Cellular and biochemical effects induced by atrazine on blood of male Japanese quail (Coturnix japonica)

Atrazine a potent endocrine disruptor herbicide is broadly used to control rapidly growing unwanted weeds in various cereals crops which induce adverse effects both in mammalian and avian species. In present study 96 mature male Japanese quail (Coturnix japonica) were procured and randomly kept in eight groups (A to H) each having 12 birds. Atrazine was administered orally at 0, 10, 25, 50, 100, 250 and 500 mg/kg body weight to all experimental groups. The mitomycin C at 2 mg/kg body weight was given to the birds of group B which served as a positive control. From each group 4 birds were randomly selected and harvested at day 15, 30 and 45. A significant (P < 0.05) decrease in serum total protein, serum albumin and serum testosterone values were recorded at day 45 in all treated groups. A significant increase in serum ALT and AST concentration was also recorded. Moreover, morphological alterations in nucleus of erythrocytes were also observed including blebbed nuclei, notched nuclei, lobed nuclei, vacuolated cells, binucleated cell, and cell with pear shaped and micronucleus. Overall, our results show that atrazine at higher doses induces significant serum biochemical alterations and changes in nucleus of erythrocytes.     

Metabolism of tetramethrin in houseflies in vivo

Houseflies susceptible to pyrethroids were found to metabolize in vivo radioactive tetramethrin (phthalthrin) or 3,4,5,6-tetrahydrophthalimidomethyl dl-trans chrysanthemumate mainly to N-(hydroxymethyl) 3,4,5,6-tetrahydrophthalimide and chrysanthemumic acid. Smaller amounts of oxidation products of tetramethrin or chrysanthemumic acid were formed. Pretreatment with piperonyl butoxide had little effect on the cleavage of the ester bond of tetramethrin, but another synergist NIA 16388 remarkably inhibited this reaction.     

The in vitro metabolism of azinphosmethyl by mouse liver

The in vitro metabolism of methoxy-¹⁴C- or ³²P-azinphosmethyl by subcellular fractions from mouse liver was studied. The major degradative activity was associated with the microsomal and soluble fractions. Since the microsomal activity required NADPH and was inhibited by carbon monoxide, it is reasonable to assume that the mixed function oxidases were involved. The microsomal system catalyzed the dearylation reaction resulting in the formation of dimethyl phosphorothioic acid and dimethyl phosphoric acid. The system was also responsible for the oxidative desulfuration of azinphosmethyl resulting in the formation of azinphosmethyl oxygen analog.     

Inhibition of termite cellulases by carbohydrate-based cellulase inhibitors: Evidence from in vitro biochemistry and in vivo feeding studies

Efficacy of three prototype termite cellulase inhibitors, cellobioimidazole (CBI), fluoromethylcellobiose (FMCB) and fluoromethylglucose (FMG) was investigated using biochemical and feeding assays. Optimal conditions for measuring endoglucanase, exoglucanase and β-glucosidase activities were first determined. The three inhibitors were then tested under optimal conditions against enzyme fractions that represented endogenous (foregut/salivary gland/midgut) and symbiotic (hindgut) cellulases. In vitro, CBI and FMCB both inhibited exoglucanase and β-glucosidase activity (I₅₀s in nM and mM range, respectively). Feeding assays showed significant impacts on both survivorship and feeding stimulation by FMCB and CBI. Enzymatic measurements on feeding assay survivors showed impacts on all three cellulase activities by CBI and lesser impacts by FMG and FMCB. Validative bioassays with the sugars glucose, maltose and cellobiose showed no feeding stimulation or mortality as occurred in feeding inhibitor bioassays. These results indicate efficacy for two cellobiose-based inhibitors, FMCB and CBI, suggesting potential for these inhibitors as novel termite control agents.     

In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs

Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O-halogenated-phenyl-O-alkyl phenylphosphonates was studied in vitro. The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The pI₅₀ of O-(2,6-dichlorophenyl) O-methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy compounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than desbromoleptophosoxon. Within a like group of chlorinated phenylphosphonates, a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in vivo delayed neurotoxic potential by the corresponding phosphonothionate exists. In vivo inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in vivo inhibitor. Ethoxyleptophos and leptophos have about equal in vivo esterase inhibitory properties. For desbromoleptophos and leptophos there is good agreement between the minimum dose causing delayed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.     

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

The effect of O,S,S,-trimethyl phosphorodithioate on the in vivo metabolism of phenthoate in the rat

The in vivo metabolism of phenthoate (O,O-dimethyl S-[α-(carboethoxy)benzyl]phosphorodithioate) was followed in rats after oral administration of a nontoxic dose of 100 mg/kg. The same metabolic study was conducted following coadministration of 0.5% O,S,S-trimethyl phosphorodithioate (OSS-Me). When administered alone, phenthoate was metabolized principally by carboethoxy ester hydrolysis and cleavage of the PO and CS bonds, resulting in at least six metabolites. The primary urinary metabolite excreted was phenthoate acid. Coadministration of 0.5% OSS-Me did not alter the types of metabolites excreted. However, a reduction of the carboxylesterase-catalyzed product (phenthoate acid) was observed, indicating that the enzyme responsible for the major pathway of phenthoate detoxication was inhibited. Alternate detoxication processes did not compensate for the reduction in carboxylesterase-catalyzed detoxication. It was concluded that inhibition of the carboxylesterase enzymes is the major cause of the potentiation of phenthoate toxicity by OSS-Me.     

Inhibition of plant pathogens in vitro and in vivo with essential oil and organic extracts of Cestrum nocturnum L.

The efficacy of the essential oil and various organic extracts from flowers of Cestrum nocturnum L. was evaluated for controlling the growth of some important phytopathogenic fungi. The oil (1000 ppm) and the organic extracts (1500 μg/disc) revealed antifungal effects against Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum in the growth inhibition range of 59.2-80.6% and 46.6-78.9%, respectively, and their MIC values were ranged from 62.5 to 500 and 125 to 1000 μg/mL. The essential oil had a remarkable effect on spore germination of all the plant pathogens with concentration and time-dependent kinetic inhibition of P. capsici. Further, the oil displayed remarkable in vivo antifungal effect up to 82.4-100% disease suppression efficacy on greenhouse-grown pepper plants. The results obtained from this study may contribute to the development of new antifungal agents to protect the crops from fungal diseases.     

Comet assay in gill cells of Prochilodus lineatus exposed in vivo to cypermethrin

Agricultural chemicals can induce genetic alterations on aquatic organisms that have been associated with effects on growth, reproduction and population dynamics. The evaluation of DNA damage in fish using the comet assay (CA) frequently involves the utilization of erythrocytes. However, epithelial gill cells (EGC) can be more sensitive, as they are constantly dividing and in direct contact with potentially stressing compounds from the aquatic environment. The aim of the present study was to evaluate (1) the sensitivity and suitability of epithelial gill cells of Prochilodus lineatus in response to different genotoxic agents through the application of the CA, (2) the induction of DNA damage in this cell population after in vivo exposure to cypermethrin. Baseline value of the CA damage index (DI) for EGC of juvenile P. lineatus was 144.68 ± 5.69. Damage increased in a dose-dependent manner after in vitro exposure of EGC to methyl methanesulfonate (MMS) and H₂O₂, two known genotoxic agents. In vivo exposure of fish to cypermethrin induced a significant increase in DNA DI of EGC at 0.150 μg/l (DI: 239.62 ± 6.21) and 0.300 μg/l (270.63 ± 2.09) compared to control (150.25 ± 4.38) but no effect was observed at 0.075 μg/l (168.50 ± 10.77). This study shows that EGC of this species are sensitive for the application of the CA, demonstrating DNA damage in response to alkylation (MMS), oxidative damage (H₂O₂), and to the insecticide cypermethryn. These data, together with our previous study on DNA damage induction on erythrocytes of this species, provides useful information for future work involving biomonitoring in regions where P. lineatus is naturally exposed to pesticides and other genotoxic agents.     

棉铃虫C型凝集素抑制多角体病毒体内增殖

为探究C型凝集素（C-type lectin）在病毒传播中的可能作用,采用半定量RT-PCR法检测了棉铃虫Helicoverpa armigera不同C型凝集素在核型多角体病毒（nuclearpolyhedrosis virus,NPV）感染后的表达变化,并通过real-time qPCR和RNA干扰技术研究了NPV在棉铃虫体内的增殖状况及对棉铃虫致死率的影响。结果显示,在感染NPV后,棉铃虫血细胞C型凝集素Ha-lectin1和Ha-lectin5基因表达变化明显,其中Ha-lectin1在血细胞和脂肪体中的表达显著上调;注射dsRNA后,Ha-lectin1基因相对于actin的表达量均值由0.19下降到0.04,下降幅度达79%,显示Ha-lectin1基因被成功干扰。随着Ha-lectin1基因的沉默,病毒滴度由0.0015快速升高到1.95,棉铃虫的死亡率也随之增加到97.7%。表明C型凝集素能够抑制NPV在棉铃虫体内的增殖。     

The apparent in vivo neuromuscular effects of the δ-endotoxin of Bacillus thuringiensis var. israelensis in mice and insects of four orders

Alkaline-dissolved crystal δ-endotoxin from Bacillus thuringiensis var. israelensis (serovar H 14) was injected into mice and seven species of insects representing the orders Lepidoptera, Orthoptera, Coleoptera, Hemiptera, and Diptera. High in vivo toxicity, at 1 to 5 ppm (μg toxin/g body wet wt), was observed with mice and some insects, including some that are not sensitive to the toxin when administered orally. Neuromuscular effects were observed when the toxin was injected directly into the body cavity of the test animals. Biochemical studies suggested that different protein fragments within the crystal δ-endotoxin may be responsible for the majority of the mosquito larvacidal activity and the neurotoxic symptoms observed in larvae of Trichoplusia ni.     

Alkylation of guanine in mice in vivo by organophosphorus insecticides. I. Trichlorphone and butonate

Following intraperitoneal administration to male mice of trichlorphone, 4 mg/animal = 160 mg/kg and butonate, 5 and 10 mg/animal = 200 and 400 mg/kg, labeled by ¹⁴C in the OCH₃-groups, nucleic acids taken from different organs and urine were analyzed for [7-¹⁴C]methylguanine. The limit of detection was 2 × 10^?8, calculated as ¹⁴C relative to the total dose. The maximum of ¹⁴C in 7-methylguanine was 2 × 10^?7 in lung, kidney, and testicles and 3 × 10^?6 in liver. The excretion rate of 7-MeG from nucleic acids is very rapid, a halflife of 2.0 hr was measured in liver from butonate and of < 24 hr was calculated in the whole body from trichlorphone, contrary to the excretion rate of 3.0–3.5 days following administration of strongly genotoxic agents. The relative amounts of [7-¹⁴C]methylguanine excreted in the urine were determined and compared with data for dichlorvos, dimethyl sulfate, and methyl methanesulfonate from the literature. Following intraperiotoneal administration, the methylating capability towards N-7 of guanine in nucleic acids is given by the ratio of about 100:10:25 for dichlorvos, butonate, and trichlorphone, respectively.     

Effect of organophosphates in vivo upon acetylcholinesterase isozymes from housefly head and thorax

Four soluble acetylcholinesterase isozymes of head and three from the thorax of housefly (Musca domestica L.) were separated by polyacrylamide gel electrophoresis. Their inhibition in vivo was studied after poisoning with an LD₅₀ of four organophosphates: malaoxon, paraoxon, diazinon and dichlorvos. The isozymes differed greatly in their degree of inhibition. Thoracic isozymes were found to be more sensitive to inhibition than head isozymes. In surviving flies, the recovery rates of head isozymes were much faster than thoracic isozymes. In vitro studies showed that thoracic isozymes differed 4.1- and 2.9-fold in their K_m and V_max.     

Precocene II metabolism: Comparative in vivo studies among several species of insects,and structure elucidation of two major metabolites

Several species of insects, exhibiting varying responsiveness to the juvenile hormone antagonist precocene II (6,7-dimethoxy-2,2-dimethylchromene), were challenged topically with a tritiated preparation of the title compound. Metabolism of [³H]precocene II was subsequently examined by withdrawing hemolymph samples from treated animals at appropriate time intervals and characterizing the extractable radiolabel chromatographically. Quantitative (or qualitative) differences observed between the respective metabolic profiles were found not correlative with specimen sensitivity to precocene. Production of two heretofore unreported metabolites, identified by spectral and chemical means as O-β-glucosides of 6- and 7-monodemethylated precocene II, was demonstrated in both sensitive and insensitive species. No evidence for the presence of a hemolymphborne, biologically effective “activated metabolite” produced in vivo by precocene-susceptible insects could be found. The latter finding may well argue for in situ bioactivation of precocene at the target tissue(s) by these sensitive insects.     

in vitro metabolism of azinphosmethyl in susceptible and resistant houseflies

The in vitro metabolism of [¹⁴C-methoxy] or [³²P]azinphosmethyl by subcellular fractions of abdomens from a resistant and a susceptible strain of houseflies was studied. The degradative activity in both strains was associated with the microsomal and soluble fractions and required NADPH and glutathione, respectively. The resistant strain possessed higher activity for both the mixed-function oxidases and the glutathione transferase than the susceptible strain, and both systems appear to be important in the resistance mechanism. The mixed-function oxidases were involved in the oxidative desulfuration as well as the dearylation of azinphosmethyl. A glutathione transferase located in the soluble fraction catalyzed the formation of desmethyl azinphosmethyl and methyl glutathione. This enzyme also demethylated azinphosmethyl oxygen analog. Although the soluble fraction exhibited both glutathione S-alkyltransferase and S-aryltransferase activity against noninsecticidal substrates, no evidence of the transfer of the benzazimide moiety from azinphosmethyl to glutathione was obtained. Sephadex G-100 chromatography of the soluble enzymes revealed a common eluting fraction responsible for both types of transferase activity.     

Structure-activity relationships of DDT-type analogs based on in vivo toxicity to the sensory nerves of the cockroach, Periplaneta americana L

Forty-three DDT-type compounds were applied in saline suspension to the crural nerve of Periplaneta americana L. and the threshold concentration (ED₅₀) to produce trains of impulses was determined together with the frequency of appearance of repetitive afterdischarge. These quantitative neurological measures were evaluated in multiple regression analyses of structural parameters including van der Waal's volume, the F and R components of Hammet's σ, and the hydrophobic constant Π. This structure-activity analysis provides an accurate estimation of the intrinsic toxicity of the DDT analogs. The results affirm previous working theories that the bulk of the functional groups within the DDT framework is the primary factor relating to activity. However, conformation is also an important parameter.     

Uptake of DDT and DDE by the quail embryo and chick

Adult female Japanese quail (Coturnix coturnix japonica) were dosed orally with pp'-DDT in order to produce residues of pp'-DDT and pp'-DDE in their eggs. Eggs were incubated and insecticide uptake by the developing embryos and chicks was compared with the results of other authors, who had injected chicken eggs with insecticide solution before incubation. Rate of uptake by quail embryos increased throughout the incubation period. At hatching and 2 days after hatching, the yolk sac was estimated to contain mean amounts of 37% and 7%, respectively, of the total residues in the chick. Quail eggs, without detectable organochlorine residues, were injected with either pp'-DDT or pp'-DDE in olive oil. At 6 days incubation significantly more DDE than DDT was found in the embryo, but this trend had stopped by 10 days incubation. Metabolism of pp'-DDT to pp'DDE was first detected after 9 days incubation. Embryonic development was slower in injected eggs than in eggs from treated females.     

Enhancement of dye-sensitized phototoxicity to house fly larvae in vivo by dietary ascorbate,diazabicyclooctane, and other additives

Several reagents reported to be singlet oxygen quenchers and/or radical scavengers and to be protectants against photooxidative damage to isolated systems in vitro were examined, in vivo, for protective effect on the larvae of the house fly (Musca domestica L.; Diptera:Muscidae). A standardized erythrosine-sensitized phototoxic test procedure was used. β-Carotene appeared to show some protective effect. Other dietary additives exhibited no measurable degree of protection to the larvae in vitro; on the contrary, mortality increased in the presence of butylated hydroxytoluene, ascorbate, and diazabicyclooctane.     

紫草色素对番茄叶霉病菌的室内抑菌活性研究

室内研究了新疆紫草色素对番茄叶霉病菌Fulvia fulva (Cooke) Cifferri的生物活性。新疆紫草色素用石油醚提取,分别用平板表面萌发法和菌落生长速率法测定其对番茄叶霉病菌孢子萌发、孢子芽管长度及菌丝生长的影响。并在离体条件下测定了其对番茄叶霉病的防治效果。结果表明,新疆紫草色素对番茄叶霉病菌抑制作用明显,其中抑制孢子萌发EC50为17.4 μg/mL,抑制孢子萌发芽管长度的EC50为1.0 μg/mL,抑制菌丝生长的EC50为200.4 μg/mL;浓度为800 μg/mL时对番茄叶霉病的离体防治效果达到90.5％。     

Effect of some ergosterol biosynthesis inhibiting fungicides on sterols and cytochrome P-450 from the Japanese quail Coturnix coturnix

Nine fungicides that inhibit ergosterol biosynthesis (diclobutrazol, fenarimol, fenpropimorph, imazalil, nuarimol, prochloraz, propiconazole, triadimefon, triadimenol) and one plant growth regulator (ancymidol) were administered to Japanese quails (Coturnix coturnix). Most of these compounds had a moderate or no effect, but prochloraz, imazalil and, to a lesser extent, propiconazole were shown to produce a dramatic increase in liver weight and cytochrome P-450 level. These three compounds were also found to be potent in-vitro inhibitors of 7-ethoxycoumarin O-de-ethylase and aniline hydroxylase, thus resulting in a biphasic effect on drug-metabolising enzymes. With these three compounds, and some others, an accumulation of lanosterol in liver was also observed, suggesting an inhibition of sterol synthesis.