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Investigating the role of oxidative stress or diet on prion disease susceptibility

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We want to study the influence of dimethoate on total PrP protein content in infected and non infected cells. We choose a simple sandwich ELISA test which can be easily performed in a P3 laboratory. Only the 96well plate is read on a spectrophotometer, and this plate can be sealed and disinfected before going out of the P3 laboratory. Technical characteristics: Our ELISA is realised with the following antibodies, given by J Grassi (CEA Saclay, France): Monoclonal mouse anti PrP SAF61, and biotinylated 3F4. Revelation is done with streptavidin-HRP conjugate (Amersham) and TMB substrate. Standard curve is done using recombinant PrP obtained by RTS system (Roche). Detection threshold is at 1ng/ml of PrP with a background signal of 0,4 OD(450nm), plateau is around 3,5 OD (450nm) with 50ng/ml. Detailed protocol - Maxisorb 96-wells plate are coated 30min at 37°C with SAF61 at 10µg/ml in PBS, saturated with PBS-1%BSA one hour at 37°C and washed 5 times with PBS-0.1%Tween20. - Samples (recombinant PrP standard, 50µg of protein from PrP knocked out mouse brain as negative control, or 50µg of protein sample from cell culture extracted with 1%DOC, 1%Triton X-100 buffer) are diluted in 50µl of PBS-1%BSA-0.1%Tween20-0.1%Triton X-100 and incubated one hour at 37°C. - After 5 washes as above, 50µl of biotinylated 4F2 at 2.5µg/ml in PBS-1%BSA-0.1%Tween20 is incubated 2 hours at 37°C and washed again. - HRP-stretavidin (Amersham ref RPN1051) is diluted 1/1600 and 50µl is incubated in each well one hour at 37°C. - Three washes in PBS-0.1%Tween20 and two with PBS are followed by 100µl of TMB substrate and incubation of 25minutes at 37°C - Reaction is stopped by 100µl H2SO4 0.5M and absorbance is read at 450nm.
In order to determine the amount of dimethoate to use, mice were treated with different amount of the drug for different times. 1 week treatments with 10 and 30 mg/kg of dimethoate let to see as tissues (livers) reacted to the external insult activating the antioxidant detoxifying enzymatic systems.All the dosed enzyme increased their specific activities (SOD, catalase, glutathione transferase-GST, reductase-GSSGRx-, peroxidase GSHPx): an acute stress was observed in terms of amount of carbonyls in proteins. This acute response indicated a tissues positive response to the stress not sufficient to detoxifying all ROS (reactive oxygen species). Longer treatments (4 and 5 weeks) with 10 mg/kg of dimethoate had a different effects on enzymatic activities. At 4 weeks levels of enzymes tended to stabilize and to return to blank levels: the enzymatic systems did not tend to counteract drug action, in fact carbonyls were higher in treated mice than in blanks (not treated). 5 weeks treatment with 10 mg/kg or 30 mg/kg of dimethoate resulted in more marked decrease in enzymatic activities: only catalase level seemed to be higher but it was not enough to counteract oxidative stress as demonstrated by protein carbonyls that were always higher than blank levels. GSH Px activity was very low: this activity is important in all tissues in detoxifying organic peroxides (Se-independent activity). Ratio GSHPx/SOD was very low during dimethoate treatments indicating an inefficient antioxidant capability of tissues. 60 and 100 mg/kg of dimethoate treatments were very toxic and therefore were excluded for further experiments. These results can be combined to evaluation of glutathione (GSH), GSH/GSSG ratio and total GSH (=[GSH] + 2x[GSSG]). One week treatment with 10 or 30 mg/kg of dimethoate resulted in small decreases in the GSH/GSSG ratio, due to higher GSSG levels, and no significant changes in total GSH levels was observed. A decrease in GSH/GSSG ratio was evident with 30 mg/kg. An oxidative stress situation is apparent (expecially at 30 mg/kg), with small oxidative damages (confirmed by protein carbonyls dosage): animal resist an acute oxidative stress with their enzyme equipment. Five week treatment with 10 or 30 mg/kg does not change dramatically the monitored parameters: GSH/GSSG ratios stabilize to a lower level (with decrements of 20-35%), small increases in carbonyl content are observed; the total GSH becomes lower with 30 mg/kg dimethoate, a sign that dimethoate is actively detoxicated. Animals reach a new "steady state" (see enzyme activities results) and resist the oxidative stress with apparently small damages. Treatment, for one or five weeks, with the intermediate doses of 15 mg/kg dimethoate confirms these results: an oxidative stress status is established and mice adapt their metabolism to this new situation.

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