Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2.

Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria.

Is acetylcholinesterase a pertinent biomarker to detect exposure of pyrethroids? A study case with deltamethrin.

The possibility to use acetylcholinesterase as biomarker of exposure to deltamethrin insecticide in the honeybee, Apis mellifera were considered. Joined actions of deltamethrin and pirimicarb (carbamate), alone or in association (dual treatment), were investigated on AChE activity in surviving and dead honeybees in order to test its reliability as biomarker. All treatments induced a reduction in tissue AChE activity in dead bees. In surviving bees, deltamethrin treatment induced an important increase of AChE activity that is not abolished by pirimicarb treatment. The analysis of AChE forms revealed an increase in the soluble form in surviving and dead bees and an increase of the membrane form in surviving bees. No direct effect of deltamethrin on soluble and membrane AChE was observed in vitro. The important increase in AChE activity in response to deltamethrin, not altered by pirimicarb treatment, suggests that AChE activity could represent a robust biomarker specific to deltamethrin exposure in living bees.

In vivo metabolic fate of [14C]-acetamiprid in six biological compartments of the honeybee, Apis mellifera L.

The in vivo metabolism of acetamiprid was studied in the honeybee, Apis mellifera L. The distribution of acetamiprid and its metabolites was monitored over a 72-h period in six biological compartments: head, thorax, abdomen, haemolymph, midgut and rectum. Honeybees were treated orally with 100 microg [14C]-acetamiprid kg(-1) bee, a dose which is about 1500 times lower than the median lethal dose. After 72 h, only 40% of the total radioactivity was eliminated, suggesting that acetamiprid and its metabolites tended to persist in the honeybee. Acetamiprid was rapidly distributed in all compartments and metabolized. Just after administration, radioactivity was mainly localized in the abdomen and subsequently in the rectum. After 72 h, the maximum amount of radioactivity (about 20% of the ingested dose) was detected again in the abdomen, whereas the lowest level of total radioactivity was detected in the haemolymph. Radioactivity in the head did not exceed 7.6% of total ingested radioactivity. More than 50% of acetamiprid was metabolised in less than 30 min, indicating a very short half-life for the compound. During the first hours, acetamiprid was mainly detected in nicotinic acetylcholine receptor-rich tissues: abdomen, thorax and head. Of the seven metabolites detected, the major ones were 6-choronicotinic acid and an unknown metabolite called U1, which was present mainly in the rectum, the thorax and the head. Our results indicate that the low toxicity of acetamiprid may reflect its rapid metabolism.

Existence of two membrane-bound acetylcholinesterases in the honey bee head.

Two acetylcholinesterase (EC 3.1.1.7) membrane forms AChE(m1) and AChE(m2), have been characterised in the honey bee head. They can be differentiated by their ionic properties: AChE(m1) is eluted at 220 mM NaCl whereas AChE(m2) is eluted at 350 mM NaCl in anion exchange chromatography. They also present different thermal stabilities. Previous processing such as sedimentation, phase separation, and extraction procedures do not affect the presence of the two forms. Unlike AChE(m1), AChE(m2) presents reversible chromatographic elution properties, with a shift between 350 to 220 mM NaCl, depending on detergent conditions. Purification by affinity chromatography does not abolish the shift of the AChE(m2) elution. The similar chromatographic behaviour of soluble AChE strongly suggests that the occurrence of the two membrane forms is not due to the membrane anchor. The two forms have similar sensitivities to eserine and BW284C51. They exhibit similar electrophoretic mobilities and present molecular masses of 66 kDa in SDS-PAGE and a sensitivity to phosphatidylinositol-specific phospholipase C in non-denaturing conditions, thus revealing the presence of a glycosyl-phosphatidylinositol anchor. We assume that bee AChE occurs in two distinct conformational states whose AChE(m2) apparent state is reversibly modulated by the Triton X-100 detergent into AChE(m1).