Cellular Localization of Dieldrin and Structure–Activity Relationship of Dieldrin Analogues in Dopaminergic Cells

The incidence of Parkinson’s disease (PD) correlates with environmental exposure to pesticides, such as the organochlorine insecticide, dieldrin. Previous studies found an increased concentration of the pesticide in the striatal region of the brains of PD patients and also that dieldrin adversely affects cellular processes associated with PD. These processes include mitochondrial function and reactive oxygen species production. However, the mechanism and specific cellular targets responsible for dieldrin-mediated cellular dysfunction and the structural components of dieldrin contributing to its toxicity (toxicophore) have not been fully defined. In order to identify the toxicophore of dieldrin, a structure–activity approach was used, with the toxicity profiles of numerous analogues of dieldrin (including aldrin, endrin, and <i>cis</i>-aldrin diol) assessed in PC6-3 cells. The MTT and lactate dehydrogenase (LDH) assays were used to monitor cell viability and membrane permeability after treatment with each compound. Cellular assays monitoring ROS production and extracellular dopamine metabolite levels were also used. Structure and stereochemistry for dieldrin were found to be very important for toxicity and other end points measured. Small changes in structure for dieldrin (e.g., comparison to the stereoisomer endrin) yielded significant differences in toxicity. Interestingly, the <i>cis</i>-diol metabolite of dieldrin was found to be significantly more toxic than the parent compound. Disruption of dopamine catabolism yielded elevated levels of the neurotoxin, 3,4-dihydroxyphenylacetaldehyde, for many organochlorines. Comparisons of the toxicity profiles for each dieldrin analogue indicated a structure-specific effect important for elucidating the mechanisms of dieldrin neurotoxicity

MPTP lesioning does not affect serum 25(OH)D levels.

<p>After MPTP lesion, serum 25(OH)D levels were measured to determine if MPTP lesion had any effect (Control/Saline Group- 40.40±1.1 ng/ml; Control/MPTP Group- 40.60±1.1 ng/ml; Vitamin D Depletion/Saline Group- 6.60±0.7 ng/ml; Vitamin D Depletion/MPTP Group- 5.91±1.4 ng/ml; n = 4 (***, p<0.0001).</p

Natural Products Discovered in a High-Throughput Screen Identified as Inhibitors of RGS17 and as Cytostatic and Cytotoxic Agents for Lung and Prostate Cancer Cell Lines

Regulator of G Protein Signaling (RGS) 17 is an overexpressed promoter of cancer survival in lung and prostate tumors, the knockdown of which results in decreased tumor cell proliferation in vitro. Identification of drug-like molecules inhibiting this protein could ameliorate the RGS17’s pro-tumorigenic effect. Using high-throughput screening, a chemical library containing natural products was interrogated for inhibition of the RGS17–Gα_o interaction. Initial hits were verified in control and counter screens. Leads were characterized via biochemical, mass spectrometric, Western blot, microscopic, and cytotoxicity measures. Four known compounds (<b>1</b>–<b>4</b>) were identified with IC₅₀ values ranging from high nanomolar to low micromolar. Three compounds were extensively characterized biologically, demonstrating cellular activity determined by confocal microscopy, and two compounds were assessed via ITC exhibiting high nanomolar to low micromolar dissociation constants. The compounds were found to have a cysteine-dependent mechanism of binding, verified through site-directed mutagenesis and cysteine reactivity assessment. Two compounds, sanguinarine (<b>1</b>) and celastrol (<b>2</b>), were found to be cytostatic against lung and prostate cancer cell lines and cytotoxic against prostate cancer cell lines in vitro, although the dependence of RGS17 on these phenomena remains elusive, a result that is perhaps not surprising given the multimodal cytostatic and cytotoxic activities of many natural products

25(OH)D depletion does not exacerbate loss of tyrosine hydroxylase staining in the striatum and nigra after MPTP lesion.

<p>A) Representative TH staining of the striatum. B) Representative TH staining of the nigra.</p

Serum 25(OH)D levels are not changed in VMAT2 LO mice.

<p>Serum 25(OH)D levels were measured in both young (2–3 month old) and old (12–15 month) VMAT2 WT and LO mice to determine loss of dopamine has any effect on 25(OH)D serum levels No differences were observed between WT and LO mice; however, older mice have higher serum 25-hydroxyvitamin D levels than young mice (***, p<0.0001).</p

Schematic of experimental design to deplete mice of 25(OH)D levels and challenge with MPTP.

<p>On day 1, mice were weighed and randomly assigned to either a group receiving vitamin D depleted chow or a group receiving control chow supplemented with vitamin D. Mice were weighed weekly to check for changes in body mass. After 36 days, 25(OH)D depletion was confirmed by ELISA assay. Then, mice were trained daily to learn the forepaw stride length task from days 44–48. On day 49, baseline behavior was measured. On day 50, MPTP injections began. Mice received a daily injection of either PBS or 15 mg/kg MPTP for 4 days. The mice were allowed to recover for 7 days. On day 60, post-MPTP behavior was measured prior to sacrificing the mice.</p

MPP+ levels are not altered by 25(OH)D depletion.

<p>Mice were fed vitamin D depleted chow for 50 days and given a single dose of MPTP (20 mg/kg). MPP+ levels were measured by HPLC.</p

Effects of 25(OH)D depletion on TH and DAT expression in MPTP-lesioned mice.

<p>A) Western analyses of striatal TH and DAT levels after MPTP lesion in vitamin D depletion mice. A representative blot is shown. β-tubulin is shown as a loading control. B,C) Densitometric analyses of striatal TH and DAT are shown (Relative values ± SEM; n = 4, ***p<0.001), respectively.</p