Effects of Food Constituents on Absorption and Bioaccessibility of Dietary Synthetic Phenolic Antioxidant by Caco-2 Cells.

One typical synthetic phenolic antioxidant 2,6-di-tert-butyl-hydroxytoluene (BHT) is widely used in foodstuff. Concerns are rising on the toxicity of BHT and its metabolites through dietary exposure. In this study, the effects of food macronutrients (i.e., lipid, carbohydrate, fiber, protein, and fasted (as control)) on absorption and bioaccessibility of BHT by Caco-2 cells were investigated. Food components decreased the absorption and bioaccessibility by Caco-2 cells. The highest absorption rate by Caco-2 cells was fasted state (first-order rate constant = 4.26 h-1), followed by carbohydrate (2.36 h-1), fiber (1.39 h-1), lipid (1.34 h-1), and protein (1.15 h-1). The order of bioaccessibility of BHT and its metabolites was fasted (100 ± 11.5%) > protein (83.1 ± 2.69%) > fiber (65.8 ± 2.67%) > carbohydrate (56.8 ± 1.58%) ≈ lipid (56.7 ± 0.82%). A solid-phase microextraction test together with a computational in vitro kinetic model suggested that the macronutrients may bind to BHT to reduce its free concentration and decrease the bioaccessibility. To our knowledge, this is the first study to report food influence on the absorption and bioaccessibility of BHT by Caco-2 cells. Results here can provide important implications for the safety regulation for dietary synthetic phenolic antioxidants.

Chemical stabilization of polymers: Implications for dermal exposure to additives.

Technical benefits of additives in polymers stand in marked contrast to their associated health risks. Here, a multi-analyte method based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) was developed to quantify polymer additives in complex matrices such as low-density polyethylene (LDPE) and isolated human skin layers after dermal exposure ex vivo. That way both technical aspects and dermal exposure were investigated. The effects of polymer additivation on the material were studied using the example of LDPE. To this end, a tailor-made polymer was applied in aging studies that had been furnished with two different mixtures of phenol- and diarylamine-based antioxidants, plasticizers and processing aids. Upon accelerated thermo-oxidative aging of the material, the formation of LDPE degradation products was monitored with attenuated total reflectance-Fourier transformed infrared (ATR-FTIR) spectroscopy. Compared to pure LDPE, a protective effect of added antioxidants could be observed on the integrity of the polymer. Further, thermo-oxidative degradation of the additives and its kinetics were investigated using LDPE or squalane as matrix. The half-lives of additives in both matrices revealed significant differences between the tested additives as well as between LDPE and squalane. For instance, 2-tert-butyl-6-[(3-tert-butyl-2-hydroxy-5-methylphenyl)methyl]-4-methylphenol (Antioxidant 2246) showed a half-life 12 times lower when incorporated in LDPE as compared to squalane. As a model for dermal exposure of consumers, human skin was brought into contact with the tailor-made LDPE containing additives ex vivo in static Franz diffusion cells. The skin was then analyzed for additives and decomposition products. This study proved 10 polymer additives of diverse pysicochemical properties and functionalities to migrate out of the polymer and eventually overcome the intact human skin barrier during contact. Moreover, their individual distribution within distinct skin layers was demonstrated. This is exemplified by the penetration of the procarcinogenic antioxidant N-phenylnaphthalen-2-amine (Neozon D) into the viable epidermis and the permeation through the skin of the neurotoxic plasticizer N-butylbenzenesulfonamide (NBBS). In addition, the analyses of additive degradation products in the isolated skin layers revealed the presence of 2-tert-butyl-4-methylphenol in all layers after contact to a polymer with substances of origin like Antioxidant 2246. Thus, attention needs to be paid to absorption of polymer additives together with their degradation products when it comes to dermal exposure assessment.

Understanding the chemistry behind the antioxidant activities of butylated hydroxytoluene (BHT): a review.

Hindered phenols find a wide variety of applications across many different industry sectors. Butylated hydroxytoluene (BHT) is a most commonly used antioxidant recognized as safe for use in foods containing fats, pharmaceuticals, petroleum products, rubber and oil industries. In the past two decades, there has been growing interest in finding novel antioxidants to meet the requirements of these industries. To accelerate the antioxidant discovery process, researchers have designed and synthesized a series of BHT derivatives targeting to improve its antioxidant properties to be having a wide range of antioxidant activities markedly enhanced radical scavenging ability and other physical properties. Accordingly, some structure-activity relationships and rational design strategies for antioxidants based on BHT structure have been suggested and applied in practice. We have identified 14 very sensitive parameters, which may play a major role on the antioxidant performance of BHT. In this review, we attempt to summarize the current knowledge on this topic, which is of significance in selecting and designing novel antioxidants using a well-known antioxidant BHT as a building-block molecule. Our strategy involved investigation on understanding the chemistry behind the antioxidant activities of BHT, whether through hydrogen or electron transfer mechanism to enable promising anti-oxidant candidates to be synthesized.

PASS-assisted design, synthesis and antioxidant evaluation of new butylated hydroxytoluene derivatives.

New multipotent antioxidants (MPAOs), namely 1,3,4-thiadiazoles and 1,2,4-triazoles bearing the well-known free radical scavenger butylated hydroxytoluene (BHT), were designed and synthesized using an acid-(base-) catalyzed intramolecular dehydrative cyclization reaction of the corresponding 1-acylthiosemicarbazides. The structure-activity relationship (SAR) of the designed antioxidants was performed along with the prediction of activity spectra for substances (PASS) training set. Experimental studies based on antioxidant activity using DPPH and lipid peroxidation assays verified the predictions obtained by the PASS-assisted design strategy. Compounds 4a-b, 5a-b and 6a-b showed an inhibition of stable DPPH free radicals at a 10(-4) M more than the well-known standard antioxidant BHT. Compounds with p-methoxy substituents (4b, 5b and 6b) were more active than o-methoxy substituents (4a, 5a and 6a). With an IC50 of 2.85 ± 1.09 µM, compound 6b exhibited the most promising in vitro inhibition of lipid peroxidation, inhibiting Fe(2+)-induced lipid peroxidation of essential oils derived from the egg yolk-based lipid-rich medium by 86.4%. The parameters for the drug-likeness of these BHT derivatives were also evaluated according to Lipinski's 'rule-of-five'. All of the BHT derivatives were found to violate one of Lipinski's parameters (Log P ≥ 5) even though they have been found to be soluble in protic solvents. The predictive TPSA and %ABS data allow for the conclusion that these compounds could have a good capacity for penetrating cell membranes. Therefore, these novel MPAOs containing lipophilic and hydrophilic groups can be proposed as potential antioxidants for tackling oxidative stress and lipid peroxidation processes.

A green mesostructured vanadosilicate catalyst and its unprecedented catalytic activity for the selective synthesis of 2,6-disubstituted p-benzoquinones.

We have developed a green method for the production of 2,6-disubstituted p-benzoquinones (DSBQs) by liquid-phase oxidations of di/tri-substituted phenols using two-dimensional hexagonally thick-walled mesoporous vanadosilicate catalysts. In particular, 2,6-di-tert-butyl-p-benzoquinone was synthesized by the oxidation of 2,6-di-tert-butylphenol, using various reaction parameters, over mesoporous VSBA-15 catalysts synthesized with various vanadium contents. A promising chemical treatment method for the preparation of green mesoporous VSBA-15(5) or W-VSBA-15(5) (W: washed) catalysts was successfully used in the presence of ammonium acetate solution to remove moderately toxic non-framework V2O5 crystallite species from the active surface, and the catalytic activity of the recovered green mesoporous VSBA-15(5) catalyst was determined. To confirm the green aspects, recyclability and hot-catalytic filtration experiments were performed. The combined results show that the green mesoporous VSBA-15(5) is a highly active, recyclable, and promising heterogeneous catalyst for the selective synthesis of DSBQs (98-100%), and has unprecedented catalytic activity compared with other mesoporous vanadosilicate catalysts.

Butylated hydroxytoluene analogs: synthesis and evaluation of their multipotent antioxidant activities.

A computer-aided predictions of antioxidant activities were performed with the Prediction Activity Spectra of Substances (PASS) program. Antioxidant activity of compounds 1, 3, 4 and 5 were studied using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and lipid peroxidation assays to verify the predictions obtained by the PASS program. Compounds 3 and 5 showed more inhibition of DPPH stable free radical at 10⁻4 M than the well-known standard antioxidant, butylated hydroxytoluene (BHT). Compound 5 exhibited promising in vitro inhibition of Fe²âº-induced lipid peroxidation of the essential egg yolk as a lipid-rich medium (83.99%, IC50 16.07 ± 3.51 µM/mL) compared to α-tocopherol (α-TOH, 84.6%, IC50 5.6 ± 1.09 µM/mL). The parameters for drug-likeness of these BHT analogues were also evaluated according to the Lipinski's "rule-of-five" (RO5). All the BHT analogues were found to violate one of the Lipinski's parameters (LogP > 5), even though they have been found to be soluble in protic solvents. The predictive polar surface area (PSA) and absorption percent (% ABS) data allow us to conclude that they could have a good capacity for penetrating cell membranes. Therefore, one can propose these new multipotent antioxidants (MPAOs) as potential antioxidants for tackling oxidative stress and lipid peroxidation processes.

Hepatic retention and toxicological responses during feeding and depuration periods in Atlantic salmon ( Salmo salar ) fed graded levels of the synthetic antioxidant, butylated hydroxytoluene.

The human safety aspects of seafood production require the expansion of vital knowledge of both nutrients and possible contaminants along the entire production chain. Thus, production of safer seafood can be achieved by using feed materials that are low in contaminants, while maintaining balanced nutrition, in order to secure optimal fish and consumer health. Our understanding of primary responses of fish health and production related diseases, as well as biological processes that influence carry-over and lowering of contaminants in farmed fish, will contribute to a sustainable production of safer seafood products. Therefore, we have studied the liver deposition and toxicological effects in salmon fed graded levels of BHT during a 12-week feeding followed by a 2-week depuration period using chemical, molecular, and catalytic assays. In general, our data showed that BHT was significantly retained in the liver and selectively modulated toxicological responses in the xenobiotic biotransformation pathways during the feeding period. Specifically, BHT produced consistent dose- and time-specific gene expression patterns for AhR2alpha, AhR2beta, CYP1A1, CYP3A, UGT1, and GSTpi. The effect of BHT on the gene expression of biotransformation enzyme did not parallel enzyme activity levels, suggesting a possible inhibition by parent BHT or its metabolites. As a safety precaution, the production of farmed Atlantic salmon in Norway requires a mandatory 2-week depuration period prior to slaughtering and market delivery to ensure the elimination of veterinary medicaments, additives, and other undesirable components. Comparison of feeding and depuration periods showed that BHT was highly retained in fish liver, as only 8-13% of fed BHT was eliminated during the 2-week depuration period. This is just a part of the total concentration in the whole fish, since BHT may have been distributed and accumulated in other organs. Since BHT or its metabolites putatively inhibited biotransformation enzymes and affected metabolism of the compound, they may have potential for toxicological and adverse health effects for both fish and fish consumers through carry-over processes from the fish products.

New analogues of butylated hydroxytoluene as anti-inflammatory and antioxidant agents.

Amine or amide derivatives bearing the 2,6-di-tert-butyl phenol moiety are synthesised. Almost all are antioxidants, reduce acute inflammation and inhibit COX-1 and lipoxygenase activity. The most potent anti-inflammatory, COX-1 inhibitor and antioxidant agent, with low toxicity, is 2,6-di-tert-butyl-4-thiomorpholin-4-ylmethyl-phenol.

Influence of quinone methide reactivity on the alkylation of thiol and amino groups in proteins: studies utilizing amino acid and peptide models.

Quinone methides (QMs) are electrophiles formed in several biological processes including direct oxidations of 4-alkylphenols by cytochromes P450. These species may be responsible for the adverse effects of certain phenolic compounds through protein alkylation, but little information is available concerning specific targets or the resulting mechanisms of cell injury. The present goal was to determine the most likely sites of adduct formation among competing protein nucleophiles utilizing QMs of varying electrophilicity. Reactions of poorly reactive, moderately reactive, and highly reactive QMs, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT-QM), 6-tert-butyl-2-(2'-hydroxyl-1',1'-dimethylethyl)-4-methylene- 2,5-cyclohexadienone (BHTOH-QM), and 2-tert-butyl-6-methyl-4-methylene-2,5-cyclohexadienone (BDMP-QM), respectively, were investigated in aqueous solutions with nucleophilic amino acids. Each QM rapidly formed a thioether derivative of cysteine with little or no competition from the addition of water (hydration). The alpha-amino groups were the primary sites of alkylation for all other amino acids examined including lysine, histidine, tyrosine, and serine, and the pseudo-first order rates were 5 to 8-fold greater than the rates of hydration. Alkylation of the side chain nitrogens of lysine and histidine occurred at about one-fourth the rate of hydration for BDMP-QM, but no reaction was detectable for BHT-QM and no reactions occurred between QMs and amino acid hydroxyl groups. The results indicate that, based on chemical reactivity, peptide alkylation should occur in the order cysteine thiol > N-terminal amino > N epsilon-lysine = NIm-histidine, with side chain modifications occurring only with the more electrophilic QMs. Reactions of QMs with the tripeptide Gly-His-Lys confirmed the results with amino acids as N alpha-glycine alkylation predominated, but side chain adducts also formed with BHTOH-QM and BDMP-QM. Human hemoglobin was treated with QMs, hydrolyzed, and assayed by HPLC-thermospray mass spectrometry. This work revealed that N epsilon-lysine was the main alkylation site, emphasizing the importance of factors, in addition to chemical reactivity, which influence protein modification by electrophiles.

[Toxicology of the synthetic antioxidants BHA and BHT in comparison with the natural antioxidant vitamin E]. / Toxikologie der synthetischen Antioxidantien BHA und BHT im Vergleich mit dem natürlichen Antioxidans Vitamin E.

The toxicology of the food preservatives butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT) as well as the naturally occurring vitamin E (alpha-tocopherol) is described. In high dosages all three compounds induce in animals impairment of blood clotting, which can be explained by an antagonism with vitamin K. Specific toxic effects to the lung have only been observed with BHT. The other described toxic effects of BHA and BHT are less characteristic and often occur only after high dosage and long-term treatment. However, BHA induces in animals tumours of the forestomach, which are dose dependent, whereas BHT induces liver tumours in long-term experiments. Because there is no indication of genotoxicity of BHA and BHT, all published findings agree with the fact that BHA and BHT are tumour promoters. In contrast to BHA and BHT, vitamin E is not carcinogenic. On the other hand, all three antioxidants have also anticarcinogenic properties. The intake of the necessary high doses as for these effects are, however, contraindicated with BHA and BHT because of their carcinogenic effects. The present overview concludes that the concentrations of BHA and BHT nowadays used in food, drugs and cosmetics are probably harmless. In addition, vitamin E can also be used in higher doses without the occurrence of adverse effects.