Copper induces apoptosis in Aedes C6/36 cells.

The Aedes albopictus C6/36 cell clone is used as a model system to study the effects of heavy metals on insect cells. Here we report on the effects of Cu(2+) on these cells. Similar to Cd(2+) and Hg(2+), Cu(2+) induces hyperpolymerization of the microtubules; moreover, with Cu(2+) this is followed by cell aggregation and massive apoptosis. This process, which is cell density dependent, is maximal between 0.75 and 1 mM; this is just under the LC(50) as determined by a membrane integrity test. At higher Cu(2+) concentrations, cell death occurs by necrosis. Apoptosis was ascertained by fluorescence and electron microscopy and by agarose gel electrophoresis. At 0.75 mM, apoptosis started at 18-hr exposure time and the amount of apoptotic cells increased almost linearly until 42 hr; then a plateau was reached with 70-80% apoptotic cells. This is the first report on Cu(2+)-induced apoptosis in insect cells. Possible induction mechanisms are discussed in the light of existing literature on vertebrate cells.

Cadmium pathology in an insect cell line: ultrastructural and biochemical effects.

Cadmium (Cd) pathology was studied in an insect cell line (Aedes albopictusC6/36) at the ultrastructural level. The most prominent pathological changes occurred at the level of the nucleus: chromatin clumping, indentations, filling and dilatation of the perinuclear cisternae and an increased amount of bound ribosomes were observed. In the cytoplasm, condensation and swelling of mitochondria, increase of both free and membrane-bound ribosomes, filling and dilatation of the rough endoplasmic reticulum, and increase of the lysosomal system were the most conspicuous effects. The increased content of the perinuclear and cytoplasmic cisternae was probably due to an increased protein synthesis or a disturbance of the protein export system. This picture differed clearly from the osmotically swollen electron-lucent cisternae that have been described in other pathological situations. The enhancement of the lysosomal system was paralleled by a slight but significant stimulation of the acid phosphatase activity in the sublethal Cd concentration range. In vitro experiments suggested that Cd probably acts directly on this enzyme. Abnormal medium acidification in cultures treated with low Cd levels was correlated with an increased production of lactic acid. Together with the morphological data, this suggested a Cd-induced impairment of the aerobic metabolism.

Uptake of HgCl2 and MeHgCl in an insect cell line (Aedes albopictus C6/36).

We studied the uptake mechanism of mercuric chloride (Hg) and methylmercuric chloride (MeHg) in Aedes albopictus C6/36 cells. The uptake kinetics, together with the effect of temperature and a metabolic inhibitor (2, 4-dinitrophenol) on the mercury accumulation, were examined. Both amounts of internalized Hg and MeHg increased linearly with the extracellular concentration. Initially, the influx rate was high for both metal species but MeHg was found to accumulate seven times faster than Hg. At longer exposure times it leveled off for Hg, while for MeHg, the intracellular concentration decreased. Hg toxicity was not significantly influenced by elevated temperatures; in contrast there was a marked decrease of the LC50/24h value for MeHg. On the other hand, Hg accumulation was temperature dependent but MeHg was not. The different toxicity and uptake rate of both mercury compounds can be explained in terms of membrane permeability and target site. For Hg the main target seems to be the plasma membrane, while MeHg readily crosses this barrier and reacts with intracellular targets. 2, 4-Dinitrophenol had no effect on the accumulation of Hg but that of MeHg was doubled. This increased MeHg accumulation might be the result of the inhibition of an active MeHg efflux mechanism; this is in agreement with the MeHg influx kinetics. Despite these differences between Hg and MeHg, which probably result from their physicochemical properties, our experiments indicate that, for both mercury species, simple diffusion is probably the main way to entrance in Aedes cells.

Effect of sublethal doses of cadmium, inorganic mercury and methylmercury on the cell morphology of an insect cell line (Aedes albopictus, C6/36).

The effect of CdCl2 (44 microM), HgCl2 (3.7 microM), and MeHgCl (2 microM) on the morphology of Aedes albopictus C6/36 cells was studied at the light microscopical level. Treatment times and metal concentrations were in the sublethal range as determined by a fluorometric dye exclusion test. The three metal species had profound effects on the cell morphology. MeHgCl treatment induced the development of a large number of short, actin-supported, tangled filopodia. Both CdCl2 and HgCl2 induced long extensions. Pretreatment with colchicine but not with cytochalasin B prevented formation of these extensions which suggests that they were supported by microtubules. This was confirmed by immunostaining for microtubules. The extensions were relatively stable towards colchicine post-treatment. To authors' knowledge, this effect has not yet been described for heavy metals. The similarity with 20-hydroxyecdysone-treated cells and the occurrence of cytoplasmic feet in insect cells is discussed.

Distribution, accumulation and depuration of administered lead in adult honeybees.

In this study the Pb concentration in honeybees was determined by graphite furnace AAS after peroral administration of PbCl2. The Pb concentration, expressed on a dry weight base was determined in relation to the distribution over the body, the accumulation time, the clearance and the exposure dose. Pb is concentrated in the digestive system with the midgut accounting for 67% and the rectum for 27% of the accumulated metal. The barrier function of this system is thus corroborated in honeybees. Pb accumulation happens slowly in young bees which feed mainly on pollen; once they switch from pollen to nectar there is a sharp rise in their Pb content. After the 26th day a limit of tissue accumulation seems to be reached. Pb accumulation is equally efficient when the contamination starts at a forager age. Pb clearance is slower than expected; after 12 days only one third of the initial lead burden has been cleared. The Pb concentration in the animals increases significantly with the Pb concentration in the sugar syrup which they are fed, except at the highest dose of 50 mg/l. The possible use of honeybees as biomonitors for Pb pollution seems promising from these results.