Dopamine (DA) metabolism in PC12 cells exposed to manganese (Mn) at different oxidation states.

The present study was aimed at assessing the role of Mn valency state in Mn-induced changes in DA metabolism by PC12 cells. Mn(ll)Cl2, Mn(lll)Acetate, and Mn(IV)O2 were used for these experiments. PC12 cells were incubated for 3, 24 and 72 hours to Mn nominal concentrations ranging from 10-8 to 10(-4) M in 24-well plates containing 2 x 10(5) cells/well. Supernatants and cellular materials were then separated and immediately processed for the analysis of dopamine (DA), and its metabolite 3,4-di-hydroxy-phenylacetic acid (DOPAC). Lactate dehydrogenase (LDH) activity and MTT cleavage were measured as indices of cell death. In parallel experiments, Mn-containing medium (10(-5) M) was removed and cells incubated for further periods with Mn-free medium to evaluate the reversibility of observed changes. At the end of the experimental periods, none of Mn-exposed cultures showed appreciable reduction in cell viability as compared to their respective controls. After exposure to Mn(II) and Mn(III), irreversible and dose-dependent decreases in the medium but not in intra-cellular DA were apparent. Indeed, 10(-4) M Mn(II) caused the disappearance of DA and DOPAC from the medium. The same effect was caused by 10(-5) M Mn(III), the dose-effect relationship being shifted towards lower dose levels. Mn(IV) induced a parallel and dose-dependent decrease of DA and DOPAC concentrations in both intra- and extra-cellular compartments. Such an effect was reversible after removal of Mn from the medium. Multiple interferences on DA metabolism are caused by Mn. Mn(II) and Mn(III) seem to block DA secretion without affecting DA turnover rate. Mn(IV) seems to cause DA depletion and aspecific (secondary) changes in secretion rates. Further studies are necessary to understand the mechanisms underlying the differential effects of various Mn compounds on DA metabolism.

Multiple interferences on catecholamine metabolism by tetrahydroisoquinolines (TIQs).

TIQs are thought to be formed by condensation between dopamine and certain metabolites of ethanol, organic solvents and anesthetic gases. Described here are experiments aimed at evaluating TIQs interference with catecholamine synthesis. Rat adrenal pheochromocytoma (PC12) cell lysates were exposed to benzyl-TIQ and phenyl-TIQ. The activities of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) were measured by HPLC-based methods following exposure to variable concentrations of TIQs. The effects of TIQs on DBH activity were also assessed in human serum. Dixon plot analyses revealed that TIQs act on TH as competitive inhibitors with different affinity. Ki for benzyl- and phenyl-TIQ were 5 and 3 microM respectively. DBH activity in serum exposed to benzyl- and phenyl-TIQ ranging from 0.2 to 20 microM rose respectively by 12.5% to 58% for benzyl- and by 7.8% to 26% for phenyl-TIQ. Such TIQs interferences with catecholamine metabolism seem to account for dopamine (DA) depletion observed in parallel in vitro experiments on PC12 cells. The dose-dependent inhibition of TH and the increased activity of DBH together with the relatively low effective doses of TIQs suggest this mechanism as a possible explanation of the selective toxicity of styrene and other solvents to dopaminergic systems observed in rabbits following experimental exposure and suspected to occur in occupationally-exposed workers.