Int. Med J Vol 3 No 2 December 2004

TERATOGENIC EFFECT OF DIMETHOATE ON CHICK EMBRYOS

Mohammed A. Alhifi[a], M. Z. Khan[b], Hossny A. Algoshai[c], V. S. Ghole[d]

ABSTRACT:

Objective To examine the teratogenic effect of the organophosphorus insecticide  Dimethoateb on the development of the brain, neural tube, heart and somites during the early embryognesis and its effect on the total blood Acetylcholine Estrase (AChE) enzyme.

Methods Chick embryo in vivo used as a model at the age of 48hrs (24 hrs incubation followed by 24 hrs exposure). 100 µl of Dimethoate injected into the air sac at dosages of 2.5ppm, 5ppm and 40 ppm after 24 hrs of incubation and the control eggs were injected with an equivalent volume of 0.9% NaCl. AChE activity was estimated after chronic exposure to Dimethoate for two weeks.

Findings Dimethoate found to have a deleterious effects and shows significant abnormal developmental effects on the heart, brain, neural tube and somites during the organogenesis and it inhibits the activity of AChE enzyme in the level of 40%.

Conclusion Dimethoate induces deformities in brain development and this suggests the defective cell proliferation. Also the central nervous system, somites and heart development were targets of Dimethoate even at low concentrations. Inhibition of AChE suggests disruption of nerve function during the embryo development.

Keywords: Dimethoate, Chick embryo, Teratogenic effect, Pesticide, Organogenesis.

INTRODUCTION:

Pesticides play an important role in the success of modern farming and food production. However, the release of pesticides to the environment arising from non-approved use, poor practice, illegal operations or misuse is increasingly contributing to environmental pollution (1). Dimethoate is an organophosphate insecticide used against a wide range of insects, including aphids, thrips, plant hoppers, and whiteflies in ornamental plants, alfalfa, apples, corn, cottons, grapefruits, grapes, lemons, melons, oranges, pares, pecans, safflower, sorghum, soybeans, tangerines, tobacco, tomatoes, watermelons, wheat and other vegetables. It is also used as residual wall spray in farm buildings for houseflies. Dimethoate has been demonstrated to be useful in livestock for the control of botflies (2).

It has been reported that Dimethoate is a teratogen to cat fetuses and induces a polydactyl deformity (3). Embryo development has been introduced as a model for the studying of the toxicants in the environment.  Klumpp used fish embryo for the monitoring of

Xiamen Harbor water quality in China (4). Sahu et al. used chick embryo development for the evaluation of the teratogenicity of the organophosphorous insecticide Dimecron, significant abnormalities were recorded (5). Also chick embryo development used by Gilani for the evaluation of the potential teratogenicity of nickel chloride (6).  It has been reported the administration of Dimethoate to mice (10-80mg/kg) on the 6^th – 16^th day of gestation did not produced fetotoxicity, fetal lethality or malformation of the fetuses (7).

In the present study, chick embryo as a model used for the investigation of the effect of Dimethoate during the early stages of development. The reasons encourage us for the use of chick embryo in this investigation as it is conservative model; in which we are saving the life of testing animals used for research experiments in a large numbers, this model is cheaper, easy to monitor and it gives quick results.

Acetyl cholinesterase (AChE), an enzyme found in the free state mainly in brain, nerve cells, muscle, lunge and erythrocytes. It plays an important role in the transmission of nerve impulse. The enzyme activity is strongly inhibited in cases of poisoning with organophosphorus compounds. The determination of AChE is of important when there is question of possible poisoning with organophosphorus compounds (8).

METHODS:

Eggs: The eggs were obtained from the IVBP (Indian Veterinary and Biological Products), Pune, India. Eggs weighing 54±1 gm were selected for experimentation.

Chemicals: Rogor (Dimethoate 30% EC) with the registration number 5-29(43)/Dimethoate (EC)-84 was used. Manufactured by Rallis India limited, Agrochemical Division. The batch was obtained from POCHA SEEDS PVT.LTD. near Sholapur Bazar, Pune 411040. The desired concentrations were prepared using sterilized 0.9% saline.

Treatment: Each egg was cleaned with 70% ethanol and marked suitably and then incubated at 38±0.5Ċ for 24 hrs. 60 - 65 % humidity of the incubator was maintained. For each treatment dose, a group of 50 eggs kept as treated and 25 eggs kept as control.  The LD50 of Dimethoate was found to be 50ppm (24 hr incubation followed by 24 hr exposure) and the screening was carried out in three steps. The first group received a treatment with the 40ppm, the second group received the treatment with 1/10^th LD50 (5ppm), and the third group received the treatment with 1/20^th LD50 (2.5ppm). The injection of 100 µl of the desired concentration of Dimethoate into the embryos aged of 24hrs was performed by making a hole under aseptic condition in the egg calcareous shell over the air chamber at the blunted end of the egg with micro-pipette. The hole was then sealed with plastic tape and then reincubated for 24hours. The control groups received the same volume of sterile normal saline. Each treatment group and its control were harvested on the second day of the injection i.e. at the age of 48 hrs the embryos were removed from their membranes, fixed with bouin's fixative and stained with heamatoxyline and eosin, then mounted on a permanent slides for further developmental studies of the somites, neural tube, brain and heart induced developmental abnormalities. 

Effect of dimethoate on the activity of Acetylechlinestrase enzyme (AChE):  the control consists of 10 embryos whereas in test 20 replicates were used. 5ppm of Dimethoate was injected in each egg from test group starting from the day 7 of incubation for 2 weeks, whereas control group was injected with sterile normal saline. On the day 21 after 12 hours of the last dose an amount of 200µl blood was drown from the blood vessels surrounding the embryonic membranes and the heamolyzate used for the assessment of the enzyme activity calorimetrically (8). 

Statistical method:

“Graph pad instat3 “Soft ware used for the analysis of the data. Krskal-Wallis test followed by Tukey-Kramer Multiple Comparison Test was used (9).

 For the AChE activity assessment student “t” Test used for the analyzing of the data.

Scheme of scoring for the damage:

The score to the abnormalities represent the degree of severity of teratogenic effect of Dimethoate. The tilted embryo and somite abnormality has been given less weightage as we have observed such abnormalities in control embryos of the same age. Similarly neural tube abnormalities also have been noted in control embryos but the frequency was less. Therefore intermittent weightage has been given, so to find abnormalities such as brain and heart abnormalities is rather rare and only can be seen when extensive damage to the embryo is caused, so these abnormalities has been given the highest score.   

RESULTS:

The LD50 of Dimethoate was found to be 50ppm. Table-1 depicts the effect of Dimethoate at different doses. The dose of 2.5ppm shows minimum teratogenic effect in embryo development as compared to the higher doses.

The dose of 5ppm (1/10^th LD50): This dose showed significant effect (P<0.001) when compared to control.  The dose of 40ppm: It was extremely significant when compared to control. Dimethoate at these concentrations caused severe abnormalities on the heart, brain, neural tube and somites during the organogenesis as described bellow:

The effect of Dimethoate on the heart development. Two types of the heart deformities have been seen, either heart miss-position or heart malformation, (Figure 2&3). At 2.5ppm and 5ppm concentrations of Dimethoate the effect on the development of the heart was not significant, whereas at the dose of 40ppm the effect was extremely significant versus the control, table-2.

 The effect of Dimethoate on the brain development: Three types of brain developmental anomalies have been detected; compacted brain, micro-cephaly or macro-cephaly, (Figure 3, 4&5). The effect on the brain development was significant at the three doses, table-3.

The effect of Dimethoate on the neural tube development. Two types of neural tube developmental anomalies detected; Opening of the Neural Tube (ONT) and Wavy Neural Tube (WNT), (Figure 5). The effect of Dimethoate was not significant at the dose of 2.5ppm and 5ppm but it was significant at 40ppm as compare to the control,  table-4.

The effect of Dimethoate on the somite development: Two types of the somite developmental anomalies have been detected, dispersed somites and less number of somites (Figure 3). The effect on the somite development was not significant at the dose of 2.5ppm compared to control, but it was significant at 5ppm and 40ppm,   table-5.

Dimethoate found to inhibit the AChE activity after repeated exposure to 5ppm of Dimethoate for two weeks and the effect was significant compared to control (P<0.0001). The inhibition of the enzyme activity was 40% of the control, table-6.

DISCUSSION

Screening of the developmental toxicity of Dimethoate carried out at the 40ppm, 5ppm (1/10^th LD50) and 2.5ppm (1/20^th LD5), so that the impact addressed at the low concentrations that the embryo might get exposed to it. Different concentrations of Dimethoate target different organs. At the dose of 2.5ppm the effect on the development of the heart, neural tube and somites was not significant (P>0.05), but the effect was significant on the development of the brain (P<0.05). Whereas at the dose of 5ppm, the effect was significant on the brain development and somite formation, (P<0.001), but it was not significant on the development of the heart and the neural tube, (P>0.05). At the dose of 40ppm Dimethoate significantly affects the development of the heart, brain, neural tube and somites, (P<0.0001). The effect on the somie development at these early stages might be results in defects in the vertebrae and skull development at the late stages as the somies will contribute to the formation of the vertebrae and the skull of the embryo. The accumulation of acetylcholine due to the inhibition of AChE may also disturb the histology of the organs like the brain and the heart.

Exposure of chick embryo to combined heavy metals and Dimethoate causes increased embryo mortality, decreased body weight, higher toxicity, and induced malformations when Dimethoate and heavy metal injected together than injection of heavy metal alone (10, 11, 12).

Administration of Dimethoate with vibration to rat fetuses during development was found to induce retardation and deformation (13). Dimethoate was found to induce morphological changes and inhibit acetylcholinestrase activity in fetal brain of rats (14).

The study revealed that chick embryo model is a promising model for the screening of the developmental toxicity of pesticides. It can be used for the study of the effect of pesticides in genotoxicity studies. This study concludes that exposure to Dimethoate results in significant inhibition of total blood AChE and this suggests disruption of nerve function during the embryo development. Deformities in brain development suggest defective cell proliferation. The central nervous system, somites and heart are targets of Dimethoate during the embryogenesis even at low concentrations. Exposure to Dimethoate during the embryonic development will retard the central nervous and the circulatory systems and their functions. Such abnormality during embryo development and its impact will remain through out the life of the animal.

REFERENCES:

1- Paul Fogg, Alistair BA Boxall, Allen Walker and Andrew A Jukes, Pesticide Degradation in a Biobed Composting Substrate. Pest Management Science 2003;   59:527-537.

2- Dimethoate, (FAO Meeting Report No. PL / 1965 / 10; WHO / Food Add. /    26. 65).

3- Khera KS Evaluation of Dimethoate (Cygon 4E) for Teratogenic Activity in the Cat. Journal of Environmental Pathology and Toxicology 1979;2(6):1283-8.

4- D.W. Klumpp, C. Humphrey, Hong Huasheng and Feng Tao Toxic Contaminants and their Biological Effects in Coastal Water of Xiamen, China. Marine Pollution Bulletin 2002;44(8):761-769.

5- Sahu CR and Ghatak S. Effect of Dimecron on Developing Chick   Embryo Malformations and other Histopathological Changes. Anatomia Histologia Embryolgia 2002;31(1):15-20.

6- Gilani SH and Marano M. Congenital Abnormalities in Nickel Poisoning in Chick Embryo. Archives of Environmental Contamination and Toxicology 1980;9(1):17-22.

7- Courtney KD. Teratogenic Evaluation of the Pesticides Baygon, Carbofuran, Dimethoate and EPN. Journal of Environmental Science and Health Part B 1985;20 (4): 373-406.

8- S. Sadasivam & A. Manickam Biochemical method, second edition, new age international (P) limited publishers 1996.

9- A. C. Wardlaw Practical statistics for experimental biology, a wiley-interscience publication 1985.

 10- Budai P, Fejes S, Varnagy L, Somlyay IM and Szabo ZK. Teratogenicity Test of Dimethoate Containing Insecticide Formulation and Cd-sulphate in Chicken Embryos after Administration as a Single Compound or in Combination. Commun Agric Appl Biol Sci. 2003;68(4 Pt B):795-8.

 11- Budai P, Fejes S, Varnagy L, Somlyay IM and Takacs I. Teratogenicity Test of Dimethoate Containing Insecticide Formulation and Heavy Elements (Cu, Cd) in Chicken Embryos after Administration as Single Compound or in Combination. Meded Rijksuniv Gent Fak Landbouwkd Toegep boil Wet 2001;66(2b):885-9.

12- Varnagy L, Budai P, Molnar E, Fuzesi I and Fancsi T. Teratogenecity Testing of BI 58 EC (38% Dimethoate) in Chicken Embryos with Special Respect to Degradation of the Active Ingredient. Acta Veterinary Hungarica 2001;49(3):355-61.

13- Weber M. The Effect of Dimethoate and Vibrations on the Fetal Development of the Rat. Anatomischer Anzeiger 1990;170(3-4):221-6.

14- Srivastava MK. And Raizada RB. Developmental Effect of Technical    Dimethoate in Rat: Maternal and Fetal Toxicity Evaluation. Indian Journal of Experimental Biology 1996;34(4):329-33.

       Tabe -1.  The developmental toxicity of Dimethoate in embryos after 24 hrs exposure.

Values are expressed as mean ± SEM. Kreskal- Wallis ((Non-parametric ANOVA) test followed by Dunn's
test.  * p value considered not significant and  ** p value considered significant

   Table -2. Abnormalities in the development of the heart induced by Dimethoate.

Values are expressed as mean ± SEM.  Kreskal- Wallis (Non-parametric ANOVA) test followed by Dunn’s
test. * p value considered not significant and ** p value considered significant.

Table -3. Abnormalities in the development of the brain induced by Dimethoate

Values are expressed as mean ± SEM. Kreskal- Wallis (Non-parametric ANOVA) test followed by Dunn’s
Multiple Comparison Test. ** p value considered significant.

Table -4. Abnormalities in the development of the neural tube induced by Dimethoate

Values are expressed as mean ± SEM. Kreskal- Wallis (Non-parametric ANOVA)  test followed by Dunn’s
Multiple Comparison Test.. * p value considered not significant and ** p value considered significant.

     Table -5. Somites abnormal development induced by Dimethoate.

Values are expressed as mean ± SEM. Kreskal- Wallis (Non-parametric ANOVA) test followed by Dunn’s
Multiple Comparison Test. * p value considered not significant and ** p value considered significant.

              Table -6. Effect of Dimethoate on the Acetylechlinestrase activity.

      Values are expressed as mean ± SEM, (Student ‘t’ test). Control n =10 and test n = 20.

p value is significantly different from control