Comorbidity of bipolar disorder with substance abuse: selection of prioritized genes for translational research.

Bipolar disorder is a highly heritable mental illness. The global burden of bipolar disorder is complicated by its comorbidity with substance abuse. Several genome-wide linkage/association studies on bipolar disorder as well as substance abuse have focused on the identification and/or prioritization of candidate disease genes. A useful step for translational research of these identified/prioritized genes is to identify sets of genes that have particular kinds of publicly available data. Therefore, we have leveraged the availability of links to related resources in the Entrez Gene database to develop a web-based resource for selecting genes based on presence or absence in particular biological data resources. The utility of our approach is demonstrated using a set of 3,399 genes from multiple eukaryotes that have been studied in the context of bipolar disorder and/or substance abuse. A web resource to automate the selection of genes that contain certain database links is available at http://compbio.jsums.edu/bpd.

Gene expression of opioid and dopamine systems in mouse striatum: effects of CB1 receptors, age and sex.

RATIONALE: Endocannabinoid, opioid, and dopamine systems interact to exhibit cannabinoid receptor neuromodulation of opioid peptides and D(4) dopamine receptor gene expression in CB(1)-cannabinoid-deficient mouse striatum. OBJECTIVE: Using CB(1)-transgenic mice, we examine primary age-sex influences and interactions on opioid and dopamine system members' gene expression in striatum. MATERIALS AND METHODS: Real-time quantitative polymerase chain reaction was used to analyze gene expression of opioid peptides [preproenkephalin (PPENK); preprodynorphin (PPDYN)], opioid receptors [delta-opioid receptor (delta-OR); mu-opioid receptor (micro-OR)] and dopamine receptor subtypes (D(1) through D(5)) in male/female CB(1)(+/+)/CB(1)(-/-) mice striata at two adult ages [young (60-90 days); old (140-300 days)]. RESULTS: (1) Increased PPENK and PPDYN, owing to genotype [CB(1)(+/+) vs. CB(1)(-/-)], depended on sex. When genotype-independent, they depended on sex (PPENK) or age (PPDYN). (2) delta-OR was age-dependent (higher in old). (3) micro-OR, owing to genotype, was age-dependent [higher in old CB(1)(-/-) males]. When genotype-independent, it depended on sex (higher in females). (4) Female D(1) was genotype-independent and age-dependent, while male D(1) was higher in old over young CB(1)(+/+) mice. (5) D(5), owing to genotype, was sex-dependent [higher in young female CB(1)(-/-) mice]. (6) D(2), genotype-independent, was higher in old over young male mice. (7) Young female D(3) was higher in CB(1)(-/-) over CB(1)(+/+) mice. Male D(3) was age-dependent (higher in old mice). (8) D(4), owing to genotype, was sex-dependent [higher in CB(1)(-/-) over CB(1)(+/+) females]. Genotype-independent D(4) was sex-dependent in young mice (higher in females) and age-dependent in males (higher in old). CONCLUSIONS: Greater striatal expression is genotype-dependent in females (opioid-peptides, D(3), D(4), D(5)) and genotype-independent in both females (PPENK, mu-OR, D(4)) and old males (PPDYN, delta-OR, D(2), D(3), D(4)).

Glucocorticoids plus opioids up-regulate genes that influence neuronal function.

(1) This study investigated the functional genomics of glucocorticoid and opioid receptor stimulation in cellular adaptations using a cultured neuronal cell model. (2) Human SH-SY5Y neuroblastoma cells grown in hormone-depleted serum were treated for 2-days with the glucocorticoid receptor-II agonist dexamethasone (30 nM); the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate (DAMGO; 1 nM); or dexamethasone (30 nM) plus DAMGO (1 nM). RNA was extracted; purified, reverse transcribed, and labeled cDNA was hybridized to a 10,000-oliogonucleotide-array human gene chip. Gene expression changes that were significantly different between treatment groups and were of interest due to biological function were verified by real-time reverse transcription polymerase chain reaction (RT-PCR). Five relevant genes were identified for which the combination of dexamethasone plus DAMGO, but neither one alone, significantly up-regulated gene expression (ANOVA, P < 0.05). (3) Proteins coded by the identified genes: FRS2 (fibroblast growth factor receptor substrate-2; CTNNB1 (beta1-catenin); PRCP (prolyl-carboxypeptidase); MPHOSPH9 (M-phase phosphoprotein 9); and ZFP95 (zinc finger protein 95) serve important neuronal functions in signal transduction, synapse formation, neuronal growth and development, or transcription regulation. Neither opioid, glucocorticoid nor combined treatments significantly altered the cell growth rate determined by cell counts and protein. (4) We conclude that sustained mu-opioid receptor stimulation accompanied by glucocorticoids can synergistically regulate genes that influence neuronal function. Future studies are warranted to determine if combined influences of glucocorticoid fluctuations and opioid receptor stimulation in vivo can orchestrate exagerated neuroadaptation to reinforcing drugs under chronic mild stress conditions.

Assessment of individual and combined toxicities of four non-essential metals (As, Cd, Hg and Pb) in the microtox assay.

Although most researches with non-essential metals (NEMs) have been done with single or individual metals, in reality, organisms are often exposed to multiple contaminants at the same time through the air, food and water. In this study, we tested the toxicity of four NEMs, As, Cd, Pb, and Hg, individually and as a composite mixture using the microtox bioassay. This assay uses the reduction of bioluminescence of the bacterium Vibrio fischeri as a measure of toxicity. The concentrations of each chemical in the mixture were based on multiples of their maximum contaminant levels (MCLs) set by the U.S. EPA. The highest concentration of exposure was 20 times the MCL, which translated into 200, 100, 40 and 300 ppb for As, Cd, Hg and Pb, respectively. The ratio for the mixture from these concentrations was 10:5:2:15 for As, Cd, Hg and Pb, respectively. Among the individual metals tested, the ranking of toxicity was Hg>Pb>Cd>As based on the EC50 values of 109, 455, 508 and 768 ppb for Hg, Pb, Cd and As, respectively. The EC50 for the composite mixture was 495% MCL which translated into nominal concentrations of 49, 25, 10 and 74 ppb for As, Cd, Hg, and Pb, respectively. Overall, the EC50 value of each NEM within the mixture was lower than the EC50 of the individual chemical; an evidence of synergism for the mixture. The individual toxic units (TU) were 0.06, 0.05, 0.09, and 0.16 for As, Cd Hg, and Pb, respectively and the summed toxic unit (TU) was 0.37 (less than 1). This study provides needed scientific data necessary for carrying out complete risk assessment of As, Cd, Hg, and Pb mixtures of some priority compounds.

CB1 knockout mice display significant changes in striatal opioid peptide and D4 dopamine receptor gene expression.

Antagonism of the CB(1) cannabinoid receptor (CB(1) receptor) by rimonabant (SR141716) reduces self-administration of alcohol and other drugs of abuse in animal models. These findings suggest that the CB(1) receptor may be a target for genetic differences that modify the salient features of rewarding drugs. In the present study, wild-type (CB(1) (+/+)) are compared to transgenic mice deficient in CB(1) receptors (CB(1) (-/-)). The goal was to investigate the influences of the cannabinoid receptor system on opioid peptide gene expression and on dopamine receptor gene expression which is commonly influenced by substances of abuse. We demonstrate using reverse transcription and real-time polymerase chain reaction (PCR) that striatal mRNA for preproenkephalin (PPENK) and preprodynorphin (PPDYN) in the CB(1) (-/-) striatum increases when compared to CB(1) (+/+). Real-time PCR analyses to evaluate D(2) and D(4) dopamine receptor gene expression in striatum isolated from CB(1) (+/+) and CB(1) (-/-) revealed a nearly 2-fold increase in D(4) receptor mRNA in the striatum from CB(1) (-/-) mice and no significant change in D(2) expression. In contrast, treatment of C57BL/6 mice with the CB(1) receptor antagonist, rimonabant, produced a reduction of both D(2) and D(4) dopamine receptor expression in the striatum. These data suggest that genetic differences in CB(1) receptor may exert a modulatory effect on D(4) dopamine receptor and opioid peptide gene expression.

Cytotoxicity of dinitrotoluenes (2,4-dNT, 2,6-DNT ) to MCF-7 and MRC-5 cells.

DNTs are considered possibly carcinogenic to humans (Group 2B) because there is inadequate evidence in humans for carcinogenicity though there is sufficient evidence in experimental animals. In this study, MCF-7 (breast) and MRC-5 (lung) cells were exposed to a serial dilution of 2,4 and 2,6 DNTs (control, 1-500 ppm) in 96 well tissue culture plates. After various time intervals (24, 48, 72 and 96 hrs) the plates were washed, and 100microl fluorescein diacetate solution (10 microg/ml in PBS) was added column wise to each well, and incubated at 37 C for 30 - 60 min before reading the fluorescence with a spectrofluorometer at excitation and emission wavelengths of 485 and 538 nm respectively. Spectrofluorometeric readings were converted to percentages of cell survival. Regression analysis was conducted to determine the relationship between cell survival and exposed concentration. Linear equations derived from the regression analysis were used to calculate the LC50 values. Results indicated that 2,6 DNT was more toxic to breast cells; LC50 values were 445 and 292 ppm at 24 and 48 hours respectively compared to 2,4 DNT showing LC50 values of 570 and 407 ppm at 24 and 48 hours, respectively. No significant differences in toxicity existed between the two chemicals with regard to lung cells. Contrary to the above observation, 2,4 DNT was more toxic to breast cells; LC50 values were 407 and 238 ppm at 24 and 48 hours respectively compared to lung cells showing LC50 values of 527 and 402 ppm at 24 and 48 hours respectively. No significant difference existed for 2,6 DNT between the two cell lines. Lungs cells were more resistant to the two chemicals.