Shift in FTIR spectrum patterns in methomyl-exposed rat spleen cells

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Abstract

Methomyl is a highly toxic carbamate insecticide which is widely used in many agricultural countries. We have applied the Fourier-transformed infrared (FTIR) spectroscopic method to study the toxicity of methomyl on cytoskeletal protein and the nucleic acid of rat spleen cells. Rats were given methomyl by gavage at 2, 6 and 8 mg/kg in single doses. Colchicine, a microtubule-disrupting agent, was given to rats at 2, 4, and 6 mg/kg in single doses and mitomycin C, an alkylating agent which acts as a DNA-cross-linking agent, was given by an intraperitoneal route to rats at 1 mg/kg. It was shown that the wavenumber of FTIR spectra at amide I and amide II in both methomyl- and colchicine-exposed rats shifted in dose response manner when compared with the control (P<0.05). The amide I and II shifts in these regions have been proposed to be the result of an alpha-helix protein conformational change. Toxic doses of mitomycin C, a DNA-cross-linking agent, did not result in this pattern. Moreover, all exposed rats showed an increase in the absorbance ratios that were related to the vibrational mode of the phosphodiester group in nucleic acid (P<0.05).

Introduction

Methomyl, a carbamate insecticide, is widely used in Thailand and many agricultural countries for crop protection. It is often cited as an agent, which may cause pesticide poisoning and it is also commonly used in suicides. Methomyl is classified as a highly hazardous (class 1B) carbamate insecticide by WHO (WHO, 1986). The toxic effect is cholinergic toxicity by cholinesterase inhibition. In addition, many carbamates can bind to tubulin, leading to interference with microtubule formation and microtubule disruption (Nakai et al., 1995, McManus and Trobetta, 1995, Holy, 1998). Recently, Lohitnavy and Sinhaseni (1998), have shown that rats orally treated with single doses of 3–7 mg/kg of methomyl showed a significant increase in serum lactate dehydrogenase (LDH) on day 1 after dosing, especially in LDH3 and LDH4 isozymes. Moreover, the splenocyte viability was significantly reduced in rats treated with an acute dose of 6 and 8 mg/kg of methomyl on days 1 and 3. The observed splenotoxicity effect was prevented by pretreatment with N-acetylcysteine (NAC), a free-radical scavenger.

Fourier-transformed infrared (FTIR) spectroscopy is normally used for the study of protein and nucleic acid structural modifications (Rice-Evans et al., 1991, Twardowski and Anazenbacher, 1994). Most importantly, much progress in research on protein–lipid interactions at interfaces has been achieved by applying biophysical techniques involving FTIR spectroscopy (Isenberg and Niggli, 1998). In the present work, we have applied the FTIR spectroscopic technique to compare the spleen cell absorption spectra of control rats with those exposed to methomyl, colchicine (a chemical known to bind to tubulin and inhibit its polymerization), or mitomycin (a DNA cross-linking agent) in order to investigate the methomyl-induced splenotoxicity.

Section snippets

Animals

Wistar male rats (103.5±6.8 g) were obtained from the National Laboratory Animal Center of Salaya Campus, Mahidol University. Six rats were housed in each cage and provided with standard rat/mouse pellets from C.P. (Thailand) Ltd. and tap water ad libitum. The animals were acclimatized for a week prior to dosing (temperature: 28±4°C; relative humidity: 40–75%).

Chemicals

Methomyl with a purity of more than 98% was obtained courtesy of DuPont (Thailand) Ltd. Colchicine, mitomycin C, sodium chloride and

Clinical signs of toxicity

The rats given methomyl at a single oral dose of 2, 6 or 8 mg/kg developed the signs of toxicity within 5 min after dosing. The severity of toxic signs depended on the dose level. These acute signs were known as results of cholinesterase inhibition, for example, nicotinic effects: generalized tremor, muscular weakness, muscular fasciculation; and muscarinic effects: increased bronchosecretion, difficult respiration, and chewing movement. These effects persisted for approximately 30 min to 1 h.

Discussion

From the results of FTIR spectroscopy of rat spleen cells from the methomyl-, colchicine- and mitomycin-exposed groups, there was no significant decrease in either the spleen weight or ratio of spleen weight to body weight for all exposures, but there was a decreasing tendency in both the spleen weight and ratio of spleen weight to body weight after methomyl- and colchicine-exposures in a dose–response manner. Interestingly, there were significant shifts in the amide I region (1652 cm−1) and

Conclusion

The shift in FTIR spectra at amide I and II regions related to alpha-helix protein conformational change in methomyl-exposed rat spleen cells may be imparted by cytoskeletal protein disruption as some carbamates do. The phosphodiester bond modification in nucleic acids related to splenotoxicity should be further studied.

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