Death of guppy fish (Poecilia reticulata) leukocytes induced by in vivo exposure to temephos and spinosad.

Temephos and spinosad are pesticides used for control of vector-borne diseases such as dengue, chikungunya and zika. However, the inadequate use of these substances has affected the health of non-target organisms. The aim of this study was to evaluate and compare, the effects of temephos and spinosad on leukocyte viability and death, using guppy fish (Poecilia reticulate) as a model organism. Guppies were exposed to temephos (10 mg/L) and spinosad (0.5 mg/L) for 7, 14, and 21 days. Afterwards, they were placed in pesticide-free fish tanks (7, 35, and 70 days) for recovery. The results showed that exposure to temephos caused leukocyte death, even at 35 days of recovery. Contrarily, the exposure to spinosad did not cause leukocyte death. This research show, for the first time, that a single dose of temephos causes apoptosis up to 56 days post-exposition, indicating that this pesticide induces chronic effects on immune response cells.

Toxicokinetics of temephos after oral administration to adult male rats.

Temephos (Tem) is the larvicide of choice to control mosquito transmission of dengue, Zika, and chikungunya. The toxicokinetic and toxicological information of temephos is very limited. The aim of this work was to determine the toxicokinetics and dosimetry of temephos and its metabolites. Male Wistar rats were orally administered temephos (300 mg/kg) emulsified with saline solution and sacrificed over time after dosing. Temephos and its metabolites were analyzed in blood and tissues by high performance liquid chromatography-diode array detector. At least eleven metabolites were detected, including temephos-sulfoxide (Tem-SO), temephos-oxon (Tem-oxon), temephos-oxon-sulfoxide (Tem-oxon-SO), temephos-oxon-SO-monohydrolyzed (Tem-oxon-SO-OH), 4,4´-thiodiphenol, 4,4´-sulfinyldiphenol, and 4,4´-sulfonyldiphenol or bisphenol S (BPS). The mean blood concentrations of temephos were fitted to a one-compartment model for kinetic analysis. At 2 h, the peak was reached (t1/2 abs = 0.38 h), and only trace levels were detected at 36 h (t1/2 elim = 8.6 h). Temephos was detected in all tissues and preferentially accumulated in fat. Temephos-sulfone-monohydrolyzed (Tem-SO2-OH) blood levels remained constant until 36 h and gradually accumulated in the kidney. Tem-oxon was detected in the brain, liver, kidney, and fat. Clearance from the liver and kidney were 7.59 and 5.52 ml/min, respectively. These results indicate that temephos is well absorbed, extensively metabolized, widely distributed and preferentially stored in adipose tissue. It is biotransformed into reactive metabolites such as Tem-oxons, Tem-dioxons, and BPS. Tem-SO2-OH, the most abundant metabolite of temephos, could be used as an exposure biomarker for toxicokinetic modeling. These results could provide critical insight into the dosimetry and toxicity of temephos and its metabolites.

Evaluation of the effects of the larvicides temephos on reproductive performance, embryofetal development and DNA integrity of Swiss mice.

Temephos is considered the gold standard by the Ministry of Health for controlling the larvae of the mosquito Aedes aegypti. The present study evaluated the effects of Temephos larvicide on the reproductive performance, embryo-fetal development and DNA integrity of Swiss mice. This study used 30 pregnant female mice: 10 were controls treated with drinking water at a dosage of 0.1 mL/10 g (body weight - b.w., administered orally - a.o.), and 20 were treated with Temephos at doses of 0.0043 mg/kg and 0.043 mg/kg (b.w., a.o.) during the gestational period. Statistical analysis showed that Temephos did not alter the biometric or reproductive parameters. Comparing the weight of the fetus to the stage of pregnancy demonstrated that the 0.0043 mg/kg dosage increased the size of the fetuses. No external malformations were detected. However, the 0.043 mg/kg dosage induced changes in the sternum, with the main change being the center of the sternum, xiphoid processes and absence of the manubrium. The other skeletal and visceral alterations did not differ from the control group and are considered variants of normality. The analysis of head measurements showed an increase in the anterior/posterior measurements of the glabella, the external occipital protuberance and the biauricular plane. The circumference and area of the head did not present significant differences. The micronucleus test showed only a 0.043 mg/kg increase in 48 h. Thus, it is considered that Temephos has a low teratogenic and genotoxic risk.

Cholinergic alterations by exposure to pesticides used in control vector: Guppies fish (Poecilia reticulta) as biological model.

Spinosad and temephos are two of the most used pesticides in Mexico for the control of vector causing disease such as dengue, chikungunya and Zika. The aim of this study was to compare the neurotoxic effects of these two pesticides using guppy fish (Poecilia reticulata) as a model organism. Guppies were exposed for 7 and 21 days to technical grade temephos and spinosad at 1.0 and 0.07 g/L, respectively, (10 and 0.5 mg/L of active substance; concentrations recommended by the Ministery of Health of the State (Secretaría de Salud de Nayarit (SSN) Mexico)). Subsequently, acetylcholinesterase activity (AChE) and acetylcholine concentrations (ACh) in muscle tissue were determined. Temephos exposure decreased AChE activity and increased ACh concentration, whereas exposure to spinosad only increased ACh concentration. Though cholinergic alterations were more severe in fish exposed to temephos, both pesticides were equally lethal during the first seven days after exposure. Nonetheless, temephos was more lethal after 21 days.

Identification of a major Quantitative Trait Locus determining resistance to the organophosphate temephos in the dengue vector mosquito Aedes aegypti.

Organophosphate insecticides (OP) have extensively been used to control mosquitoes, such as the vector Aedes aegypti. Unfortunately, OP resistance has hampered control programs worldwide. We used Quantitative Trait Locus (QTL) mapping to evaluate temephos resistance in two F1 intercross populations derived from crosses between a resistant Ae. aegypti strain (RecR) and two susceptible strains (MoyoD and Red). A single major effect QTL was identified on chromosome 2 of both segregating populations, named rtt1 (resistance to temephos 1). Bioinformatics analyses identified a cluster of carboxylesterase genes (CCE) within the rtt1 interval. qRT-PCR demonstrated that different CCEs were up-regulated in F2 resistant individuals from both crosses. However, none exceeded the 2-fold expression. Primary mechanisms for temephos resistance may vary between Ae. aegypti populations, yet also appear to support previous findings suggesting that multiple linked esterase genes may contribute to temephos resistance in the RecR strain as well as other populations.

Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos.

Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide-induced responses in the mosquito.

Enzymatic Alterations and Genotoxic Effects Produced by Sublethal Concentrations of Organophosphorous Temephos in Poecilia reticulata.

The responses of biochemical and genetic parameters were evaluated in tissues of Poecilia reticulata exposed to sublethal and environmentally relevant concentrations of 0.005, 0.01, or 0.02 mg/L of the organophosphorous (OP) pesticide temephos (TE) for 168 h. Activities of enzymes brain acetylcholinesterase (AChE) and liver carboxylesterase (CbE) were determined. Nuclear abnormalities (NA) and micronucleus (MN) frequency in gill erythrocytes were also measured. No mortality was observed over the experimental period; however, brain AChE activities were decreased significantly in guppies in all TE treatment groups after 72 h of exposure. Hepatic CbE activities of fish were increased in all TE treatment groups at 96, 120, and 144 h of exposure. The frequencies of MN and NA in fish gill erythrocytes displayed a marked rise after 168 h of exposure to concentrations of 0.01 or 0.02 mg/L TE. Thus, determination of these parameters may be employed as potential indices of exposure to TE using this sentinel organism for monitorining.

Ecotoxicity and environmental risk assessment of larvicides used in the control of Aedes aegypti to Daphnia magna (Crustacea, Cladocera).

Dengue transmitted by mosquitoes of the genus Aedes, species aegypti, is a major public health concern in Brazil. The chemical control of the mosquito larvae has been performed with the larvicide temephos since 1967. However, vector resistance was reported to temephos in several Brazilian states, and the Ministry of Health ordered the replacement of this larvicide by diflubenzuron (DFB), an inhibitor of chitin synthesis. Both insecticides are diluted in water with larvae and are able to reach aquatic environments in which they subsequently adversely damage nontarget organisms. The aims of this study were to (1) determine the acute toxicity (EC50) and environmental risk (RQ) of DFB and temephos to the microcrustacean Daphnia magna, and (2) evaluate the chronic toxicity (no-observed-effect concentration [NOEC] and lowest-observed-effect concentration [LOEC]) of these larvicides to D. magna. The experiments were performed according to a completely randomized design. The estimated 48-h EC50 of temephos was 0.15 µg/L (lower limit = 0.1 and upper limit = 0.2 µg/L) and the 48-h EC50 of DFB was 0.06 µg/L (lower limit = 0.03 and upper limit = 0.1 µg/L). RQ values were 4.166.7 to DFB and 6.666.6 to temephos. NOEC and LOEC values were respectively 2.5 and 5 ng/L for DFB, and respectively 6.2 and 12.5 ng/L for temephos. Thus, temephos and DFB are classified as highly toxic to Daphnia magna and pose a high environmental risk to this species. Mortality of D. magna was observed at concentrations lower than those used in the field to control A. aegypti larvae.

Effect of Wolbachia on insecticide susceptibility in lines of Aedes aegypti.

Two stable infections of Wolbachia pipientis, wMelPop and wMel, now established in Aedes aegypti, are being used in a biocontrol program to suppress the transmission of dengue. Any effects of Wolbachia infection on insecticide resistance of mosquitoes may undermine the success of this program. Bioassays of Ae. aegypti were conducted to test for differences in response to insecticides between Wolbachia infected (wMelPop, wMel) and uninfected lines. Insecticides screened were bifenthrin, the pyrethroid commonly used for adult knockdown, as well as larvicides: Bacillus thuringiensis var. israelensis, the organophosphate, temephos and the insect growth regulator, s-methoprene. While differences in response between lines were detected for some insecticides, no obvious or consistent effects related to presence of Wolbachia infection were observed. Spreading Wolbachia infections are, therefore, unlikely to affect the efficacy of traditional chemical control of mosquito outbreaks.

Spinosad toxicity to Simulium spp. larvae and associated aquatic biota in a coffee-growing region of Veracruz State, Mexico.

Spinosad is a naturally derived insecticide that has shown potential as a mosquito larvicide. To determine the activity of spinosad against blackflies, late-instar larvae from a community comprising Simulium triittatum (63.6%) and seven other species, including three known vectors of onchocerciasis in Mexico (S. metallicum, S. ochraceum, and S. callidum), were subjected to concentration-mortality laboratory bioassays following World Health Organization guidelines. Cephalic capsule measurements confirmed the relatively homogeneous distribution of experimental larvae. The 50% lethal concentration of spinosad was estimated at 1.48 ppm spinosad (95% confidence interval: 1.07-2.33) for a 10-min exposure period, whereas larvae treated with 0.05 ppm of the organophosphate temephos experienced 61% mortality. Immature aquatic insects were identified to genus and tested for their susceptibility to spinosad in the laboratory. After exposure to 12 ppm spinosad for 10 min, ephemeropterans, odonates, trichopterans, and hemipterans did not experience significantly increased mortality over that of untreated controls, whereas a significant increase in mortality was observed in spinosad-treated Plecoptera (P < 0.001). Tilapia and trout fry exposed to 12 ppm spinosad for 10 min did not experience increased mortality at 24-h postexposure over that of the controls. We conclude that spinosad is less toxic than temephos to these blackfly species, but is likely to have a low impact on nontarget members of the aquatic community.

Sensitivity of two Mesocyclops (Crustacea, Copepoda, Cyclopidae), from tropical and temperate origins, to the herbicides, diuron and paraquat, and the insecticides, temephos and fenitrothion.

Ecotoxicological assessment in the tropics is based mainly on knowledge gained from temperate organisms, although many studies have shown the need for models that are more appropriate to tropical regions. The toxicity of two herbicides and two insecticides to nauplii of two freshwater zooplankton species, Mesocyclops aspericornis (von Daday in Inseln Zool Jb Syst 24:175-206, 1906) from a tropical reservoir and a Mesocyclops sp. from a temperate pond, were compared. Both strains were sensitive to paraquat, temephos and fenitrothion, tropical M. aspericornis being generally more tolerant (EC50s of 207, 1,450 and 1,840 µg L(-1) for paraquat, temephos and fenitrothion, respectively) than the temperate Mesocyclops sp. (EC50s of 152, 45 and 1,017 µg L(-1) for paraquat, temephos and fenitrothion, respectively) whereas diuron was only slightly toxic (LOEC = 446 µg L(-1) for both strains). The nauplii mortality increased with the exposure time for both species during each experiment. Further isolation of model organisms from tropical ecosystems is needed, to meet the requirements for a tropical risk assessment scheme.

Temephos Decreases Sperm Quality and Fertilization Rate and Is Metabolized in Rat Reproductive Tissues at Low-Dose Exposure.

Temephos is an organophosphorus pesticide used in control campaigns against vectors that transmit diseases, including dengue, a public health concern. The WHO classifies temephos in category III and its safe concentration (low-observable-adverse-effect level) in male rats is 100 mg/kg/day for up to 44 days. Temephos inhibits acetylcholinesterase (AChE) and is metabolized in different tissues, probably by mixed-function oxidases; one of its metabolites is bisphenol S (BPS), which is considered an endocrine disruptor. The aim of this study was to evaluate the effects of temephos on sperm function and its biotransformation in the testis, epididymis, and other tissues to explore its toxicity in rats treated with 100 mg/kg/day/5 or 7 days (gavage). AChE activity was inhibited 70% starting on day 3 and 13 or 41% mortality was observed at 5 or 7 days, respectively. After 7 days, temephos significantly decreased sperm motility (30%) and viability (10%) and increased (10%) lipoperoxidation, and the sperm DNA exhibited no damage. Temephos was distributed and metabolized in all tissues, with the highest levels observed in the adipose tissue and temephos levels were 16-fold higher in the epididymis than in the testis. Notably, BPS was observed in the testis. At 5 days, decreased sperm motility (12.5%) and viability (5.7%) were observed and sperm fertilization decreased (30%). These results suggest that temephos decreases sperm quality and fertilization capacity at recommended safe concentrations and that it is metabolized in male reproductive tissues. This pesticide places the reproductive health of exposed people at risk, suggesting the need to reevaluate its toxicity.

A Challenge for a Unique Dengue Vector Control Programme: Assessment of the Spatial Variation of Insecticide Resistance Status amongst Aedes aegypti and Aedes albopictus Populations in Gampaha District, Sri Lanka.

BACKGROUND: To date, dengue is considered an important public health problem in Sri Lanka. Irrational use of insecticides without evidence-based applications has primed the development of resistance in mosquito vectors. METHOD: The present study investigated the resistance status of Aedes aegypti and Aedes albopictus to commonly used insecticides in three selected Medical Officer of Health (MOH) areas (i.e., Attanagalla, Dompe, and Negombo) in Gampaha District, Western Province of Sri Lanka. Entomological surveys were performed using ovitraps and larval collections. Larval bioassays were carried out to determine the LC50, LC90, and LC95 and susceptibility status for organophosphate temephos, whereas adult bioassays were performed to test the 0.03% deltamethrin and 0.8% malathion susceptibility. RESULTS: The study revealed that the temephos concentrations required to control Ae. aegypti (13.7-17.7 times) and Ae. albopictus (4.6-7.6 times) are higher than the diagnostic concentration (0.012 mg/L) proposed by the World Health Organization. The highest resistance levels were observed for both Ae. aegypti (14 ± 1.87) and Ae. albopictus (36 ± 1.87) collected from the Negombo MOH area. Therefore, the WHO recommended diagnostic concentration is no longer effective in controlling Ae. aegypti and Ae. albopictus larvae in these areas. Both the dengue vectors have evolved a high level of insecticide resistance to malathion and deltamethrin in the Gampaha District except Ae. albopictus mosquitoes in rural areas. Further, vectors in rural areas are indicated susceptible (>98%) to pyrethroids and emergence of resistance (<97%) for organophosphate insecticides. CONCLUSION: The results of this study warrant the vector management authorities on the proper application of insecticides and rational use in vector control. The susceptibility status of vector mosquitoes should be continuously monitored especially in dengue-endemic areas parallel to the routine surveillance programme. Further molecular studies are strongly recommended to determine the Knockdown Resistance (kdr) mutations among Aedes populations.

Combination larvicidal action of Solanum xanthocarpum extract and certain synthetic insecticides against filarial vector, Culex quinquefasciatus (SAY).

The combination activities of temephos, fenthion and petroleum ether extract of Solanum xanthocarpum were observed for their larvicidal activities against Culex quinquefasciatus. The combination of temephos and S. xanthocarpum was studied at ratios of 1:1, 1:2, and 1:4. Similar ratios were also used for the combination of fenthion and S. xanthocarpum. The temephos/plant extract combination acted antagonistically. The combination of fenthion and plant extract acted synergistically against the target organisms at a ratio of 1:1, which showed the best results of: LC50 0.0144 and 0.0056 ppm and LC90 0.0958 and 0.0209 ppm at 24 and 48 hours, respectively. The present study will be helpful in developing a commercial formulation for effective vector management.

Resistance to insecticides and synergism by enzyme inhibitors in Aedes albopictus from Punjab, Pakistan.

The widespread use of insecticides has ecological consequences such as emergence of insecticide resistance and environmental pollution. Aedes albopictus is a major vector of dengue virus in the Punjab province, Pakistan. Control of Ae. albopictus with insecticides along with source eradication is critical in the prevention and control of dengue fever but is threatened by the development of insecticide resistance. Here, field strains of Ae. albopictus from eight cities of Punjab were evaluated for resistance against temephos, deltamethrin and permethrin. For temephos, high resistance (RRLC50 > tenfold) was found in larvae of the Rawalpindi strain, moderate resistance (RRLC50 = five- to tenfold) in Multan, Faisalabad, Sialkot, Lahore and Sheikhupura strains, and low resistance (RRLC50 < fivefold) in Kasur and Sahiwal strains. In the case of deltamethrin, high resistance was seen in adults of the strain from Faisalabad, moderate resistance in the strains from Sialkot, Sheikhupura, Lahore and Kasur, and low resistance in Sahiwal, Multan and Rawalpindi strains. For permethrin, adults of all the field strains exhibited high levels of resistance. In synergism bioassays, toxicity of all the insecticides in the field strains significantly enhanced when tested in combination with piperonyl butoxide or S,S,S-tributylphosphorotrithioate, suggesting the probability of metabolic-based mechanisms of resistance. In conclusion, field strains of Ae. albopictus from Punjab exhibit resistance to temephos, deltamethrin and permethrin, which might be associated with metabolic mechanisms of resistance.

Comparative efficacy of Eucalyptus globulus (Labill) hydrodistilled essential oil and temephos as mosquito larvicide.

In this study Eucalyptus globulus essential oil was tested on major mosquito species and toxicity was compared with temephos. Mortality was calculated after 24 h and 48 h post treatment. In 24 h treatment essential oil show strong larvicidal activity with LC50 and LC90 values were 30.198ppm, 103.389ppm for Anopheles stephensi, 13.578ppm, 106.755ppm for Aedes aegypti; and, 7.469ppm, 32.454ppm for Culex quinquefasciatus and 48 h post treatment LC50 and LC90 values were, 12.576, 49.380ppm for Anopheles stephensi, 7.926, 34.470ppm for Aedes aegypti and 4.408, 21.048ppm for Culex quinquefasciatus. Chemical composition of essential oil using GC-MS and FT-IR analysis shows the presence of 1,8-cineol, (71.7%); α-pinene, (9.14%) as a major compounds. Our findings suggest that essential oil from Eucalyptus globulus leaves can be used for control of mosquito larvae.

Detrimental effects of temephos on male fertility: An in vitro study on a mouse model.

The World Health Organization recommends temephos as a nonsystemic organophosphorus pesticide due to its low mammalian toxicity compared with other chemical compounds. Although several studies have reported that temephos may be toxic under certain conditions, little research effort has been made to evaluate its effects on mammalian fertility. Therefore, the present study was designed to evaluate the effect of temephos on sperm functions and male fertility. Initially, cauda epididymis from mouse spermatozoa was incubated with temephos (0, 0.1, 1, 10, and 100 µM). Then, sperm motility and motion kinematics, capacitation status, intracellular adenosine triphosphate level, lactate dehydrogenase level, protein kinase A (PKA) activity, and degree of tyrosine phosphorylation were analyzed. Finally, the rates of fertilization and early embryonic development were evaluated. Sperm motility and motion kinematics were found to be significantly altered in temephos groups. In addition, the acrosome reaction and capacitation significantly increased and decreased in the 100 µM temephos group, respectively. Intracellular adenosine triphosphate levels significantly decreased in the 1, 10, and 100 µM temephos groups compared with that in the control group. Moreover, PKA activity and tyrosine phosphorylation significantly decreased in most temephos groups. Further, the rates of fertilization and early embryonic development significantly decreased in all temephos groups. Taken together, it was determined that temephos had harmful effects on male fertility. Therefore, the reproductive toxicity of temephos should be considered before its use.

Effect of temephos on cholinesterase activity in the earthworm Eisenia fetida (Oligochaeta, Lumbricidae).

In this study, adult Eisenia fetida earthworms were exposed to the sub-lethal concentrations of temephos using the contact filter paper test procedure. Since temephos is an organophosphate pesticide, its effects on earthworms were determined by measuring ChE inhibition--a known biomarker of exposure. The ChE activity was measured after a short time of exposure--1 and 2 h. As expected, the lowest ChE activity (72.70% and 38.03% inhibition) was measured at the highest concentration of temephos (120 ng cm(-2)) applied. More interestingly, at the 0.12 ng cm(-2) concentration the ChE activity increased up to 36.28% of activity in the control in all three conducted experiments. Dose-response curves showed an inverted U-shape characteristic for hormesis. This hormetic-like effect could be important for health status of an earthworm.

Insecticide Resistance and Fitness: The Case of Four Aedes aegypti Populations from Different Brazilian Regions.

BACKGROUND: Chemical control is still a major strategy to constrain vector density and mitigate pathogen transmission. However, insecticide overuse poses a high selective pressure, favouring the spread of resistance alleles in natural populations. In an insecticide-free environment, a fitness cost is expected in resistant insects when compared to susceptible counterparts. This study investigates whether insecticide resistance to an organophosphate (temephos) and a pyrethroid (deltamethrin) is associated with fitness traits in four Aedes aegypti wild populations sampled every three months over one year. FINDINGS: We measured development time from larvae to adult, female survival, wing length, fecundity, and adult resistance to starvation in field insecticide resistant Ae. aegypti populations four times over a year. These results were confronted with resistance levels to temephos and deltamethrin and with potentially related mechanisms, including a kdr mutation in the pyrethroid target site. No differences in fitness cost were found after contrasting mosquitoes from the same population collected throughout a year, irrespective of differences in insecticide resistance levels. Additionally, significant differences were not observed among field populations. However, compared to the reference strain Rockefeller, field females survived significantly less. Moreover, larval development was equal or slower in three out of four field populations. In no case differences were evidenced in starvation tolerance, wing length, and fecundity. CONCLUSIONS: Overall, field resistant mosquitoes seemed to have a slight fitness disadvantage when compared with the Rockefeller susceptible strain which might represent a potential fitness cost of insecticide resistance. However, after comparing Ae. aegypti from the same population but sampled at different moments, or from different field populations, mosquito life-history traits varied independently of resistance ratios. The metabolic deviations necessary to overcome the adverse effects of insecticides may cause an energy trade-off that affects energy allocation and ultimately basic demands of insect biology. The extent of fitness cost due to insecticide resistance is critical information to delay the evolution of resistance in wild vector populations.

Inhibition of miR-128 Abates Aß-Mediated Cytotoxicity by Targeting PPAR-γ via NF-κB Inactivation in Primary Mouse Cortical Neurons and Neuro2a Cells.

PURPOSE: Alzheimer's disease (AD) is the sixth most common cause of death in the United States. MicroRNAs have been identified as vital players in neurodegenerative diseases, including AD. microRNA-128 (miR-128) has been shown to be dysregulated in AD. This study aimed to explore the roles and molecular mechanisms of miR-128 in AD progression. MATERIALS AND METHODS: Expression patterns of miR-128 and peroxisome proliferator-activated receptor gamma (PPAR-γ) messenger RNA in clinical samples and cells were measured using RT-qPCR assay. PPAR-γ protein levels were determined by Western blot assay. Cell viability was determined by MTT assay. Cell apoptotic rate was detected by flow cytometry via double-staining of Annexin V-FITC/PI. Caspase 3 and NF-κB activity was determined by a Caspase 3 Activity Assay Kit or NF-κB p65 Transcription Factor Assay Kit, respectively. Bioinformatics prediction and luciferase reporter assay were used to investigate interactions between miR-128 and PPAR-γ 3'UTR. RESULTS: MiR-128 expression was upregulated and PPAR-γ expression was downregulated in plasma from AD patients and amyloid-ß (Aß)-treated primary mouse cortical neurons (MCN) and Neuro2a (N2a) cells. Inhibition of miR-128 decreased Aß-mediated cytotoxicity through inactivation of NF-κB in MCN and N2a cells. Moreover, PPAR-γ was a target of miR-128. PPAR-γ upregulation attenuated Aß-mediated cytotoxicity by inactivating NF-κB in MCN and N2a cells. Furthermore, PPAR-γ downregulation was able to abolish the effect of anti-miR-128 on cytotoxicity and NF-κB activity in MCN and N2a cells. CONCLUSION: MiR-128 inhibitor decreased Aß-mediated cytotoxicity by upregulating PPAR-γ via inactivation of NF-κB in MCN and N2a cells, providing a new potential target in AD treatment.