Histone deacetylase inhibitors and candidate gene expression: An in vivo and in vitro approach to studying chromatin remodeling in a clinical population

Objective

The emerging field of psychiatric epigenetics is constrained by the dearth of research methods feasible in living patients. With this focus, we report on two separate approaches, one in vitro and one in vivo, developed in our laboratory.

Method

In the first approach, we isolated lymphocytes from 12 subjects and cultured their cells with either 0.7 mM valproic acid (VPA), 100 nM Trichostatin A (TSA), or DMSO (control) for 24 h based upon previous dose response experiments. We then measured GAD67 mRNA expression using realtime RT-PCR, total acetylated histone 3 (H3K9,K14ac) levels using Western blot analysis, and attachment of H3K9,K14ac to the GAD67 promoter using ChIP. In the second approach, we measured GAD67 mRNA and total H3K9,K14ac levels in lymphocytes from 11 schizophrenia and 7 bipolar patients before and after 4 weeks of clinical treatment with Depakote ER^® (VPA).

Results

In the first approach, VPA induced a 383% increase in GAD67 mRNA, an 89% increase in total H3K9,K14ac levels, and a 482% increase in H3K9,K14ac attachment to the GAD67 promoter. TSA induced comparable changes on all measures. In the second approach, bipolar subjects had significantly higher baseline levels of H3K9,K14ac compared to subjects with schizophrenia. Subjects with clinically relevant serum levels of VPA (?65 μg/mL) showed a significant increase in GAD67 mRNA expression.

Conclusions

Our results utilizing two separate approaches for examining chromatin remodeling in real clinical time provide possible means to investigate epigenetic events in living patients.     

α7 and non-α7 nicotinic acetylcholine receptors modulate dopamine release in vitro and in vivo in the rat prefrontal cortex

Nicotine enhances attentional and working memory aspects of executive function in the prefrontal cortex (PFC) where dopamine plays a major role. Here, we have determined the nicotinic acetylcholine receptor (nAChR) subtypes that can modulate dopamine release in rat PFC using subtype-selective drugs. Nicotine and 5-Iodo-A-85380 (β2* selective) elicited [³H]dopamine release from both PFC and striatal prisms in vitro and dopamine overflow from medial PFC in vivo . Blockade by dihydro-β-erythroidine supports the participation of β2* nAChRs. However, insensitivity of nicotine-evoked [³H]dopamine release to α-conotoxin-MII in PFC prisms suggests no involvement of α6β2* nAChRs, in contrast to the striatum, and this distinction is supported by immunoprecipitation of nAChR subunits from these tissues. The α7 nAChR-selective agonists choline and Compound A also promoted dopamine release from PFC in vitro and in vivo , and their effects were enhanced by the α7 nAChR-selective allosteric potentiator PNU-120596 and blocked by specific antagonists. DNQX and MK801 inhibited [³H]dopamine release evoked by choline and PNU-120596, suggesting crosstalk between α7 nAChRs, glutamate and dopamine in the PFC. In vivo, systemic (but not local) administration of PNU-120596, in the absence of agonist, facilitated dopamine overflow in the medial PFC, consistent with the activation of extracortical α7 nAChRs by endogenous acetylcholine or choline. These data establish that both β2* and α7 nAChRs can modulate dopamine release in the PFC in vitro and in vivo . Through their distinct actions on dopamine release, these nAChR subtypes could contribute to executive function, making them specific therapeutic targets for conditions such as schizophrenia and attention deficit hyperactivity disorder.     

Screening of Chemical Penetration Enhancers for Transdermal Drug Delivery Using Electrical Resistance of Skin

Purpose  A novel technique is presented for identifying potential chemical penetration enhancers (CPEs) based on changes in the electrical resistance of skin. Methods  Specifically, a multi-well resistance chamber was designed and constructed to facilitate more rapid determination of the effect of CPEs on skin resistance. The experimental setup was validated using nicotine and decanol on porcine skin in vitro. The multi-well resistance chambers were capable of operating at 37°C in order to simulate the physiological temperature of the human body. Further, the utility of the multi-well resistance chamber technique was validated using standard Franz diffusion cells. Electrical resistance measurements were used to evaluate the potency of seven new potential CPEs, identified using virtual screening algorithms. From the resistance measurements, the chemicals 1-dodecyl-2-pyrrolidinone (P), menthone (M) and R(+)-3-amino-1-hydroxy-2-pyrrolidinone (C) were identified as the better penetration enhancers among the seven tested. Further, traditional permeation experiments were performed in Franz diffusion cells to confirm our findings. Results  The permeation test results indicated that, of the three CPEs deemed potentially viable using the newly-developed resistance screening technique, both P and M increased the permeation of the test drug (melatonin) through skin in 48 h. Conclusion  In summary, this resistance technique can be used to effectively pre-evaluate potential CPEs, thereby reducing the time required to conduct the permeability studies.     

Cleistanthin A,a diphyllin glycoside from Cleistanthus collinus,is cytotoxic to PHA‐stimulated (proliferating) human lymphocytes

An ideal anticancer drug would be one that preferentially kills tumor cells with the least toxicity to normal cells. Cleistanthin A, a diphyllin glycoside of the tropical plant Cleistanthus collinus, was found to possess cytotoxic and tumor regressing properties. To find out whether this compound acts selectively on proliferating cells it was tested against quiescent and proliferating human lymphocytes. Mitogen‐stimulated and unstimulated human lymphocytes were treated with cleistanthin A. A cytotoxicity assay using MTT was used to assess the viability of the cells. Percentage viability of the unstimulated and treated cells were normalized to that of the untreated and unstimulated cells and percentage viability of stimulated and treated cells were normalized to that of stimulated and untreated cells. Quiescent lymphocytes were refractory to the action of cleistanthin A. Only proliferating cells were killed. Cell death was proportional to the percentage of cells in the proliferating stage and was also dose‐dependent. Quiescent lymphocytes pretreated with cleistanthin A had the ability to proliferate upon subsequent stimulation with PHA. These results indicate that cleistanthin A does not affect the viability of quiescent cells. Also, it did not affect the proliferating potential of quiescent cells. However, this compound drastically affected proliferating cells by reducing their viability to 10–20%. Our results therefore indicate that the antiproliferative property of cleistanthin A could be used in regimens for treating tumors with extensive proliferative potencies. Drug Dev. Res. 51:187–190, 2000. © 2001 Wiley‐Liss, Inc.     

Potential beneficial effect of naringenin on lipid peroxidation and antioxidant status in rats with ethanol‐induced hepatotoxicity

Objectives The aim was to study the effect of naringenin, a biologically active compound, on tissue antioxidant status and lipid peroxidation in ethanol‐induced hepatotoxicity in rats. Methods Rats were divided into four groups: Groups 1 and 2 received isocaloric glucose and 0.5% carboxymethyl cellulose; groups 3 and 4 received 20% ethanol equivalent to 6 g/kg daily for 60 days. In addition, groups 2 and 4 were given naringenin (50 mg/kg) daily for the last 30 days of the experiment. Key findings The results showed significantly elevated levels of serum aspartate and alanine transaminases, γ‐glutamyl transpeptidase, tissue thiobarbituric acid reactive substances, conjugated dienes, lipid hydroperoxides and protein carbonyl content, and significantly lowered activities/levels of antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione‐S‐transferase, reduced glutathione and vitamins C and E in ethanol‐treated rats compared with control rats. Administration of naringenin to rats with ethanol‐induced liver injury significantly decreased the levels of serum aspartate and alanine transaminases, γ‐glutamyl transpeptidase, tissue thiobarbituric acid reactive substances, conjugated dienes, lipid hydroperoxides and protein carbonyl content and significantly elevated the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione‐S‐transferase, and the levels of reduced glutathione and vitamins C and E in the tissues compared with unsupplemented ethanol‐treated rats. Histological changes observed in the liver correlated with the biochemical findings. Conclusions Taken together these findings suggest that naringenin has a therapeutic potential in the abatement of ethanol‐induced hepatotoxicity.     

Impact of Essentiale L on ethanol-induced changes in rat brain and erythrocytes

INTRODUCTION: The present study was designed to investigate the effect of Essentiale L, a mixture of polyenylphospholipids from soybeans, on oxidative stress in various brain regions, on erythrocytes (RBC) and on RBC membrane composition in ethanol-administered rats. METHODS: Adult male albino rats of body weight 150-170 g were divided into four groups and administered either isocaloric glucose (5 g/kg body weight/day) or ethanol (6 g/kg body weight/day) through oral gavage. Essentiale L was administered to a set of ethanol-fed rats and the control rats at a dosage of 300 mg/kg body weight/day through oral gavage. The treatment protocol was carried out for 45 days. At the end of the experimental period, the animals were sacrificed, and the biochemical parameters related to the lipid profile, oxidative stress and thiol status were assayed in the brain regions, RBC and RBC membrane. RESULTS: Ethanol administration resulted in increased levels of lipid peroxidation products in RBC and different brain regions, such as the cortex, cerebellum, striatum, hippocampus and hypothalamus, and depletion of enzymatic and nonenzymatic antioxidants and alterations in oxidised glutathione/glutathione (GSSG/GSH) ratio and thiol groups (protein-bound and total), signifying oxidative stress. Ethanol-treated rats also showed significant alterations in protein content and lipid composition in RBC membranes. Significant differences in the relative proportions of hexose, hexosamine and sialic acid of the membranes were observed. Administration of Essentiale L prevented all the alterations induced by ethanol and returned their levels to near-normal. CONCLUSION: These findings suggest that Essentiale L, a therapeutic adjunct for liver diseases, also has bioprotective effects on nonhepatic tissues and cells.     

Alzheimer’s Disease and Type 2 Diabetes: Multiple Mechanisms Contribute to Interactions

Obesity, metabolic syndrome, and type 2 diabetes (T2D) are related disorders with widespread deleterious effects throughout the body. One important target of damage is the brain. Persons with metabolic disorders are at significantly increased risk for cognitive decline and the development of vascular dementia and Alzheimer’s disease. Our review of available evidence from epidemiologic, clinical, and basic research suggests that neural dysfunction from T2D-related disease results from several underlying mechanisms, including metabolic, inflammatory, vascular, and oxidative changes. The relationships between T2D and neural dysfunction are regulated by several modifiers. We emphasize 2 such modifiers, the genetic risk factor apolipoprotein E and an age-related endocrine change, low testosterone. Both factors are independent risk factors for Alzheimer’s disease that may also cooperatively regulate pathologic interactions between T2D and dementia. Continued elucidation of the links between metabolic disorders and neural dysfunction promises to foster the development of effective therapeutic strategies.