Filogenia da sensibilidade da acetilcolinesterase cerebral de peixes ao metil-paraoxon como um possível marcador ambiental / Phylogeny of the sensitivity of fish brain acetylcholinesterase to methyl-paraoxon as a possible marker environmental

Todos os animais com células nervosas e musculares possuem a enzima acetilcolinesterase (EC 3.1.1.7, AChE) e essa seqüência de aminoácidos está presente em muitas outras proteínas, com ou sem atividade catalítica, fato que permite que essa proteína seja utilizada em estudos de filogenia e de evolução. A grande diferença de afinidade entre substratos e inibidores é utilizada como um biomarcador para estudos genéticos de vários grupos de animais, especialmente insetos. Peixes possuem uma grande diferença na sensibilidade da AChE ao metil-paraoxon (MP) em relação aos animais terrestres e essa diferença pode estar relacionada à evolução. As constantes cinéticas de inibição (CCI) para o mecanismo de inibição progressivamente irreversível da AChE cerebral ao MP foram determinadas em duas fontes de AChE como um modelo para avaliação do potencial de uso das CCI como biomarcador para estudos evolucionários e filogenéticos. As CCI da AChE cerebral de seis exemplares de tainha (Mugil liza), coletados em duas lagoas da costa do Estado do Rio de Janeiro (Araruama e Saquarema), em tempos diferentes (2005 e 2007, respectivamente), foram determinadas em dois laboratórios distintos (CESTEH - FIOCRUZ e Dept. Bioquímica - UERJ). As CCI, medidas separadamente em cada exemplar, indicaram que essas constantes são preservadas em todos os exemplares de uma mesma espécie e que a metodologia empregada pode ser conduzida em laboratórios distintos sem grandes variações. A AChE cerebral de tainha foi tomada como um exemplo de enzima menos sensível (IC50 = 2118nM) e a de galinha comercial (Gallus gallus domesticus) como um exemplo de uma enzima muito mais sensível ao MP (IC50 = 26nM)...

The enzyme acetylcholinesterase (EC 3.1.1.7, AChE) is present in all animalswith neurons and muscle cells, and many other proteins have the same sequence, with or without catalytic activity, allowing them to be used for phylogenetic and evolutionary studies. The great difference between substrates and inhibitor affinities makes it useful as a biomarker for genetic studies of various animals groups, especially insects. There are great differences among fish in AChE sensitivity to methyl-paraoxon (MP) in relation to terrestrial animals, and these differences can be related to evolution. The inhibition kinetic constants (IKC) for progressive irreversible inhibition of brain AChE with MP were determined in these two AChE sources as models for evaluating IKC as potential biomarkers in evolutionary and phylogenetic studies, especially among fish. IKC of brain AChE from six specimens of Mugil liza, a very common coastal fish, was collected from two of Brazil's lagoons in Rio de Janeiro State during 2005 at Araruama and 2007 at Saquarema. First samples were assayed at CESTEH - Fundação Oswaldo Cruz and the latter at Dept. Bioquímica - Universidade do Estado do Rio de Janeiro. The IKC was measured separately for each fish showing that these constants weremaintained for all animals of the same species and that this methodology can beused in different laboratories without variations. The cerebral AChE of tainha was used as an example of a less sensitive enzyme (Concentration which inhibits 50% of enzyme activity after 30 minutes of incubation, or IC50, = 2118nM). The commercial hen (Gallus gallus domesticus) was used an example of a very highly sensitiveenzyme to MP (IC50 = 26nM)...

Effect of methyl parathion formulation on estrous cycle and reproductive performance in albino rats.

The animals were injected intraperitoneally with graded doses of methyl parathion at 1.5 to 3 mg/kg body weight for 15 days from the day of estrus. Results indicated that the methyl parathion treatment showed irregular estrous cycles, affect the duration of each estrous cycle, proestrus and diestrus were significantly changed in 2.5 and 3 mg treatment groups. But there was no significant change in the number and duration of each estrous cycle, duration of proestrus and diestrus in 1.5 and 2 mg methyl parathion treatment groups. However, there was a significant decrease in the duration of estrus, while there was no significant change in the duration of metestrus in all methyl parathion treatment rats when compared with those of the corresponding parameters of the control. There was no significant effect on number of live pups on day 1 and 5 except in 3 mg methyl parathion treatment group where it was significantly decreased. There was no significant change in reproductive indices like pregnancy, parturition, live birth and viability in all the methyl parathion treatment rats except the viability index in the highest dose.

Correlation of time course of blood cholinesterase activity and toxic manifestations of acute methylparathion in antidote treated rats.

Study was conducted to find out the correlation between red blood cholinesterase (RBC ChE) and plasma butyryl cholinesterase (BuChE) activities and toxic signs of oral methylparathion (MPT) and their recovery pattern with or without atropine treatment in female rats. Enzyme activity was estimated before and after an oral dose of MPT (7.5 mg/kg-1) at various time intervals upto 120 hr. Antidote groups received atropine (10 mg/kg-1, i.p.), either alone or with diazepam (2.5 mg/kg-1, i.p.), at the onset of toxic signs. Inhibition of enzyme activity served as definite index of acute toxicity of MPT. RBC ChE activity correlated with the intensity of toxic signs in no-antidote rats, while in atropine treated groups, there was no correlation. BuChE levels did not correlate with toxic signs in any of the groups except in the fatal group. The resynthesis of both the enzymes was complete in 120 hr study and did not synchronize with the recovery pattern of animals from toxic signs. Compared to BuChE, RBC ChE activity was found to be a more sensitive indicator for the diagnosis of severity of MPT toxicity.

Delayed effects of acute oral and chronic inhalational exposure to methylparathion on learning and memory in rats.

Impairment of acquisition phase of the learning process was observed in rats even at 3 weeks after single oral exposure to the near-lethal dose of commercial grade methylparathion (MP) followed by atropine resuscitation. Though there was a trend towards memory impairment in this group of animals, memory retention was not significantly affected. Chronic inhalational exposure to MP (one exposure/day for 3 weeks) did not significantly alter learning or memory. No significant alteration in red blood cell or brain acetylcholinesterase levels were observed in either of the two groups. It appears that behavioural effects can persist even 3 weeks after exposure to acute near-lethal doses of the pesticide, as occurs in the clinical situation of suicidal attempts; while repeated exposure to no-observed-effect-level doses as occurs in farm and factory workers, may not be associated with behavioural changes.

Methyl parathion induced regional alterations in the regulatory proteins during critical stage of central nervous system development in albino rat pups.

Sublethal doses of methyl parathion (O,O-dimethyl-O-nitrophenyl- thiophosphate) injected intraperitoneally to 15 and 21 day old rat pups induced regional alterations in the central nervous system (CNS) in the levels of total RNA, total proteins, modulatory protein Calmodulin (CaM), in the activity levels of membrane bound enzyme Ca(2+)-ATPase and phospholipids. Levels of RNA and total proteins increased considerably in 15 days old methyl parathion treated (MPT) rat pups. Contrary to this the RNA and total protein content exhibited remarkable decrease in 21 day old methyl parathion treated animals. Calmodulin level showed an increase in cerebral cortex and brain stem and decrease in cerebellum and spinal cord in 15 day old methyl parathion treated rat pups. Whereas the level of Calmodulin decreased in cerebral cortex and cerebellum and increased in brain stem and spinal cord in 21 day old methyl parathion treated rat pups. Activity levels of calcium dependent ATPase showed significant inhibition in all the regions of Central Nervous System (CNS) of 15 and 21 day old methyl parathion treated rat pups. Phospholipids showed a general increase in all the regions of Central Nervous System on methyl parathion exposure. In the light of these observations, it has been suggested that the molecular regulatory mechanisms involving Ca2+/CaM are rendered inefficient due to toxic impact of methyl parathion.

Repeated dermal toxicity of technical HCH and methyl parathion (50EC) to female rats (Rattus norvigicus).

Repeated dermal application of hexachlorocyclohexane (HCH; 100 mg/kg/day) or methyl parathion (2 mg/kg/day) individually or in combination for 7, 15 and 30 days produced pathomorphological changes in skin, liver, kidney and brain of female rats along with significant enzymatic alterations in the activity of transaminase, alkaline phosphatase lactic dehydrogenase and acetylcholinesterase. The two insecticides in combination though produced severe toxicity on day 30 than at other periods, the changes were not suggestive of any additive or potentiation effect at the test doses.