An optimized method for the measurement of acetaldehyde by high-performance liquid chromatography.

BACKGROUND: Acetaldehyde is produced during ethanol metabolism predominantly in the liver by alcohol dehydrogenase and rapidly eliminated by oxidation to acetate via aldehyde dehydrogenase. Assessment of circulating acetaldehyde levels in biological matrices is performed by headspace gas chromatography and reverse phase high-performance liquid chromatography (RP-HPLC). METHODS: We have developed an optimized method for the measurement of acetaldehyde by RP-HPLC in hepatoma cell culture medium, blood, and plasma. After sample deproteinization, acetaldehyde was derivatized with 2,4-dinitrophenylhydrazine (DNPH). The reaction was optimized for pH, amount of derivatization reagent, time, and temperature. Extraction methods of the acetaldehyde-hydrazone (AcH-DNP) stable derivative and product stability studies were carried out. Acetaldehyde was identified by its retention time in comparison with AcH-DNP standard, using a new chromatography gradient program, and quantitated based on external reference standards and standard addition calibration curves in the presence and absence of ethanol. RESULTS: Derivatization of acetaldehyde was performed at pH 4.0 with an 80-fold molar excess of DNPH. The reaction was completed in 40 minutes at ambient temperature, and the product was stable for 2 days. A clear separation of AcH-DNP from DNPH was obtained with a new 11-minute chromatography program. Acetaldehyde detection was linear up to 80 µM. The recovery of acetaldehyde was >88% in culture media and >78% in plasma. We quantitatively determined the ethanol-derived acetaldehyde in hepatoma cells, rat blood and plasma with a detection limit around 3 µM. The accuracy of the method was <9% for intraday and <15% for interday measurements, in small volume (70 µl) plasma sampling. CONCLUSIONS: An optimized method for the quantitative determination of acetaldehyde in biological systems was developed using derivatization with DNPH, followed by a short RP-HPLC separation of AcH-DNP. The method has an extended linear range, is reproducible and applicable to small-volume sampling of culture media and biological fluids.

Effects of ethanol on mouse embryonic stem cells.

BACKGROUND: Fetal alcohol syndrome (FAS) reflects a constellation of congenital abnormalities caused by excess maternal consumption of alcohol. It is likely that interference with embryonic development plays a role in the pathogenesis of the disorder. Ethanol-induced apoptosis has been suggested as a causal factor in the genesis of FAS. Mouse embryonic stem (mES) cells are pluripotent cells that differentiate in vitro to cell aggregates termed embryoid bodies (EBs), wherein differentiation capacity and gene expression profile are similar to those of the early embryo. METHODS: To investigate the effects of ethanol during differentiation, mES cells were cultured on a gelatin surface in the presence of leukemia inhibitory factor which maintains adherent undifferentiated cells or in suspension to promote formation of EBs. All cells were treated (1-6 days) with 80 mM ethanol. The pluripotency and differentiation of mES cells were evaluated by western blotting of stage-specific embryonic antigen (SSEA-1), transcription factors Oct-3/4, Sox-2, and Nanog, using alkaline phosphatase staining. Apoptosis (early to late stages) was assessed by fluorescence-activated cell sorting using TdT-mediated biotin-dUTP nick-end labelling assay and fluorescein isothiocyanate-Annexin V/propidium iodide staining. RESULTS: Ethanol increased apoptosis during in vitro differentiation of mES cells to EBs, whereas undifferentiated cells were not affected. Ethanol exposure also interfered with pluripotency marker patterns causing an upregulation of SSEA-1 under self-renewal conditions. In EBs, ethanol delayed the downregulation of SSEA-1 and affected the regulation of transcription factors during differentiation. CONCLUSION: Our findings suggest that ethanol may contribute to the pathogenesis of FAS by triggering apoptotic pathways during differentiation of embryonic stem cells and deregulating early stages of embryogenesis.

Suppression of heavy drinking and alcohol seeking by a selective ALDH-2 inhibitor.

BACKGROUND: Inherited human aldehyde dehydrogenase 2 (ALDH-2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH-2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH-2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However, daidzin can reduce drinking in some rodents without necessarily increasing acetaldehyde. Therefore, a selective ALDH-2 inhibitor might affect other metabolic factors involved in regulating drinking. METHODS: Aldehyde dehydrogenase 2 inhibitors were synthesized based on the co-crystal structure of ALDH-2 and daidzin. We tested the efficacy of a highly selective reversible ALDH-2 inhibitor, CVT-10216, in models of moderate and high alcohol drinking rats. We studied 2-bottle choice and deprivation-induced drinking paradigms in Fawn Hooded (FH) rats, operant self-administration in Long Evans (LE), FH, and inbred P (iP) rats and in cue-induced reinstatement in iP rats. We also assayed blood acetaldehyde levels as well as dopamine (DA) release in the nucleus accumbens (NAc) and tested possible rewarding/aversive effects of the inhibitor in a conditioned place preference (CPP) paradigm. RESULTS: CVT-10216 increases acetaldehyde after alcohol gavage and inhibits 2-bottle choice alcohol intake in heavy drinking rodents, including deprivation-induced drinking. Moreover, CVT-10216 also prevents operant self-administration and eliminates cue-induced reinstatement of alcohol seeking even when alcohol is not available (i.e., no acetaldehyde). Alcohol stimulates DA release in the NAc, which is thought to contribute to increased drinking and relapse in alcoholism. CVT-10216 prevents alcohol-induced increases in NAc DA without changing basal levels. CVT-10216 does not show rewarding or aversive properties in the CPP paradigm at therapeutic doses. CONCLUSION: Our findings suggest that selective reversible ALDH-2 inhibitors may have therapeutic potential to reduce excessive drinking and to suppress relapse in abstinent alcoholics.

Mitochondrial fusion and Bid-mediated mitochondrial apoptosis are perturbed by alcohol with distinct dependence on its metabolism.

Environmental stressors like ethanol (EtOH) commonly target mitochondria to influence the cell's fate. Recent literature supports that chronic EtOH exposure suppresses mitochondrial dynamics, central to quality control, and sensitizes mitochondrial permeability transition pore opening to promote cell death. EtOH-induced tissue injury is primarily attributed to its toxic metabolic products but alcoholism also impairs tissues that poorly metabolize EtOH. We embarked on studies to determine the respective roles of EtOH and its metabolites in mitochondrial fusion and tBid-induced mitochondrial apoptosis. We used HepG2 cells that do not metabolize EtOH and its engineered clone that expresses EtOH-metabolizing Cytochrome P450 E2 and alcohol dehydrogenase (VL-17A cells). We found that fusion impairment by prolonged EtOH exposure was prominent in VL-17A cells, probably owing to reactive oxygen species increase in the mitochondrial matrix. There was no change in fusion protein abundance, mitochondrial membrane potential or Ca2+ uptake. By contrast, prolonged EtOH exposure promoted tBid-induced outer mitochondrial membrane permeabilization and cell death only in HepG2 cells, owing to enhanced Bak oligomerization. Thus, mitochondrial fusion inhibition by EtOH is dependent on its metabolites, whereas sensitization to tBid-induced death is mediated by EtOH itself. This difference is of pathophysiological relevance because of the tissue-specific differences in EtOH metabolism.

MICU1 regulation of mitochondrial Ca(2+) uptake dictates survival and tissue regeneration.

Mitochondrial Ca(2+) uptake through the recently discovered Mitochondrial Calcium Uniporter (MCU) is controlled by its gatekeeper Mitochondrial Calcium Uptake 1 (MICU1). However, the physiological and pathological role of MICU1 remains unclear. Here we show that MICU1 is vital for adaptation to postnatal life and for tissue repair after injury. MICU1 knockout is perinatally lethal in mice without causing gross anatomical defects. We used liver regeneration after partial hepatectomy as a physiological stress response model. Upon MICU1 loss, early priming is unaffected, but the pro-inflammatory phase does not resolve and liver regeneration fails, with impaired cell cycle entry and extensive necrosis. Ca(2+) overload-induced mitochondrial permeability transition pore (PTP) opening is accelerated in MICU1-deficient hepatocytes. PTP inhibition prevents necrosis and rescues regeneration. Thus, our study identifies an unanticipated dependence of liver regeneration on MICU1 and highlights the importance of regulating MCU under stress conditions when the risk of Ca(2+) overload is elevated.

The effect of controlled drinking in alcoholic cardiomyopathy.

BACKGROUND: Cardiomyopathy is a potentially fatal complication of alcohol abuse. In alcoholic persons who develop cardiac dysfunction, abstinence is thought to be essential to halt further deterioration of cardiac contractility. Some evidence indicates that reducing alcohol intake may also be beneficial. OBJECTIVE: To evaluate the effect of moderate "controlled" drinking on cardiac function in patients with alcoholic cardiomyopathy. DESIGN: 4-year prospective cohort study. SETTING: A university hospital in Barcelona, Spain. PATIENTS: 55 alcoholic men with cardiomyopathy who had been drinking a minimum of 100 g of ethanol per day for at least 10 years. MEASUREMENTS: Evaluation of ethanol intake and nutrition, clinical assessment of cardiac status, and sequential echocardiography and radionuclide cardiac angiography. RESULTS: After the first year of evaluation, all patients with cardiomyopathy who abstained from alcoholic beverages demonstrated significant improvement in left ventricular function (average increase in left ventricular ejection fraction, 0.131 [95% CI, 0.069 to 0.193]). Patients who drank 20 to 60 g of ethanol per day showed a comparable mean improvement of 0.125 (CI, 0.082 to 0.168). In contrast, left ventricular ejection fraction deteriorated further in most patients who continued to abuse alcohol (>80 g/d). After 4 years, left ventricular ejection fraction had continued to improve in both abstinent patients and those who controlled their drinking. Ten patients who had continued to consume more than 80 g of ethanol per day died during the study. CONCLUSION: In patients with alcoholic cardiomyopathy, both abstinence and controlled drinking of up to 60 g of ethanol per day (four standard drinks) were comparably effective in promoting improvement in cardiac function.

Angiotensin-converting enzyme gene polymorphism is associated with vulnerability to alcoholic cardiomyopathy.

BACKGROUND: Chronic alcohol abuse has a dose-dependent toxic effect on the myocardium, leading to alcoholic cardiomyopathy. The fact that only a minority of persons with chronic alcoholism have this condition suggests the possibility of a genetic vulnerability. In this context, polymorphism of the angiotensin-converting enzyme (ACE) gene has been implicated in cardiac dysfunction. OBJECTIVE: To compare the ACE genotypes of alcoholic persons who have cardiomyopathy with those of comparable alcohol abusers who have normal cardiac function. DESIGN: Case-control study over a 2-year period. SETTING: An academic tertiary referral hospital in Barcelona, Spain. PATIENTS: 30 alcoholic men with symptomatic cardiomyopathy and 27 alcoholic men with normal cardiac function. MEASUREMENTS: Ethanol intake, cardiac status, left ventricular ejection fraction (LVEF), and ACE gene polymorphism. RESULTS: The DD ACE genotype was present in 57% of alcoholic persons with an LVEF less than 0.50 and in 7% of those with normal cardiac function. Compared with persons who had an I allele, the odds ratio for development of left ventricular dysfunction in alcoholic persons with the DD genotype was 16.4. CONCLUSIONS: Vulnerability to cardiomyopathy among chronic alcohol abusers is partially genetic and is related to presence of the ACE DD genotype. This finding demonstrates genetic susceptibility to alcohol-induced myocardial damage.

Different effects of red wine and gin consumption on inflammatory biomarkers of atherosclerosis: a prospective randomized crossover trial. Effects of wine on inflammatory markers.

BACKGROUND: No intervention studies have explored the anti-inflammatory effects of different alcoholic beverages on markers of atherosclerosis. We embarked on a randomized, crossover, single-blinded trial to evaluate the effects of wine and gin on inflammatory biomarkers of atherosclerosis. METHODS AND RESULTS: Forty healthy men (mean age, 37.6 years) consumed 30 g ethanol per day as either wine or gin for 28 days. Before and after each intervention, we measured the expression of lymphocyte function-associated antigen 1 (LFA-1), Mac-1, very late activation antigen 4 (VLA-4), and monocyte chemoattractant protein (MCP-1) in monocytes, as well as the soluble vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), interleukin-1alpha (IL-1alpha), C-reactive protein (hs-CRP) and fibrinogen. After either gin or wine consumption, plasma fibrinogen decreased by 5 and 9%, respectively, and cytokine IL-1alpha by 23 and 21%. The expression of LFA-1 (-27%), Mac-1 (-27%), VLA-4 (-32%) and MCP-1 (-46%) decreased significantly after wine, but not after gin. Wine reduced the serum concentrations of hs-CRP (-21%), VCAM-1 (-17%) and ICAM-1 (-9%). CONCLUSIONS: Both wine and gin showed anti-inflammatory effects by reducing plasma fibrinogen and IL-1alpha levels. However, wine had the additional effect of decreasing hs-CRP, as well as monocyte and endothelial adhesion molecules.

Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism: implications for breast cancer prevention.

Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with "stemness," more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This "two-compartment" metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert "low-risk" breast cancer patients to "high-risk" status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results also show that antioxidants [such as N-acetyl cysteine (NAC)] can effectively reverse or prevent ethanol-induced oxidative stress in cancer-associated fibroblasts, suggesting a novel strategy for cancer prevention. We also show that caveolin-1 and MCT4 protein expression can be effectively used as new biomarkers to monitor oxidative stress induced by ethanol.

Mitochondrial dysfunction in breast cancer cells prevents tumor growth: understanding chemoprevention with metformin.

Metformin is a well-established diabetes drug that prevents the onset of most types of human cancers in diabetic patients, especially by targeting cancer stem cells. Metformin exerts its protective effects by functioning as a weak "mitochondrial poison," as it acts as a complex I inhibitor and prevents oxidative mitochondrial metabolism (OXPHOS). Thus, mitochondrial metabolism must play an essential role in promoting tumor growth. To determine the functional role of "mitochondrial health" in breast cancer pathogenesis, here we used mitochondrial uncoupling proteins (UCPs) to genetically induce mitochondrial dysfunction in either human breast cancer cells (MDA-MB-231) or cancer-associated fibroblasts (hTERT-BJ1 cells). Our results directly show that all three UCP family members (UCP-1/2/3) induce autophagy and mitochondrial dysfunction in human breast cancer cells, which results in significant reductions in tumor growth. Conversely, induction of mitochondrial dysfunction in cancer-associated fibroblasts has just the opposite effect. More specifically, overexpression of UCP-1 in stromal fibroblasts increases ß-oxidation, ketone body production and the release of ATP-rich vesicles, which "fuels" tumor growth by providing high-energy nutrients in a paracrine fashion to epithelial cancer cells. Hence, the effects of mitochondrial dysfunction are truly compartment-specific. Thus, we conclude that the beneficial anticancer effects of mitochondrial inhibitors (such as metformin) may be attributed to the induction of mitochondrial dysfunction in the epithelial cancer cell compartment. Our studies identify cancer cell mitochondria as a clear target for drug discovery and for novel therapeutic interventions.

Oligonucleotide-mediated gene targeting in human hepatocytes: implications of mismatch repair.

Gene therapy using viral vectors for liver diseases, particularly congenital disorders, is besought with difficulties, particularly immunologic reactions to viral antigens. As a result, nonviral methods for gene transfer in hepatocytes have also been explored. Gene repair by small synthetic single-stranded oligodeoxynucleotides (ODNs) produces targeted alterations in the genome of mammalian cells and represents a great potential for nonviral gene therapy. To test the feasibility of ODN-mediated gene repair within chromosomal DNA in human hepatocytes, two new cell lines with stably integrated mutant reporter genes, namely neomycin and enhanced green fluorescent protein were established. Targeting theses cells with ODNs specifically designed for repair resulted in site-directed and permanent gene conversion of the single-point mutation of the reporter genes. Moreover, the frequency of gene alteration was highly dependent on the mitotic activity of the cells, indicating that the proliferative status is an important factor for successful targeting in human hepatocytes. cDNA array expression profiling of DNA repair genes under different cell culture conditions combined with RNA interference assay showed that mismatch repair (MMR) in actively growing hepatocytes imposes a strong barrier to efficient gene repair mediated by ODNs. Suppression of MSH2 activity in hepatocytes transduced with short hairpin RNAs (shRNAs) targeted to MSH2 mRNA resulted in 25- to 30-fold increase in gene repair rate, suggesting a negative effect of MMR on ODN-mediated gene repair. Taken together, these data suggest that under appropriate conditions nonviral chromosomal targeting may represent a feasible approach to gene therapy in liver disease.

Curricular reform may improve students' performance on externally administered comprehensive examinations.

AIM: To determine whether changes in the format of teaching pathology and the introduction of active learning principles can improve medical students' performance on external examinations and enhance clinical skills. METHOD: The sophomore Pathology Course at Jefferson Medical College (JMC) in Philadelphia, Pennsylvania, USA, was completely restructured in 1986, with greater emphasis placed on independent study, small group teaching, and case study discussion. We used the scores of JMC medical students on the National Board of Medical Examiners (NBME) Part I Examination to compare the performance of JMC students who completed their medical education before curricular change (entering classes 1982-1984) with the performance of subsequent generations of students who were taught according to the reformed curriculum (entering classes 1985-1988). RESULTS: The two groups of students were comparable in terms of standard social and psychometric parameters, such as mean age at matriculation, female/male ratio, ratio of minority students in the class, premedical college grade point averages, and mean scores on the preadmission Medical College Admissions Test. JMC students who studied pathology prior to the curricular reform received on the pathology subsection of the NBME Part I Examination reform scores that were close to the national average. In contrast, mean scores for students who studied pathology after curricular changes were significantly higher than the national average (P<0.001). Based on their pathology subscores, the number of JMC students scoring below the cutoff line for passing (380 points) decreased significantly after the curricular reform, whereas the number of high-scoring students whose scores ranked them in the 90th percentile nationally increased. Curricular reform was also associated with an increase in overall student satisfaction. CONCLUSION: Curricular changes that include an emphasis on active learning can improve the performance of medical students on externally administered, objective examinations. We have shown that the means of the medical school class can be improved, the number of failing students reduced, and the number of high-scoring students increased. The improvement of students' scores was not limited to the first class after curricular reform, but persisted throughout the entire observation period of four years.

Alcohol and mitochondria in cardiac apoptosis: mechanisms and visualization.

Apoptosis of myocytes is likely to contribute to a variety of heart conditions and could also be important in the development of alcoholic heart disease. A fundamental pathway to apoptosis is through mitochondrial membrane permeabilization and release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol. The authors' results show that prolonged exposure of cultured cardiac cells to ethanol (35 mM for 48 hr) promotes Ca2+-induced activation of the mitochondrial permeability transition pore (PTP). PTP-dependent mitochondrial membrane permeabilization is followed by release of cytochrome c and execution of apoptosis. The authors propose that chronic ethanol exposure, in combination with other stress signals, may allow for activation of the PTP by physiological calcium oscillations, providing a trigger for cardiac apoptosis during chronic alcohol abuse. Coincidence of apoptosis promoting factors occurs in only a small fraction of myocytes, but because of the absence of regeneration, even a modest increase in the rate of cell death may contribute to a decrease in cardiac contractility. Detection of apoptotic changes that are present in only a few myocytes at a certain time in the heart is not feasible with most of the apoptotic assays. Fluorescence imaging is a powerful technology to visualize changes that are confined to a minor fraction of cells in a tissue, and the use of multiphoton excitation permits imaging in situ deep in the wall of the intact heart. This article discusses potential mechanisms of the effect of alcohol on mitochondrial membrane permeabilization and visualization of mitochondria-dependent apoptosis in cardiac muscle.

Alterations in insulin-like growth factor-I signaling in cardiomyocytes from chronic alcohol-exposed rats.

BACKGROUND: Insulin-like growth factor-I (IGF-I) is a required cytokine for the development and maintenance of the cardiovascular system, and it may play a role in certain pathophysiological conditions. METHODS: Adult male rats were fed a liquid diet that contained 36% alcohol for 4 to 8 months, and their littermates served as isocaloric pair-fed controls, so we could examine IGF-I signaling in rat cardiomyocyte preparations. RESULTS: Recently, our laboratory reported that IGF-I activates protein kinase-C (PKC)-alpha and that PKC-alpha activity is required for IGF-I-dependent activation of Erk1/Erk2 and IGF-I-dependent protein synthesis. But in chronic alcohol-exposed rats, there is loss of PKC-alpha activation by IGF-I, represented as a reduction in PKC-alpha translocation to the membrane. In reverse transcription-polymerase chain reaction experiments, both the alcoholic and control animals expressed the IGF-I receptor (IGF-1R, alpha subunit) and IGF-I mRNAs in approximately equal amounts. However, in the alcoholic cardiomyocyte protein preparations, there was a higher basal level of IGF-1R autophosphorylation of its internal tyrosine kinase domain, and IGF-I-activated autophosphorylation was reduced in the alcoholic protein preparations. Previously, we have demonstrated that acute IGF-I exposure enhances the rate of Mn2+ influx through activated nitrendipine-sensitive cardiac Ca2+ channels, and that this enhancement of channel activity is PKC-dependent. Here, we report that in alcohol-exposed myocytes, IGF-I-induced augmentation of the cardiac Ca2+ channel activity was absent. IGF-I increased the rate of protein synthesis in the control animals by 56% as determined in protein synthesis experiments that measured the rate of 14C-L-phenylalanine incorporation over time, and this effect was blocked by preincubation with Gö6976, a specific inhibitor of PKC-alpha. However, in the alcohol-exposed cardiomyocytes, IGF-I did not increase the rate of protein synthesis. CONCLUSION: These results suggest that IGF-I-dependent PKC-alpha activation and IGF-I-dependent protein synthesis are altered in the hearts of chronic alcohol-exposed rats. This may be the result of the IGF-1R being in a chronically activated state, and it may alter the normal function of PKC-alpha.