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Purification and characterization of a novel pyrethroid hydrolase from Aspergillus niger ZD11.

The pyrethroid pesticides residues on foods and environmental contamination are a public safety concern. Pretreatment with pyrethroid hydrolase has the potential to alleviate the conditions. For this purpose, a fungus capable of using pyrethroid pesticides as a sole carbon source was isolated from the soil and characterized as Aspergillus niger ZD11. A novel pyrethroid hydrolase from cell extract was purified 41.5-fold to apparent homogeneity with 12.6% overall recovery. It is a monomeric structure with a molecular mass of 56 kDa, a pI of 5.4, and the enzyme activity was optimal at 45 degrees C and pH 6.5. The activities were strongly inhibited by Hg(2+), Ag(+), and rho-chloromercuribenzoate, whereas less pronounced effects (5-10% inhibition) were observed in the presence of the remaining divalent cations, the chelating agent EDTA and phenanthroline. The purified enzyme hydrolyzed various insecticides with similar carboxylester. trans-Permethrin is the preferred substrate.

Aspergillus niger↗

Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112.

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.

Amino Acid Sequence↗

[Air travel--a disease risk? ].

Civil air traffic moves in an altitude that is dangerous for human beings without technical aids. Low temperature and humidity, reduced air pressure combined with oxygen pressure, seasonal depending high concentration of ozone and cosmic radiation may also be risk factors. Further risks on long haul flights may be jet lag, risk of economy class syndrome and contact with permethrin, because of disinsection before take-off out of tropical regions. The aim of an airline is to offer good cabin air quality, comfort and security for all passengers, may they be healthy, old, disabled or sick. Medical incidences on board arise and passengers expect modern equipment such as doctor's kit and defibrillators.

Aerospace Medicine↗

The German Environmental Survey 1990/1992 (GerES II): reference concentrations of selected environmental pollutants in blood, urine, hair, house dust, drinking water and indoor air.

The German Environmental Survey (GerES) is a large-scale, representative population study that has been carried out three times up to now with a time interval of about 7 years. GerES I was performed in 1985/1986, GerES IIa in 1990/1991 in West Germany, and GerES IIb in 1991/1992 in East Germany, the former German Democratic Republic (GDR). In GerES II, blood, urine, and scalp hair samples of 4021 adults aged 25-69 years and of 736 children aged 6-14 years were analysed as well as environmental samples (house dust, drinking water, indoor and personal air, diet). Characteristics of the frequency distributions of the substances analysed in the different media were calculated. The geometric mean (GM) for lead, cadmium, and mercury in the blood of adults amounted to 45.3, 0.36, and 0.51 microg/l, respectively. The corresponding values of arsenic, cadmium, and mercury in urine were 6.3, 0.29, and 0.54 microg/l, respectively. The concentrations of lead in blood, cadmium in blood and urine, and mercury in blood are lower in children than in adults. The GM ofpentachlorophenol (PCP) in urine of adults was 2.67 microg/l and in urine of children, 4.15 microg/l. These results of GerES II were compared with the so-called HBM values which represent health-based exposure guidelines and have been defined by the Human Biomonitoring Commission (HBC) of the Federal Environmental Agency, interalia for lead in blood, cadmium in urine, mercury in blood and urine, and PCP in urine. They also provided asound basis for the setting of reference values to describe the status of the German population. A total of 1.8% and 0.6% of the German females in child-bearing age had a level of lead in blood higher than HBM-I (100 microg/l) and HBM-II ( 150 microg/l), respectively. One percent of the children had a blood lead level above HBM-I. House dust and drinking water were analysed to characterise exposure in the domestic environment. Arsenic, cadmium, and lead deposition in homes amounted to 5.4 ng/(m2 day), 11.7 ng/(m2 day), and 0.29 microg/(m2 day), respectively. In the content of vacuum cleaner bags, concentrations were 2.1,0.9, and 5.9 microg/g. PCP, lindane and permethrin could be detected in the house dust of most German households. The pollutant load of the drinking water is significantly influenced by the corrosion of pipe materials and fittings. The new EC limit value of 10 microg/l for lead was exceeded in 7.7% of the first draw samples. The relatively high percentage (14%) of samples from East Germany that exceeded the current German guideline value for formaldehyde of 0.1 ppm in indoor air may be explained by the widespread use of contaminated particleboard in the former GDR.

Adolescent↗

Transfer efficiencies of pesticides from household flooring surfaces to foods.

The transfer of pesticides from household surfaces to foods was measured to determine the degree of excess dietary exposure that occurs when children's foods contact contaminated surfaces prior to being eaten. Three household flooring surfaces (ceramic tile, hardwood, and carpet) were contaminated with an aqueous emulsion of commercially available pesticides (diazinon, heptachlor, malathion, chlorpyrifos, isofenphos, and cis- and trans-permethrin) frequently found in residential environments. A surface wipe method, as typically used in residential exposure studies, was used to measure the pesticides available on the surfaces as a basis for calculating transfer efficiency to the foods. Three foods (apple, bologna, and cheese) routinely handled by children before eating were placed on the contaminated surfaces and transfers of pesticides were measured after 10 min contact. Other contact durations (1 and 60 min) and applying additional contact force (1500 g) to the foods were evaluated for their impact on transferred pesticides. More pesticides transferred to the foods from the hard surfaces, that is, ceramic tile and hardwood flooring, than from carpet. Mean transfer efficiencies for all pesticides to the three foods ranged from 24% to 40% from ceramic tile and 15% to 29% from hardwood, as compared to mostly non-detectable transfers from carpet. Contact duration and applied force notably increased pesticide transfer. The mean transfer efficiency for the seven pesticides increased from around 1% at 1 min to 55- 83% when contact duration was increased to 60 min for the three foods contacting hardwood flooring. Mean transfer efficiency for 10-min contact increased from 15% to 70% when a 1500 g force was applied to bologna placed on hardwood flooring. Contamination of food occurs from contact with pesticide-laden surfaces, thus increasing the potential for excess dietary exposure of children.

Air Pollution, Indoor↗

Pesticides and their metabolites in the homes and urine of farmworker children living in the Salinas Valley, CA.

In support of planning efforts for the National Children's Study, we conducted a study to test field methods for characterizing pesticide exposures to 20 farmworker children aged 5-27 months old living in the Salinas Valley of Monterey County, California. We tested methods for collecting house dust, indoor and outdoor air, dislodgeable residues from surfaces and toys, residues on clothing (sock and union suits), food, as well as spot and overnight diaper urine samples. We measured 29 common agricultural and home use pesticides in multiple exposure media samples. A subset of organophosphorus (OP), organochlorine (OC) and pyrethroid pesticides were measured in food. We also analyzed urine samples for OP pesticide metabolites. Finally, we administered four field-based exposure assessment instruments: a questionnaire; food diary; home inspection; and a self-administered child activity timeline. Pesticides were detected more frequently in house dust, surface wipes, and clothing than other media, with chlorpyrifos, diazinon, chlorthal-dimethyl, and cis- and trans-permethrin detected in 90% to 100% of samples. Levels of four of these five pesticides were positively correlated among the house dust, sock, and union suit samples (Spearman's rho=0.18-0.76). Pesticide loading on socks and union suits was higher for the group of 10 toddlers compared to the 10 younger crawling children. Several OP pesticides, as well as 4,4'-DDE, atrazine, and dieldrin were detected in the food samples. The child activity timeline, a novel, low-literacy instrument based on pictures, was successfully used by our participants. Future uses of these data include the development of pesticide exposure models and risk assessment.

Agriculture↗

Rapid preconcentration method for the determination of pyrethroid insecticides in vegetable oils and butter fat and simultaneous determination by gas chromatography-electron capture detection and gas chromatography-mass spectrometry.

A simple and rapid solid-phase extraction (SPE)-GC method for the preconcentration and quantification of pyrethroids at low nanogram levels in oils and high fat content samples is presented. The method was studied using seven highly persistent pyrethroid insecticides, viz., cypermethrin, deltamethrin, fenvalerate, cyfluthrin, allethrin, cyhalothrin and permethrin. Preconcentration was achieved by treating the oil samples with methyltrioctylammonium chloride and subsequent elution of the pyrethroid molecules from a graphitized carbon black SPE cartridge using 5 ml of acetonitrile. Pyrethroid quantification was achieved by GC with electron capture detection. Recoveries of the pyrethroids at fortification levels of 0.05-0.5 ppm were 94-105%. Storage on graphitized carbon black for 30 d lowered the recovery of the pyrethroids by only 3-6%. The method compared well with results obtained by a GC-MS method. The relative standard deviation at a concentration level of 0.05-0.2 microgram ml-1 ranged from 1.31 to 5.16%. The limit of detection achieved was 0.002 microgram ml-1 without any additional clean-up and with little interference from lipids during analysis.

Animals↗

Multiresidue method for the analysis of multiclass pesticides in agricultural products by gas chromatography-tandem mass spectrometry.

A multiresidue method is described for determining 55 organophosphorus and organochlorinated compounds and pyrethroids commonly used in crop protection. Pesticide residues are extracted from samples with a mixture of ethyl acetate and sodium sulfate, obtaining a final preconcentration of I mg sample (ml extract)(-1). No additional clean-up steps are necessary. Analysis is performed by gas chromatography by using a combination of positive chemical ionisation (PCI) and electron impact (EI) ionisation modes and tandem mass spectrometry (GC-PCI/EI-MS-MS). Good sensitivity and selectivity of the method are obtained with limits of detection (LODs) ranging from 0.07 to 4.21 microg kg(-1) in all the cases, except for methamidophos, permethrin, cypermethrin and difenconazol. Average recoveries between 52 and 114% are obtained and good linearity is observed in the studied ranges (r > or = 0.994). The RSD values are < or = 29% in all the cases. The method has been applied to the analysis of 178 vegetable samples, as a part of the monitoring programme of the Association of Producers and Exporters of Fruits and Vegetables of Almería (COEXPHAL) and quality control systems applied during the assays have demonstrated a good performance and stability with time.

Food Contamination↗

Demodex folliculorum and topical treatment: acaricidal action evaluated by standardized skin surface biopsy.

A standardized skin surface biopsy was performed in 34 patients suffering from skin diseases with high Demodex folliculorum density (Dd) > 5D/cm2 before, during and after topical treatment. The patients were randomized into six comparable groups to study six topical treatments: metronidazole 2%, permethrin 1%, sublimed sulphur 10%, lindane 1%, crotamiton 10% and benzyl benzoate (BB) 10%. Their acaricidal activity was measured according to three criteria: (i) for each treatment, decrease of Dd to under the normal threshold (< or = 5 D/cm2); (ii) for each treatment, a significant decrease in Dd; and (iii) comparison of the relative difference in Dd between treatments. These three criteria converged to establish the acaricidal activity of BB on D. folliculorum; the efficacy of crotamiton was demonstrated by the second criterion. An important irritating effect was observed with BB and sulphur.

Administration, Cutaneous↗

Scabies presenting as a necrotizing vasculitis in the presence of lupus anticoagulant.

A 75-year-old man presented with a progressive eruption of 6 weeks' duration. Purpura and focal areas of cutaneous infarction were present on the legs. There were scabies burrows on the wrists and hands, and a mite was extracted. Histology of a skin biopsy showed a necrotizing leucocytoclastic vasculitis. Circulating lupus anticoagulant was detected. Treatment consisted of topical 5% permethrin cream and systemic steroids.

Aged↗

Crusted (Norwegian) scabies in a patient with dystrophic epidermolysis bullosa.

A 13-year-old girl with severe non-mutilating recessive dystrophic epidermolysis bullosa (EB) was admitted to hospital because of a Staphyloccus aureussepsos, deterioration of her general condition and worsening of her skin disease, which itched severely. In addition to the blisters and erosions normally seen, she was covered from head to toe with scales and hyperkeratotic crusts. Despite intensive topical therapy, her skin condition did not improve significantly until scabies was detected and treated 1 week after admission. Because of the huge number of mites found and the crusted appearance, a diagnosis of crusted (Norwegian) scabies was made. She was successfully treated with two doses of ivermectin orally and one application of lindane ointment. Permethrin cream was not tolerated. In this patient crusted scabies may have developed because of: (i) a modified host response due to malnourishment; (ii) inability to scratch because of the absence of fingernails; and (iii) abnormal scratching behaviour because of the vulnerability of EB skin, or a combination of these factors. Limited isolation measures were taken on admission and full measures were taken immediately after the diagnosis of crusted scabies was made. Prophylactic treatment of ward personnel was not undertaken. Fortunately, there was not an outbreak of scabies in the hospital.

Adolescent↗

Rosacea-like demodicidosis associated with acquired immunodeficiency syndrome.

We present a 35-year-old patient with acquired immunodeficiency syndrome who had demodicidosis on his face, characterized by multiple papules and papulopustules, associated pruritus, numerous mites on skin-surface biopsy and in biopsy specimens, and rapid response to topical treatment with permethrin. It seems likely that Demodex infestation does not manifest unless local or systemic immune function is altered, leading to the proliferation of the organism and subsequent disease.

AIDS-Related Opportunistic Infections↗

Induction of cytochrome P450 CYP6B7 and cytochrome b5 mRNAs from Helicoverpa armigera (Hubner) by pyrethroid insecticides in organ culture.

An organ culture system derived from Helicoverpa armigera has been used to study the expression of cytochrome P450 and cytochrome b5 mRNAs. Northern analysis showed that levels of the mRNAs for cytochrome P450s, CYP6B2, CYP6B6 and CYP6B7, and cytochrome b5 in control tissue were commensurate with those in the tissue of whole larvae. Substantial induction of cytochrome P450, CYP6B7 and cytochrome b5 mRNAs by alpha-pinene, and the pyrethroids, fenvalerate, cypermethrin and permethrin were observed in fat body culture. Neither mRNA was induced, either in midgut or integument organ cultures. In contrast, the relatively water-soluble compound phenobarbital, could induce CYP6B7 mRNA but not cytochrome b5 mRNA in fat body cultures. As for pyrethroids, phenobarbital had no effect on the other tissues in culture. These results confirm a previous conclusion that pyrethroids could induce CYP6B7 mRNA, which was based upon a very slight induction observed in living insects. Because many cytochrome P450 substrates can act as their inducers, these results support a previous conclusion that CYP6B7 could be the enzyme that is involved in pyrethroid resistance in H. armigera.

Animals↗

Cytochrome P450 CYP6X1 cDNAs and mRNA expression levels in three strains of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae) having different susceptibilities to pyrethroid insecticide.

Three cDNAs, cloned from both pyrethroid-susceptible and -resistant strains of Lygus lineolaris, contained a 1548 nucleotide open reading frame encoding a 516 amino acid residue protein. Predicted cytochrome P450s from cDNAs were classified as the first three new members of subfamily CYP6X, CYP6X1v1 for a susceptible strain and CYP6X1v2 and CYP6X1v3 for two resistant strains. Putative cytochrome P450 CYP6X1s from L. lineolaris were highly similar (up to 42% amino acid sequence identity) to several insect CYP6s that are responsible for reduced sensitivity to pyrethroid insecticides. A total of twenty-six nucleotide substitutions were revealed between cDNAs of susceptible and resistant strains. Two nucleotide substitutions resulted in amino acid changes, Asp373 to Ala373 and Ser487 to Ala487, between susceptible and resistant strains. The resistant laboratory strain contained 2.1-fold higher cytochrome P450 mRNA per microgram total RNA than the susceptible laboratory strain. Topical treatment with 10 ng permethrin elevated cytochrome P450 mRNA levels by approximately 2-fold. The results of this study indicated that cytochrome P450 gene mutation, coupled with up-regulation, was present only in the pyrethroid resistant strains, and was possibly related to resistance development in the tarnished plant bug.

Alleles↗

Pyrethroid resistance in tropical bedbugs, Cimex hemipterus, associated with use of treated bednets.

When Tanzanian villages were provided with pyrethroid-treated bednets, bedbugs (Cimicidae) disappeared; however, after about 6 years they have re-appeared in these villages. Using a newly devised test-kit, susceptibility tests of bedbugs Cimex hemipterus (Fabricius) from five of these villages showed that there is resistance to permethrin and alphacypermethrin in bedbugs from each of the villages, in contrast to those from five villages without treated nets. Circumstantial evidence indicates that bedbug resistance to pyrethroid insecticides may evolve more readily in villages with incomplete coverage rates of treated bednets, allowing bedbug infestations to become re-established. Bedbugs have not returned to a village where nearly all the beds have been provided with pyrethroid-treated bednets for 14 years.

Animals↗

Origin and prevention of airport malaria in France.

Since 1969, 63 cases of airport malaria have been reported in Western Europe, 24 of which occurred in France. Most were due to Plasmodium falciparum. In 1994, 7 cases occurred in and around Roissy Charles de Gaulle airport (CDG), showing 4 types of contamination: among employees working on airstrips or opening containers, among residents living near the airport, among people living at some distance from the airport after a secondary transport of vectors, and by vectors transported in luggage. In-flight or stop-over infection is not considered as airport malaria. The infective anophelines originated from airports where malaria transmission occurs, mostly in subsaharan Africa. A tentative list is given taking into account aerial traffic with France. Surveys in the airports of Dakar (Senegal), Cotonou (Benin), Abidjan (Cote d'Ivoire) and Yaoundé (Cameroun) found potential vectors in all of these from July to September. After 1994, the Contrôle Sanitaire aux Frontières (CSF) in charge at CDG concentrated its efforts on the flights at risk, as well as information and sensitization of airline companies, which resulted in 73% and 87% of the flights at risk being properly disinsected in 1995 and 1996. Despite pyrethroid resistance in Anopheles gambiae s.s. in West Africa, the efficacy of aircraft spraying with permethrin aerosols is still acceptable. However, surveillance of resistance should be improved and search for nonpyrethroid insecticides suitable for aircraft strongly encouraged.

Africa, Western↗

A new strategy for treating nets. Part 2: users' perceptions of efficacy and washing practices and their implications for insecticide dosage.

The conventional way to treat a mosquito net with pyrethroid insecticide is to apply a standard dosage every 6-12 months, and to avoid washing the net until just before retreatment. In some places, nets are normally washed much more often than this, and it may then be more appropriate to apply smaller amounts of insecticide after each wash. The choice of strategy must take into account not only biological effectiveness, but also users' perceptions of this effectiveness and their net-washing habits. We used focus groups to compare users' responses to nets treated with different dosages and chemicals. One hundred current net users in urban Dares-Salaam were each given a net that had been pretreated either with permethrin (200 or 500 mg/m2), or with lambdacyhalothrin (3 or 15 mg/m2), or with water. Neither participants nor investigators knew which group had received which treatment. Focus group discussions were held after 2, 8 and 12 weeks. Participants greatly preferred treated nets. Low doses were perceived to be less effective, especially after 8 and 12 weeks. After 12 weeks most participants had washed their nets, despite requests to the contrary Dirty nets were regarded as unhealthy and socially unacceptable. Few participants experienced side-effects or expressed fears about the safety of treatment. We conclude that asking people to refrain from washing their nets is unrealistic. A 'low-dose frequent-treatment' strategy of insecticide application may be more appropriate in the long run. At first, however, low doses give perceptibly inferior protection. An initial high (loading) dose, followed by frequent lower (maintenance) dosages, might solve this problem.

Adolescent↗

Head louse infestations: epidemiologic survey and treatment evaluation in Argentinian schoolchildren.

BACKGROUND: Our aim was to demonstrate that the treatment of individual cases is effective, but not sufficient, to control endemic Pediculus capitis, and that eradication of the epidemiologic school focus may lower significantly the prevalence of infestation. Statistical data on the degree of infestation relating to socio-economic and cultural variables were also updated. Therapeutic effects and educational impact were evaluated. METHODS: An educational and motivational program was designed for pupils, parents, and teachers: 326 children and 15 adults were subjected to clinical and parasitologic evaluation. The recorded parameters included the age, sex, hair style and length, presence of other dermatologic diseases, degree of infestation, clinical remission, parasitologic remission, dwelling type and features, need to share a bed with co-dwellers, availability of home tap water supply, level of family income, and periodic medical controls. The entire population received treatment with neutral shampoo and rinsing cream containing 1% permethrin. Exclusion criteria were the presence of acute scalp inflammation and a history of pyrethrin and/or pyrethroid sensitivity. Statistical analysis was performed as required on data expressed as frequencies, percentages, means, and standard deviations by chi-square and Fisher exact tests. RESULTS: The overall infestation prevalence rate was 81.5%, the highest values corresponding to children from 6 to 11 years of age, with a slight predominance in males (55.4% vs. 44.6%). A significantly greater rate of clinical remission was observed in subjects enjoying home tap water supplies (p < 0.01). CONCLUSIONS: The model of research plus action adopted allows the following conclusions to be drawn: (i) individual and isolated treatments for pediculosis are useful, but will not by themselves allow for the epidemiologic control of this parasitosis; (ii) massive, complete, and simultaneous treatments lead to a significant decrease in infestation prevalence; (iii) educational measures tending to foster collective awareness enable greater epidemiologic surveillance to be achieved; (iv) careful inspection of the entire scalp is essential with the use of a powerful light source and lenses with high magnification, as the parasite has no predilection for any given area; (iv) socio-economic and cultural conditions are not relevant for infestation, although a good home tap water supply is essential for treatment.

Adolescent↗