Dieldrin-induced neurotoxicity involves impaired mitochondrial bioenergetics and an endoplasmic reticulum stress response in rat dopaminergic cells.

Mitochondria are sensitive targets of environmental chemicals. Dieldrin (DLD) is an organochlorine pesticide that remains a human health concern due to high lipid bioaccumulation, and it has been epidemiologically associated to an increased risk for Parkinson's disease (PD). As mitochondrial dysfunction is involved in the etiology of PD, this study aimed to determine whether DLD impaired mitochondrial bioenergetics in dopaminergic cells. Rat immortalized dopaminergic N27 cells were treated for 24 or 48h with one dose of either a solvent control, 2.5, 25, or 250µM DLD. Dopaminergic cells treated with 250µM DLD showed increased Casp3/7 activity at 24 and 48h. DLD also caused a dose dependent reduction in cell viability of ∼25-30% over 24h. No significant effects on cell viability, apoptosis, nor cytotoxicity were detected at 24 or 48h with 2.5µM DLD. Following a 24h exposure to 2.5 and 25µM DLD, viable cells were subjected to a mitochondrial stress test using the Seahorse XFe24 Extracellular Flux Analyzer. Following three independent experiments conducted for rigor, dopaminergic cells that were treated with 2.5 and 25µM DLD consistently showed a reduction in maximum respiration and spare capacity compared to the control group. Molecular responses were measured to determine mechanisms of DLD-induced mitochondrial dysfunction. There were no changes in transcripts associated with mitochondrial membrane potential and permeability (e.g. Ant, Hk1, Tspo, Vdac), nor PI3 K/Akt/mTor signaling or mitochondrial-associated apoptotic factors (Bax, Bcl2, Casp3). However, transcript levels for Chop/Gadd153 (DNA Damage Inducible Transcript 3), an apoptotic gene activated following endoplasmic reticulum (ER) stress, were 3-fold higher in N27 cells treated with DLD, suggesting that DLD-induced mitochondrial dysfunction is related to ER stress. Dopamine cells were also assessed for changes in tyrosine hydroxylase (TH) protein, which did not differ among treatments. This study demonstrates that DLD impairs oxidative respiration in dopamine cells, and ER stress is hypothesized to be associated with the DLD-induced mitochondrial dysfunction. This is important as ER stress is also linked to PD. This study presents mechanistic insight into pesticide-induced mitochondrial dysfunction using a chemical that is reported to be associated to a higher risk for neurodegenerative disease.

Critical parameters in the MCF-7 cell proliferation bioassay (E-Screen).

The MCF-7 cell proliferation bioassay has grown in popularity as a rapid test for detecting potentially oestrogenic compounds. Several MCF-7 cell sublines with different sensitivities to oestrogens are currently used, with maximal proliferation responses ranging from two- to 10-fold above those of hormone-free controls. In the highly responsive MCF-7 BUS cell line, we evaluated critical assay parameters for test performance, including growth conditions, initial seeding densities and differences in growth stimulation in medium containing human serum or fetal calf serum as well as appropriate solvents for oestrogen-mimicking compounds. Modifications significantly reduced the labour-intensive steps and overall assay costs without affecting the sensitivity of the assay. Using this optimized test regimen, the responsiveness of treated MCF-7 BUS cells was consistently increased up to 11-fold over hormone-free controls. The specificity was characterized by examining the effects of oestradiol-17beta, the anti-oestrogen ICI 182,780, and dieldrin, a recognized xeno-oestrogen. The improved proliferation bioassay will be a useful tool in identifying potential xeno-oestrogens.

Inhibition of gap junctional-mediated intercellular communication in vitro by aldrin, dieldrin, and toxaphene: a possible cellular mechanism for their tumor-promoting and neurotoxic effects.

Several mechanisms have been postulated to be responsible for the pleiotropic effects of toxic chemicals. Although the cytotoxicity and mutagenicity of chemicals are well studied and relatively easily detected, the noncytotoxic and nonmutagenic (i.e., epigenetic) mechanisms of chemical toxicity are less well understood. An in vitro assay, using cocultures of Chinese hamster cells to measure metabolic cooperation between V79 6-thioguanine-sensitive (6TGs) and resistant (6TGr) cells, has been developed to detect noncytotoxic and nonmutagenic chemicals that inhibit, quantitatively, gap junctional communication. The insecticides aldrin, dieldrin, and toxaphene, known to have pleiotropic toxic effects in animals, were shown to inhibit gap junctional communication. Interpretation of results suggests that chemical inhibition of gap junctional communication could be a possible mechanism to explain their tumor-promoting and neurotoxic effects.

Cellular lesions in the central nervous system of Periplaneta americana following insecticide treatment in vitro and in vivo. I. Nerve cell bodies and the neuropile.

Cellular lesions in the metathoracic ganglion of P. americana following insecticide treatment have been examined. Treatment of the isolated ganglia with dieldrin (10 microM) and bioresmethrin (5 microM) induced mitochondrial damage in the neuropile and nerve cell bodies. The mitochondria in treated nerve cells were swollen with broken cristae and devoid of normal morphological appearance. Following in vivo treatment of cockroaches with these insecticides, this type of mitochondrial damage was observed even at the onset of poisoning. In the prostrate cockroaches, however, the mitochondrial swelling was accompanied by the accumulation of electron dense granules. In addition, the neuropiles of insecticide-treated ganglia contained secondary lysosomes which increased in size and number with the progress of poisoning and showed signs of depletion of synaptic vesicles. The action of dieldrin upon the ultrastructure was completely abolished by pretreatment of ganglia with 10 mM Mg2+. On the other hand, pretreatment of ganglia with tetrodotoxin and pentobarbital-sodium had very little effect on the action of dieldrin though these compounds blocked the action of bioresmethrin. The results of this study suggest that cellular lesions in the insect CNS, caused by dieldrin, are due to an enhanced uptake of calcium into nerve terminals which may occur independent of membrane depolarization. The effects of bioresmethrin upon the ultrastructure of the CNS are apparently mediated by nerve excitation and membrane depolarization. It is concluded that treatment of intact cockroaches with dieldrin and bioresmethrin initiates catabolic processes in the nerve cells leading to cellular lesions which are indicative of neuronal degeneration.

Cellular lesions in the central nervous system of Periplaneta americana following insecticide treatment in vitro and in vivo. II. Glial cells.

Glial cell damage in the central nervous system of P. americana following insecticide treatment has been studied. Treatment of isolated metathoracic ganglia with 10 microM dieldrin caused an increase in the electron opacity of the inner and outer glial cells without effecting such a change in the structure of glial cells in the ventral connectives. Glial cells in the ganglia and ventral connectives treated with 5 microM bioresmethrin were found to be swollen and even more electron lucent than the control cells. In the prostrate cockroaches (24 hr after the treatment) the action of bioresmethrin and dieldrin upon peripheral glial cells was identical. Both insecticides caused vacuolation and darkening of outer glial cells; their effects extended to include glial cells in the ventral connectives. At the onset of poisoning, 20-30 min following the application of dieldrin, outer glial cells exhibited slight increase in electron opacity while the inner glial cells showed increase in lysosomal activity. The observed action of dieldrin upon the ultrastructure of glial cells was prevented by pretreatment of the nervous tissue with 10 mM Mg2+. Though tetrodotoxin and sodium-pentobarbital had very little effect upon the action of dieldrin, these drugs blocked bioresmethrin-induced alterations in the fine structure of glial cells. The results of this study suggest that alterations in the ultrastructure of insect neuroglia following treatment with insecticides tested in this study are probably due to perturbations in the neural element of the nervous system.

Effect of dieldrin on rat liver plasma membrane enzymes.

Dieldrin, an organochlorine insecticide, when administered orally to male albino rats at a dose of 5 mg/kg body weight/day for 15 days inhibits the enzyme Mg2+ -ATPase and stimulates the activity of 5'-nucleotidase and NADH-dehydrogenase in liver plasma membrane. L-ascorbic acid supplementation offers some protection in relation to the inhibition of Mg2+ -ATPase.