Copper reduces striatal protein nitration and tyrosine hydroxylase inactivation induced by MPP+ in rats.

Striatal administration of 1-methyl-4-phenylpyridinium (MPP(+)), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson's disease. Copper acts as a prosthetic group of several antioxidant enzymes and recent data show that copper attenuated MPP(+)-evoked neurotoxicity. We evaluated the effect of copper (as a supplement) upon proteins nitration (60 kDa) and tyrosine hydroxylase (TH) inactivation induced by MPP(+) (10 microg/8 microL) injection into the rat striatum. Copper pretreatment (10 micromol/kg i.p.) prevented both MPP(+)-induced proteins nitration and TH inactivation. Copper treatment also prevented the dopamine-depleting effect of MPP(+) injection. Those results were accompanied by a significant reduction of enzymatic activity of the constitutive nitric oxide synthase (cNOS), whereas, the protein levels of the three isoforms of NOS remained unchanged. Results indicate that the effect of copper against MPP(+)-induced proteins nitration and TH inactivation in the striatum of rat may be mediated by a reduction of cNOS activity.

Stereospecific hydrolysis of a phosphoramidate as a model to understand the role of biotransformation in the neurotoxicity of chiral organophosphorus compounds.

Calcium-dependent and EDTA-resistant hydrolyses of R and S isomers of O-hexyl O-2,5-dicholorophenyl phosphoramidate (HDCP) were observed in serum and subcellular fractions of liver, kidney and brain from hen, rat and rabbit. In serum, the Ca(2+)-dependent hydrolysis was much higher in rabbit than in other species. Liver showed a higher activity than kidney and brain. The S-HDCP isomer was hydrolysed to a higher extent than the other isomer. The fact that this stereospecificity favours the S-isomer is more clearly observed in rabbit serum, and in rat and rabbit liver particulate fractions. In such tissues and species, the EDTA-resistant hydrolysis was not stereospecific. Soluble fractions of rat brain and of hen liver, kidney and brain, showed a lower total activity but with a higher proportion of EDTA-resistant activity and a higher hydrolysis of the R-HDCP isomer. The Ca(2+)-dependent stereoselective biodegradation of S-HDCP is dominant in the most active tissues in rabbit and rat. It can therefore be concluded that S-HDCP would be biodegraded faster than R-HDCP. Furthermore, R-HDCP is the isomer that will remain at a higher proportion to be available for interaction with the target of neurotoxicity.

Importancia de la hidrólisis estereoespecífica en la evaluación de riesgos tóxicos de insecticidas fosforamidatos / The importance of stereospecific hydrolysis in the risk assessment of phosphoramidate insecticides

Los principales efectos tóxicos de los insecticidas organofosforados derivan de la inhibición y envejecimiento de la acetilcolinesterasa y la esterasa diana de la neuropatía. Las fosfotriesterasas son esterasas capaces de hidrolizar insecticidas organofosforados. En este trabajo se estudia en varios tejidos de conejo, rata y gallina la estereoespecificidad de las fosfotriesterasas implicadas en la hidrólisis del insecticida O-hexil O-2,5-diclorofenil fosforamidato. También se ha caracterizado la capacidad de los isómeros R y S de dicho fosforamidato para inhibir y envejecer la esterasa diana de neuropatía. Entre todas las especies estudiadas los tejidos de conejo mostraron los mayores niveles de actividades hidrolizantes de insecticida, siendo la gallina la especie con menores niveles de esta actividad. En conejo y rata la mayoría de las fosfotriesterasas de los tejidos estudiados mostraron preferencia por la hidrólisis del isómero S del compuesto. Sin embargo, no se pudieron extraer conclusiones claras respecto a la estereoespecificidad de la hidrólisis en gallina. El isómero S del O-hexil O-2,5-diclorofenil fosforamidato inhibió la esterasa diana de la neuropatía de cerebro de gallina con I50 de 7.6 nM, siendo 190 nM el valor para la inhibición por el correspondiente isómero R. La citada esterasa inhibida por el compuesto de configuración S pudo ser reactivada por KF transcurridas 22 horas de la inhibición, reactivación que no ocurrió cuando el inhibidor fue el compuesto de configuración R. Así pues, los niveles de fosfotriesterasas y su estereoespecificidad van a determinar qué isómero alcanza las dianas tóxicas y en qué concentración y por tanto cuáles serán los efectos tóxicos que se manifiestan. Se concluye que se deben considerar los niveles de fosfotriesterasas y su estereoespecificidad en la evaluación de riesgos tóxicos de insecticidas fosforamidatos (AU)

Dichlorophenyl phosphoramidates as substrates for avian and mammalian liver phosphotriesterases: activity levels, calcium dependence and stereospecificity.

The present study shows the existence of both Ca2+-dependent and EDTA-resistant hydrolysing activities against HDCP and paraoxon in the particulate and soluble fractions of hen, rat and rabbit liver. HDCP was more extensively hydrolysed than paraoxon in both subcellular fractions and each of three individuals of the three animal species under study in spite of wide interindividual variations. However the ratio of HDCP versus paraoxon hydrolysing activity (HDCPase/paraoxonase), although within the same order of magnitude, cannot be considered as constant as it ranges one- to seven-fold between individuals of the same species. Also there is no constant ratio of Ca2+-dependent/EDTA-resistant activities. Rabbit liver showed the highest rates of Ca2+-dependent hydrolysis for both organophosphorus compounds whereas the hen paraoxonase activity was not inhibited by EDTA. The stereospecific hydrolysis of HDCP was mostly a Ca2+-dependent one, the S-HDCP isomer being hydrolysed faster than the R-HDCP one. The suggestion is made that HDCP could be conveniently used to measure PTE activity in the liver.

Enzyme concentration as an important factor in the in vitro testing of the stereospecificity of the enzymatic hydrolysis of organophosphorus compounds.

A report is made of important differences in the Ca(2+)-dependent hydrolysis of the chiral phosphoramidate O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) when recorded using different quantities of hen liver microsomes. In a colorimetric microassay using the microsomes from 5mg tissue in the presence of HDCP stereoisomers and 2.5mM calcium, the R-HDCP isomer was hydrolysed at a rate similar to or slightly faster than S-HDCP isomer (14% v. 11%), while the S-HDCP stereoisomer was hydrolysed faster than R-HDCP (17% v. 25% and 21% v. 43%) when HDCP isomers hydrolysis was assayed in the presence of the microsomes from 10 or 20mg, respectively. This stereospecific hydrolysis was verified assaying racemic HDCP and quantities of liver microsomes from 10 to 80mg of tissue, using a chiral chromatographic method; thus, the increase in the ratio of remaining R-HDCP/S-HDCP was dependent on the amount of liver microsomes (range one- to threefold). This study demonstrates that the concentration of the subcellular fraction in in vitro assays is a critical factor to be taken into account in securing a more realistic approximation to the stereospecific enzymatic processes occurring in biological systems. Our data concerning the hydrolysis of HDCP by liver microsomes at high enzyme concentrations afford a better fit to the in vivo toxicological response with HDCP than assays performed with the most commonly used highly diluted preparations.

Dapsone attenuates kainic acid-induced seizures in rats.

We tested the ability of dapsone (4,4'-diamino-diphenyl sulfone) to attenuate kainic acid-induced seizures. We observed that 9.375 and 12.5 mg/kg doses of dapsone administered 30 min before a single kainic acid (10 mg/kg) i.p. injection were able to decrease the time of electroencephalographic seizures by 52% and 82%, respectively, as compared with rats administered with 10 mg/kg kainic acid only. The 12.5 mg/kg dose of dapsone was also able to diminish both kainic acid-evoked body and head shakes (58%) and kainic acid-induced mortality (75%). These results suggest that dapsone could be used in clinical trials as anticonvulsant.

Combined D-penicillamine and prussian blue as antidotal treatment against thallotoxicosis in rats: evaluation of cerebellar lesions.

Rats were treated with a single dose of thallium acetate (32 mg/kg i.p.) and the antidotal effect of D-penicillamine and prussian blue given alone or in combination was assessed by means of evaluation of the thallium-induced cerebellar histological lesions. After thallium poisoning (24 h), antidotes were administered for 4 days as follows: D-penicillamine (DP) 25 mg/kg, i.p. twice daily; prussian blue (PB), 50 mg/kg p.o., twice daily. Mortality among the treatment groups was as follows: control, 87.5%; DP, 100%; PB, 56.25%; DP+PB, 25%. Three days after these treatments, rats treated with the combination DP+PB presented a significantly lower number of altered Purkinje cells in cerebellum as compared with those of the thallium alone treated animals, indicating adequate protection by this antidote treatment against thallium neurotoxicity. Prussian blue protected against thallium-induced neurotoxicity to a lesser extent as compared with the effects obtained by the DP+PB protection. DP did not protect against thallium-induced alterations of Purkinje cells. These results confirm the efficacy of the combined antidotal treatment of DP and PB against thallium toxicity in rats, and support the possible application in human cases of thallotoxicosis.

Relationship between physicochemical properties of prussian blue and its efficacy as antidote against thallium poisoning.

In the present work we established a relationship between some physicochemical properties of two different batches of Prussian blue (PB) and their in vivo efficacy as an antidote against thallium poisoning. The physicochemical properties studied were crystallite size and thallium-adsorbing capacity. One of the batches was synthesized and the other was obtained from commercial sources. The synthesized PB batch with the smallest crystallite size had both the highest adsorption capacity and antidotal efficacy. Synthesized PB protected 100% of the animals against one LD50 thallium dose, whereas the commercial PB batch protected only 80%. Thallium content in blood and tissues (liver, kidney, brain) was also analysed after antidotal PB treatment in rats previously intoxicated with a sublethal dose of T1+. Animals treated with synthesized PB showed significantly less thallium in blood and tissue contents than those values of commercial PB-treated rats, indicating better antidotal properties of the synthesized PB. According to the present study we suggest an in vivo evaluation of the compound before distribution of the product to toxicological units, if X-ray diffractometric analysis is not available, in order to identify and determine the crystallite size of the compound as it plays an important role in the efficacy of PB.

D-penicillamine and prussian blue as antidotes against thallium intoxication in rats.

D-penicillamine (DP) and prussian blue (PB), given alone and in combination, were evaluated in rats as treatments against acute thallotoxicosis. Animals were poisoned by intraperitoneal (i.p.) injection of thallium(I) acetate at different doses (16, 30, 40, 50 and 70 mg/kg). Later (24 h), treatments were administered until day 5, as follows: D-penicillamine (DP), 25 mg/kg, i.p. route, twice daily; prussian blue (PB), 50 mg/kg, oral route, twice daily. LD50 values were estimated for each treatment with the following results: control, 32 mg/kg; DP, 27 mg/kg; PB, 42 mg/kg; PB + DP, 64 mg/kg. Thallium content was analyzed in six body organs and eight brain regions after treatments. PB administration induced significant elimination of thallium from all tissues. DP treatment diminished thallium content in body organs, but increased it in brain regions, indicating a redistributive effect of DP. DP + PB treatment decreased thallium content in all body organs and brain regions. Renal thallium content in the DP + PB group was significantly lower than that of PB alone group, suggesting accelerated urinary excretion of thallium as a result of DP action. Results indicate that DP administered alone may be dangerous because of its redistributive effect, but given in combination with PB may be useful as treatment against thallium poisoning.