Liming Tao

Synthesis, crystal structure, and insecticidal activities of highly congested hexahydroimidazo[1,2-a]pyridine derivatives: effect of conformation on activities.

A series of hexahydroimidazo[1,2-a]pyridine derivatives were designed and synthesized through aza-Diels-Alder reactions and evaluated for insecticidal activities. Compounds 6a-d with endo-conformation were endowed with excellent insecticidal activities against cowpea aphid ( Aphis craccivora ) and armyworm ( Pseudaletia separata Walker), whereas exo-compounds 7a-d showed only low activities against cowpea aphid. The difference in activities between the endo- and exo-conformations indicated that conformation was the determinant of life or death of the insects for these compounds.

Effects of sublethal concentrations of the chitin synthesis inhibitor, hexaflumuron, on the development and hemolymph physiology of the cutworm, Spodoptera litura.

Abstract The effects of sublethal concentrations 0.1, 0.5, and 1.2 µg mL-1of the chitin synthesis inhibitor, hexaflumuron, on larval growth and development, the count and proportion of hemocytes, and carbohydrate content (trehalose and glyceride) in hemolymph were investigated in the cutworm, Spodoptera litura (Fabricious) (Lepidoptera: Noctuidae). When 3rdinstar larvae were subjected to the sublethal concentrations, there were dose-dependent effects on larval weight and length of each instar larvae, percent pupation and the duration of development. Most of the larvae died during the molting process at all concentrations. Few individuals from 0.5 and 1.2 µg mL -1concentrations could develop to the 6thinstar, while the pupae emerging from the 0.1 µg mL -1concentrations did not exceed 16% of the number of the initial larvae. In 5thinstar S. litura, the total number of hemocytes was significantly increased at 24 hours post—treatment, whereas the proliferation of hemocytes was inhibited, plasmatocyte pseudopodia contracted, and granulocyte expanded at 96 hours post—treatment. The increases of plasmatocyte count and the decreases of granulocyte count were dose—dependent. The longer treatment time of the sublethal concentrations increased the content of total carbohydrate and trehalose in hematoplasma, and was dose—dependent in hemocytes. The content of glyceride in hemolymph was significantly higher at 24 hours post—treatment, but gradually returned to normal levels at 96 hours post—treatment as compared with the control. The results suggested that sublethal concentrations of hexaflumuron reduced S. litura larval survival and interfered with hemolymph physiological balances.

Spinosad induces programmed cell death involves mitochondrial dysfunction and cytochrome C release in Spodoptera frugiperda Sf9 cells.

Spinosad, a reduced-risk insecticide, acts on the nicotinic acetylcholine receptors and the gamma-aminobutyric acid receptor in the nervous system of target insects. However, its mechanism of action in non-neural insect cells is unclear. This study aimed to evaluate mitochondrial functional changes associated with spinosad in Spodoptera frugiperda (Sf9) insect cells. Our results indicate that in Sf9 cells, spinosad induces programmed cell death and mitochondrial dysfunction through enhanced reactive oxygen species production, mitochondrial permeability transition pore (mPTP) opening, and mitochondrial membrane potential collapse, eventually leading to cytochrome C release and apoptosis. The cytochrome C release induced by spinosad treatment was partly inhibited by the mPTP inhibitors cyclosporin A and bongkrekic acid. Subsequently, we found that spinosad downregulated Bcl-2 expression and upregulated p53 and Bax expressions, activated caspase-9 and caspase-3, and triggered PARP cleavage in Sf9 cells. These findings suggested that spinosad-induced programmed cell death was modulated by mitochondrial dysfunction and cytochrome C release.

Natural pyrethrins induces apoptosis in human hepatocyte cells via Bax- and Bcl-2-mediated mitochondrial pathway

Natural pyrethrins have been widely used for pest control in organic farming and for residential indoor pest managements. Although the specific mechanisms underlying their activity are incompletely understood, natural pesticides are considered the safest based on their target specificity and rapid degradation in the environment. Here, we used inxa0vitro bioassays to characterize the cytotoxic effects of natural pyrethrins and attempted to delineate the cellular and molecular mechanisms of their cytotoxicity against human hepatocytes. The results demonstrate that natural pyrethrins reduce cell viability and enhance apoptosis in HepG2 cells. In addition, the current data indicate that natural pyrethrins cause a reduction in the mitochondrial membrane potential (Δψm), increase reactive oxygen species production, and up-regulate the Bax/Bcl-2 expression, leading to the release of cytochrome-c into the cytosol, activation of caspase-9 and caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). Taken together, the results indicate that natural pyrethrins has potentially exert adverse effects on human health by inducing caspase-dependent apoptosis in hepatocytes through Bax- and Bcl-2-mediated mitochondrial pathway.

Synergistic effect of non-ionic surfactants Tween 80 and PEG6000 on cytotoxicity of insecticides

The use of surfactants in the development of a suitable formulation for insecticides should improve the solubility behavior, the permeability and the efficiency against pests meanwhile decrease the toxic risks of insecticides on human health. Cytotoxicity of insecticides including abamectin, chlorfluazuron, hexaflumuron, chlorpyrifos, and tebufenozide was assessed on human HepG2 and lepidopteran Tn5B1-4 cells utilizing insecticide alone and in combination with nontoxic concentrations of nonionic surfactants Tween 80 and PEG6000. The results showed avermection revealed high cytotoxicity, chlorfluazuron and hexaflumuron possessed median cytotoxicity, and chlorpyrifos and tebufenozide had little cytotoxicity on HepG2 and Tn5B1-4 cells. The co-incubation with Tween 80 and PEG6000 powerfully counteracted the cytotoxicity of avermectin. Tween 80 enhanced, whereas PEG6000 compressed, the cytotoxicity of chlorfluazuron on Tn5B1-4 cells, and also improved a bit of the cytotoxicity of chlorpyrifos or tebufenozide on HepG2 cells. PEG6000 was more suitable to be used as surfactant in improving insecticide solubility and reducing the cytotoxicity. The present investigation demonstrates the necessity of utilizing surfactants to weaken the cytotoxicity of insecticides.

Detection on emamectin benzoate-induced apoptosis and DNA damage in Spodoptera frugiperda Sf-9 cell line.

Emamectin benzoate (EMB), an important macrocyclic lactone insecticide that belongs to the avermectin family and possesses excellent potency in controlling pests, is non-carcinogenic and non-mutagenic conducted in rats and mice, but EMB-induced cytotoxicity and genotoxicity in arthropod insect have been seldom reported yet. In the present paper, we quantified the cytotoxicity of EMB through the detections on cell viability, DNA damage, and cell apoptosis in Spodoptera frugiperda Sf-9 cells in vitro. The results showed that EMB caused a concentration- and time-dependent reduction on the viability of Sf-9 cells, and the median inhibitory concentrations (IC50) were 3.34μM at 72h of exposure. The dual acridine orange/ethidium bromide staining showed that exposure to EMB induced a significant time- and concentration-dependent increase on cell apoptosis. The alkaline comet assay revealed that EMB induced significant increases on single-strand DNA breaks, and the percentage of γH2AX-positive cells represented a time- and concentration-dependent formation of DNA double-strand breaks in Sf-9 cells. Interestingly, the similar cytotoxic actions of EMB also went for the human cancerous HeLa cells as a control cell group. Data demonstrated the potential cytotoxic effect of EMB on Sf-9 cells that was significantly greater than the effect of hydrogen peroxide at the same concentrations.

Avermectin Confers Its Cytotoxic Effects by Inducing DNA Damage and Mitochondria-Associated Apoptosis

Avermectin (AVM) has been widely used in agriculture and animal husbandry on the basis of its broad spectrum of effective anthelmintic activity and specificity targets. However, AVM induction of cytotoxicity through DNA damage is remains elusive. Here we investigate the cytotoxic effects of AVM in human nontarget cells in vitro. We clarify that AVM inhibited the viability of HeLa cells and enhanced apoptosis. We have used alkaline comet assay and γH2AX foci formation to detect DNA damage of HeLa cells. As expected, we found AVM caused DNA double-strand breaks in HeLa cells, as measured by significance of comet assay parameters (e.g., tail DNA) and increases of γH2AX foci in HeLa cells. Moreover, established assays of cytotoxicity were performed to characterize the mechanism of AVM toxicity on HeLa cells. The results demonstrated the collapse of mitochondrial membrane potential, and up-regulating the expression level of Bax/Bcl-2 resulted in a release of cytochrome c into cytosol as well as the activation of caspase-9/-3 and cleavage of poly(ADP-ribose) polymerase (PARP). We conclude that AVM has a potential risk to human health by inducing human cell DNA damage and mitochondria-associated apoptosis.

Spinosad induces autophagy of Spodoptera frugiperda Sf9 cells and the activation of AMPK/mTOR signaling pathway

Spinosad, a high-selectivity neural toxin, has been widely used in agricultural production. However, the mode of action of spinosad on insect non-neural cells is not yet clear and hence requires further investigation. Therefore, to reveal the cytotoxic mechanisms of spinosad, we investigated whether and how it can induce autophagic cell death. After treating Sf9 cells with spinosad, the resulting autophagosome was observed by transmission electron microscopy and monodansylcadaverine staining. Interestingly, spinosad induced the accumulation of Beclin-1, degradation of p62, and intensification of LC3-B formation and translocation and thus autophagy, whereas, 3-MA treatment reverted the phenotype. Under ATP depletion conditions, spinosad induced autophagy of Sf9 cells and activation of the AMPK/mTOR signaling pathway.

Staurosporine shows insecticidal activity against Mythimna separata Walker (Lepidoptera: Noctuidae) potentially via induction of apoptosis.

Staurosporine (STS), a wide-spectrum kinase inhibitor, is widely used in studies of apoptosis in mammalian cells. However, its physiological and mechanistic effects have never been clearly defined in insect cells, and other applications of STS have rarely been reported. The present study reveals the insecticidal activity of STS on larvae of Mythimna separata Walker, and the apoptotic mechanism induced by STS on lepidopteran Sf9 cell lines. We demonstrate that the viability of Sf9 cells is inhibited by STS in a time- and concentration-dependent manner. Intracellular biochemical assays show that STS-induced apoptosis of Sf9 cells coincides with a decrease in the mitochondrial membrane potential, the release of cytochrome c into the cytosol, a significant increase of the Bax/Bcl-2 ratio, and a marked activation of caspase-9 and caspase-3. These results indicate that a mitochondrial-dependent intrinsic pathway contributes to STS induced caspase-3 activation and apoptosis in Sf9 cells which is homologous to the mechanisms in mammalian cells. This study contributes to our understanding of the mechanism of insect cell apoptosis and suggests a possible new application of STS as a potential insecticide against Lepidopteran insect pests in agriculture.

Cytotoxic effects of tebufenozide in vitro bioassays

Tebufenozide is considered an environmentally friendly pesticide due to its specificity on target insects, but the effects on human are well studied. Studies on the toxicity of tebufenozide at molecular and cellular level is poorly understood. The present study reveals non-selective cytotoxic effects of tebufenozide, and the apoptotic mechanism induced by tebufenozide on HeLa and Tn5B1-4 cells. We demonstrate that the viability of HeLa and Tn5B1-4 cells is inhibited by tebufenozide in a time- and concentration-dependent manner. Intracellular biochemical assays showed that tebufenozide-induced apoptosis of two cell lines concurrent with a decrease in the mitochondrial membrane potential and an increase reactive oxygen species generation, the release of cytochrome-c into the cytosol and a marked activation of caspase-3. These results indicate that a mitochondrial-dependent intrinsic pathway contributes to tebufenozide induced apoptosis in HeLa and Tn5B1-4 cells and suggests potential threats to ecosystems and human health.