Sublethal effects of an organophosphorus insecticide (RPR-II) on biochemical parameters of tilapia, Oreochromis mossambicus.

The effect of exposure to sublethal concentrations (0.017 mg L(-1), 1/10 of LC50) of the novel organophosphate (OP) insecticide, 2-butenoic acid-3-(diethoxyphosphinothioyl) methyl ester (RPR-II) on biochemical parameters in Oreochromis mossambicus was studied during exposure for 3, 7, 15, 30 and its recovery response after seven days. Acetylcholinesterase (AChE) activity of brain, gill and muscle was inhibited by 67%, 77% and 73% respectively on day-30. The plasma and kidney alanine aminotransferase (AlaAT), and aspartate aminotransferase (AspAT) activity increased, while decreases were observed in gill and liver. Increases in acid phosphatase (AcP), and alkaline phosphatase (AP) activities were observed in plasma, gill, and kidney, and reductions of 20% and 61% in liver AcP and AP, respectively. Depletion of glycogen was observed in all tissues, an indication of typical stress related response of the fish with pesticide. Lactate dehydrogenase (LDH) activity decreased in liver and muscle, indicating tissue damage but a significant increase in LDH activity in gill and brain was observed. Depletion of glutathione (GSH) was observed in all tissues, thereby enhancing lipid peroxidation resulting in cell damage. The induction in hepatic glutathione-S-transferase (GST) levels indicates protection against the toxicity of xenobiotic-induced lipid peroxidation. There was a significant recovery in the above biochemical parameters, in all tissues of fish after a recovery period of seven days. These results revealed that the OP insecticide RPR-II is highly toxic and affects the intermediary metabolism of O. mossambicus.

Hematological and clinical chemistry changes induced by subchronic dosing of a novel phosphorothionate (RPR-V) in Wistar male and female rats.

A novel phosphorothionate [2-butenoic acid-3-(diethoxy phosphinothioyl)-ethyl ester; RPR-V] synthesized at Indian Institute of Chemical Technology (Hyderabad, India) was studied using subchronic doses of 0.033 (low), 0.066 (medium), and 0.099 (high) mg kg(- 1) in male and female rats daily for 90 days. Continuous treatment with RPR-V caused significant (p < 0.05) decreases in body-weight gain, feed intake, hemoglobin (Hb), hematocrit (Hct), and total erythrocyte count (TEC), whereas total leukocyte count (TLC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were increased. Similarly, RPR-V caused significant elevation in serum clinical chemistry parameters calcium, phosphorus, creatinine, and chloride contents, whereas protein and glucose levels were depressed in both male and female treated rats after 45 and 90 days of treatment. These alterations were significant when compared with two-way ANOVA showing that these changes were dose- and time-dependent. The effects of low dose were generally not statistically significant, whereas medium and high doses caused significant effects. The changes in male rats were not significant when compared with female rats showing no sexual dimorphism by this compound. Recovery was observed after 28 days post-treatment (withdrawal study), indicating that the compound entered into the system was eliminated from the body, and the blood parameters were improved. Hematological and clinical chemistry parameters can be detected rapidly and hence can be used for prediction and diagnosis of pesticide toxicity. Alterations in these parameters show toxic stress in the treated animals especially on blood and blood-forming organs.

Effects of a novel organophosphorus pesticide (RPR-V) on extra hepatic detoxifying enzymes after repeated oral doses in rats.

The effects of a novel organophosphorous pesticide, 2-butenoic acid-3-(diethoxy phosphinothionyl) ethyl ester (RPR-V) on glutathione S-transferases (GST), UDP-glucuronyl transferases (UDPGT) and the level of glutathione (GSH) were evaluated in rats after repeated oral administration at 33 microg kg(-1)day(-1) (low), 66 microg kg(-1)day(-1) (mid) and 99 microg kg(-1)day(-1) (high) for 90 days and at 28 days (withdrawal) post-treatment. GSH level and GST in kidney; GSH level in brain decreased significantly at mid and high doses on 45th and 90th day (P < 0.05). However, UDPGT activity in brain and kidney increased significantly at mid and high doses on 45th and 90th day (P < 0.05). Interestingly, the withdrawal study revealed that the effect was reversible 28 days after the treatment when all the enzymes reverted to levels close to those of controls. These results suggest that RPR-V has the potential to modulate the extrahepatic detoxifying enzymes and thereby interact with other physiological processes in the exposed organisms.

Effects of monocrotophos and its analogs in acetylcholinesterase activity's inhibition and its pattern of recovery on euryhaline fish, Oreochromis mossambicus.

Acute toxicity of monocrotophos (MCP) and its newly synthesized thiol analogs 2-butenoic acid-3-(diethoxyphosphinothionyl) methyl ester (RPR II) and 2-butenoic acid-3-(diethoxyphosphinothionyl) ethyl ester (RPR-V) was studied on euryhaline fish, Oreochromis mossambicus. The median lethal concentrations of MCP, RPR-II, and RPR-V are 11.506, 0.167, and 0.174 mgL(-1), respectively. Both the analogs were found to be 65-fold more toxic than MCP. Inhibitory and recovery patterns of brain, gill, and muscle acetylcholinesterase (AChE) was studied in vivo after exposure to LC(50) (96 h) concentrations. Recovery study was performed of regular intervals of day-1, -7, -14, -21, and -28 to establish the time course of 50% and 100% recovery in neurotransmitter enzyme of brain, gill, and muscle tissues. The sensitivity of the tissue AChE was in the order gill>brain>muscle. The relative toxicity of these compounds with regard to AChE was RPR-II>RPR-V>MCP. The MCP-treated fishes recovered at a faster rate than for RPR-V, followed by RPR-II.

Biochemical enzyme activity in different tissues of rats exposed to a novel phosphorothionate (RPR-V).

The effects of a novel phosphorothionate (RPR-V) synthesized at Indian Institute of Chemical Technology, Hyderabad, was studied using three sub-chronic doses of 0.033 (low), 0.066 (medium) and 0.099 (high) mg kg(-1) in male and female rats for a period of 90 days. This long term and repeated treatment of RPR-V revealed that the membrane bound target enzymes Aspartate aminotransferase and Alanine aminotransferase increased significantly in serum and kidney, whereas these enzymes significantly decreased in liver and lung tissues when measured after 45 and 90 days of treatment. This compound also caused significant inhibition of RBC Acetylcholinesterase, target enzyme of organophosphorus compounds revealing its effect on normal synaptic transmission. Two way Anova studies disclosed that the alterations were mostly dose and time dependent, sexual dimorphism was not observed when the activities of male rats were compared with female rats. Enzyme recoveries were recorded after 28 days of post treatment, high degree positive correlation was observed with regard to these enzymes between serum versus kidney, whereas in case of serum versus liver and lung tissues high degree negative correlation was recorded. These enzyme profiles elucidates that they increased in serum but they decreased significantly in liver and lung indicating necrosis of these tissues. However, in case of kidney the level of these enzymes increased significantly with parallel to serum, which is suggestive of an increase synthesis of these enzymes, may be an adaptive mechanism due to the stress of the toxicant. These biomarker enzymes can be detected rapidly and hence may be used for the prediction and diagnosis of pesticide insults.

Long-term effects of a novel phosphorothionate (RPR-II) on detoxifying enzymes in brain, lung, and kidney rats.

The effects of a phosphorothionate, 2-butenoic acid-3-(diethoxyphosphinothioyl) methyl ester (RPR-II), on the activities of glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) and the level of glutathione (GSH) were evaluated in rats after administration of RPR-II at 0.014 (low), 0.028 (medium), and 0.042 (high) mgkg(-1)day(-1) for 90 days and also at 28 days (withdrawal) after stopping treatment. Brain GST activity and GSH level decreased significantly at the high dose on the 45th and 90th days of treatment. Dose- and time-dependent decreases in GST activity and GSH was level were observed in lung at medium and high doses and in kidneys at all three doses on both the 45th and 90th days. UDPGT activity increased significantly in kidneys at the medium and high doses at 45 and 90 days. Brain and lung did not display any significant variations in UDPGT activity when compared with the control. Interestingly, the withdrawal study revealed that the effect was reversible within 28 days of cessation of treatment, when enzyme activity reverted to levels close to those of controls. The study revealed that RPR-II affected the GSH- and GST-dependent detoxification system of the treated tissues of rat and its potential to modulate the enzymes is in the order kidneys>lung>>brain. The present subacute study suggests that RPR-II may bring about physiological upsets by altering GSH- and GST-dependent events in different tissues of exposed organisms.

Acute and sub-acute effects of 2-butenoic acid-3-(diethoxy phosphinothioyl) methyl ester (RPR-II) on testis of albino rat.

Acute and sub-acute toxic effects of a novel phosphorothionate coded as RPR-II on testis of albino rats were studied. In acute study rats received a single dose of 12.3 mg/kg of RPR-II and sacrificed after 24 hr. For sub-acute study 0.58 mg/kg/day was administered orally to rats for 10 and 21 days. Acute exposure of rats to RPR-II brought no change either in the gonadosomatic index (GSI) or in the structure of testis or in the serum levels of testosterone. Testis glutathione (GSH) level and glutathione S-transferase (GST) activity was significantly decreased whereas, acid phosphatase (AcP) levels increased significantly at 24 hr post-treatment. On 7th day (withdrawal period) after the cessation of the treatment the GSH, GST, AcP, and AkP levels reached to near control. The sub-acute study revealed a significant decrease in GSI on 10th and 21st day of the treatment. In contrast, a time-dependent and significant increased in GSH level and GST activity was observed on 100th and 21st day of post-treatment, except GSH level on 10th day, which was declined. Due to RPR-II treatment the testis AcP and alkaline phosphatase (AkP) levels were significant at both 10th and 21st day of medication but AcP levels were increased whereas AkP levels decreased. The histopathological studies on day 10th showed considerable loss of spermatozoids in testis and at 21st day complete derangement of cellular organization was observed. Testosterone levels decreased significantly on 10th day and remained significantly low at 21st day. However, withdrawal studies showed a recovery in testis of rat treated with RPR-II. GST, GSH, GSI, AcP and AkP values recovered, testosterone levels were also well recovered but recovery in testis structure remained at a low profile. The present study suggests that RPR-II may cause testicular toxicity in rats affecting the normal functioning of testis and it also gave some new information in withdrawal studies.

Alterations in hepatic detoxifying enzymes induced by new organophosphorus insecticides following subchronic exposure in rats.

The present study examined the structure-toxicity relationship of two novel phosphorothionates, a methyl ester (RPR-II) and an ethyl ester (RPR-V), with regard to their interaction with certain biochemical indices in rat. Male rats were treated orally with 10% (low), 20% (medium) and 30% (high) doses of the two compounds (14, 28 and 42 microg kg(-1) day (-1) of RPR-II and 33, 66 and 99 microg kg(-1) day(-1) of RPR-V) daily for 90 days. The activity of hepatic glutathione S-transferase (GST) and UDP-glucuronyl transferase (UDPGT) and the level of glutathione (GSH) were estimated at 0, 45 and 90 days of treatment as well as 28 days after cessation of treatment. RPR-II caused statistically significant depletion of GSH after 45 and 90 days of treatment at the high dose, whereas RPR-V depleted GSH only after 90 days at the high dose. RPR-II inhibited GST after 45 and 90 days at medium and high doses, whereas RPR-V caused inhibition of GST after 45 and 90 days only at the high dose. Significantly, UDPGT activity was increased only by the high dose of RPR-II after 90 days. However, a dose and time-dependent increase in UDPGT activity was observed at all three doses of RPR-V after 45 and 90 days. There was no modulation in any of the three indices at low doses of the two organophosphorus insecticides. The withdrawal study revealed that induced changes in hepatic parameters were reversible 28 days after cessation of treatment. The results indicated that the two insecticides had different potential to modulate hepatic GST, UDPGT and GSH due to subchronic exposure and that these metabolic alterations are quite reversible after withdrawal of treatment.

Effects of phosphorothionate on the reproductive system of male rats.

Acute and Sub-acute toxic effects of a new pesticide phosphorothionate coded as RPR-V on testis of albino rat were studied. For the acute study, rats received a single dose of 30 mg/kg of RPR-V and sacrificed after 24 hours. For the Subacute study, 1.42 mg/kg/day was administered orally to rats for 10 days and 21 days. Acute exposure of rats to RPR-V brought no change either in the Gonadosomatic Index (GSI) or in the structure of testis or in the serum levels of Testosterone. Similarly, no significant change was observed in the Glutathione S-transferase (GST) activity. But, in testis there was significant increased in the reduced Glutathione (GSH) and Acid Phosphatase (AcP), whereas Alkaline Phosphatase (AkP) levels decreased significantly at 24hr post treatment. On 7th day (withdrawal period) after the cessation of the treatment the GSH, AcP, and AkP levels reached to near control. The sub-acute study revealed a significant post treatment. Due to RPR-V treatment the testis AcP levels increased significantly at 21st day of medication but AkP levels decreased both at 10th and 21st day of post treatment. Histopathological studies showed that after 10th day testis showed considerable loss of spermatozoids and at 21st day complete derangement of cellular organization was observed. Testosterone levels decreased significantly after 10th day and remained significantly low at 21st day. However, withdrawal studies showed a recovery in testis of rat treated with RPR-V. GST, GSH, GSI, AcP and AkP values were recovered, testosterone levels were also recovered but recovery in testis structure remained at a low profile.

Subacute effects of a phosphorothionate pesticide on mixed function oxidases of Wistar rats.

Subacute oral toxicity of a newly developed phosphorothionate insecticide (2-butenoic acid-3-(diethoxy-phosphinothioyl) methyl ester), coded as RPR-2, was studied in male rats by oral (multiple) intubation of low (0.014 mg kg(-1) day(-1)), medium (0.028 mg kg(-1) day(-1)), and high (0.042 mg kg(-1) day(-1)) dose for 90 days. The medium and high dose produced toxic symptoms along-with some mortality (20%) occurred in the high dose treated rats. The medium and high doses caused significant inhibition in cytochrome P-450 activity in liver, lung, kidney and brain tissues at 45 and 90 days. The high dose caused significant decrease in cyt.b5 activity of all the four tissues at 45 and 90 days. Whereas, medium dose brought such effect in liver and lung at 45 and 90 days. Kidney and brain cyt.b5 activity decreased significantly at 90th day due to medium dose. Low dose also caused inhibition in cyt.b5 activity in brain at 90th day. Cytochrome P-450 reductase activity was decreased significantly in liver,

Inhibition of acetylcholinesterase and different ATPases by a novel phosphorothionate (RPR-II) in rat brain.

A novel phosphorothionate (2-butenoic acid-3-(diethoxy phosphinothioyl)-methyl ester (RPR-II), synthesized at the Indian Institute of Chemical Technology, Hyderabad, targets its effect on rat brain acetylcholinesterase (AChE) and Na(+)-K(+), Mg(2+), and Ca(2+) ATPases, as evident in this investigation. Three subchronic doses 0.014 (low), 0.028 (medium), and 0.042 (high) mg kg(-1) were administered to rats daily for a period of 90 days RPR-II caused statistically significant dose- and time-dependent inhibition in brain AChE and also in Na(+)-K(+), Mg(2+), and Ca(2+) ATPases in both male and female rats after 45 and 90 days of treatment. The low dose was generally insignificant while the medium and high doses were significantly effective. Females were more susceptible than males with regard to brain AChE, Na(+)-K(+), and Mg(2+) ATPases, which indicates sexual dimorphism in the treated rats. Interestingly, after 28 days post-treatment, recovery of these enzymes was observed. The relative sensitivities of these enzymes indicated that brain AChE was more sensitive than any of the ATPases, but among the ATPases Na(+)-K(+) ATPase was more susceptible than Ca(2+) or Mg(2+) ATPases. This compound, besides inhibiting the target of organophosphates, AChE, also inhibited different ATPases, suggesting both synaptic transmission and nerve conduction were affected.

Acid and alkaline phosphatase activities in a novel phosphorothionate (RPR-11) treated male and female rats. Evidence of dose and time-dependent response.

The effect of a novel phosphorothionate, the methyl ester of 2-butenoic acid-3-diethoxy phosphinothioyl (RPR-II) was studied on membrane bound target enzymes Acid (AcP) and Alkaline (AkP) Phosphatases in different tissues of male and female albino Wistar rats. Three sub-chronic doses 0.014 (low), 0.028 (medium) and 0.042 (high)mg/kg-1 were administered to the rats daily for a period of 90 days. The long term and repeated administration of RPR-II caused significant increase of AcP and AkP in serum and kidney (AcP), whereas these enzymes simultaneously decreased significantly in liver, kidney (female rat AkP) and lung tissues in both male and female rats after 45 and 90 days of treatment. However, the kidney AcP increased significantly in both the sexes which is suggestive of an increase in synthesis of this enzyme which may be an adaptive mechanism to the toxicant stress. The changes in serum, liver, kidney and lung of both male and female rats by this compound were statistically significant when compared with two way Anova showing that they are dose and time dependent. The alterations in male rats were statistically insignificant when compared with female rats showing no sexual dimorphism by this compound. Recovery was observed after 28 days of post treatment (withdrawal study) indicating reversal of the toxic symptoms once the toxicant is removed. High degree negative correlation was observed for serum versus liver and lung and in other cases substantial correlation was observed. The changes observed in these enzymes showed that liver was most susceptible followed by lung and kidney. There are marker enzymes and their increase in different tissues might be due to the increased permeability of plasma membrane or cellular necrosis, showing the stress condition of the treated rats. This investigation elucidates the effect of these biomarker enzymes which increased in blood, might be due to the necrosis of liver, kidney and lung tissues by this compound.

Biochemical alterations induced by a new phosphorothionate (RPR-II) in tissues of male and female rats.

This study was conducted to investigate the effect of a new phosphorothionate, the methyl ester of 2-butenoic acid-3-diethoxy phosphinothioyl (RPR-II) on membrane bound target enzymes aspartate amino transferase (ASAT), alanine amino transferase (ALAT) and RBC acetylcholinesterase (AChE) in different tissues of male and female albino wistar rats when treated orally with 0.014 (low), 0.028 (medium) and 0.042 (high) mg/kg daily for a period of 90 days. Repeated administration of RPR-II caused significant increase of ASAT and ALAT enzymes in serum, liver and kidney and significant decrease was recorded in lung in both male and female rats when measured after 45 and 90 days of treatment. This compound also caused significant inhibition of RBC AChE indicating its effect on nerve synapsis. Females were more susceptible than males with regard to ASAT and ALAT levels in serum and liver and also in kidney ASAT, whereas reverse trend was recorded in lung ALAT, suggesting sexual dimorphism in the treated rats. These studies also indicated that the levels of these affected enzymes were recovered to normal conditions after 28 days of post treatment (withdrawal study). Positive correlation was observed with regard to these enzymes between serum, liver and kidney, whereas in case of serum and lung a negative correlation was recorded. These enzymes profile elucidates lung necrosis whereas in other tissues the level of enzymes increased showing an adaptive mechanism due to the chemical stress.

Effects of a new phosphorothionate (RPR-V) on ATPases and acetylcholinesterase in rat brain by subchronic dosing.

The effect of a new phosphorothionate, the ethyl ester of 2-butenoic acid 3-diethoxy phosphinothioyl (RPR-V) synthesized at the Indian Institute of Chemical Technology, Hyderabad was studied using peroral doses of 0.033, 0.066 and 0.099 mg kg(-1) in male and female rats daily over 90 days. This repeated administration of RPR-V caused significant inhibition of acetylcholinesterase, Na+-K+, Mg2+ and Ca2+-ATPases in the brain of male and female rats when measured after 45 and 90 days of treatment. The effects of the low dose were generally not statistically significant, whereas medium and high doses caused significant effects. Females were more susceptible with regard to brain AChE, but the reverse was seen with Mg2+-ATPase, suggesting sexual dimorphism. Enzyme recoveries were seen 28 days after the final dose. Since RPR-V not only inhibited AChE but also ATPases, it is possible that both synaptic transmission and nerve conduction were affected.

Teratism induced in the developing chick by RPR-V, an organophosphate.

Fertile White Leghorn chicken eggs were injected on day 4 of incubation with 0, 0.25, 0.50 or 1.00 mg RPR-V/egg and opened on day 20 or allowed to hatch. The following parameters were examined: hatchability, growth and development, external malformations and skeletal deformities. As the dose was increased, the hatchability decreased, and the incidence of deformities increased. Staining of the skeleton with Alizarin red showed clear deformities in the embryos. The results suggest that RPR-V has teratogenic effects on chick embryos when injected on day 4 of incubation.

Interaction of monocrotophos and its novel thion analogues with microsomal cytochrome P-450: in vivo and in vitro studies in rat.

The binding of monocrotophos (MCP) and its two thion analogues (coded as RPR-II and RPR-V) to rat hepatic microsomal cytochrome P-450 (HMC) were investigated in vitro by difference spectroscopy. These three organophosphorus insecticides were found to bind stoichiometrically to HMC with very high affinity (Ks 34-50 microM). RPR-V showed the highest binding affinity followed by RPR-II and MCP. Association of these compounds with HMC occurred within 2 min of addition in the cuvette and therefore, appeared to be tight binding ligands of cytochrome P-450. In vivo studies at equitoxic doses of the three compounds 24 h after oral treatment in rats revealed that they all caused reduction in MC content in liver, lung, kidney and brain, as against induction in cardiac and splenic cytochrome P-450. These in vivo results suggest organ specificity in modulating the microsomal cytochrome P-450 (MC) content of hepatic and extra-hepatic tissues by the three compounds. Apparently, their binding affinity with HMC is strongly correlated with their LD50 value and has a substantial co-relationship with the cytochrome P-450 level in the liver.

A comparative study of blood changes and brain acetylcholinesterase inhibition by monocrotophos and its analogues in rats.

The effects of monocrotophos and its newly synthesized analogues, RPR-II and RPR-V, on hematology and blood chemistry 24 hr post-treatment were studied in rats given doses of 0.96, 1.23, and 3.0 mg/kg po, respectively. It was found that monocrotophos caused a significant increase in the mean WBC count. RPR-V, a significant decrease in hematocrit and RBC count, whereas RPR-II did not alter any hematological parameter. The activities of membrane-bound enzymes in serum were not significantly changed by all three compounds except for a statistically significant increase of 38% in SGOT activity by RPR-II. Only monocrotophos caused a significant inhibition of the brain acetylcholinesterase activity. In vitro studies with partially purified preparations of rat brain cholinesterase revealed that monocrotophos was the most potent anticholinesterase agent, followed by RPR-II and RPR-V. All three compounds caused inhibition of rat brain cholinesterase by decreasing the Vmax and increasing the Km values, indicating a mixed type of inhibition. The two analogues appeared to be less neurotoxic than monocrotophos.

Hepatic and extra hepatic glutathione depletion and glutathione-S-transferase inhibition by monocrotophos and its two thiol analogues.

Effect of monocrotophos (MCP) and its thiol analogues (coded as RPR-2 and RPR-5) on hepatic and extra-hepatic glutathione (GSH) depletion and glutathione-S-transferase (GST) inhibition was studied at 0.96, 1.23 and 3.0 mg/kg respectively 24 h after medication in rats. All the three compounds caused tissue specific depletion of GSH from hepatic and extra-hepatic tissues. Cytosolic GST activity was significantly inhibited in all the tissues, MCP being the most potent inhibitor. Both in vitro and in vivo data indicate that hepatic GST inhibiting potential of the three compounds lies in the order MCP greater than RPR-5 greater than RPR-2. In vitro effect of 3 compounds on GSH activation kinetics of GST demonstrate competitive inhibition by MCP and non-competitive inhibition by the two analogues. However, CDNB activation kinetics of the enzymes revealed mixed inhibition by all 3 compounds. The present study suggests that monocrotophos and its thiol analogues may bring about physiological upsets by altering GSH and GST dependent events in different tissues of exposed organisms.