gait, acetylcholinesterase, and neurotoxic esterase in young chicks after in ovo exposure. Toxicology 49, 253-261 (1988). M. Farage-Elawar, M. F. Ehrich, B. S. Jortner and H. P. Misra, Effects of multiple oral doses of two carbamate insecticides on esterase levels in young and adult chickens. Pestic. Biochem. Physiol. 32(3), 262-268 (1988). G. M. Anderson, Liquid chromatographic analysis of monoamines and their metabolites. In Neurornethods: Amines and their metabolites, ed. by A. A. Boulton, G. B. Baker and J. M. Baker, pp. 129-196. Humana Press, Clifton, NJ (1985). 0. H. Lowry, N. J. Rosenbrough, A. L. Farr and R. J. Randall, Protein measurement with the fohn phenol reagent. J. Biol. Chem. 193, 265-275 (1951). M. B. Newby-Schmidt and S. Norton, Detection of subtle effects on the locomotor ability of chickens. Neurobehav. Toxicol. Teratol. 3, 45-48 (1981). W. J. Dixon, Ratios involving extreme values. Ann. Math. Stat. 22, 68-78 (1951). J. H. Zar, Biostatistical Analysis, 2nd Edn, pp. 194-195. Prentice-Hall. Englewood Cliffs (1974). W. D. Blaker, Computer program for the parametric and nonparametric comparison of several groups to a control. Comput. Biol. Med. 17, 37-44 (1987). V. A. Butygin and K. A. Viatchannikov, The effect of prolonged administration of small doses of Sevin on the serotonin content in the blood, brain tissue and enterochromaffinic cells of the duodenum in white rats. Zdravookhr. Beloruss. 15, 44-47 (1969). A. Hassan and J. A. Santolucito, Pharmacological effects of carbaryl. Il-Modification of serotonin metabolism in the rat brain. Experimentia 27, 287-288 (1971). S. J. Bursian and F. W. Edens, The effect of acute carbaryl administration on various neurochemical and blood chemical parameters in the Japanese quail. Toxicol. Appl. fharmacol. 46, 463-473 (1978). C. P. Carpenter, C. S. Weil, P. E. Palm, M. W. Woodside, J.H. Ill Nair and H. F. Smith, Mammalian toxicity of 1naphthyl-N-methylcarbarnate (Sevin insecticide). J. Agric. food Chem. 9, 30-39 (1961).
Chick Embryo Exposure to Carbamates Alters
Neurochemical Parameters and Behavior M. Farage-Elawar" and W. D. BlakerS VAiMD Regional College of Veterinary Medicine, Department of Biomedical Sciences, Blacksburg, VA 24061, USA
Key words: carbamates; locomotion; dopamine; DOPAC; HVA; 5-HT, S-HIAA; chicks; in o v o .
Recent evidence has shown that exposure to pesticides can lead to long-term neurophysiological and functional deficits. We have demonstrated previously that locomotion in chicks exposed to some organophosphates and carbamates could be altered persistently without concomitant central or peripheral esterase inhibition. Furthermore, histopathology of the ataxic chickens showed no lesions in either the central or peripheral systems. In this study, we examined whether locomotion alterations seen in chicks exposed in ovo to carbaryl and aldicarb are accompanied by perturbations in particular central neurotransmitter systems. Carbaryl and aldicarb were injected in ovo on day 15 of incubation at 6, 16 and 65 mg kg-' egg weight and at 0.2, 0.4 and 3.5 mg kg-' egg weight, respectively. Neurotransmitter levels (assayed by high performance liquid chromatography with electrochemical detection) and locomotion were measured at various times ( 1 4 3 days) after dosing. At the lower doses of both carbaryl and aldicarb, a trend towards prolonged decreases in cerebral dopamine and homovanillic acid was seen. The high dose of carbaryl significantly reduced dopamine and the high dose of both compounds significantly decreased homovanillic acid and 5-hydroxyindoleacetic acid. Persistent locomotion alterations were observed only at the higher doses of both carbaryl and aldicarb when the specific neurotransmitters measured returned to normal levels.
0-(methylcarbamoyl)oxime), or Temiks, is a systemic oxime carbamate that is used on a variety of crops, INTRODUCTION including cotton, sugar cane, potatoes, peanuts and ornamental plants. Carbaryl (1-naphthyl-Nmethylcarbamate), or Sevins, is used extensively for Organophosphorus esters (OPs) and carbamate (CA) the control of ectoparasites on poultry, including compounds are used extensively as insecticides owing control of chicken mites and lice. Carbaryl has been to their acute neurotoxicity. These two classes of assumed to be a safe pesticide owing to the easy insecticides inhibit not only the acetylcholinesterase diagnosis of the acute toxicity and the rapid reversibility (AChE) in the nervous system of insects but also in of acute symptoms once the exposure is removed. brain, red blood cells, plasma, liver, pancreas, intestine and skeletal muscles of mammals. This inhibition Thus, although it has been assumed that carbaryl has results in an accumulation of acetylcholine at the no long-term toxic effects in humans, carbamates as post-synaptic membranes, mimicking the muscarinic, well as OPs have been shown to cause brain alterations.x,lo.lI nicotinic and central nervous system actions of acetylcholine. These symptoms in animals include Most studies of the effects of carbaryl on neurochemvomiting, diarrhoea, sweating, cyanosis, twitching of ical parameters have been performed on adult mamthe muscles and hypera~tivity."~ mals, Almost no studies have been done on chickens The acute toxicity of both the OPs and CA have been or chick embryos and none have investigated the effect studied extensively. Recent evidence from humans of aldicarb. We have demonstrated that aldicarb and and animals exposed to these insecticides shows that carbaryl cause alterations in the gait of young chicks additional effects, including behavioral and functional without affecting several esterases in the central nerdeficits, do occur. Exposure to OPs and CA have been vous system (CNS) and the peripheral nervous system reported to cause a number of neurophysiological (PNS).'*.''." Since no direct correspondence has been problems in humans, such as difficulty in cognition, found between the inhibition of esterases and effects on memory and communication."" Additionally. alterthe gait of chicks, we investigated possible correlations ations in the locomotion were shown to persist in between brain neurotransmitter levels and altered chicks exposed to some OPs and carbamates. These locomotion. alterations occur even after the recovery of the neuroIt is well accepted that the action of CNS neurotoxic esterase (NTE), AChE and other e s t e r a ~ e s . ' ~ - ~ ' transmitter systems, particularly the dopaminergic sysAldicarb (2-methyl-2-(methylthio)propionaldehyde tem, contributes to the control of motor behavior in a variety of species. Since our previous work showed that gait alteration is not associated with esterase * Present address: The Procter and Gamble Company. Ivorydale Technical Center, 5299 Spring Grove Avenue. Cincinnati.OH 45217. perturbation, we were interested in investigating IJSA. whether locomotion alterations seen in the chicks that ?Present address: Department of Biology. Furman University, were exposed to carbaryl and aldicarb will be associated Greenville, SC 29613. USA. 026(L437X/92/060421-06$08.00 (Q 1992 by John Wiley & Sons. Ltd.
Received 2 July 1991 Accepfed (revised) 14 February 1992
422
M. FARAGE-ELAWAR AND W. D. BLAKER
with perturbations of specific neurotransmitters. The neurotransmitters that were measured in this study were chosen to correlate with other studies that were performed on mammals and as representative examples of the many CNS neurotransmitter systems. The two chemicals carbaryl and aldicarb were of interest in this study because: (i)
they are the two most popular carbamate insecticides; (ii) carbaryl has been implicated in behavioral and functional deficits in humans;&'" (iii) aldicarb is shown to have the lowest L D ~ "of any pesticide registered in the USA; and (iv) they have been tested extensively in our previous studies. 12-"
EXPERIMENTAL Animals
Thirty to fifty fertile chicken eggs per dose group, hybrids between Peterson strain roosters and Hubbard hens, were obtained from Rocco hatchery, Harrisonburg, VA. The eggs were incubated at 38°C and candled on days 7 and 14 of incubation to discard infertile and dead eggs. After hatching, chicks were fed a corn-soybean diet, without antibiotics or other additives, designed for chicks at Virginia Tech. Both food and water were provided ad libitum. They were identified individually by wing bands. Chicks were kept in thermostatically controlled starter batteries for 2.5 weeks and then moved to cages containing 4-6 birds each. Chemicals and treatment
Aldicarb (2-methyl-2-(methylthio)propionaldehydeU (methylcarbamoyl)oxime), or Temiko (990/, pure, purchased from Chem Serv., PA), or carbaryl (1naphthyl-N-methylcarbamate), or SevinB (99% pure, a gift from Rhone-Poulonc, Research Triangle Park, NC), were dissolved in acetone, diluted in corn oil and then the acetone was evaporated by constant stirring at room temperature. The control solution of the corn oil was prepared similarly but without the carbamates. On day 15 of incubation, eggs were injected with either corn oil, aldicarb or carbaryl. Day 15 of incubation was chosen because the chick embryo is well developed at this period of time and thus chemicals would not interfere with organogenesis. The doses of aldicarb were 0.2, 0.4 and 3.5 mg kg-' egg weight (ew) and for carbaryl 6, 16 and 65 mg kg-' ew. These doses were chosen to correspond to our previous studies. The injection volume was 0.1 ml per egg and all doses were injected at the same time into the albumin through the small end of the egg. The injection site was first swabbed with 70% ethanol and, immediately prior to dosing, a small hole sufficient in size to insert a 22-g needle was made in the shell. After injection, the holes were sealed with melted paraffin. The chicks were allowed 7 days after hatching in order to acclimatize before initiation of gait analysis.
Biochemical assays
At a different time period after hatching, the brain was removed and the cerebrum and subcortical-telencephalon were rapidly dissected on ice. They were stored at -70°C and the levels of dopamine, its two metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), serotonin (5-HT) and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ED).'* Protein measurements of the acid precipitates of the individual brain samples were done according to the method of Lowry et al.'" Behavioral assays
Locomotion was evaluated for eight chicks per group at different time intervals after 7 days of acclimatization. The method of Newby-Schmidt and Norton,2" with some modifications, was used for evaluation of locomotion. The feet of each chick were dipped into water-soluble, non-toxic paint, using different colors to distinguish the left and right feet. A pathway 15-20 inches wide and 3-4 feet long, closed on one side by a wood board and on the other by a wall, was designed so that chicks could only see ahead of them. White paper lined the pathway, and a cage at the end containing other chicks provided impetus for the chick to walk towards the cage. Measurements made for each stride were: distance between consecutive placements of the same foot (stride length); the distance between one foot and the preceding and succeeding placements of the opposite foot (hypotenuse); and the stride width. The sine of the angle of placement was then derived by dividing the stride width by the hypotenuse. Statistics
Outlier values in each data group were eliminated by using the criteria of the Dixon gap test.21 Typically, < 2% of the values are eliminated. Either Student's t-test (for comparing two groups) or the Dunnett multiple comparison test (for comparing several treatment groups to a control g r o ~ p ) was ~ ~ ,used ~ ~ to compare effects of treatment on the length, width and sine of the angle of stride and on the brain monamine
Table 1. Effect of aldicarb and carbaryl treatment on per cent hatchability"
Group Control Aldicarb
Ca rbaryl
Dose (mg kg-' ew)
0.2 0.4 3.5 6 16
65 a
YO Hatchability 84 91 87 57 97 97 77.5
Thirty to fifty eggs per dose group.
423
CHICK EMBRYO EXPOSURE TO CARBAMATES
1500
I
b
levels. A P value of < 0.05 (two-sided) was considered to be statistically significant. controls
3.5 mg kg-'
Aldicarb
RESULTS
mg kg-'
Carbaryl
Per cent hatchability
65
A dose-response relationship was found with both carbaryl and aldicarb (Table 1). The lower dose of carbaryl was accompanied by 97% hatchability, and 77.5% hatchability was found at the highest dose. Lower doses of aldicarb did not affect the per cent hatchability, whereas the highest dose caused a reduction (Table 1).
1000
c,
L: -4 P
$ >.
V
m 5W
Growth
0 . 0
10
20
In ovo injection of aldicarb and carbaryl on day 15 of incubation to chick embryos did not affect significantly the body weight of the chicks after hatching. Figure 1 shows the body weight from days 7 to 43 after hatching of chicks that received 3.5 mg kgg' ew aldicarb and 65 mg kg-' ew carbaryl as embryos.
40
30
Days after hatching
Biochemical results
Figure 1. Body weights of control and treated chick embryos after hatching. Treated chick embryos received either 3.5 mg kg - l egg weight aldicarb or 65 mg kg-' egg weight carbaryl in ovo on day 15 of incubation. Controls were injected with equivalent volumes of corn oil.
The results shown in Table 2 show a decrease in HVA with 0.4 mg kg-l ew aldicarb treatment and a trend for such a decrease with carbaryl treatment. Importantly, in the same samples there were no effects on the levels of 5-HT or its metabolite 5-HIAA. Thus, with the
Table 2. Effect of in ovo injection of aldicarb and carbaryl on day 15 of incubation on monoamine levels in the cerebrum of chicks 1 day after hatching" Group
Dose (mg kg-' ew) Dopamine
Control Aldicarb Carbaryl
13.8 2 2.2 12.6 0.7 9 . 6 1.5 ~ 9 . 4 f 0.5 13.5f 1.6
*
0.2 0.4 6 16
DOPAC
(7) (7) (7) (6) (7)
1.2 ? 0.1 1.0 f 0.1 0.9 t 0.1 1.Of 0.1 1.1 0.1
*
5-HT
HVA
(7) (7) (6) (6) (6)
4.1 f 0.1 4.0 ? 0.4 2.8 f 0.2 3.3 ? 0.1 3.6* 0.3
(7) (7) (6Ib (5) (7)
*
25 2 24 2 2 23 1 24 ? 1 23 f 2
*
5-HIAA (7) (7) (6) (7) (7)
*
2.7 0.4 2.1 t 0.1 2.7 2 0.4 2.4 t 0.2 3 . 0 ? 0.4
(5) (5) (4) (6) (5)
a Results expressed as prnol rng-' protein ? S E M . The numbers in parentheses represent sample size. Significantly different from control; P < 0.05.
Table 3. Effect of in ovo injection of aldicarb and carbaryl on day 15 of incubation on monoamine levels in the cerebrum of chicks 2 days after hatching' Group
n
Dose (mg kg-' ew) Dopamine
Control Aldicarb Carbaryl
6 7 5
3.5 65
a
11.6 f 0.9 11.7 5 1.0 8.5 f 0.8
DOPAC
HVA
5-HT
5-HIM
8.8 ? 1.3 6.5 2 0.8 7.4 f 0.8
4.5 3.7 2.5
42.8 39.4 32.7
5.7 0.3 4 . 8 2 0.4 3.8f 0.7"
Results expressed as pmol mg-' protein 2 SEM Significantly different from control; P < 0.05. Significantly different from control; P < 0.01.
f 0.4
f 0.3b 5
0.2'
2.6 3.5 f 4.0
f ?
*
424
M. FARAGE-ELAWAR A N D W. D . BLAKER
Table 4. Effect of in ovo injection of aldicarb and carbaryl on day 15 of incubation on monoamine levels in the subcortical-telencephalon of chicks 2 days after hatching" Group
n
Dose (rng kg-l ew)
Dopamine
HVA
5-HT
5-HIAA
Control Aldicarb Carbaryl
6 7 5
3.5 65
93212 9955 9425
14.752.1 16.220.8 13.0k0.9
5628 5423 5323
5.1 f0.6 5.0f0.3 5.020.5
a
Results expressed as pmol m g - l protein
SEM.
5
Table 5. Effect of in ovo injection of aldicarb and carbaryl on day 15 of incubation on monoamine levels 43 days after hatching Group
Dose (mgikg ew)
Subcortical telencephalon Dopamine HVA
Doparnine
Control Aldicarb Carbaryl
3.5 65
72 84 70
30 43 37
f f
2
6 (7) 9 (5) 10 (5)
Results expressed as pmol per mgsize.
a
18
-
16
-
20
37 47 39
protein
2
6 (6) 8 (4) ? 7 (5)
5
?
f
f
5
Cerebrum HVA
3 (7) 7 (5) 4 (6)
7.1 7.0 5.7
f
2
2
1.1 (4) 0.6 (4) 0.4 (6)
SEM. Numbers in parentheses represent sample
T
T.
7 6 1
14
12 10
PI
2 +I
-
5-
4-
3-
8-
-
6-
0
3.5 mg kg-'
3.5 mg kg-l A l d i c a r b
65
I
Controls
Controls
-
2-
~-
PI
2
mg kg-'
'
Carbaryl
1
65
i "I-,
0
10
.
I
20
.
I
30
Aldicarb
mg k g - l C a r b a r y l
.
I
4 0
-
I
5 0
Days a f t e r h a t c h i n g Days a f t e r h a t c h i n g
Figure2. Stride length versus days after hatching in chicks treated in ovo on day 15 of incubation with either 3.5 rng kg-' egg weight aldicarb or 65 mg kg-l egg weight carbaryl. Results expressed as means f SEM. The points significantly different from controls are denoted by an asterisk. Locomotion was evaluated for eight chicks per group.
Figure3. Stride width versus days after hatching in chicks treated in ovo on day 15 of incubation with either 3.5 mg kg-' egg weight aldicarb or 65 mg kg-' egg weight carbaryl. Results expressed as means f SEM. The points significantly different from controls are denoted by an asterisk. Locomotion was evaluated for eight chicks per group.
425
CHICK EMBRYO EXPOSURE TO CARBAMATES
lower ranges of aldicarb and carbaryl administration there are indications of a perturbation of the dopamine system but not of the 5-HT system. When aldicarb and carbaryl were given at the higher doses of 3.5 and 65 mg k g ' ew, respectively, a significant reduction in HVA and 5-HIAA (with carbaryl only) was seen 2 days after the chicks have hatched (Table 3). No effect was seen in any of the monoamine levels in the subcortical-telencephalon of 2-day-old chicks (Table 4). When dopamine and HVA were measured in the subcortical-telencephalon and cerebrum 43 days after hatching. no significant differences in the levels of neurotransmitters were seen between treated and control groups (Table 5 ) . Behavioral results
The locomotion of the low-treated and control chicks were measured from days 7 to 22 after hatching. The lower doses of both carbaryl and aldicarb did not affect the stride length, stride width or the sine of the angle of placement (data not shown). The higher doses of both compounds caused an alteration in the gait of the chicks. The stride length of the treated chicks was significantly shorter than that of the controls and this effect lasted until the end of the experiment (Fig. 2).
u a
I
0.8
-
0.7
-
0.6
-
0.5
-
0.4
-
0.3
-
0.2
-
A'
- '
rd r(
a 0 al
4
tn
2
L
W
0 P)
2
rn
0.1
-
-
controls
3.5 mg kg 65
mg kg
-1
-1
Aldicarb Carbaryl
A significant effect on the stride width was seen also with both treatments (Fig. 3). Similarly, the sine of the angle of the treated chicks significantly increased from days 7 to 43 after hatching (Fig. 4).
DISCUSSION
Most studies have focused on the acute toxicity of these pesticides, measuring AChE levels in the central and peripheral nervous systems. When blood ChE is 95% inhibited, ACh in the brain rises to 2-3-fold in acute carbaryl poisoning. Multiple oral dosages of > 1 mg carbaryl kg-' day-' causes an increase in the levels of 5-HT in the blood and brain of rats for 2-4 months. followed by a decrease after 6 months of dosing.'" Changes in catecholamines also have been associated with sublethal single and repeated doses of carbaryl. When carbaryl is administered as a single oral dose of 60 mg kg-' to rats, an increase in the brain levels of 5-HT and its primary metabolite 5-HIAA is observed.'-5 Bursian and EdensZ6 demonstrated an increase in brain dopamine concentrations and an increase in norepinephrine (NE) in Japanese quail given single intramuscular injections of 30 mg carbaryl kg-'. Studies conducted with mammalian species indicate that carbaryl affects ChE activity, catecholamine and indolamine metabolism and plasma glucose level^.'^ Our study demonstrated that in ovo administration of aldicarb and carbaryl at certain doses causes longterm alterations in locomotion of the chicks with reductions in certain neurotransmitter metabolites in the CNS early after hatching. Other studies show that the same behavioral effects can be seen without having alterations in some esterase activities. ':-I7 In our study, even when the levels of neurotransmitters returned to normal, locomotion alterations persisted. Whether the initial alterations in certain neurotransmitters lead to such long-term locomotion alterations remains unknown. More studies should be conducted to investigate further the possible correlations between toxicantinduced behavioral effects seen in animals and humans and alterations in neurotransmitter systems. Certain CNS disorders, such as Alzheimer's disease and Parkinson's disease. are known to be associated with sDecific neurotransmitter alterations. Since xenobiotics such as pesticides are being implicated in long-term neurophysiological behavioral changes and neurotransmitter alterations. further investigation is needed to seek out and substantiate the exact nature of such correlations of pesticide exposure and CNS deficits. u
0.0 0
10
20
30
40
50
Days a f t e r h a t c h i n g
Figure4. Sine of the angle of placement versus days after hatching in chicks treated in ovo on day 15 of incubation with either 3.5 rng kg -' egg weight aldicarb or 65 m g kg -' egg weight carbaryl. Results expressed as means ? SEM. The points significantly different from controls are denoted by an asterisk. Locomotion was evaluated for eight chicks per group.
Acknowledgements The authors would like to thank Mr Gregory Thornwall for his technical assistance.
426
M. FARAGE-ELAWAR AND W. D . BLAKER
REFERENCES
1. R. L. Metcalf, Organic Insecticides. Interscience Publishers, New York (1955). 2. T. R. Fukuto, M. A. H. Fahmy and R. L. Metcalf, Alkaline hydrolysis, anticholinesterase and insecticidal properties of some nitro-substituted phenyl carbamates. J. Agric. food Chem. 15, 273-281 (1967). 3. T. R. Fukuto, R. L. Metcalf, R. L. Jones and R. 0. Myers, Structure reactivity and biological activity of Q (diethylphosphory1)oximes and Q(methylcarbamoyl) oximes of substituted acetophenones and substituted benzaldehydes. J. Agric. Food Chem., 17, 923-930 (1969). 4. F. Matsamara, Toxicology of Insecticides, 2nd Edn. Plenum Press, New York (1985). 5. R. D. O'Brien, Insecticides: Action and Metabolism. Academic Press. New York (1967). 6. D. R. Metcalf and J. H. Holmes, EEG, psychological and neurological alterations in humans with organophosphorus exposure. Ann. NY Acad. Sci. 160 (11, 357-365 (1969). 7. F. H. Duffy, J. L. Burchfiel, P. H. Bartels, M. Gaon and V. M. Sim, Long term effects of an organophosphate upon human electroencephalogram. Toxicol. Appl. Pharmacol. 47, 161-176, (1979). 8. F. H. Duffy and J. L. Burchfiel, Long term effects of the organophosphate sarin on EEG's in monkeys and humans. Neurotoxicology 1, 667-689 (1980). 9. E. P. Savage, Pesticides may alter brain function. Sci. News 129(6), 88 (1986). 10. R. A. Branch and E. Jacqz, Subacute neurotoxicity following long-term exposure to carbaryl. Am. J. Med. 80, 741-745 (1986). 11. R. A. Branch and E. Jacqz, Is carbaryl as safe as its reputation? Does it have a potential for causing chronic neurotoxicity in humans? Am. J. Med. 80, 659-664 (1986). 12. M. Farage-Elawar,Enzyme and behavioral changes in young chicks as a result of carbaryl treatment. J. Toxicol. Environ. Health 26, 119-131 (1989). 13. M. Farage-Elawar, Toxicity of aldicarb in young chicks. Neurotoxicol. Teratol. 10(6), 549-554 (1989). 14. M. Farage-Elawar, Effects of in ovo injection of carbamates on chick embryo hatchability, esterase enzyme activity and locomotion of chicks. J. Appl. Toxicol. 10, 197-201 (1990). 15. M. Farage-Elawar and B. Magnus Francis, Effect of multiple dosing of fenthion. fenitrothion, and desbromoleptophos
16.
17.
18.
19. 20. 21. 22. 23. 24.
25. 26.
27.
in young chicks. J. Toxicol. Environ. Health 23, 217-228 (1988). M. Farage-Elawar and B. Magnus Francis, Effects of fenthion, fenitrothion and desbromoleptophos on gait, acetylcholinesterase, and neurotoxic esterase in young chicks after in ovo exposure. Toxicology 49, 253-261 (1988). M. Farage-Elawar, M. F. Ehrich, B. S. Jortner and H. P. Misra, Effects of multiple oral doses of two carbamate insecticides on esterase levels in young and adult chickens. Pestic. Biochem. Physiol. 32(3), 262-268 (1988). G. M. Anderson, Liquid chromatographic analysis of monoamines and their metabolites. In Neurornethods: Amines and their metabolites, ed. by A. A. Boulton, G. B. Baker and J. M. Baker, pp. 129-196. Humana Press, Clifton, NJ (1985). 0. H. Lowry, N. J. Rosenbrough, A. L. Farr and R. J. Randall, Protein measurement with the fohn phenol reagent. J. Biol. Chem. 193, 265-275 (1951). M. B. Newby-Schmidt and S. Norton, Detection of subtle effects on the locomotor ability of chickens. Neurobehav. Toxicol. Teratol. 3, 45-48 (1981). W. J. Dixon, Ratios involving extreme values. Ann. Math. Stat. 22, 68-78 (1951). J. H. Zar, Biostatistical Analysis, 2nd Edn, pp. 194-195. Prentice-Hall. Englewood Cliffs (1974). W. D. Blaker, Computer program for the parametric and nonparametric comparison of several groups to a control. Comput. Biol. Med. 17, 37-44 (1987). V. A. Butygin and K. A. Viatchannikov, The effect of prolonged administration of small doses of Sevin on the serotonin content in the blood, brain tissue and enterochromaffinic cells of the duodenum in white rats. Zdravookhr. Beloruss. 15, 44-47 (1969). A. Hassan and J. A. Santolucito, Pharmacological effects of carbaryl. Il-Modification of serotonin metabolism in the rat brain. Experimentia 27, 287-288 (1971). S. J. Bursian and F. W. Edens, The effect of acute carbaryl administration on various neurochemical and blood chemical parameters in the Japanese quail. Toxicol. Appl. fharmacol. 46, 463-473 (1978). C. P. Carpenter, C. S. Weil, P. E. Palm, M. W. Woodside, J.H. Ill Nair and H. F. Smith, Mammalian toxicity of 1naphthyl-N-methylcarbarnate (Sevin insecticide). J. Agric. food Chem. 9, 30-39 (1961).
