Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions

Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b₅ knockout mouse livers to validate P450 activation and establish the role for b₅ in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or “pyrethrome.” Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450–insecticide interactions and aiding the development of unique tools for disease control.Pyrethroids are synthetic analogs of pyrethrins, botanical chemicals derived from chrysanthemum flowers (1). They are highly potent insecticides with low mammalian toxicity that are used worldwide in ∼3,500 registered products in agricultural, medicinal, veterinary, and public health sectors. Importantly, they are the only class of insecticide recommended for insecticide-treated nets for malaria control. More than 254 million insecticide-treated nets were distributed across Africa between 2008–2010 (2). Similar to antibiotics, pyrethroids are critical for controlling a diverse spectrum of diseases. Unfortunately, similar to antibiotics, such intense exposure affects health and drives the rapid evolution of insecticide resistance (3).Pyrethroids are structurally highly diverse (4) but share a common architecture comprising a cyclopropane acid group coupled to an alcohol moiety, as exemplified by deltamethrin (Fig. 1A). Traditionally, they are divided into two classes (type 1 and type 2), depending on the absence (type 1) or presence (type 2) of an α-cyano group (Fig. 1B). Pyrethroids work by blocking the voltage-gated sodium channels, causing paralysis in arthropods, and resulting in death (3). Resistance is commonly associated with target site modification or metabolic resistance, in which increased rates of biotransformation, generally by P450s, esterases, and GSTs, reduce toxic potency (3).Open in a separate windowFig. 1.Conversion of deltamethrin into PyABPs. (A) Structure of deltamethrin with constituent acid and alcohol moieties. Primary sites of P450 hydroxylation are indicated by bold arrows at the 2′ and 4′ positions, and minor routes of hydroxylation are indicated with open arrows (1). (B) Conversion of deltamethrin to a PyABPP involves the addition of an alkyne warhead and a clickable handle. The structures of the general probe and the PyABPs synthesized are illustrated. Alkyne warhead groups were located in the 2′ or 4′ positions, whereas alkyne click handles replaced the cyano group (type 1) or terminal bromides (type 2). The general P450 probe 2-EN is boxed parallel to its type 1 pyrethroid analog, P2.Although the toxicity and metabolism of pyrethroids in mammals and insects have been extensively characterized (1), the role of specific enzymes and pathways involved in pyrethroid clearance is unclear. In insects, P450s are key enzymes involved with metabolic degradation, with constitutive overexpression of specific P450s leading to pyrethroid resistance (5, 6). Similarly, in mammals, the toxic potency of pyrethroids is inversely related to their rates of metabolic elimination (7), with both P450 oxidation and carboxyl esterase-mediated hydrolysis playing major roles. Humans have 57 P450 genes, rodents ∼80 P450 genes, and insects up to ∼200 P450 genes (8). Where genome information exists, genetic and microarray-based studies of pyrethroid-resistant versus susceptible populations have been used to identify P450s potentially capable of pyrethroid metabolism (3, 5). However, relatively few P450s have been functionally validated through recombinant P450 expression. Thus, probes able to identify pyrethroid-metabolizing enzymes directly would aid our understanding of the fundamental processes of insecticide–organism interactions, expanding our understanding of the risks of pyrethroid exposure to mammals and the enzymatic mechanisms of metabolism and resistance used by insects.Activity-based protein profiling (ABPP) has been described for the functional profiling of P450s (9, 10). The activity-based probes (ABPs) work in a mechanism-dependent manner to covalently label P450s, whereby the labeling events are detectable by adding a fluorescent reporter group via copper-catalyzed azide-alkyne cycloaddition (“click chemistry”) onto the probe−P450 adducts (9, 10). Furthermore, affinity tags can also be incorporated to pull down and identify probe–P450 adducts. The major advantage of ABPPs is their ability to directly assess enzyme activity. In this article, we have designed and synthesized a group of seven pyrethroid mimetic ABPs (PyABPs) on the basis of the deltamethrin scaffold (Fig. 1B) for the targeted identification of pyrethroid-metabolizing P450s in highly divergent organisms. We have investigated their reactivity profiles against pyrethroid metabolizing and nonmetabolizing recombinant mosquito P450s and mouse and rat liver microsomes. We show that PyABPs can be used to reveal pyrethroid structure–activity relationships, and they also have been used to identify pyrethroid-reactive P450s and related detoxification enzymes in rat liver microsomes, demonstrating their potential for directly assessing pyrethroid-metabolizing enzyme activity.     

Evaluation of long-lasting insecticidal nets after 2 years of household use

Development of long-lasting insecticidal nets (LLINs) may eliminate the need for insecticide retreatment of ITNs. While two LLINs (Olyset, Sumitomo Chemical Co., Japan; and PermaNet 1.0, Vestergaard-Frandsen, Denmark) have received recommendations from the World Health Organization Pesticide Evaluation Scheme, field-testing under normal use has been limited. We used a survival analysis approach to compare time to net failure of conventional polyester bednets treated only with deltamethrin to two LLINs and two candidate LLINs (Olyset; PermaNet; Insector, Athanor, France; and Dawa, Siamdutch Mosquito Netting Co., Thailand). Additionally, we evaluated nets treated with a process designed to increase the wash-durability of permethrin-treated nets through the addition of cyclodextrin (a starch) in the treatment process. Houses in western Kenya were randomly assigned to one of the six net types and nets were distributed to cover all sleeping spaces. Households were visited monthly to assess reported side effects in inhabitants and washing frequency. Nets were evaluated for insecticidal activity by periodic WHO cone bioassays with mortality assessed at 24 h. Nets with bioassay mortality <70% were assayed monthly until failure, defined as the first of two consecutive bioassay mortality rates <50%. Time to failure was analyzed using an extended Cox Proportional Hazards model controlling for the cumulative number of washes. We distributed 314 nets to 177 households in June-July 2002; 22 nets (7.0%) were lost to follow-up and 196 (62.4%) failed during the first 2 years of the evaluation. Controlling for cumulative number of washes, PermaNet 1.0 [Hazard Ratio (HR) 0.14, 95% Confidence Interval (CI) 0.06-0.31] had a significantly lower risk of failure than conventional nets while Insector had a significantly higher risk of failure (HR 2.57, 95% CI 1.06-4.15). The risks of failure of the remaining nets (Olyset: HR 1.29, 95% CI 0.79-2.10; Dawa: HR 0.58, 95% CI 0.32-1.18; cyclodextrin: HR 0.65, 95% CI 0.40-1.1) were not significantly different from that of a conventional net. PermaNet 1.0 performed significantly better than conventional nets and should be recommended to malaria control programs.     

Insecticide-treated bednets reduce mortality and severe morbidity from malaria among children on the Kenyan coast

New tools to prevent malaria morbidity and mortality are needed to improve child survival in sub-Saharan Africa. Insecticide treated bednets (ITBN) have been shown, in one setting (The Gambia, West Africa), to reduce childhood mortality. To assess the impact of ITBN on child survival under different epidemiological and cultural conditions we conducted a community randomized, controlled trial of permethrin treated bednets (0.5 g/m²) among a rural population on the Kenyan Coast. Between 1991 and 1993 continuous community-based demographic surveillance linked to hospital-based in-patient surveillance identified all mortality and severe malaria morbidity events during a 2-year period among a population of over 11 000 children under 5 years of age. In July 1993, 28 randomly selected communities were issued ITBN, instructed in their use and the nets re-impregnated every 6 months. The remaining 28 communities served as contemporaneous controls for the following 2 years, during which continuous demographic and hospital surveillance was maintained until the end of July 1995. The introduction of ITBN led to significant reductions in childhood mortality (PE 33%, CI 7–51%) and severe, life-threatening malaria among children aged 1–59 months (PE 44%, CI 19–62). These findings confirm the value of ITBN in improving child survival and provide the first evidence of their specific role in reducing severe morbidity from malaria.     

Free distribution of insecticide treated bed nets to pregnant women in Kinshasa: an effective way to achieve 80% use by women and their newborns

Objective  To determine whether long lasting insecticide treated bed nets (LLINs) distributed free of charge to pregnant women at their first antenatal clinic visit in Kinshasa, DRC are used from the time of distribution to delivery and 6 months after delivery.
Methods  Women were enrolled into a cohort study at their first antenatal care (ANC) visit and provided LLINs free of charge. Reported use of these nets was then measured at the time of delivery ( n  = 328) and in a random sample of women ( n  = 100) 6 months post-delivery using an interviewer administered, structured questionnaire.
Results  At baseline, only 25% of women reported having slept under a bed net the night before the interview. At the time of delivery, after being provided an LLIN for free, this increased to 79%. Six months post-delivery ( n  = 100), 80% of women reported sleeping under a net with a child under the age of 5 the night before the interview.
Conclusions  Freely distributed bed nets are acceptable, feasible and result in high usage. Free distribution of bed nets during antenatal clinic visits may be a highly effective way to rapidly increase the use of bed nets among both pregnant women and their newborn infants in areas with high levels of ANC attendance.     

Insecticide-treated bednets for the prevention of Plasmodium falciparum malaria in Cambodia: a cluster-randomized trial

OBJECTIVES: To validate and quantify the impact of insecticide-treated bednets (ITN) on malaria morbidity and mortality in Cambodia. METHODS: A paired, cluster-randomized trial of ITN was conducted in Rattanakiri, North East Cambodia. Thirty-four villages with a total population of 10,726 were randomized to receive deltamethrin-impregnated bednets or to control (no net provision). Cross-sectional surveys measured Plasmodium falciparum prevalence at baseline and 10 months after ITN distribution. Village malaria volunteers in control and intervention villages treated dipstick-positive P. falciparum cases with artesunate and mefloquine. The resulting passive surveillance data were used as an estimate of the incidence of clinical P. falciparum infections. RESULTS: There was a protective efficacy of 28% in P. falciparum incidence (adjusted rate ratio 0.72, 95% CI 0.47-1.08) and 9% in P. falciparum prevalence (adjusted prevalence ratio 0.91, 95% CI 0.65-1.28) in ITN relative to control villages; however, neither of these estimates reached statistical significance. Individual-level analysis indicated a greater reduction in P. falciparum prevalence among under 5-year-olds (adjusted OR = 0.63, 95% CI 0.26-1.53) compared to older individuals (interaction P = 0.042). The protective efficacy of 35% (95% CI -28, 67%) with respect to clinical P. falciparum incidence in under 5-year-olds was more pronounced than the corresponding estimates for prevalence but was again not significant. CONCLUSIONS: Lack of statistical significance in the results is likely to be due to a lack of power. The analysis provides further evidence for ITN effectiveness in South East Asia, particularly among individuals under 5 years of age.     

A health facility based case-control study of effectiveness of insecticide treated nets: potential for selection bias due to pre-treatment with chloroquine

Case control studies offer an attractive way to assess the effectiveness of insecticide treated nets (ITN) under programme conditions but have the drawback of being susceptible to bias in the choice of controls. We evaluated the potential for pre-treatment with chloroquine to result in misclassification of cases and controls and affect estimates of ITN effectiveness in case control studies in urban and rural clinics in Eastern Afghanistan. During the one-month study, use of ITN showed no effect against malaria in the urban clinic (adjusted odds ratio OR 1.08; 95% CI 0.73-1.6) and the protective effect seen in the rural clinic was not significant (OR 0.62; 95% CI 0.2-2.4). Levels of pre-treatment were high in both clinics: 24% in urban and 19% in rural clinic attenders. In the urban clinic attenders the level of pre-treatment between bed net users and non-users was not significantly different (OR 1.07, 95% CI 0.70-1.64); therefore the misclassification of cases as controls did not introduce any selection bias. Amongst rural clinic attenders, bed net users were less likely to pre-treat with chloroquine than users (OR 0.33, 95% CI 0.14-0.77); this introduced a selection bias that resulted in an underestimation of the effectiveness of bed nets. Case control studies using health facility data are liable to selection bias especially in areas of high pre-treatment rates with chloroquine. Generalisation of results over a wide geographic region, or between urban and rural settings, may not be appropriate.     

Malaria control by residual insecticide spraying in Chingola and Chililabombwe,Copperbelt Province,Zambia

Malaria is endemic in the whole of Zambia and is the leading cause of morbidity and mortality. Prior to 1980, effective malaria control was achieved in the northern mining towns of Chingola and Chililabombwe by means of annual residual spraying programmes. In the 1970s, incidence rates were as low as 20/1000 p.a., but by 2000 had increased to 68/1000 p.a. in Chingola and to 158/1000 p.a.in Chililabombwe. Konkola Copper Mines (KCM) initiated a malaria control programme in which all dwellings in the two towns and within a 10-km radius were sprayed with either dichlorodiphenyltrichloroethane or a synthetic pyrethroid (Icon by ZENECA or Deltamethrin by Aventis). Houses were sprayed in November and December 2000, at the start of the peak transmission period. There was a statistically significant reduction in malaria incidence recorded at KCM health facilities in the two towns, representing a protective incidence rate ratio of 0.65 (95% CI 0.44, 0.97) when comparing the post-spraying period with the corresponding period of the previous 2 years. This reduction followed a single round of house spraying during a year with higher rainfall than the preceding two and in an area where chloroquine was first-line treatment. This house-spraying programme is an example of private/public sector collaboration in malaria control.     

四川省疟疾防治中的环境因素分析

目的了解环境因素变化对疟疾流行和防治的影响。方法调查农村历年水稻耕作方式 ,历年农药使用情况 ,农村人居环境以及经济变化情况等。对比分析这些变化对媒介按蚊孳生繁殖和疟疾流行的影响。结果水稻推行湿润灌溉和农药使用量逐年加大 ,人房和牛房的媒介按蚊密度分别下降 71 .43 %和 86.73 % ,农村人居环境改善 ,人蚊接触减少 ,疟疾病例减少 ,流行强度减弱 ,流行季节高峰削弱 ,流行面逐渐缩小。结论水稻耕作方式的变化和农药的使用 ,有利于控制媒介按蚊孳生繁殖 ;农村人居环境改善可遏制疟疾流行     

Socio-economic factors associated with the purchasing of insecticide-treated nets in Afghanistan and their implications for social marketing

Malaria is often a major health problem in war-torn countries in the tropics owing to the collapse of health services and the vulnerability of displaced populations to epidemics. Insecticide-treated nets (ITN) represent one of the few options for obtaining protection against malaria in unstable settings deficient in health infrastructure. Social marketing of subsidized ITN by a consortium of non-governmental organizations began in Afghanistan in 1993 and has continued every year since then despite regular political turmoil. Almost 350,000 nets have been sold and approximately 1.2 million people protected. In 2000 we examined the determinants of ITN purchasing among households in Nangarhar province, eastern Afghanistan, as part of an effort to increase ITN uptake. The survey was conducted using a structured questionnaire to collect data on socio-economic characteristics and malaria beliefs and practices among more than 400 net-owning and non-net-owning households. A composite socio-economic index was created using principal components analysis, and survey households were divided into socio-economic quartiles. ITN were 4.5 times more likely to be purchased by families from the richest quartile and 2.3 times more likely to be purchased from the upper-middle quartile than from the two lower quartiles. Even so, a significant minority from the lower quartiles did prioritize and buy ITN. In conflict affected countries where livelihoods are compromised, it is necessary to target subsidies at the most impoverished to make ITN affordable and to improve overall coverage.     

高效液相色谱法测定人体血中氯喹含量

为了深入了解氯喹药代动力学有关情况,用高效液相色谱检测4名志愿者口服氯喹总量分别为600mg、600mg、900mg和1200mg后,血药浓度168h后尚有141μg/L、134μg/L、208μg/L和298μg/L,均明显高于有效杀灭疟原虫20μg／L浓度。用单剂600mg和首日600mg,次日300mg氯喹治疗间日疟现症病人各1例,追踪观察28h均无症状复发和原虫再现,获得痊愈。结果表明口服氯喹在人体内清除非常缓慢,维持有效杀灭疟原虫时间长,是治疗间日疟、三日疟和卵型疟的首选药。     

Community factors associated with malaria prevention by mosquito nets: an exploratory study in rural Burkina Faso

Malaria-related knowledge, attitudes and practices (KAP) were examined in a rural and partly urban multiethnic population of Kossi province in north-western Burkina Faso prior to the establishment of a local insecticide-treated bednet (ITN) programme. Various individual and group interviews were conducted, and a structured questionnaire was administered to a random sample of 210 heads of households in selected villages and the provincial capital of Nouna. Soumaya, the local illness concept closest to the biomedical term malaria, covers a broad range of recognized signs and symptoms. Aetiologically, soumaya is associated with mosquito bites but also with a number of other perceived causes. The disease entity is perceived as a major burden to the community and is usually treated by both traditional and western methods. Malaria preventive practices are restricted to limited chloroquine prophylaxis in pregnant women. Protective measures against mosquitoes are, however, widespread through the use of mosquito nets, mosquito coils, insecticide sprays and traditional repellents. Mosquito nets are mainly used during the rainy season and most of the existing nets are used by adults, particularly heads of households. Mosquito nets treated with insecticide (ITN) are known to the population through various information channels. People are willing to treat existing nets and to buy ITNs, but only if such services would be offered at reduced prices and in closer proximity to the households. These findings have practical implications for the design of ITN programmes in rural areas of sub-Saharan Africa (SSA).     

Dynamics of insecticide resistance in the malaria vector Anopheles gambiae s.l. from an area of extensive cotton cultivation in Northern Cameroon

Objective To explore temporal variation in insecticide susceptibility of Anopheles gambiae s.l. populations to the four chemical groups of insecticides used in public health and agriculture, in close match with the large‐scale cotton spraying programme implemented in the cotton‐growing area of North Cameroon. Methods Mosquito larvae were collected in 2005 before (mid June), during (mid August) and at the end (early October) of the cotton spraying programme. Larvae were sampled in breeding sites located within the cotton fields in Gaschiga and Pitoa, and in Garoua, an urban cotton‐free area that served as a control. Insecticide susceptibility tests were carried out with 4% DDT (organochlorine), 0.4% chlorpyrifos methyl (organophosphate), 0.1% propoxur (carbamate), 0.05% deltamethrin and 0.75% permethrin (pyrethroids). Results Throughout the survey, An. gambiae s.l. populations were completely susceptible to carbamate and organophosphate, whereas a significant decrease of susceptibility to organochlorine and pyrethroids was observed during spraying in cotton‐growing areas. Tolerance to these insecticides was associated with a slight increase of knockdown times compared to the reference strain. Among survivor mosquitoes, the East and West African Kdr mutations were detected only in two specimens of An. gambiae s.s. (n = 45) and not in Anopheles arabiensis (n = 150), suggesting metabolic‐based resistance mechanisms. Conclusions Environmental disturbance due to the use of insecticides in agriculture may provide local mosquito populations with the enzymatic arsenal selecting tolerance to insecticides.     

The immunology of malaria—a view from the bush

During the past few years a great deal has been learnt about malaria parasites and the immune responses that they evoke in their hosts. However, this new knowledge has so far had little impact on the practical problems of malaria control in tropical developing countries where malaria is still responsible for much mortality and morbidity. In Africa the malaria situation is now more serious than it was 20 years ago. Exciting new developments in molecular biology suggest that this situation may change in a few years time but attempts should be made to put the advances that have been made to the best practical use as soon as possible rather than delaying until even better technologies have been developed. This approach will require close collaboration between immunologists in sophisticated laboratories in industrialised countries and smaller laboratories in the countries of the developing world where malaria is still a major problem.     

江苏省恶性疟防治研究

为了解江苏省恶性疟的流行特点和媒介传疟作用，制定有效的防治对策以尽快控制和消灭恶性疟，我们通过多年大量的现场流行病学调查和实验研究，证实嗜人按蚊是江苏省传播恶性疟的主要媒介。根据调查和研究结果，在全省恶性疟流行区采取了大范围持续的室内滞留喷洒灭蚊，和药物浸泡蚊帐巩固灭蚊，同时抓好传染源和易感人群的综合防治措施，结果在较短时间内控制了恶性疟的流行，并大幅度降低了间日疟发病率。经多年疟疾监测证实，已连续８年未发生当地感染的恶性疟病例，达到基本消灭恶性疟的标准，取得了显著的防治效果。     

Exchange Transfusion for Severe Malaria

Background: Exchange transfusion (ET) is a controversial ancillary treatment of severe falciparum malaria. Patients and Methods: We conducted a retrospective analysis of severe malaria treated in our institution. Nine cases of ET were identified between 1991 and 1998 and compared to 12 controls with similar parasitemia. Results: Groups were similar at admission except for an increased age in the ET group (p < 0.02). All patients received iv quinine. Outcome was similar in both groups (two deaths in the ET group, three in the control group). However, in patients with parasitemia > 30%, the death rate was significantly lower in ET patients than in controls (0/4 vs 3/3, p < 0.029). Conclusion: Despite definitive data from controlled trials, we suggest that ET should be considered in severe malaria cases with very high parasitemia and severity criteria or worsening clinical condition despite adequate chemotherapy. Received: March 13, 2000 · Revision accepted: February 16, 2001     

Parasite-specific lactate dehydrogenase for the diagnosis of Plasmodium falciparum infection in an endemic area in West Uganda

The measurement of parasite lactate dehydrogenase (pLDH) has been presented as an easy and rapid method for the diagnosis of malaria in humans. In order to evaluate the sensitivity and specificity of such a test we examined blood samples from 429 Ugandan patients. While pLDH activity was significantly linked to parasitaemia, sensitivity and specificity were found to be rather low at 58.8 and 62.2% respectively. The positive and negative predictive values failed to meet necessary standards. We conclude that the methods of measurement of pLDH activity in malaria infection, although potentially useful for the fast diagnosis of malaria, need to be improved to be of true value in endemic areas.