Toxicity of the amphetamine metabolites 4-hydroxyamphetamine and 4-hydroxynorephedrine in human dopaminergic differentiated SH-SY5Y cells.

Amphetamine (AMPH) is a psychostimulant used worldwide by millions of patients in the clinical treatment of attention deficit hyperactivity disorder, narcolepsy or even obesity, and is also a drug of abuse. 4-Hydroxynorephedrine (4-OHNE) and 4-hydroxyamphetamine (4-OHAMPH) are two major metabolites known to persist in the brain longer than AMPH. The contribution of AMPH metabolites for its neurotoxicity is undetermined. We evaluated the toxicity of AMPH and its metabolites 4-OHNE and 4-OHAMPH, obtained by chemical synthesis, in human dopaminergic differentiated SH-SY5Y neurons. Cells were exposed to AMPH (concentration range 0-5mM) or 4-OHAMPH or 4-OHNE (concentration range 0-10mM) for 24 or 48h, and the viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) leakage assays. Results showed that for both AMPH and the metabolites a concentration-dependent toxicity was observed. The toxic concentration 50% (TC50) for AMPH and 4-OHNE following 24h exposure was circa 3.5mM and 8mM, respectively. For 4-OHAMPH the TC50 was not reached in the tested concentration range. N-acetyl cysteine, cycloheximide, l-carnitine, and methylphenidate were able to reduce cell death induced by AMPH TC50. Acridine orange/ethidium bromide staining showed evident signs of late apoptotic cells and necrotic cells following 24h exposure to AMPH 3.50mM. The 4-OHAMPH metabolite at 8.00mM originated few late apoptotic cells, whereas 4-OHNE at 8.00mM resulted in late apoptotic cells and necrotic cells, in a scenario similar to AMPH. In conclusion, the AMPH metabolite 4-OHNE is more toxic than 4-OHAMPH, nonetheless both are less toxic than the parent compound in vitro. The most toxic metabolite 4-OHNE has longer permanence in the brain, rendering likely its contribution for AMPH neurotoxicity.

Developmental toxicity of 4-substituted amphetamines in mice.

Despite overwhelming and tragic evidence of their detrimental and dangerous consequences, amphetamines remain significant drugs of abuse and addiction. The effects of 4-substituted amphetamines: 4-hydroxyamphetamine (4-HA), 4-methoxyamphetamine (4-MEA), 4-ethoxyamphetamine (4-ETA), 4-propoxyamphetamine (4-PPA), and 4-benzyloxyamphetamine (4-BEA) on intrauterine development, pregnancy outcome, postnatal growth, and survival were compared in Swiss-Webster mice. Single daily doses (0, 50, or 100 mg/kg) of an aqueous solution of different amphetamines were administered on pregnancy days 6 through 18. The 50 mg/kg doses of all amphetamines were well tolerated by the mothers and did not produce any overt signs of maternal toxicity or death. However, a few mothers died on different days of gestation after receiving 100 mg/kg of 4-HA, 4-MEA, 4-ETA, and 4-BEA. The mothers that failed to deliver naturally (3 d after the due date) were killed and their uteri were examined for live/dead fetuses and resorption sites. In comparison with respective controls, the incidence of resorptions was markedly higher in the 4-MEA- and 4-ETA-dosed groups. Delivery was prolonged in the 4-PPA- and 4-BEA-treated dams. Apparently well-formed but dead pups were delivered by 4-HA-, 4-PPA-, and 4-BEA-dosed mice. Marked reductions in average litter size and weight occurred after intrauterine exposure to 100 mg/kg 4-BEA. Treatment with 4-ETA, 4-PPA, and 4-BEA not only resulted in a high incidence of cannibalism within 24 h after birth but also caused an increase in cumulative pup mortality during the first 3 weeks of age. Body weight gain was significantly lower in 3-week-old offspring exposed to 4-HA and 4-PPA than in the controls. The findings suggest that 4-substituted amphetamines exhibit a wide variation in their effects on maternal toxicity and pregnancy wastage, and produce adverse effects on parturition, pup survival, and postnatal development.