The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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It is also used in animal feed, cosmetics, pharmaceuticals, rubber, biodiesel, and petroleum products [ 13 ]. Some studies on BHA have revealed beneficial effects, with anti-tumor and nephroprotective potential [ 14 , 15 ]. Nevertheless, BHA has been shown to be an endocrine disrupter and a carcinogen in rats [ 16 , 17 ]. It can also perturb adipogenesis and increase the incidence of obesity [ 11 , 17 ].
The effects of BHT and BHA on various organs liver, lung, kidney, blood system, and reproductive system have been reviewed [ 18 ]. TBHQ is also added to a wide range of foods, such as unsaturated vegetable oils, residual frying oils, lard, and infant formula, as well as in biodiesel [ 19 , 20 ].
Several studies documented the chemoprotective effects of TBHQ. However, other in vitro studies, indicated that it can cause DNA damage and in vivo tests suggest it may be carcinogenic, cytotoxic, and genotoxic [ 19 , 21 ]. Despite the large number of scientific papers describing the controversial effects of BHT and its analogs using in vitro and in vivo animal models, only very few studies have described these synthetic antioxidants in humans [ 1 , 5 , 23 , 24 ].
The potential for human exposure of BHT and its analogs, as well as the lack of information surrounding this exposure, support the need to study their biotransformation reactions, especially concerning the formation of reactive metabolites. Oxidative metabolites, glutathione GSH adducts, glucuronides, and sulfate conjugates have been characterized by liquid chromatography coupled to a quadrupole-time-of-flight high-resolution tandem mass spectrometer.
Isotope-labeled BHT analogs were employed to aid in the structural elucidation of metabolites. The metabolism of these two compounds was also studied to confirm metabolic pathways. A biphenyl solid-core column using 5 mM ammonium acetate and acetonitrile as mobile phases A and B, respectively, yielded significant signal increase over using formic acid as an additive, as well as ameliorating peak shapes for many of the metabolites found in this study.
BHT eluted with a retention time of Otherwise, this molecule is quite resistant to fragmentation, and when collision energy was increased to form other structurally characteristic fragments, most of the signal was lost and no clear product ions were observed.
Both human and rat microsomes yielded similar metabolic profiles for all tested compounds and incubations conditions. Throughout the manuscript, representative chromatograms from the human incubations are shown. All metabolites were confirmed with accurate mass measurements within 5 ppm of the theoretical exact masses. When BHT was incubated under oxidative conditions, several metabolites were detected, as shown in Figure 2.
To investigate whether the oxygen is added to the para -methyl or on a t -butyl group, isotope-labeled BHTs were also incubated Table 2. While for the isomer at Di-hydroxylated BHT was also detected at BHT-aldehyde via the oxidation of the p -methyl group was also detected at Another two oxidative metabolites were detected at 9.
The isomer eluting at 9. The second isomer Table 1 , at It is also used as a synthetic phenolic antioxidant in plastics and food packaging [ 30 ]. DBP was detected in oxidative incubations at This metabolite has been confirmed by the synthetic DBP standard, which was also incubated under oxidative conditions Figure S1. Numerous metabolites of BHT have been described and metabolic pathways proposed [ 6 , 31 ]. A major metabolic pathway is initiated by the oxidation of the p -methyl group, leading to the formation BHT-aldehyde and BHT-acid by stepwise oxidation.
Another metabolic pathway is initiated by the oxidation of the t -butyl group. Thompson et al. The oxidative pathways of BHT have also been studied in vivo in different species, including the oxidation of the p -methyl group as a major metabolic route in rat, rabbit, and monkey, while the oxidation of t -butyl groups has been described as the predominant pathway in human and mouse [ 33 ].
Zhang et al. These two metabolites are proposed to result from the formation of the quinone methide intermediate followed by the addition of GSH on the methylene carbon.
This hypothesis is supported by results from incubations with isotope-labeled BHT analogs. Tajima et al. This GSH adduct was also described by Madsen et al. This supports the structure where the SG group replaces the methyl group. Glutathione is able to scavenge radicals by its electron-donating ability, enabling it to neutralize such reactive species. This GSH adduct had not been described in previous studies. This metabolite had not been reported previously.
Armstrong et al. BHA dimer was also found to form in rat intestine and by incubating BHA with rat intestine peroxidase, as well as horseradish peroxidase [ 37 ]. No dimer was detected for TBHQ under our conditions, which may be explained by its preference to form the quinone reactive metabolite. Peters et al. They also suggested that these conjugates could represent nephrotoxic metabolites, and may be responsible for the tumor-promoting effects of TBHQ and BHA [ 38 ]. The first is suggested to form via an epoxide followed by GSH addition and loss of a water molecule, while the second is likely formed via the ortho -quinone reactive metabolite.
These two novel metabolites have not been previously characterized. These same sulfate conjugates were previously detected in human urine using GC-MS [ 39 ]. The major metabolic pathway for BHA is reported to be via the conjugation of the free hydroxyl group with both glucuronic acid and sulfate [ 39 ].
Conning et al. The oxidative metabolism for these two compounds was also studied to help support the complex metabolic pathway proposed for BHT. Using an untargeted high-resolution tandem mass spectrometry approach to decipher the metabolism of analogous compounds, while incorporating isotope labeling, proved to be a powerful method to elucidate structures of all detected metabolites.
Adding an equal volume of cold acetonitrile quenched the reaction. MetabolitePilot 2. PeakView 2. Likewise, a United Nations Environment Program assessment noted that BHT had a moderate to high potential for bioaccumulation in aquatic species though the assessment deemed BHT safe for humans [9].
International regulations are stronger. The State of California requires warning labels on products containing BHA, notifying consumers that this ingredient may cause cancer. Baur, A. Wada, H. Fact sheet for Butylhydroxyanisol.
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