Insong J. Lee

Mechanism of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy)-mediated mitochondrial dysfunction in rat liver

Despite numerous reports citing the acute hepatotoxicity caused by 3,4‐methylenedioxymethamphetamine (MDMA) (ecstasy), the underlying mechanism of organ damage is poorly understood. We hypothesized that key mitochondrial proteins are oxidatively modified and inactivated in MDMA‐exposed tissues. The aim of this study was to identify and investigate the mechanism of inactivation of oxidatively modified mitochondrial proteins, prior to the extensive mitochondrial dysfunction and liver damage following MDMA exposure. MDMA‐treated rats showed abnormal liver histology with significant elevation in plasma transaminases, nitric oxide synthase, and the level of hydrogen peroxide. Oxidatively modified mitochondrial proteins in control and MDMA‐exposed rats were labeled with biotin‐N‐maleimide (biotin‐NM) as a sensitive probe for oxidized proteins, purified with streptavidin–agarose, and resolved using 2‐DE. Comparative 2‐DE analysis of biotin‐NM‐labeled proteins revealed markedly increased levels of oxidatively modified proteins following MDMA exposure. Mass spectrometric analysis identified oxidatively modified mitochondrial proteins involved in energy supply, fat metabolism, antioxidant defense, and chaperone activities. Among these, the activities of mitochondrial aldehyde dehydrogenase, 3‐ketoacyl‐CoA thiolases, and ATP synthase were significantly inhibited following MDMA exposure. Our data show for the first time that MDMA causes the oxidative inactivation of key mitochondrial enzymes which most likely contributes to mitochondrial dysfunction and subsequent liver damage in MDMA‐exposed animals.

Evaluation of the transport, in vitro metabolism and pharmacokinetics of Salvinorin A, a potent hallucinogen

Salvinorin A is an unregulated potent hallucinogen isolated from the leaves of Salvia divinorum. It is the only known non-nitrogenous kappa-opioid selective agonist, and rivals synthetic lysergic acid diethylamide (LSD) in potency. The objective of this study was to characterize the in vitro transport, in vitro metabolism, and pharmacokinetic properties of Salvinorin A. The transport characteristics of Salvinorin A were assessed using MDCK-MDR1 cell monolayers. The P-glycoprotein (P-gp) affinity status was assessed by the P-gp ATPase assay. In vitro metabolism studies were performed with various specific human CYP450 isoforms and UGT2B7 to assess the metabolic characteristics of Salvinorin A. Cohorts (n = 3) of male Sprague Dawley rats were used to evaluate the pharmacokinetics and brain distribution of Salvinorin A (10 mg/kg, intraperitoneal (i.p.) over a 240-min period. A validated UV-HPLC and LC/MS/MS method was used to quantify the hallucinogen concentrations obtained from the in vitro and in vivo studies, respectively. Salvinorin A displayed a high secretory transport in the MDCK-MDR1 cells (4.07 +/- 1.34 x 10(-)5 cm/s). Salvinorin A also stimulated the P-gp ATPase activity in a concentration (5 and 10 microM)-dependent manner, suggesting that it may be a substrate of (P-gp). A significant decrease in Salvinorin A concentration ranging from 14.7 +/- 0.80% to 31.1 +/- 1.20% was observed after incubation with CYP2D6, CYP1A1, CYP2C18, and CYP2E1, respectively. A significant decrease was also observed after incubation with UGT2B7. These results suggest that Salvinorin A maybe a substrate of UGT2B7, CYP2D6, CYP1A1, CYP2E1, and CYP2C18. The in vivo pharmacokinetic study showed a relatively fast elimination with a half-life (t1/2) of 75 min and a clearance (Cl/F) of 26 L/h/kg. The distribution was extensive (Vd of 47.1 L/kg); however, the brain to plasma ratio was 0.050. Accordingly, the brain half-life was relatively short, 36 min. Salvinorin A is rapidly eliminated after i.p. dosing, in accordance with its fast onset and short duration of action. Further, it appears to be a substrate for various oxidative enzymes and multi-drug resistant protein, P-gp.

Mechanisms of MDMA (Ecstasy)-Induced Oxidative Stress, Mitochondrial Dysfunction, and Organ Damage

Despite numerous reports about the acute and sub-chronic toxicities caused by MDMA (3,4-methylenedioxymethamphetamine, ecstasy), the underlying mechanism of organ damage is poorly understood. The aim of this review is to present an update of the mechanistic studies on MDMA-mediated organ damage partly caused by increased oxidative/nitrosative stress. Because of the extensive reviews on MDMA-mediated oxidative stress and tissue damage, we specifically focus on the mechanisms and consequences of oxidative-modifications of mitochondrial proteins, leading to mitochondrial dysfunction. We briefly describe a method to systematically identify oxidatively-modified mitochondrial proteins in control and MDMA-exposed rats by using biotin-N-maleimide (biotin-NM) as a sensitive probe for oxidized proteins. We also describe various applications and advantages of this Cys-targeted proteomics method and alternative approaches to overcome potential limitations of this method in studying oxidized proteins from MDMA-exposed tissues. Finally we discuss the mechanism of synergistic drug-interaction between MDMA and other abused substances including alcohol (ethanol) as well as application of this redox-based proteomics method in translational studies for developing effective preventive and therapeutic agents against MDMA-induced organ damage.

Estradiol and progesterone-mediated regulation of P-gp in P-gp overexpressing cells (NCI-ADR-RES) and placental cells (JAR)

The effect of progesterone and estrogen treatment on the expression and function of P-glycoprotein (P-gp) was evaluated in JAR cells and a P-gp overexpressing cell line, NCI-ADR-RES. Western blot analysis and real-time Q-PCR were used to evaluate P-gp protein and MDR1 mRNA expression respectively in the cells following incubation with progesterone (P4) and/or beta-estradiol (E2). Cellular uptake studies of the P-gp substrates, saquinavir and paclitaxel, were performed to evaluate function. Treatment with either E2 or P4 resulted in a significant increase in P-gp protein levels in the NCI-ADR-RES cells at concentrations of or greater than 100 nM or 10 nM, respectively. JAR cells also had increased levels of P-gp with 100 nM of P4 but were much more sensitive to E2 showing increased P-gp at a concentration of 1 nM. Furthermore, E2 or P4 treatment resulted in a significant decrease in cellular uptake of the P-gp substrates tested in these cells lines. Based on mRNA quantitation, a transient increase (2-fold) in MDR1 levels was observed at 8 h postincubation with either E2 or P4, while MDR1 levels remained high in the JAR cells treated with E2 for 72 h postincubation. The addition of actinomycin D, a transcription inhibitor negated the increase in P-gp by P4 and E2. P4 and E2 increase P-gp expression and function in NCI-ADR-RES and JAR cells with the ERalpha-expressing cells (JAR) much more sensitive to E2. Furthermore, transcriptional regulation by E2 and P4 likely contributes to the modulation of P-gp levels.

NMR metabolomic analysis of caco-2 cell differentiation.

The high-resolution (1)H NMR spectra as applied to Caco-2 cells during their differentiation into enterocyte like cells are presented. The data clearly reveal differences in the metabolic profiles over time as the Caco-2 cells differentiate. In the (1)H NMR spectra, the aliphatic regions from 4.5 to 1.0 ppm are dominated by peaks from myo-inositol, creatine, taurine, glutamine, glutamate, phosphatidylcholine, choline, alanine and lactate. While a majority of metabolites are present at both the early undifferentiated state and the late differentiated states, the levels of certain metabolites are seen to change dramatically, and in particular, the ratio of myo-inositol and taurine. The NMR spectrum from 10 to 5 ppm shows the aromatic amino acids (Phe, Tyr), NAD, ATP and ribose signals. The appearance of glucose resonances in the differentiated cells (30 days old) spectra suggests that these cells become gluconeogenic. Our study represents a novel method to analyze the differentiation of Caco-2 cells using a metabolomic approach. The results indicate, for the first time, that taurine and glucose biosynthesis occurs in these cells and thus by extension may occur in the intestine. This metabolomic approach can therefore be used to detect novel biological pathways as well as yield useful markers for differentiation.

Distribution of Saquinavir, Methadone, and Buprenorphine in Maternal Brain, Placenta, and Fetus During Two Different Gestational Stages of Pregnancy in Mice

Efflux transporters such as P-glycoprotein (P-gp) play a critical role in the maternal-to-fetal and blood-to-brain transfer of many drugs. Using a mouse model, the effects of gestational age on P-gp and MRP expression in the placenta and brain were evaluated. P-gp protein levels in the placenta and brain were greater at mid-gestation (gd 13) than late-gestation (gd 18). Likewise, brain MRP1 levels were greater at mid-gestation, whereas, placental levels were greater at late-gestation. To evaluate these effects on drug disposition, concentrations of [(3)H]saquinavir, [(3)H]methadone, [(3)H]buprenorphine, and the paracellular marker, [(14)C]mannitol were measured in plasma, brain, placenta, and fetal samples after i.v. administrations to nonpregnant and pregnant mice. Following i.v. administration, [(3)H]saquinavir placenta-to-plasma and fetal-to-plasma ratios were significantly greater in late-gestation mice versus mid-gestation. Furthermore, late-gestation mice experienced significant increases in the [(3)H]saquinavir and [(3)H]methadone brain-to-plasma ratios 60 min after dosing relative to mid-gestation (p < 0.05). No significant differences were observed in these tissue-to-plasma ratios for buprenorphine or mannitol. Repeated dosing (three doses, once daily) decreased the differential uptake of [(3)H]saquinavir in brain but potentiated it in the fetus. These results suggest that differential expression of P-gp and possibly MRP1 contributes to the gestational-induced changes in brain and fetal uptake of saquinavir.

Increased oxidative‐modifications of cytosolic proteins in 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy)‐exposed rat liver

It is well established that 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) causes acute liver damage in animals and humans. The aim of this study was to identify and characterize oxidative modification and inactivation of cytosolic proteins in MDMA‐exposed rats. Markedly increased levels of oxidized and nitrated cytosolic proteins were detected 12 h after the second administration of two consecutive MDMA doses (10 mg/kg each). Comparative 2‐DE analysis showed markedly increased levels of biotin‐N‐methylimide‐labeled oxidized cytosolic proteins in MDMA‐exposed rats compared to vehicle‐treated rats. Proteins in the 22 gel spots of strong intensities were identified using MS/MS. The oxidatively modified proteins identified include anti‐oxidant defensive enzymes, a calcium‐binding protein, and proteins involved in metabolism of lipids, nitrogen, and carbohydrates (glycolysis). Cytosolic superoxide dismutase was oxidized and its activity significantly inhibited following MDMA exposure. Consistent with the oxidative inactivation of peroxiredoxin, MDMA activated c‐Jun N‐terminal protein kinase and p38 kinase. Since these protein kinases phosphorylate anti‐apoptotic Bcl‐2 protein, their activation may promote apoptosis in MDMA‐exposed tissues. Our results show for the first time that MDMA induces oxidative‐modification of many cytosolic proteins accompanied with increased oxidative stress and apoptosis, contributing to hepatic damage.

Drug Interaction Between Ethanol and 3,4-Methylenedioxymethamphetamine (“ecstasy”)

Alcohol (ethanol) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are frequently co-abused, but recent findings indicate a harmful drug interaction between these two agents. In our previous study, we showed that MDMA exposure inhibits the activity of the acetaldehyde (ACH) metabolizing enzyme, aldehyde dehydrogenase2 (ALDH2). Based on this finding, we hypothesized that the co-administration of MDMA and ethanol would reduce the metabolism of ACH and result in increased accumulation of ACH. Rats were treated with MDMA or vehicle and then administered a single dose of ethanol. Liver ALDH2 activity decreased by 35% in the MDMA-treated rats compared to control rats. The peak concentration and the area under the concentration versus time curve of plasma ACH were 31% and 59% higher, respectively, in the MDMA-ethanol group compared to the ethanol-only group. In addition, the MDMA-ethanol group had 80% higher plasma transaminase levels than the ethanol-only group, indicating greater hepatocellular damage. Our results not only support a drug interaction between MDMA and ethanol but a novel underlying mechanism for the interaction.

Regulation of Gene Expression in Brain Tissues of Rats Repeatedly Treated by the Highly Abused Opioid Agonist, Oxycodone: Microarray Profiling and Gene Mapping Analysis

Although oxycodone is the most often used opioid agonist, it remains one of the most understudied drugs. We used microarray analysis to better understand the global changes in gene expression in brain tissues of rats repeatedly treated with oxycodone. Many genes were significantly regulated by oxycodone (e.g., Fkbp5, Per2, Rt1.Dα, Slc16a1, and Abcg2). Validation of the microarray data by quantitative real-time-polymerase chain reaction (Q-PCR) indicated that there was a strong significant correlation (r = 0.979, p < 0.0000001) between the Q-PCR and the microarray data. Using MetaCore (a computational platform), many biological processes were identified [e.g., organic anion transport (p = 7.251 × 10−4) and regulation of immune response (p = 5.090 × 10−4)]. Among the regulated genes, Abcg2 mRNA was up-regulated by 2.1-fold, which was further confirmed by immunoblotting (1.8-fold up-regulation). Testing the Abcg2 affinity status of oxycodone using an Abcg2 ATPase assay suggests that oxycodone behaves as an Abcg2 substrate only at higher concentrations (≥500 μM). Furthermore, brain uptake studies demonstrated that oxycodone-induced Abcg2 up-regulation resulted in a significant (p < 0.05) decrease (∼2-fold) in brain/plasma ratios of mitoxantrone. These results highlight markers/mediators of neuronal responses and identify regulatory pathways involved in the pharmacological action of oxycodone. These results also identify genes that potentially modulate tolerance, dependence, immune response, and drug-drug interactions. Finally, our findings suggest that oxycodone-induced up-regulation of Abcg2 enhanced the efflux of the Abcg2 substrate, mitoxantrone, limiting its brain accumulation and resulting in an undesirable drug-drug interaction. Extrapolating these results to other Abcg2 substrates (e.g., daunorubicin and doxorubicin) indicates that the brain uptake of these agents may be affected if they are administered concomitantly with oxycodone.

Repeated administration of oxycodone modifies the gene expression of several drug metabolising enzymes in the hepatic tissue of male Sprague-Dawley rats, including glutathione S-transferase A-5 (rGSTA5) and CYP3A2.

Objectives Clinical use and illicit abuse of the potent opioid agonist oxycodone has dramatically increased over the past decade. Yet oxycodone remains one of the least studied opioids, particularly its interactions on the genomic level. The aim of this study was to examine potential alterations in gene expression of drug metabolising enzymes in the liver tissue of male Sprague‐Dawley rats chronically treated with oxycodone.