Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
Parasitol Res. 2011 Sep;109(3):531-7. doi: 10.1007/s00436-011-2280-0. Epub 2011 Feb 19.
Previous studies have shown that permethrin resistance in our selected PMD-R strain of Aedes aegypti from Chiang Mai, Thailand, was associated with a homozygous mutation in the knockdown resistance (kdr) gene and other mechanisms. In this study, we investigated the metabolic mechanism of resistance of this strain compared to the PMD strain which is susceptible to permethrin. The permethrin susceptibility of larvae was determined by a dose-response bioassay. Two synergists, namely piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate (BNPP), were also added to determine if the resistance is conferred by oxidase or esterase enzymes, respectively. The LC(50) value for PMD-R (25.42 ppb) was ∼25-fold higher than for PMD (1.02 ppb). The LC(50) was reduced 3.03-fold in PMD-R and 2.27-fold in PMD when the oxidase inhibitor (PBO) was added, but little or no reduction was observed in the presence of BNPP, indicating that oxidative enzymes play an important role in resistance. However, the LC(50) previously observed in the heterozygous mutation form was reduced ∼eightfold, indicating that metabolic resistance is inferior to kdr. The levels of cytochrome P450 (P450) extracted from fourth instar larvae were similar in both strains and were about 2.3-fold greater in microsomal fractions than in crude supernatant and cytosol fractions. Microsome oxidase activities were determined by incubation with each of three substrates, i.e., permethrin, phenoxybenzyl alcohol (PBOH), and phenoxybenzaldehyde (PBCHO), in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NAD(+)), PBO, and BNPP. It is known that hydrolysis of permethrin produces PBOH which is further oxidized to PBCHO by alcohol dehydrogenase (ADH) and then to phenoxybenzoic acid (PBCOOH) by aldehyde dehydrogenase (ALDH). When incubated with permethrin, a small amount of PBCOOH was detected in both strains (about 1.1-1.2 nmol/min/mg protein), regardless of the addition of NADPH. The addition of PBO resulted in about 70% and 50% reduction of PBCOOH in PMD and PMD-R, respectively. The addition of BNPP reduced PBCOOH about 50% and 35% in PMD and PMD-R, respectively. Using PBOH as substrate increased PBCOOH ∼16-fold and ∼40-fold in PMD and PMD-R, respectively. Using PBCHO as substrate increased PBCOOH ∼26-fold and ∼50-fold in PMD and PMD-R, respectively. The addition of NADPH, and particularly NAD(+), increased the level of PBCOOH. Together, the results have indicated the presence of a metabolic metabolism involving P450, ADHs, and ALDHs in both PMD and PMD-R strains, with greater enzyme activity in the latter.
先前的研究表明，我们在泰国清迈选择的埃及伊蚊 PMD-R 品系对氯菊酯的抗性与击倒抗性 (kdr) 基因的纯合突变和其他机制有关。在这项研究中，我们研究了与对氯菊酯敏感的 PMD 品系相比，该品系的代谢抗性机制。幼虫对氯菊酯的敏感性通过剂量反应生物测定来确定。还添加了两种增效剂，即增效醚 (PBO) 和双(4-硝基苯基)-磷酸酯 (BNPP)，以确定抗性是否由氧化酶或酯酶分别赋予。PMD-R 的 LC(50) 值 (25.42 ppb) 比 PMD (1.02 ppb) 高约 25 倍。当加入氧化酶抑制剂 (PBO) 时，PMD-R 的 LC(50) 降低了 3.03 倍，PMD 的 LC(50) 降低了 2.27 倍，但在 BNPP 存在下几乎没有或没有观察到降低，表明氧化酶在抗性中起着重要作用。然而，在杂合突变形式中观察到的 LC(50) 降低了约 8 倍，表明代谢抗性不如 kdr。从第四龄幼虫中提取的细胞色素 P450 (P450) 的水平在两种菌株中相似，并且在微粒体部分中比在粗上清液和胞质部分中高约 2.3 倍。通过在存在或不存在烟酰胺腺嘌呤二核苷酸磷酸 (NADPH)、烟酰胺腺嘌呤二核苷酸 (NAD(+))、PBO 和 BNPP 的情况下用三种底物，即氯菊酯、苯氧苄醇 (PBOH) 和苯氧苯甲醛 (PBCHO) 孵育来确定微粒体氧化酶活性。已知氯菊酯的水解产生 PBOH，其进一步被醇脱氢酶 (ADH) 氧化为 PBCHO，然后被醛脱氢酶 (ALDH) 氧化为苯氧苯甲酸 (PBCOOH)。用氯菊酯孵育时，在两种菌株中都检测到少量的 PBCOOH(约 1.1-1.2 nmol/min/mg 蛋白)，而与是否添加 NADPH 无关。添加 PBO 导致 PMD 和 PMD-R 中 PBCOOH 的减少分别约为 70%和 50%。添加 BNPP 分别导致 PMD 和 PMD-R 中 PBCOOH 的减少约 50%和 35%。使用 PBOH 作为底物分别使 PMD 和 PMD-R 中的 PBCOOH 增加了约 16 倍和 40 倍。使用 PBCHO 作为底物分别使 PMD 和 PMD-R 中的 PBCOOH 增加了约 26 倍和 50 倍。添加 NADPH，特别是 NAD(+)，增加了 PBCOOH 的水平。总的来说，这些结果表明 PMD 和 PMD-R 菌株中存在涉及 P450、ADHs 和 ALDHs 的代谢代谢，后者的酶活性更高。
