DMF-Activated Nrf2 Ameliorates Palmitic Acid Toxicity While Potentiates Ferroptosis Mediated Cell Death: Protective Role of the NO-Donor S-Nitroso-N-Acetylcysteine.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease that can develop into an aggressive form called nonalcoholic steatohepatitis (NASH), which ultimately progresses to cirrhosis, hepatocellular carcinoma (HCC), and end-stage liver failure. Currently, the deterioration of NAFLD is attributed to specific lipid toxicity which could be due to lipotoxicity and/or ferroptosis. In the current study, we evaluated the involvement of the nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf-2), which is a main activator of phase II metabolism in the two types of lipid-induced toxicity in hepatocytes, lipotoxicity by saturated fatty acids, and in ferroptosis, and the effect of NO donor treatment. AML12 cells were exposed to 600 µM palmitic acid to induce lipotoxicity or treated with 20 µM erastin or 5 µM RSL3 for ferroptosis. In SFA-lipotoxicity, pretreatment with the Nrf2 activator dimethyl fumarate (DMF) managed to ameliorate the cells and the oxidative stress level while aggravating ferroptosis due to emptying the thiol pool. On the other hand, the nitric oxide (NO)-donor, S-nitroso-N-acetylcysteine (NAC-SNO) proved to be effective in the prevention of hepatocytes ferroptosis.

S-Nitroso-N-acetylcysteine (NAC-SNO) vs. nitrite as an anti-clostridial additive for meat products.

Nitrite is added to meat products as a preservative and it acts as a bacteriostatic compound against Clostridium botulinum growth. Nitric-oxide (˙NO), myoglobin and S-nitroso-compounds seem to be the main molecules generated from nitrite in meat products, which by decomposition to ˙NO, form the main anti-clostridial factor. The growth of C. sporogenes from activated spores in the presence of 0.5-2.5 mM NAC-SNO was compared to nitrite, both at 37 °C for 5 days and at room temperature for 28 days. The present study demonstrates that NAC-SNO under the same conditions and concentrations, in meat products, acts as an anti-clostridial compound similar to nitrite. In contrast to nitrite which must be activated in meat by heating, NAC-SNO generates the anti-clostridial factor directly, without heating, as was evaluated in an unheated bacteriological medium. The toxic effect of NAC-SNO and nitrite in methaemoglobinaemia and generation of N-nitrosamines in vivo, in mice, were also determined. Mice were gavage fed milk containing 45 mg per kg per bw of nitrite or an equimolar equivalent of NAC-SNO in the presence of 50 mg per kg per bw of N-methylaniline. Nitrite generated methaemoglobinaemia and carcinogenic N-nitrosoamines (N-nitrosomethylaniline); however, NAC-SNO under the same conditions and concentrations generates much less methaemoglobin and no detectable N-nitrosoamines in the blood, in vivo.

S-Nitroso-N-acetylcysteine (NAC-SNO) as an Antioxidant in Cured Meat and Stomach Medium.

The stability of lipids in meat products depends on the initial concentration of hydroperoxides, the catalytic involvement of metal ions and myoglobin, endogenous antioxidants, and biological and technological factors. Ground meat was treated with additives, sealed in vacuum bags, heated to 75 °C, and stored opened to air at 4 °C. S-Nitroso-N-acetylcysteine (NAC-SNO) at concentration like nitrite used by the industry prevents lipid peroxidation in the product, even after storage for 1 month at 4 °C. The same simulated treatments at different concentrations of both compounds show that NAC-SNO acts as an antioxidant ∼4-fold better than nitrite at pH 6.2 or 3.0. Ascorbic acid significantly improves nitrite antioxidant effect. NAC-SNO was found to prevent, much better than nitrite, accumulation of reactive aldehydes and hydroxynonenal protein modification. In condition like those used by the industry for meat products processing, NAC-SNO acts better than nitrite to provide antioxidant protection without the side effect of N-nitrosation, oxidation, and the loss of nutrient generated by nitrite.

S-Nitroso-N-acetylcysteine Generates Less Carcinogenic N-Nitrosamines in Meat Products than Nitrite.

Nitrite reacts with secondary amines to form N-nitrosamines (N-NA), which lead to gastrointestinal cancers. The aim of this study was to compare nitrite with S-nitrosocysteine (Cys-SNO) and S-nitroso-N-acetylcysteine (NAC-SNO) with respect to N-NA formation, which was evaluated by determining the conversion of N-methylaniline to N-nitrosomethylaniline. Under neutral and acidic pH conditions, N-NA formation rate was nitrite > Cys-SNO > NAC-SNO. In the presence of copper or nucleophiles, NAC-SNO generated much less N-NA than Cys-SNO. Nitrite and Cys-SNO produced higher amounts of N-NA in the presence of oxygen, whereas NAC-SNO was almost oxygen insensitive. In meat in the stomach medium, NAC-SNO produced much lower amounts of N-NA than other additives. In heated meat, Cys-SNO and NAC-SNO generated the nitrosyl-hemochrome pink pigment, better than nitrite. In conclusion, NAC-SNO was much less reactive for N-NA formation than nitrite and Cys-SNO in conditions relevant to meat production and stomach digestion.

Redox homeostasis in stomach medium by foods: The Postprandial Oxidative Stress Index (POSI) for balancing nutrition and human health.

Red-meat lipid peroxidation in the stomach results in postprandial oxidative stress (POS) which is characterized by the generation of a variety of reactive cytotoxic aldehydes including malondialdehyde (MDA). MDA is absorbed in the blood system reacts with cell proteins to form adducts resulting in advanced lipid peroxidation end products (ALEs), producing dysfunctional proteins and cellular responses. The pathological consequences of ALEs tissue damage include inflammation and increased risk for many chronic diseases that are associated with a Western-type diet. In earlier studies we used the simulated gastric fluid (SGF) condition to show that the in vitro generation of MDA from red meat closely resembles that in human blood after consumption the same amount of meat. In vivo and in vitro MDA generations were similarly suppressed by polyphenol-rich beverages (red wine and coffee) consumed with the meal. The present study uses the in vitro SGF to assess the capacity of more than 50 foods of plant origin to suppress red meat peroxidation and formation of MDA. The results were calculated as reducing POS index (rPOSI) which represents the capacity in percent of 100g of the food used to inhibit lipid peroxidation of 200g red-meat a POSI enhancer (ePOSI). The index permitted to extrapolate the need of rPOSI from a food alone or in ensemble such Greek salad, to neutralize an ePOSI in stomach medium, (ePOS-rPOSI=0). The correlation between the rPOSI and polyphenols in the tested foods was R2=0.75. The Index was validated by comparison of the predicted rPOSI for a portion of Greek salad or red-wine to real inhibition of POS enhancers. The POS Index permit to better balancing nutrition for human health.

Lipid Peroxidation in a Stomach Medium Is Affected by Dietary Oils (Olive/Fish) and Antioxidants: The Mediterranean versus Western Diet.

Red meat is an integral part of the Western diet, and high consumption is associated with an increased risk of chronic diseases. Using a system that simulated the human stomach, red meat was interacted with different oils (olive/fish) and lipid peroxidation was determined by measuring accumulation of malondialdehyde (MDA) and lipid peroxides (LOOH). Olive oil decreased meat lipid peroxidation from 121.7 ± 3.1 to 48.2 ± 1.3 µM and from 327.1 ± 9.5 to 77.3 ± 6.0 µM as assessed by MDA and ROOH, respectively. The inhibitory effect of olive oil was attributed to oleic acid rather than its polyphenol content. In contrast, fish oils from tuna or an ω-3 supplement dramatically increased meat lipid peroxidation from 96.2 ± 3.6 to 514.2 ± 6.7 µM MDA. Vitamin E inhibited meat lipid peroxidation in the presence of olive oil but paradoxically increased peroxidation in the presence of fish oil. The inhibitory properties of oleic acid may play a key role in the health benefits of the Mediterranean diet.