Amelioration of Phytanic Acid-Induced Neurotoxicity by Nutraceuticals: Mechanistic Insights.

Phytanic acid (PA) (3,7,11,15-tetramethylhexadecanoic acid) is a methyl-branched fatty acid that enters the body through food consumption, primarily through red meat, dairy products, and fatty marine foods. The metabolic byproduct of phytol is PA, which is then oxidized by the ruminal microbiota and some marine species. The first methyl group at the 3-position prevents the ß-oxidation of branched-chain fatty acid (BCFA). Instead, α-oxidation of PA results in the production of pristanic acid (2,10,14-tetramethylpentadecanoic acid) with CO2. This fatty acid (FA) builds up in individuals with certain peroxisomal disorders and is historically linked to neurological impairment. It also causes oxidative stress in synaptosomes, as demonstrated by an increase in the production of reactive oxygen species (ROS), which is a sign of oxidative stress. This review concludes that the nutraceuticals (melatonin, piperine, quercetin, curcumin, resveratrol, epigallocatechin-3-gallate (EGCG), coenzyme Q10, ω-3 FA) can reduce oxidative stress and enhanced the activity of mitochondria. Furthermore, the use of nutraceuticals completely reversed the neurotoxic effects of PA on NO level and membrane potential. Additionally, the review further emphasizes the urgent need for more research into dairy-derived BCFAs and their impact on human health.

Fetal alcohol exposure impairs learning and memory functions and elevates levels of various biochemical markers of Alzheimer's disease in the brain of 12-month-old rats.

BACKGROUND: Alcohol drinking during pregnancy often adversely affects brain development among offspring, inducing persistent central nervous system dysfunction. However, it is unknown whether fetal alcohol exposure (FAE) promotes the biochemical characteristics of Alzheimer's disease in offspring. METHODS: We used a first- and second-trimester human equivalent rat model of FAE that involves feeding a liquid diet containing 6.7% v/v ethanol from gestational days 7 through 21 in Fischer-344 rats. Control rats were fed an isocaloric liquid diet or rat chow ad libitum. Pups were weaned on postnatal day 21 and housed by sex. They were used for behavioral and biochemical studies at about 12 months of age. Only one male or one female offspring from a litter was included in each experimental group. RESULTS: Fetal alcohol-exposed offspring had poorer learning and memory functions than controls. The experimental animals, both male and female, also had elevated levels of acetylcholinesterase (AChE) activity, hyperphosphorylated-tau protein, ß-amyloid (Aß) and Aß1-42 proteins, ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and Unc-5 netrin receptor C (UNC5C) proteins in the cerebral cortex and hippocampus at 12 months of age. CONCLUSIONS: These findings show that FAE increases the expression of some of the biochemical and behavioral phenotypes of Alzheimer's disease.

miRNA-383 and miRNA-384 Suppress Proopiomelanocortin Gene Expression in the Hypothalamus: Effects of Early Life Ethanol Exposure.

INTRODUCTION: Early life ethanol exposure is known to program hypothalamic proopiomelanocortin (POMC) neurons to express a reduced level of POMC and its control of stress axis functions throughout the life span. In this study, we tested whether miRNAs contribute to the ethanol-induced suppression of Pomc gene expression during the developmental period. METHODS: In in vivo studies, POMC-EGFP male mice were fed with 2.5 g/kg ethanol using milk formula (AF), pair-fed isocaloric milk formula, or left in the litter during postnatal days (PNDs) 2-6. In in vitro studies, mHypoA-POMC/GFP cells were treated with ethanol (50 mM) for a 24-h period. Hypothalamic tissues or cell extracts were used for measurement of miRNAs and POMC mRNA. RESULTS: Determination of genome-wide microRNA expression profile identified 40 miRNAs significantly altered in hypothalamic tissues of AF mice. In silico analysis further identified miRNA-383, -384, and -488 have putative binding sites at the POMC 3'UTR. However, only miR-383 and miR-384 are identified to be responsive to ethanol. Administration of miR-383 or -384 inhibitor oligos suppressed ethanol-stimulated miR-383 or -384 expression and restored Pomc mRNA and protein expression in AF mice. mHypoA-POMC/GFP cells when treated with ethanol showed elevated levels of miR-383 and miR-384 and reduced level of Pomc mRNA. Treatment with miR-383 or -384 mimic oligos reduced the level of Pomc mRNA, while treatment with miR-383 or -384 inhibitor oligos increased the level of Pomc mRNA. Reporter assay further confirms the binding specificity of miR-383 and miR-384 to Pomc 3'UTR. CONCLUSION: These data suggest that miR-383 and miR-384 suppress Pomc gene expression and may contribute to the ethanol-induced alteration of the stress axis functions.

Epigenetic insight into effects of prenatal alcohol exposure on stress axis development: Systematic review with meta-analytic approaches.

We conducted a systematic review with meta-analytic elements using publicly available Gene Expression Omnibus (GEO) datasets to determine the role of epigenetic mechanisms in prenatal alcohol exposure (PAE)-induced hypothalamic-pituitary-adrenal (HPA) axis dysfunctions in offspring. Several studies have demonstrated that PAE has long-term consequences on HPA axis functions in offspring. Some studies determined that alcohol-induced epigenetic alterations during fetal development persist in adulthood. However, additional research is needed to understand the major epigenetic events leading to alcohol-induced teratogenesis of the HPA axis. Our network analysis of GEO datasets identified key pathways relevant to alcohol-mediated histone modifications, DNA methylation, and miRNA involvement associated with PAE-induced alterations of the HPA axis. Our analysis indicated that PAE perturbated the epigenetic machinery to activate corticotrophin-releasing hormone, while it suppressed opioid, glucocorticoid receptor, and circadian clock genes. These results help to further our understanding of the epigenetic basis of alcohol's effects on HPA axis development.

Neuroprotective Effects of Natural Antioxidants Against Branched-Chain Fatty Acid-Induced Oxidative Stress in Cerebral Cortex and Cerebellum Regions of the Rat Brain.

Valproic acid (VPA) is short branched-chain fatty acid (BCFA) derived from valeric acids which are naturally produced by Valeriana officinalis (flowering plant). Neurotoxicity caused by BCFA-like VPA may be mediated by oxidative stress, according to research involving the cerebral cortex and cerebellum. In the present study, we explored the possible protective effect of different antioxidants such as melatonin, quercetin, and piperine on VPA exposure by using a supernatant preparation of the cerebral cortex and cerebellum regions of the rat brain. The present study revealed that melatonin, quercetin, and piperine significantly prevented VPA-induced oxidative stress in the cerebral cortex and cerebellum regions. VPA was also observed to lower the level of reduced glutathione, and this effect was significantly mitigated by these antioxidants. Melatonin, quercetin, and piperine also ameliorated and altered the activities of AChE, Na+, K+ATPase, and MAO in the cerebral cortex and cerebellum. Results of this study also suggest that prior treatment of antioxidants like melatonin, quercetin, and piperine helps in combating the oxidative stress induced by VPA in the cerebral cortex and cerebellum region of the rat brain. Thus, sufficient dietary intake of these antioxidants by individuals at high risk of VPA exposure could prove beneficial in combating the adverse effect of VPA.

Transgenerational inheritance of fetal alcohol effects on proopiomelanocortin gene expression and methylation, cortisol response to stress, and anxiety-like behaviors in offspring for three generations in rats: Evidence for male germline transmission.

Previously it has been shown that fetal alcohol exposure increases the stress response partly due to lowering stress regulatory proopiomelanocortin (Pomc) gene expression in the hypothalamus via epigenetic mechanisms for multiple generations in mixed-breed rats. In this study we assess the induction of heritable epigenetic changes of Pomc-related variants by fetal alcohol exposure in isogenic Fischer 344 rats. Using transgenerational breeding models and fetal alcohol exposure procedures, we determined changes in hypothalamic Pomc gene expression and its methylation levels, plasma corticosterone hormone response to restraint stress, and anxiety-like behaviors using elevated plus maze tests in fetal alcohol-exposed offspring for multiple generations in isogenic Fischer rats. Fetal alcohol-exposed male and female rat offspring showed significant deficits in POMC neuronal functions with increased Pomc gene methylation and reduced expression. These changes in POMC neuronal functions were associated with increased plasma corticosterone response to restraint stress and increased anxiety-like behavior. These effects of fetal alcohol exposure persisted in the F1, F2, and F3 progeny of the male germline but not of the female germline. These data suggest that fetal alcohol exposure induces heritable changes in Pomc-related variants involving stress hyperresponsiveness and anxiety-like behaviors which perpetuate into subsequent generations through the male germline via epigenetic modifications.

Melatonin attenuates branch chain fatty acid induced apoptosis mediated neurodegeneration.

Valproic acid (VPA)-a short branched chain fatty acid (BCFA), is widely recognized as an anticonvulsant and a mood-stabilizing drug, but various adverse effects of VPA have also been investigated. However, the impact of BCFAs aggregation on brain cells, in the pathogenesis of neurodegeneration remains elusive. The objective of this study is to understand the cellular mechanisms underlying VPA-induced neuronal cell death mediated by oxidative stress, and the neuroprotective role of exogenous melatonin treatment on VPA-induced cell death. Neurotoxicity of VPA and protective role exerted by melatonin were assessed in vitro in SH-SY5Y cells and in vivo in the cerebral cortex and cerebellum regions of Wistar rat brain. The results show that melatonin pre-treatment protects the cells from VPA-induced toxicity by exerting an anti-apoptotic and anti-inflammatory effect by regulating apoptotic proteins and pro-inflammatory cytokines. The findings of the present study emphasize novel insights of melatonin as a supplement for the prevention and treatment of neuronal dysfunction induced by VPA.

Valproic Acid Induced Neurotoxicological Manifestations and its Mitigation by Melatonin in Rat Brain Synaptosomes.

BACKGROUND AND AIMS: A short branched chain fatty acid, valproic acid (VPA), has been used worldwide for decades in the intervention of seizure disorders, neuropathic pain and migraine. However, several adverse effects of VPA have been reported over the years. The aim of our investigation was to evaluate the adverse effects of VPA on synaptic functions by using synaptosomal preparation of rat brain as an in vitro model and the possible protective role of melatonin against VPA induced neurotoxicity. Melatonin is an antioxidant and scavenger of free radicals secreted by the pineal gland. METHODS: In the present investigation, synaptosomes prepared from rat brain were co-treated with melatonin (10 µmol) and VPA (5 mmol) for 2 h under in vitro conditions. RESULTS: In this study, co-treatment of melatonin with VPA significantly restored the elevated levels of lipid peroxidation (LPO) and protein oxidation. In addition, melatonin prevented VPA induced alterations in non-enzymatic antioxidant defence reduced glutathione (GSH) and activities of synaptosomal integral enzymes such as AChE, Na+, K+ -ATPase and MAO. A significant increase in the generation of reactive oxygen species (ROS) induced by VPA was observed and melatonin ameliorated elevated level of ROS generation. Moreover, the enhanced level of NO and diminished activity of synaptosomal mitochondrial membrane potential was completely prevented by melatonin treatment. CONCLUSION: Our results corroborate the use of melatonin as a nutraceutical and mitigatory agent against VPA induced neurotoxicity in brain synaptosomes.

Phytanic acid induced neurological alterations in rat brain synaptosomes and its attenuation by melatonin.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) (Phyt) is a saturated branched chain fatty acid which originates after the breakdown of chlorophyll molecule, phytol. It plays an important role in a variety of metabolic disorders with peroxisomal impairments. The aim of our investigation was to evaluate the adverse effects of Phyt on synaptic functions by using synaptosomal preparation of rat brain as an in vitro model and the possible protective role of melatonin against Phyt-induced neurotoxicity. Melatonin is an antioxidant, secreted by the pineal gland. Melatonin and its metabolites have neuroprotective effects on cellular stress, by reducing reactive oxygen species (ROS) and reactive nitrogen species (RNS). In the present investigation, synaptosomes prepared from rat brain were co-treated with melatonin (10µM) and Phyt (50µM) for 2h. Co-treatment of Phyt with melatonin significantly restored the altered levels of protein carbonyl (PC) contents and lipid peroxidation (LPO). It also replenished the Phyt-induced alterations on the levels of non-enzymatic antioxidant defence reduced glutathione (GSH), enzymatic antioxidants such as catalase (CAT) and superoxide dismutase (SOD) and synaptosomal integral enzymes such as AChE, Na+, K+-ATPase and MAO. We observed that Phyt induced oxidative stress in synaptosomes as indicated by an elevation in the generation of ROS and melatonin was able to inhibit the elevated ROS generation. Moreover, the neurotoxic effects elicited by Phyt on NO level and membrane potential were totally prevented by the treatment of melatonin. The results of our investigation emphasize the potential use of melatonin as a nutraceutical and mitigatory agent against Phyt-induced oxidative stress.

Phytanic Acid-Induced Neurotoxicological Manifestations and Apoptosis Ameliorated by Mitochondria-Mediated Actions of Melatonin.

Phytanic acid, a saturated branched chain fatty acid and a major constituent of human diet, is predominantly found in dairy products, meat, and fish. It is a degradation product from the phytol side chain of chlorophyll. Degradation of PA is known to occur mainly in peroxisomes via α-oxidation and in mitochondria via ß-oxidation. Due to its ß-methyl group present at the 3-position of the carbon atoms, PA cannot be ß-oxidized. Although alteration in the metabolism of PA may play an important role in neurodegeneration, the exact mechanism behind it remains to be evaluated. In this study, we have described the potential of PA to induce neurotoxicity as an in vitro model (neuronal cell line, SH-SY5Y cells). Cells were pretreated with melatonin (10 µM) for 1 h followed by with and without PA (100 µM) for 24 h. In the present study, our data has confirmed that PA markedly increased both intracellular reactive oxygen species and reactive nitrogen species levels. Our results have shown that PA treatment did not induce cell death by cleavage of caspase-3/PARP-1 mediated by mitochondria through intrinsic pathways; however, PA induced nitric oxide-dependent apoptosis in SH-SY5Y cells. Additionally, melatonin pretreatment reduced the cell death in SH-SY5Y cells. Melatonin also effectively exerted an antiapoptotic and anti-inflammatory action by regulating Bax, Bcl-2, p-NFκB, and iNOS expressions in SH-SY5Y cells. These results suggested that melatonin acted as an antioxidative and antiapoptotic agent by modulating ROS, apoptotic proteins, and inflammatory responses under BCFA-induced neurotoxic conditions. The protective effects of melatonin depend on direct scavenging activity of free radicals and indirect antioxidant effects. Further deciphering of the cellular and molecular mechanism associated with neuroprotection by melatonin is warranted in BCFA-induced neurotoxicity.

Nephroprotective activities of quercetin with potential relevance to oxidative stress induced by valproic acid.

Valproic acid (VPA) is ubiquitously used as a major drug in the intervention of epilepsy and in the control of several kinds of seizures. Cellular toxicities are the serious dose-limiting side effects of VPA when applied in the treatment of diseases. Oxidative stress has been proven to be involved in VPA-induced toxicity. Accumulating evidence intimates that oxidative stress caused by free radicals and in kidney cells contributes to the pathogenesis of VPA-induced nephrotoxicity. The pathogenesis of these forms of VPA nephrotoxicity is still not clear. The aim of our investigation was to evaluate the nephrotoxic potential of VPA and protective effects of quercetin (QR) against VPA-induced nephrotoxicity by using rat kidney tissue preparation as an in vitro model. Oxidative stress indexes such as lipid peroxidation (LPO) and protein carbonyl (PC) content were appraised. The levels of oxidative stress markers, LPO, and PC were significantly elevated. Nonenzymatic antioxidants effect was also demonstrated as a significant increase in reduced glutathione (GSH) and nonprotein thiol level (NP-SH). VPA exposure altered the activities of glutathione metabolizing enzymes such as glutathione-S-transferase, glutathione peroxidase, and glutathione reductase. Pre-treatment with QR could reverse the VPA-induced effects in kidney tissue preparation of rat. Based on reno-protective and antioxidant action of QR, we suggest that this flavonoid compound could be considered as a potential safe and effective approach in attenuating the adverse effect of VPA-induced nephrotoxicity.

Manganese pre-treatment attenuates cadmium induced hepatotoxicity in Swiss albino mice.

Cadmium (Cd) is a soft, malleable bluish-white metal with low melting point, a ubiquitous heavy metal and an environmental pollutant, found in soil, water and air. The presence of Cd in the components of the environment such as air, soil and groundwater is to a large part due to human activity, and the general population is exposed mainly by contaminated drinking water or food. Manganese (Mn) is a component in many enzymes, which play an important role in counteracting oxidative stress. In vitro experiments have revealed the ability of Mn to scavenge oxygen free radicals generated in differently mediated lipid peroxidation (LPO) conditions. The aim of the present study was to investigate the in vivo preventive effect of Mn(2+) pre-treatment on acute Cd-intoxication with regard to oxidative stress biomarker and antioxidant defense system in liver of Swiss albino mice. On exposure to Cd a significant increase in LPO levels, decrease in thiol content and induction in glutathione metabolizing enzyme were observed. Mn pre-treatment attenuated the modulation caused in the above-mentioned parameters due to acute Cd exposure in mice. In conclusion, the results from this study demonstrate that the protective effect of Mn in Cd-induced systemic toxicity in mice. Further investigations are required on the relation between Mn accumulation and resistance to oxidative stress and on the factors influencing Mn/Cd transport in rodents are needed to elucidate the molecular basis of this protective effect.

Cadmium-induced hepatotoxicity and its abrogation by thymoquinone.

Cadmium (Cd(2+) ) causes alteration of the cellular homeostasis and oxidative damage. The aim of the present study was to investigate the possible protective role of thymoquinone (TQ), a predominant bioactive component present in black seed oil (Nigella sativa) on the hepatotoxicity of Cd(2+) with special reference to its protection against perturbation of nonenzymatic and enzymatic antioxidants. The effect of TQ pretreatment was examined in postnuclear supernatant prepared from liver of Swiss albino mice under in vitro conditions. CdCl(2) treatment (5 mM) resulted in a significant increase in antioxidant enzymatic activities. It also caused a significant (p < 0.001) increase in protein carbonyl and reduced glutathione content. Pretreatment with TQ (10 µM) showed a significant protection as manifested by noticed attenuation of protein oxidation and rejuvenation of the depleted antioxidants of cellular fraction. These results strengthen the hypothesis that TQ exerts modulatory influence on the antioxidant defense system on being subjected to toxic insult.

Postnuclear supernatant: an in vitro model for assessing cadmium-induced neurotoxicity.

Cadmium (Cd) is a toxic heavy metal commonly found in industrial workplaces, a food contaminant and a major constituent of cigarette smoke. Most of the organs are susceptible to Cd-induced toxicity, including brain. Postnuclear supernatant (PNS) has been accepted as an in vitro model for assessing xenobiotic induced toxicity. The goal of the present study was to validate PNS as an in vitro model for investigating the effect of Cd-induced neurotoxicity. Neurotoxic induction by Cd was established in a dose-dependent manner in PNS in vitro. Enzymatic and non-enzymatic antioxidants were used as biomarkers of exposure. Antioxidant enzymatic activity was measured as a significant increase in activities of catalase, superoxide dismutase, and glutathione S-transferase. On exposure to Cd, a significant increase in acetylcholinesterase and decrease in sodium-potassium ATPase activity was also observed. Non-enzymatic effect was also demonstrated as a significant elevation in reduced glutathione and non-protein thiol activity, but there was no significant increase or decrease in the concentrations of protein thiol. In accordance with the toxicity of Cd towards the studied brain structure, Cd-induced oxidative stress has been a focus of toxicological research as a possible mechanism of neurotoxicity. Our results suggest that PNS preparations can be used as a model for future investigation of xenobiotic-induced neurotoxicity under in vitro conditions.