We aimed to manipulate the metabolism of Saccharomyces cerevisiae to produce lactic acid and search for the potential influence of acid transport across the plasma membrane in this process. Saccharomyces cerevisiae W303-1A is able to use l-lactic acid but its production in our laboratory has not previously been detected. When the l-LDH gene from Lactobacillus casei was expressed in S. cerevisiae W303-1A and in the isogenic mutants jen1∆, ady2∆ and jen1∆ ady2∆, all strains were able to produce lactic acid, but higher titres were achieved in the mutant strains. In strains constitutively expressing both LDH and JEN1 or ADY2, a higher external lactic acid concentration was found when glucose was present in the medium, but when glucose was exhausted, its consumption was more pronounced. These results demonstrate that expression of monocarboxylate permeases influences lactic acid production. Ady2 has been previously characterized as an acetate permease but our results demonstrated its additional role in lactate uptake. Overall, we demonstrate that monocarboxylate transporters Jen1 and Ady2 are modulators of lactic acid production and may well be used to manipulate lactic acid export in yeast cells.
Cardiovascular complications associated with 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse have increasingly been reported. The indirect effect of MDMA mediated by a sustained high level of circulating biogenic amines may contribute to the cardiotoxic effects, but other factors, like the direct toxic effects of MDMA and its metabolites in cardiac cells, remain to be investigated. Thus, the objective of the present in vitro study was to evaluate the potential cardiotoxic effects of MDMA and its major metabolites 3,4-methylenedioxyamphetamine (MDA), N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA) using freshly isolated adult rat cardiomyocytes. The cell suspensions were incubated with these compounds in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 4 h. alpha-MeDA, N-Me-alpha-MeDA, and their respective aminochromes (oxidation products) were quantified in cell suspensions by HPLC-DAD. The toxic effects were evaluated at hourly intervals for 4 h by measuring the percentage of cells with normal morphology, glutathione (GSH), and glutathione disulfide (GSSG); intracellular Ca(2+), ATP, and ADP; and the cellular activities of glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. No toxic effects were found after exposure of rat cardiomyocytes to MDMA or MDA at any of the tested concentrations for 4 h. In contrast, their catechol metabolites N-Me-alpha-MeDA and alpha-MeDA induced significant toxicity in rat cardiomyocytes. The toxic effects were characterized by a loss of normal cell morphology, which was preceded by a loss of GSH homeostasis due to conjugation of GSH with N-Me-alpha-MeDA and alpha-MeDA, sustained increase of intracellular Ca(2+) levels, ATP depletion, and decreases in the antioxidant enzyme activities. The oxidation of N-Me-alpha-MeDA and alpha-MeDA into the toxic compounds N-methyl-alpha-methyldopaminochrome and alpha-methyldopaminochrome, respectively, was also verified in cell suspensions incubated with these MDMA metabolites. The results obtained in this study provide evidence that the metabolism of MDMA into N-Me-alpha-MeDA and alpha-MeDA is required for the expression of MDMA-induced cardiotoxicity in vitro, being N-Me-alpha-MeDA the most toxic of the studied metabolites.
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