L-carnitine protects DNA oxidative damage induced by phenylalanine and its keto acid derivatives in neural cells: a possible pathomechanism and adjuvant therapy for brain injury in phenylketonuria.

Although phenylalanine (Phe) is known to be neurotoxic in phenylketonuria (PKU), its exact pathogenetic mechanisms of brain damage are still poorly known. Furthermore, much less is known about the role of the Phe derivatives phenylacetic (PAA), phenyllactic (PLA) and phenylpyruvic (PPA) acids that also accumulate in this this disorder on PKU neuropathology. Previous in vitro and in vivo studies have shown that Phe elicits oxidative stress in brain of rodents and that this deleterious process also occurs in peripheral tissues of phenylketonuric patients. In the present study, we investigated whether Phe and its derivatives PAA, PLA and PPA separately or in combination could induce reactive oxygen species (ROS) formation and provoke DNA damage in C6 glial cells. We also tested the role of L-carnitine (L-car), which has been recently considered an antioxidant agent and easily cross the blood brain barrier on the alterations of C6 redox status provoked by Phe and its metabolites. We first observed that cell viability was not changed by Phe and its metabolites. Furthermore, Phe, PAA, PLA and PPA, at concentrations found in plasma of PKU patients, provoked marked DNA damage in the glial cells separately and when combined. Of note, these effects were totally prevented (Phe, PAA and PPA) or attenuated (PLA) by L-car pre-treatment. In addition, a potent ROS formation also induced by Phe and PAA, whereas only moderate increases of ROS were caused by PPA and PLA. Pre-treatment with L-car also prevented Phe- and PAA-induced ROS generation, but not that provoked by PLA and PPA. Thus, our data show that Phe and its major metabolites accumulated in PKU provoke extensive DNA damage in glial cells probably by ROS formation and that L-car may potentially represent an adjuvant therapeutic agent in PKU treatment.

Environmental enrichment reduces cocaine neurotoxicity during cocaine-conditioned place preference in male rats.

Environmental enrichment (EE) has a neuroprotective role and prevents the development of cocaine addiction behavior in rats. Studies showing the role of EE in cocaine toxicity are nonexistent. We hypothesized that rats exposed to EE are protected from cocaine-induced changes in the redox profile and DNA damage after undergoing conditioned place preference (CPP). Ten male Wistar rats were placed in EE cages equipped with toys, a ladder and tunnels, and ten were provided clean, standard laboratory housing (non-EE). EE and non-EE rats were randomly allocated to the classical CPP cocaine vs. saline (COC/Saline) group, where cocaine (15 mg/kg; i.p.) was tested alternately with saline. Afterwards, intracellular reactive species and antioxidant enzymes were evaluated and the comet essay was performed in the prefrontal cortex and hippocampus of rats. As expected, EE rats spent less time in the cocaine-paired chamber, and as a new result, less cocaine-induced DNA damage was observed in the two brain structures. Altogether, our results demonstrate that EE decreases neurotoxicity in brain regions linked to cocaine addiction but does not extinguish it completely.

In vitro model to study cocaine and its contaminants.

Cocaine is one of the most popular illicit drug worldwide. Due its great addictive potential, which leads to euphoria and hyperactivity, it is considered a public health concern. At the central nervous system, the drug acts inhibiting catecholamine re-uptake. It is now known that in addition to the toxicity of the drug itself, the contaminants present in the street drug have raised concern about the harmful effects on health. Toxicological in vivo and in vitro studies have demonstrated the toxic effects of cocaine correlated with the generation of reactive oxygen species (ROS), which in turn lead to oxidative damage to the cells. Therefore the aim of this work was to propose an in vitro model that reunites the main parameters of toxicity of the cocaine already observed in the literature so far, and we tested this model using cocaine and seizure cocaine sample (SCS), kindly provided by Federal Police of Brazil. For that, we used a C6 glioblastoma cells and evaluated cell death, oxygen reactive species induction, oxidation of macromolecules as membrane lipids and DNA and loss of mitochondrial membrane potential after cocaine exposure. The results showed that cocaine can decrease cellular viability in a dose-dependent way in the C6 cell immortalized and astrocytes primary culture. Cocaine also induced cellular death by apoptosis. However, in the seizure cocaine sample (SCS), the predominant cell death was due to necrosis. Using dichlorofluorescein (DCF) assay, we confirmed ROS production after cocaine exposition. In agreement with these findings, occurred an increasing in MDA production, as well as increased superoxide dismutase (SOD) and catalase (CAT) activity. The induction of DNA damage was observed after cocaine. Our results demonstrate the occurrence of mitochondrial dysfunction by depolarization of mitochondrial membrane as a consequence of cocaine treatment. In summary, these results demonstrated that cocaine can induce reactive oxygen species formation, leading to oxidative stress. As a consequence of this unbalance, DNA damage, lipidic peroxidation and loss of mitochondrial membrane occurred, which could be an answer to cell death observed.

Cocaine induces DNA damage in distinct brain areas of female rats under different hormonal conditions.

We evaluated levels of neuronal DNA damage after acute or repeated cocaine treatment in different brain areas of female rats after ovariectomy or sham surgery. Rats in the control and acute groups were given saline i.p., whereas in the repeated group were given 15 mg/kg, i.p., cocaine for 8 days. After a 10 day washout period, the control group was given saline i.p., whereas rats in the acute and repeated groups were given a challenge dose of 15 mg/kg, i.p., cocaine. After behavioural assessment, rats were killed and the cerebellum, hippocampus, hypothalamus, prefrontal cortex and striatum were dissected for the Comet assay. Acute cocaine exposure induced DNA damage in all brain areas. This effect persisted after repeated administration, except in the hypothalamus, where repeated treatment did not cause increased DNA damage. Sexual hormones exhibited a neuroprotective effect, decreasing cocaine-induced DNA damage in cycling rats in all brain areas.