Toxicokinetics of Malathion in Larval Stages of the Toad Bufo arenarum (Hensel): Effect of Exogenous Spermidine

The aim of this work was to study the absorption, biotransformation, and excretion of malathion (¹⁴C-methoxy) and its metabolites in larval stages of the toad Bufo arenarum (Hensel). Also, changes in malathion metabolization by the action of the exogenous polyamine spermidine were studied. Malathion clearance from the media was uniexponential, and spermidine reduced the uptake in the larvae, causing an increase in the apparent half-life of the toxicant. Concomitant with this effect, spermidine increased the level of induction of mixed-function oxidases due to malathion and caused a progressively higher malaoxon/malathion ratio. As a consequence of the higher conversion to the active metabolite malaoxon, spermidine also provoked a significant enhancement in the inhibitory effect of Malathion on acetylcholinesterase activity. [methoxy¹⁴C]malathion metabolites, such as carboxylesterase and glutathione S-transferase products, were detected in the toad larvae and in the media. The excreted products of carboxylesterase activity were about 70% of the total radioactivity, and the glutathione S-transferase products (methyl glutathione) were 20–30% of the total radioactivity. No significant variations in the levels of excreted products due to the action of exogenous spermidine were detected. Malathion inhibited carboxylesterase activity, independent of the presence of spermidine in the media. In turn, glutathione S-transferase activity was induced by spermidine, but was not affected by the exposure to low concentrations of malathion for 48 h. We conclude that the presence of spermidine in the medium modifies malathion toxicokinetics, increasing its toxicity in B. arenarum larvae.     

Inhibition of mycelial growth and sporidial formation in the wheat pathogenNeovossia indica by a polyamine biosynthetic inhibitor

DL-α-Difluoromethylornithine (DFMO), a specific suicide inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17), strongly inhibited mycelial growth and sporidial formation of the wheat pathogen,Neovossia indica, in vitro, while DL-α-difluoromethylarginine (DFMA), the analogous suicide inhibitor of arginine decarboxylase (EC 4.1.1.19), did not. The inhibited mycelial growth and sporidial formation were not only restored by putrescine (polyamine) addition, but were actually enhanced in the putrescine + DFMO cultures. Besides altering mycelial growth and morphology, DFMO also reduced the cell size drastically. The inhibition of fungal polyamine biosynthesis is discussed in relation to selective control of plant disease.     

Eugenol reduces growth and increases activity of S-adenosylmethionine decarboxylase in the phytopathogenic fungusBotrytis fabae

This study was undertaken to examine the possibility that eugenol-induced reductions in growth ofBotrytis fabae are associated with alterations in polyamine metabolism.B. fabae was grown in liquid medium amended with different concentrations of eugenol. Changes in fungal biomass, and activities of enzymes of polyamine biosynthesis and catabolism were studied. An examination was also made of the incorporation of radioactivity from ornithine into polyamines. Activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC) and flux of label from ornithine into the polyamine spermine were greatly increased inB. fabae grown in the presence of eugenol. However, no significant changes were observed in polyamine catabolism or in the concentrations of free polyamines in treated fungal tissue. http://www.phytoparasitica.org posting May 17, 2005.     

Fungicidal activity of the polyamine analogue,keto-putrescine

The polyamine analogue, keto-putrescine, was shown to provide substantial control of infections by six economically important plant pathogens, with EC₅₀ values ranging between 57 and 82 mg litre^?1. However, keto-putrescine was relatively less effective in vitro against Phytophthora infestans, Pyricularia oryzae and Pyrenophora avenae, with EC₅₀ values ranging from 145 to 340 mg litre^?1. In contrast, the polyamine analogue N-acetylputrescine had to effect on in-vitro growth of P. avenae or P. oryzae or infection of barley seedlings with powdery mildew. When P. avenae was grown in the presence of keto-putrescine, there were small, insignificant reductions in ornithine decarboxylase activity, with more substantial, significant reductions in S-adenosylmethionine decarboxylase activity. These changes were accompanied by a 43% reduction in spermidine concentration and increased concentrations of spermine and cadaverine.     

Fungicidal activity of the synthetic putrescine analogue, (E)-l,4-diaminobut-2-ene,and derivatives

The putrescine analogue. (E)-1,4-diaminobut-2-ene (E-BED), synthesized as the dihydrochloride salt, controlled five economically important crop pathogens, Erysiphe qraminis DC f.sp. hordei Marchal. Uromyces viciae-fabae (Pers.) Schroet, Botrytis fabae Sardina, Podosphaera leucotricha (Ell. & Ev.) Salm. and Phytophthora infestans (Mont) De Bary. The Z-isomer. Z-BED. was also fungicidal, although less so than E-BED. Post-inoculation treatment with E-BED gave greater control of powdery mildew infection on barley and rust and chocolate spot on broad bean than did pre-inoculation application. It was also effective in vitro against Botrytis cinerea Pers. ex Fr., Pyricularia oxyzae Br. & Cav. and Pyrenophora avenae Ito & Karibay. When P. avenae was grown in the presence of E-BED dihydrochloride at 81·5 mg litre^?1, growth was reduced by 58% and there were significant reductions in soluble ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activity. These changes were accompanied by a sevenfold increase in putrescine concentration, a 60% increase in spermine concentration and a 32% reduction in spermidine concentration within the fungal tissue.     

Polyamines can inhibit paraquat toxicity and translocation in the broadleaf weed Arctotheca calendula

Reduced paraquat transport from the site of application to the site of action in the chloroplast seems a likely mechanism for paraquat resistance in several weed species including Arctotheca calendula. Recently, it has been shown that paraquat translocation in A. calendula is correlated with paraquat-induced injury and is reduced in paraquat-resistant A. calendula. Studies with leaf slices have shown that some polyamines when applied concomitantly with paraquat can reduce the toxic effects of paraquat. This study examined the effects of three polyamines, putrescine, cadaverine, and spermidine, on paraquat translocation to examine the possibility that paraquat translocation in susceptible plants would be reduced in the presence of polyamines due to competition of the polyamines with cellular paraquat uptake. Two polyamines, spermidine and cadaverine were effective in reducing paraquat translocation in susceptible A. calendula inducing these plants to perform more like resistant A. calendula in terms of translocation. Quantification of the polyamine contents of resistant and susceptible A. calendula showed that resistant plants have higher constitutive spermidine levels than susceptible plants, which infers a possible role of either polyamines or a polyamine transporter in paraquat resistance.     

Effect of leaf scald (<Emphasis Type="Italic">Xanthomonas albilineans</Emphasis>) on polyamine and phenolic acid metabolism of two sugarcane cultivars

Polyamine and phenolic acid levels as well as activities of some enzymes of their biosynthetic metabolism were examined in two sugarcane (Saccharum officinarum) cultivars differing in susceptibility to leaf scald, a disease caused by the bacterium, Xanthomonas albilineans. Juice obtained from both infected cultivars showed significantly increased levels of free putrescine and ornithine decarboxylase activity. However, the pathogen induced different changes in the two cultivars in subsequent metabolic steps. Whereas acid insoluble conjugated spermidine completely disappeared from the highly susceptible cv. C 439-52, an increase in acid insoluble conjugated polyamines was observed in the moderately susceptible cv. L 55-5. Phenolic acid metabolism also differed in the two cultivars. Since total phenolic acid content and phenylalanine ammonium lyase activity was greater in both cultivars after infection, distribution of phenolic acids between free or conjugated forms diverted into different pathways. The level of susceptibility of the two cultivars is discussed in terms of changes in these compounds.     

Germination and appressiorial formation by uredospores ofUromyces viciae-fabae exposed to inhibitors of polyamine biosynthesis

An examination was made of the effects of three polyamine biosynthesis inhibitors on germination and appressorium formation by uredospores of the bean rust fungusUromyces viciae-fabea on artificial membranes. The ornithine decarboxylase inhibitor -difluoromethylornithine had no effect on uredospore germination, even when used at 2mM, whereas appressorium formation was reduced by 63% at 0.5 mM and by 99% at 2mM. Methylglyoxal bis(guanylhydrazone), an inhibitor of S-andenosylmethionine decarboxylase, reduced germination when used at 0.025 mM, and at this concentration, appressorium formation was completed prevented. Uredospore germination was unaffected by as much as 3 mM cyclohexylamine, an inhibitor of spermidine esynthase, while appressorium formation was reduced at 1 mM and completely prevented at 3.3 mM. These results support previous suggestions that inhibitors of polyamine biosynthesis exert their main effect on the early stages of fungal development on the leaf surface.Abbreviantions CHA cyclohyxylamine - DFMO -difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone)     

Studies on the induction of rat uterine ornithine decarboxylase by DDT analogs. I. Comparison with estradiol-17β activity

The effect of DDT analogs and estradiol-17β on uterine ornithine decarboxylase activity in the immature intact and ovariectomized rat was studied. Pretreatment with various doses of o,p′DDT [1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane] or estradiol-17β caused a marked increase in the specific activity of ornithine decarboxylase in the 20,000g supernatant fraction of uterine homogenates but not in liver homogenates. Doses as low as 0.5 mg of o,p′DDT or 0.002 μg of estradiol-17β stimulated uterine ornithine decarboxylase activity in the ovariectomized rat. The peaks of activity after treatment with o,p′ DDT and estradiol-17β occurred at 6 and 5 hr, respectively. The level of ornithine decarboxylase activity in untreated groups was consistently lower in ovariectomized rats than in intact immature animals. Treatment with o,p′ DDT (10 mg/100 g body weight) of ovariectomized and intact immature rats demonstrated at 131-fold and an about 20-fold increase in uterine ornithine decarboxylase activity, respectively. Treatment of ovariectomized rats with cycloheximide or actinomycin D effectively blocked the increase in ornithine decarboxylase caused by o,p′ DDT. Similar results were obtained with cycloheximide in the intact immature rat. Animals subjected to both adrenalectomy and ovariectomy demonstrated an increase in ornithine decarboxylase activity when treated with either estradiol-17β or o,p′ DDT. Dose-response curves obtained for estradiol-17β and o,p′ DDT suggest a similar mechanism of action for the two compounds. Graphic analysis of the dose-response curves for estradiol-17β and o,p′ DDT demonstrated an ED₅₀ of 0.038 μg/100 g body weight and 1.8 mg/100 g body weight, respectively. The examination of various DDT analogs in intact and ovariectomized animals showed that o,p′ DDT was the most potent inducer of ornithine decarboxylase. The order of decreasing potency of DDT analogs was o,p′ DDT, o,p′ DDD. p,p′ DDT, p,p′ DDD, and p,p′ DDE.     

Role of a microsomal carboxylesterase in reducing the action of malathion in eggs of Triatoma infestans

A microsomal malathion carboxylesterase present in Triatoma infestans eggs was active from the first day of embryonic development. This microsomal egg malathionase (MEM) showed a unique band in polyacrilamide gel electrophoresis (PAGE) when malathion was used as substrate. In vivo metabolism of [¹⁴C]malathion during all embryonic development showed a 10% degradation due to carboxylesterases. The in vitro evaluation of the same metabolic pathway catalyzed by the microsomal fraction of T. infestans eggs showed partial inhibition by paraoxon. α- and β-malathion monoacids were identified as the main metabolites of the in vivo and in vitro metabolic pathways. The carboxylesterase band that appeared in PAGE (MEM) from the first day of embryonic development could be the main cause of malathion tolerance in T. infestans eggs.     

Azinphos-methyl and carbaryl resistance in adults of the codling moth (Cydia pomonella (L.), Lepidoptera: Tortricidae) from Northeastern Spain

The resistance of Cydia pomonella (L.) to organophosphates is widespread throughout the pome fruit growing areas. The lethal effects of two insecticides inhibitors of the acetylcholine esterase, azinphos-methyl and carbaryl, were evaluated in adults of five and four field populations of the codling moth, respectively. The lethal concentrations (LC₅₀ and LC₉₀) of these insecticides were determined in a susceptible strain from Spain (S_Spain). Topical bioassays using the approximate LC₉₀ values (3000 mg (a.i.)/L of carbaryl and 2000 mg (a.i.)/L of azinphos-methyl) that were obtained in S_Spain were tested as diagnostic concentrations. The enzymatic activities of mixed-function oxidases (MFO), glutathione S-transferases (GST) and esterases (EST) were measured to investigate their potential role in the detoxification of these insecticides.Carbaryl and azinphos-methyl caused ?53% and ?39% corrected mortality, respectively, in field populations, although the diagnostic concentrations applied were twofold and fourfold higher than the maximum concentration registered in Spain, respectively. The activities of MFO and GST were 7.3- to 16.1-fold higher and 2.5- to 3.7-fold higher in all the field populations compared to those in S_Spain, respectively.     

Sex-related response to organophosphorus and carbamate insecticides in adult Oriental fruit moth, Grapholita molesta

During the development of a resistance-monitoring bioassay that uses males as the life-stage tested, the relationship of adult female and male susceptibility of Grapholita molesta to different classes of insecticides was investigated. Preliminary results indicated that more males survived diagnostic doses of azinphos-methyl than females. Additional research revealed that, although the body mass of adult male G molesta was only 69% of that of female moths (5.67 and 8.20 mg, respectively), their LC50 values were 2.6, 4.1 and 10.3 times higher than those of females for azinphosmethyl, malathion and parathion-methyl, respectively. However, female G molesta moths were more tolerant to methomyl than were male moths. There was no indication that this sex-related response occurred in G molesta larvae. The results presented here raise concerns regarding the use of pheromone traps for determining whether insecticide treatments are required and as part of resistance monitoring programs.     

Synthesis and antifungal activity of five classes of diamines

Examples of five classes of diamines were synthesized and tested for antifungal activity. Two classes, the bis(cyclohexylmethyl)diamines and the bis(benzyl)diamines, were most effective in reducing mycelial growth of the oat leaf stripe pathogen Pyrenophora avenae Ito & Kuribay when used at a concentration of 250microM. The bis(benzyl)diamine BBD5 and the hydroxypyridylethylamine HPE2 both reduced powdery mildew infection of barley seedlings by greater than 70% when applied as a post-inoculation spray at 250 microM. Several of the compounds examined, and especially BBD5 and HPE2, reduced the formation of spermidine but greatly increased spermine levels. These changes in P avenae treated with BBD5 and HPE2 were also accompanied by greatly elevated activity of polyamine oxidase. It is suggested that the antifungal activity of these compounds may be related to the accumulation of spermine and specifically to its toxicity.     

Dual role of esterases in insecticide resistance in the green rice leafhopper

The role of esterases as related to insecticide resistance was studied in an organophosphorus (OP)-resistant strain of the green rice leafhopper. As judged by p-nitrophenyl acetate hydrolysis, 21, 5, and 74% of the esterase activity was located in nuclei/mitochondria, microsomes, and the soluble fraction, respectively. All the fractions were active in hydrolyzing malathion, paraoxon, and fenvalerate. Hydrolysis of malathion and fenvalerate increased with time while that of paraoxon reached a plateau within 15 min. Since a considerable amount of p-nitrophenol was detected in the paraoxon reaction at 0°C and at zero time, the formation of p-nitrophenol may be due to phosphorylation of the esterases rather than phosphorotriesterase action. The results suggest a dual role for esterases in resistance mechanisms; a catalyst for hydrolysis of malathion and fenvalerate, and a binding protein for the oxygen analogs of other OP insecticides, both of which would protect the intrinsic target, acetylcholinesterase, from inhibition. Chromatofocusing of the soluble fraction resolved five esterase peaks, I–V. These esterases were active toward the three general substrates as well as for the three insecticides tested, except for Peak I in which the overall activity was too low. Thin-layer agar gel electrophoresis showed that the chromatofocusing peaks I–V corresponded to the electrophoretic bands E₁–E₅, some of which were previously shown to be associated with OP resistance. The dual role of these esterases may explain the cross-resistance between malathion and other OP insecticides as well as synergism between OP and carbamate insecticides.     

Abscisic acid is a potent inhibitor of growth and sporidial formation inneovossia indica cultures: Dual mode of actionvia loss of polyamines and cellular turgidity

The inhibition of polyamine biosynthesis inNeovossia indica (Mitra) Mundkur by D,L— α-difluoromethylornithine (DFMO) caused an effective reduction of mycelial growth and sporidial production underin vitro conditions, which was reversed by ornithine application. Abscisic acid proved to be similarly effective, not only in inhibiting mycelial growth and sporidial formation but also the germination of teliospores, which constitute the primary inoculum of the pathogen. ABA-mediated inhibition resulted in decreased polyamine levels and loss of cellular turgidity of mycelial cultures. Scanning electron microscopy of ABA-treated cultures revealed extremely shrunken hyphae, in marked contrast to the turgid controls. It is suggested that the manipulation of ABA levels and/or tissue sensitivity in wheat could be a strategy to combat ‘Karnal’ bunt, the disease caused byN. indica.     

In vitro metabolism of insecticides during midgut penetration

A study of the metabolism of ¹⁴C-labeled dieldrin, DDT, malathion, and carbaryl during penetration of the isolated midgut of two insects (Blaberus discoidalis and Manduca sexta) and a section of the intestine of a mammal (Mus musculus) is reported. There was appreciable metabolism of malathion during penetration, including differences in the activation reaction to malaoxon, between insects and mammals. Metabolism was relatively slow during penetration of carbaryl and the chlorinated hydrocarbon insecticides, and little difference in metabolic patterns was noted among the three species. The penetration studies were supported by experiments in which insecticides were incubated with intact and homogenized midgut preparations.     

Phytotoxicity of Roundup Ultra 360 SL in aquatic ecosystems: Biochemical evaluation with duckweed (Lemna minor L.) as a model plant

Glyphosate-based herbicides (e.g. Roundup Ultra 360 SL) are extensively used in aquatic environment. Although glyphosate is more environmental favorable than many other herbicides, it may be exceptionally dangerous for aquatic ecosystems through high water solubility. Thus, the aim of the work was quantification of influence of Roundup Ultra 360 SL (containing isopropylamine salt of glyphosate as an active ingredient) on biomass and chlorophyll content within duckweed (Lemna minor L.). Moreover, changes in polyamine content and activity of such antioxidative enzymes as catalase (CAT) and ascorbate peroxidase (APX) were assayed in order to determine the biochemical mechanisms of L. minor response to the herbicide treatment. Obtained results showed that phytotoxicity of the herbicide was connected with decrease in chlorophyll-a, b and a+b content, and reduction of biomass growth. Roundup, similarly to some abiotic and biotic stressors, caused over-accumulation of putrescine, spermidine and total polyamines (PAs) within duckweed tissues. In addition an increase in CAT and APX activities suggested that stress generated by the herbicide treatment was at least partially connected with oxidative burst. Intensity of the duckweed responses to the herbicide was dependent on the applied herbicide level and/or duration of treatment.     

Screening insecticides against the tobacco whitefly (Bemisia tabaci) on cotton,using a leaf cage laboratory method

Over 20 insecticides were screened against the tobacco whitefly,Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae), on cotton. AsB. tabaci feeds only on live plants, a suitable laboratory test method incorporating young cotton plants was developed. Both cover sprays and systemic insecticides applied to the soil were tested. Triazophos, pyridaphenthion, azinphos-methyl, bendiocarb and flucythrinate were found to be effective and persistent. The best of the systemic insecticides was aldicarb, followed by butocarboxim.