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Mechanism of Odap Toxicity in One-day-old Chicks

M. ZAKARIA SHAMIM
Department of Biochemistry, University of Dhaka, Dhaka -1000, Bangladesh.

M. SALIM HOSSAIN
Department of Biochemistry, University of Dhaka, Dhaka -1000, Bangladesh.

KHALEDA lSLAM
Department of Biochemistry, University of Dhaka, Dhaka -1000, Bangladesh.

HARUN K.M. YUSUF
Department of Biochemistry, University of Dhaka, Dhaka -1000, Bangladesh.

FERNAND LAMBEIN
Laboratory for Physiological Chemistry, University of Gent, B-9000 Gent. Belgium.

Abstract/ Executive Summary:

The neurotoxin ODAP (ß--N-oxalyl-α, ß-diamino propionic acid ) of Lathyrus satiuus prod uces lathyrism in humans. One-day-old chicks, when given a single dose (0.1 mmol) of the toxin, exhibit characteristic symptoms including dizziness, the head moving downwards, the neck bending to a side and the body moving around itself, within 4 - 6 h of the administration. The chicks recover within 24 h. Homoarginine, a natural inhibitor of nitric oxide synthase, is found to provide a remarkable protection against ODAP toxicity. When given simultaneously with ODAP at equimolar dose (0.1 mmol), 90 - 95% of the chicks are found normal showing no signs of toxicity while the remainder also recover within 12 h after an initial stage of mild intoxication. HPLC analysis of brain and serum extracts showed that ODAP caused a moderate but significant rise in the concentrations of cirtrulline and GABA for up to 12 h. On the other hand, in the animals given ODAP + homoarginine, there was no increase in citrulline but a sharp rise in GABA over the same period. The results suggest that ODAP, like glutamate, exerts its neurotoxici ty th ro ugh the nitric oxide pathway. Estimation of homoarginine in Lathyrus seeds of varying ODAP contents shows a clear cut inverse relationship between the two.

Keywords: Lathyrus toxin, ODAP, Nitric oxide, Day-old chicks

Location:

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

To study the mechanism of ODAP Toxicity in one - day- old chicks

Methodology:

One-day-old chicks were used in the study. ODAP was chemically synthesized and checked for its purity (> 96% pure). Homoarginine, citrulline and other neuroactive amino acids (aspartate, glutamate, GABA) were purchased from Sigma. HPLC-grade methanol and acetonitrile were from E . Merck. A Waters High Performance Liquid Chromatograph ( HPLC) was used for quantitative analysis of tissue amino acids. Forty eight one-day-old chicks, purchased from a local farm, were divided into three groups. Chicks of one group (n = 18) were fed a dose of 0.1 mmol (in 0.5 ml water) of ODAP each by a rubber tube. Chicks of the second groups (also n = 18) received a dose of 0.1 mmol of ODAP + 0.1 mmol of homoarginine (in 0.5 ml water). The third group (control, n = 12) was given 0 .5 ml of normal saline. Six chicks from the control were sacrificed by decapitation each at 0 and 24 h of the experiment. Then six chicks from the other groups were sacrificed after 6, 12 and 24 h of the treatment. Their blood and brain were collected. Serum was prepared and deproteinized by adding 4 vol absolute ethanol to 1 vol of serum . After centrifugation at 3000 rpm for 10 min, the clear supernatant was used for amino acid analysis after derivatization with phenylisothiocyanate (PITC) by the method of Heinrickson and Meredith.(⁶) Brain tissue (six brains pooled from each group) was quickly homogenized in 5 vol ice cold normal saline and centrifuged at 10,000 g for 20 min. The supernatant was deproteinized and analyzed by HPLC as for serum. Glutamate, aspartate, GABA and citrulline in serum and brain extracts were determined by HPLC equipped with a Pye-Unicam LC UV detector 484 and Hewlet Packard 745-B integrator. A C18 reversed-phase column from C18HL (300 x 3.9 mm with particle size, 3 mm) was used for the instrument. The mobile phase buffer used was sodium acetate trihydrate, pH 6.4. For homoarginine and citrulline, acetonitrile was added to this buffer, 12% (v/v). Khesari seeds of varying ODAP content were collected from the Bangladesh Institute of Nuclear Agriculture (BINA) and their ODAP content was determined by o-phthalaldehyde (OPT) method(⁷)and homoarginine content by HPLC.(⁸).

Source of Information: Dhaka Univ. J. Biol. Sci. 11( 1) : 1-7, 2002 (January)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The results showed that homoarginine provided protection against ODAP toxicity. Other related amino acids like arginine or citrulline were totally ineffective. To see if ODAP toxicity was through production of nitric oxide (measurable by determination of citrulline) and the protective effect of homoarginine was through inhibition of NOS, citrulline was measured in the brain of the animals. To assess any associated changes in the levels of the neuroactive amino acids, glutamate, asparate and GABA were also measured. It was found that ODAP caused significant increase in the level of cirtrulline and GABA in the brain at all times for up to 12 h compared to the controls. On the other hand in the homoarginine-treated chicks, while there was a sharp rise in GABA level during the first 12 h with subsequent fall at 24 h, there was almost no change in citrulline level which was comparable to that of the controls throughout . Similar pattern of changes was seen in the serum as well. Changes in the concentrations of glutamate and asparate were not consistent and are not reported here .

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