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Abstract Organophosphorus poisoning is still an important health attention in Egypt where agriculture is the main livelihood of people. With the advance of times, pesticides are, nowadays routinely used for modern cultivation method. These are readily available as over the counter preparations in village shops and act as a common agent for suicidal purpose after trivial family problems. The high rate of organophosphate compound poisoning can be attributed to the uncontrolled trade of this material, its inappropriate conservation, and its widespread use by the public and consequent availability. The biochemical evaluation demonstrated that the enzymatic activity of cholinesterase was strongly inhibited by exposure to OP, whereas that of catalase increased significantly. This increase can be explained by the stimulation of antioxidant defense system. A healthy cell has a mortal enemy called free radical which is unstable and reacts quickly with the nearest stable molecule abstracting its electron to attain stability. When the attacked molecule loses its electron, it becomes a free-radical itself, these formations of free-radicals continue on and on if antioxidants are not available. Free radical production becomes excessive and causes molecular damage. Propagation and initiation of free radicals chain reaction can be minimized by the donation of hydrogen from the antioxidants. The destructive effects of free radicals can be prevented with the addition of anti-oxidants in the diet or by anti-oxidant supplements. A good antioxidant complex supplement actually has advantages over diet sources in that the complex has many different specific types of anti-oxidants which seek out and destroy free radicals at many various cellular sites. Catalase is one of the cellular defense mechanisms against cytotoxic oxygen species (H2O2). Catalase is present in cells of aerobic organism. Catalase converts two molecules of the strong oxidant, hydrogen peroxide, to molecular oxygen and two molecules of water. The present findings indicate that cells continually suffer from oxidative stress in spite of over-activity of antioxidant defense mechanism as indicated by increase in CAT activity. The higher levels of antioxidant enzymes may be necessary to detoxify increased concentration of ROS that are generated from oxidative stress because of OP toxicity. The body could not compensate in a short period. Therefore, the antioxidants at suitable doses should be given in order to reduce oxidative damage, which could be effective in speedy recovery of acute OP poisoning cases. Clinical Part During the study period, out of 2558 cases of poisoning admitted to MPCC, 468 cases were due to organophosphorus insecticides poisoning. Incidence was more common in females (58.1%). Sex ratio (M: F) was 1:1.39 and 21-30 years was the age group which commonly affected. Unmarried persons were more involved. Most of the cases (71.8%) belonged to rural area. The most common mode was suicidal (88.5%) due to financial and domestic problems, while 6.4% of cases were accidental and 5.1% were occupational. The common route of exposure was ingestion of poison (94.7%). The most common manifestations were miosis 83.3%, followed by vomiting 82.7% and abdominal pain 78.6%. Nearly two thirds of the patients had bradycardia and more than half of the studied cases manifested with sweating representing 57.7%. Disturbed consciousness level was found in 24.3% of cases and only 17.3% had diarrhea. Most patients were mildly affected (60.5%), 31.4% were moderate and lastly 8.1% were severe. Mortality was significantly higher in severe toxicity group. Hypokalemia was the most common electrolyte imbalance. Most cases (86.1%) arrived to hospital in less than 4 hours, followed by 11.5% arrived 4 to 8 hours after consumption of poison. Only 2.4 % of cases came after 8 hours. Late hospital arrival increased the severity of poisoning. Female suicide (62.6%) exceeded that of the male (37.4%), while males outnumbered females in accidental and occupational poisoning. There was highly significant relation between plasma cholinesterase level on admission and severity grades of poisoning, which can be helpful for predicting the outcome in OP poisoning cases. Mild cases were hospitalized for 2.7 ± 1.4 days, while moderate cases were hospitalized for 4.8 ± 2.5 days. Severe cases stayed for longer periods in hospital with a mean value of 11.6 ± 5.9 days. Plasma cholinesterase levels were significantly decreased as compared to controls and increased up to normal level after treatment with atropine and toxogonin. Catalase activity was highly significantly increased in OP patients as compared to controls. Activity of CAT was inhibited but not altered significantly after treatment with atropine and oxime. Experimental part The toxic effect of profenofos on male adult albino rats was studied with estimation of two biomarkers cholinesterase and catalase activities at different time intervals 1 hour, 6, 24 hours and 7 days after single oral dose (1/10 LD50). The animals were divided equally into four main groups as follows: •group (I):Control group •group (II):Organophosphorus toxicity group Profenofos was administered as a single sub-lethal oral dose 20 mg/Kg (1/10 LD50). •group (III): Organophosphorus + antidotes treated group Profenofos was administered as a single oral dose 20 mg/Kg. Then the rats were treated with 17 mg/kg atropine and 30 mg/kg toxogonin IP. •group (IV): Organophosphorus + antidotes + antioxidants treated group Profenofos was administered as well as atropine and toxogonin as mentioned above. The animals were treated concomitantly with vitamin C 200 mg/kg and vitamin E 150 mg/kg once daily for 7 days orally. Toxicity manifestations Rats administered profenofos showed signs of toxicity related to cholinesterase inhibition (miosis, lacrimation, salivation, muscle tremors, muscle weakness, diarrhea) which decreased both in intensity and frequency with passage of time. Observed improvement occurred with atropine and oxime treatment. However, this improvement was more prominent in vitamin C and E given group. Body weight changes of rats There was body weight gain of all studied groups after 7 days; the lowest body weight gain was recorded in group II (7.2%) and higher weight gain (8.1%) in group III, while the highest weight gain among the toxicity groups was found in group IV treated with antidotes and antioxidants (14.4%). Cholinesterase activity Serum cholinesterase activity of rats after 1 hour The activity of serum cholinesterase of rats orally received profenofos was suppressed and the difference was statistically highly significant in relation to controls. The activity was 41.14% of the control in group II. The application of treatment in group III and IV caused an increase in cholinesterase activity compared to the poisoned animals of group II but the difference was statistically non-significant. Cholinesterase activities were 46.01% of the control in group III and 47.65% in group IV. Serum cholinesterase activity of rats after 6 hours An increase was observed in the values of serum cholinesterase after 6 hours. It was 51.29% of the control in OP group. Again an increase in the enzyme level was observed in antidotes treated group (57.22%) with the highest increase (61.75%) in rats treated with antidotes and antioxidants. A greater efficiency in the restoration of cholinesterase activity in group IV caused a significant difference in relation to group II. Serum cholinesterase activity of rats after 24 hours Further increase was observed in serum cholinesterase after 24 hours. Rats of group II had 68.32% of the control activity. Higher values were recorded in animals receiving treatment; 83.64% in group III and 88.23% in group IV. The difference between group IV and the controls was attenuated. Serum cholinesterase activity of rats after 7 days After 7 days with regaining of cholinesterase activity, much improvement was recorded in serum cholinesterase of rats of all groups. Serum cholinesterase of group II (OP) was 92.05% of the control activity, while that of group III and IV was 94.77% and 98.72% respectively. The difference between the groups became non-significant. As regards brain cholinesterase, it was inhibited by profenofos. Antidotes caused increase cholinesterase activity in brain tissues with further increase in rats treated with additional vitamin C and E. Brain cholinesterase activity of rats after 1 hour As regards brain cholinesterase, it was inhibited by profenofos to 50.28% of the control activity in group II. The difference was statistically highly significant compared to control rats with non-significant elevation after treatment as compared to OP exposed rats. As compared to control group, the levels were 55.05% and 56.56% in group III and IV respectively. Brain cholinesterase activity of rats after 6 hours Higher level of brain cholinesterase activity was recorded after 6 hours related to control. It was 60.94% of the control activity in OP exposed group. More elevation was recorded in group III (77.84%) and in group IV (86.6%). Brain cholinesterase activity of rats after 24 hours After 24 hours, more increase was recorded of brain CHE (72.07%, 83.99% and 89.51% of the control) in groups II, III and IV respectively. Brain cholinesterase activity of rats after 7 days After a week in this experimental condition, brain cholinesterase activity did not show statistical differences between the groups. The percentages of activity were 90.56% in group II, 91.58% in group III and 98.15% in group IV of the control activity. Catalase activity High CAT activities were detected in rats poisoned with profenofos. Less elevation was reported in rats receiving antidotal treatment. Treatment with vitamin C and E exerts an antioxidative effect by decreasing lipid peroxidation and CAT activity in blood and brain. Serum catalase activity of rats after 1 hour The present study resulted in highly significant increased CAT activities in treated rats in relation to control. Activities of groups II, III and IV were 157.95%, 151.6% and 148.54% of the control respectively. Serum catalase activity of rats after 6 hours High CAT activities were detected in rats poisoned with profenofos in group II (139.18% of the control). Less elevation was reported in rats receiving treatment (129.9% for group III and 123.45% for group IV). Serum catalase activity of rats after 24 hours Comparing enzyme activities of animals after 24 hours, it was noticed persistent elevation of CAT activity (130.86%) in OP exposed rats with less value in group III and IV (122.78% and 111.23% of the control respectively). The values of group IV became non-significant in relation to the control. Serum catalase activity of rats after 7 days The values of activity in rats determined after 7 days showed downward tendency towards control values. The activities in group II were 121.75% and 110.05% group III with nearly normalization in animals of group IV (103.57%). The addition of vitamin C and E to routine treatment caused significant decrease in serum catalase activity in relation to group II. Brain catalase activity of rats after 1 hour High brain CAT activities were observed in rats poisoned with profenofos. The activity increased significantly to144.15% of the control in group II. Less elevation was recorded in group III and in group IV (133.34% and129.15% of the control respectively). Brain catalase activity of rats after 6 hours Persistent increased CAT activity was detected in profenofos treated rats in relation to control (142.36%). Less elevation was recorded in group III (133.05%) and (116.78%) in group IV. Brain catalase activity of rats after 24 hours Brain catalase activity of rats in groups II, III and IV were 136.37%, 129.55 % and 115.64% of the control respectively after 24 hours. Brain catalase activity of rats after 7 days After 7 days, CAT activities in profenofos treated rats were 123.66% of the control. The activities were 122.74 % in antidotal treated group and 104.17 % after treatment with antioxidants. The differences between groups were non-significant. Histological examination of brain (Hx& E) Exposure to profenofos causes important degenerative changes in brain tissue in the form of congestion of cerebral blood vessels, edema and pyknotic nuclei. Rats treated with antidotes showed milder histopathological features. Marked improvement was observed in animals treated with a combination of vitamins C and E. Seven days after the intoxication, it was observed that structural changes were regressed. Mild vascular congestion and persistent vaculation were noted in group II. Section from group III showed mild vascular congestion and little perivascular edema. In group IV, nearly normal morphological appearance of the nerve cells was observed with residual fine vacuolations. PAS reaction for detection of mucopolysaccharides Cerebral cortex of control group showed very strong PAS reaction, while section of rats treated with OP (group II) and dissected after 24 hours showed weak PAS reaction. Animals of group III showed weak to moderate PAS reaction after 24 hours. Cerebral cortex of rats of group IV stained moderately. After 7 days, higher staining was noted in different groups. The cells moderately stained in group II. While, some of the cells strongly stained in group III with much improvement was seen in group IV. Toluidine blue stain for detection of Nissl’s granules Control animals showed very strong blue staining of Nissl’s granules. Cells of rat cerebral cortex treated with OP were seen with faintly stained cytoplasm without clear Nissl’s granules in the degenerated cells. Sections of albino rat cerebral cortex treated with OP and antidotal therapy showing nerve cells containing Nissl’s granules moderately blue stained and others weakly stained after 24 hours, while weak reaction was detected in few degenerated cells in group IV. After 7 days, OP treated group showed moderate staining. Antidotal therapy showed restoration of most of cells to nearly their darkly stained cytoplasm containing Nissl’s granules, while sections of animals treated with additional antioxidants showed strongly stained cytoplasm. Immunohistochemical stain for Glial Fibrillar Acidic Protein (GFAP) Astrocytes did not express GFAP at immunohistochemically detectable levels, or express only very low levels in control group, while they showed positive immunoreaction for GFAP in profenofos toxicity group after 24 hours. Animals treated with antidotal therapy showed less prominent GFAP immunohistochemical staining compared to OP group. Vitamins C and E given to rats in group IV caused weak GFAP marking after 24 hours. After 7 days, sections of rat cerebral cortex treated with OP showed strong positive immunoreactivity with extensive overlapping and interdigitations of processes of astrocytes. Antidotal therapy caused less astrocytic density compared to OP group with moderately reactive astrocyte processes which do not overlap. Addition of antioxidants showed GFAP marking nearly similar to control group. The study concluded that oxidative stress plays an essential role in profenofos mediated injury and the combination of vitamins C and E ameliorated profenofos biochemical and histological deficits through their free radical scavenging effect. The addition of vitamin C and E in repeated doses to routine treatment of OP poisoning can play supplementary role and enhance the effectiveness of therapy. |