Irreversible Cytotoxicity Research Articles

Effects of haloperidol metabolites on neurotransmitter uptake and release: possible role in neurotoxicity and tardive dyskinesia

This research explored the effects of haloperidol (HP) metabolites on biogenic amine uptake and release, and compared them to those of MPTP and its toxic metabolite, MPP +. In synaptosome preparations from mouse striatum and cortex, the HP metabolites haloperidol pyridinium (HPP +), reduced haloperidol pyridinium (RHPP +), and haloperidol tetrahydropyridine (HPTP) inhibited the presynaptic uptake of dopamine and serotonin, with greater affinity for the serotonin transporter. HPP + was the most potent inhibitor of dopamine uptake, and HPTP of serotonin uptake, both with IC 50 values in the low micromolar range. RHPP + was less active than the other metabolites, but was more active than the parent compound, HP. Inhibition of uptake was reversed when free drug was removed by centrifugation and then resuspension of the synaptosomes in fresh buffer, suggesting that inhibition of uptake was due to interaction with the transporters and was not due to irreversible cytotoxicity. HPP + showed noncompetitive inhibition of both serotonin and dopamine uptake, suggesting that it has a relatively slow dissociation rate for its interaction with the transporter proteins. In experiments on amine release, HPP + and HPTP were four-fold less potent than MPP + for releasing preloaded dopamine from striatal synaptosomes, and only MPP +-dependent release was antagonized by the uptake blocker, mazindol. In contrast, RHPP + displayed little ability to release either amine neurotransmitter. HPTP was about two-fold more potent than MPP + for releasing serotonin from cortical synaptosomes, whereas HPP + was less active than MPP +. The specific serotonin transport blocker fluoxetine was only able to antagonize release induced by MPP +. These results suggest that HP metabolites bind to the transporters for dopamine and serotonin, but are not transporter substrates. In contrast to their potent effects on amine release, HPP + and HPTP were unable to release preloaded GABA from cortical synaptosomes. The implications of these results concerning a possible role of HP metabolites in the development of tardive dyskinesia are discussed.

A sensitive fluorometric assay for determining hydrogen peroxide-mediated sublethal and lethal endothelial cell injury.

A rapid and sensitive quantitative fluorometric assay was developed to measure the response of endothelial cells to hydrogen peroxide (H2O2). The response of an endothelial cell-derived cell line, EA-hy-926, or human umbilical vein endothelial cells to H2O2 was determined using calcein-AM, a dye which becomes fluorescent upon cleavage by intracellular esterase(s). The ability of the cells to take up and convert calcein-AM was measured directly in the wells of 96-well flat-bottomed tissue culture plates with cell monolayers using a computerised microplate fluorimeter, or in cell suspensions using flow cytometry. The results obtained by these techniques were compared with each other and with a standard 51Cr release cytotoxicity assay. We found that calcein-AM is a highly sensitive probe for measuring H2O2-mediated cell injury, as it can not only detect the irreversible cytotoxicity measured by 51Cr release assay, but can also distinguish sublethal and reversible injury seen at low H2O2 concentrations.

Benzoyl peroxide cytotoxicity evaluated in vitro with the human keratinocyte cell line, RHEK-1

The human keratinocyte cell line, RHEK-1, was used to evaluate the cytotoxicity of benzoyl peroxide (BZP). As determined with the neutral red (NR) cytotoxicity assay, the 24-h midpoint (NR 50) toxicity values, in mM, were 0.11 for BZP and 29.5 for benzoic acid, the stable metabolite of BZP. Irreversible cytotoxicity occurred after a 1-h exposure to 0.15 mM BZP and greater. When exposed to BZP for 7 days, a lag in growth kinetics was first observed at 0.06 mM BZP. Damage to the integrity of the plasma membrane was evident, as leakage of lactic acid dehydrogenase occurred during a 4-h exposure to BZP at 0.05 mM and greater. Intracellular membranes were also affected, as extensive vacuolization, initially perinuclear but then spreading throughout the cytoplasm, was noted in BZP-stressed cells. The generation of reactive free radicals from BZP was suggested by the following: the intracellular content of glutathione was lowered in cells exposed to BZP; cells pretreated with the glutathione-depleting agent, chlorodinitrobenzene, were hypersensitive to a subsequent challenge with BZP; lipid peroxidation by BZP was inducible in the presence of Fe 2+; and cells previously maintained in a medium amended with vitamin E, an antioxidant, were more resistant to BZP, showed less lipid peroxidation in the presence of BZP + Fe 2+ and did not develop the extensive intracellular vacuolization as compared to non-vitamin E maintained cells.

Inhibition of epidermal growth factor receptor functions by tyrosine kinase inhibitors in NIH3T3 cells

Epidermal growth factor (EGF) induces transformed phenotypes in EGF receptor-overexpressing NIH3T3 (ER12) cells. Tyrosine kinase inhibitors such as erbstatin and its stable analogue methyl 2,5-dihydroxycinnamate inhibited the EGF-induced phenotypes changes in these cells; while 5′- O-methylerbstatin, an inactive analogue, did not. Methyl 2,5-dihydroxycinnamate inhibited intracellular tyrosine kinase activity in EGF-treated ER12 cells. Methyl 2,5-dihydroxycinnamate also delayed the EGF-induced DNA synthesis from the quiescent phase ER12 cells without showing irreversible cytotoxicity. It inhibited the DNA synthesis most efficiently at the early G 1 phase. Thus, tyrosine kinase inhibitors may modify malignant phenotypes in EGF receptor-overexpressing neoplasms.

(+)-Catechin inhibits the invasion of malignant fibrosarcoma cells into chick heartin vitro

(+)-Catechin, a flavonoid extractable from higher plants and trees, inhibits the invasion of malignant MO4 fibrosarcoma cells into embryonic chick heart fragments in vitro. This inhibition is maximal at a drug concentration of 0·5 mM. The growth of the MO4 cells is only partly inhibited at such a concentration, and the effect can hardly be ascribed to an irreversible cytotoxicity of the drug to the host tissue. Pretreatment of the host tissue with the drug seems to be a prerequisite for the inhibitory action, which suggests that the anti-invasive effect is at least partly mediated by the condition of the heart tissue. We hypothesize that the collagen-stabilizing effect of (+)-catechin is the key to the explanation of its anti-invasive properties.

(+)-Catechin inhibits the invasion of malignant fibrosarcoma cells into chick heart in vitro.

(+)-Catechin, a flavonoid extractable from higher plants and trees, inhibits the invasion of malignant MO4 fibrosarcoma cells into embryonic chick heart fragments in vitro. This inhibition is maximal at a drug concentration of 0.5 mM. The growth of the MO4 cells is only partly inhibited at such a concentration, and the effect can hardly be ascribed to an irreversible cytotoxicity of the drug to the host tissue. Pretreatment of the host tissue with the drug seems to be a prerequisite for the inhibitory action, which suggests that the anti-invasive effect is at least partly mediated by the condition of the heart tissue. We hypothesize that the collagen-stabilizing effect of (+)-catechin is the key to the explanation of its anti-invasive properties.

Dopaminergic influences on prolactin synthesis and release from rat anterior pituitary cultures

Dopaminergic influences on prolactin release from lactotrophs have been studied using the rat anterior pituitary cell culture. The prolactin inhibiting activity of hypothalamic extracts was examined in relation to dopamine. Dopamine inhibited prolactin secretion from the rat anterior pituitary cell culture in a dose dependent fashion. The median effective dose was 2 x 10 -7 M and the maximal inhibition (70-90 ~o of the control value) was shown by 10 -5 M dopamine. Further increase in dopamine concentration did not result in any further inhibition of prolactin secretion. Dopamine antagonists such as haloperidol and (§ could inhibit the effect of dopamine on prolactin release. A Schild plot from the relationship between dopamine and (+)butaclamol in prolactin release in the anterior pituitary cell culture showed that pAz value of (+)butaclamol is 8.9 with a slope of 1.00. High concentrations of haloperidol or (+) butaclamol (both above 10-SM) caused a marked inhibition in prolactin release initially, followed by some irreversible cytotoxicity on the anterior pituitary cells. The major damage