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Abstract 2-Hydroxybenzylamine (2-HOBA), a naturally occurring compound found in buckwheat, can protect cells and tissues from oxidative stress. In this study, 2-HOBA acetate was orally administered to male and female rats for 90 consecutive days at doses of 100, 500, and 1000 mg·kg BW−1·d−1 (n = 20 per sex/group). Subchronic administration of 2-HOBA was well tolerated at all dose levels. 2-HOBA-treated male rats were slightly heavier in the last weeks of the study, but this difference was very small (<5%), did not show a dose–response relationship, and was not observed in female rats. Similarly, some statistically significant changes in serum biochemistry and hematology parameters were noted, but these were not considered to be of biological or toxicological significance. Sporadic differences in organ weights were observed between groups, but all were small (<10%) and unlikely to indicate toxicity. The incidence of histopathological lesions was similar between treated and control groups across all organs. Based upon these findings, the no-observed-adverse-effect level was determined to be ≥ 1000 mg·kg BW−1·d−1, which was the highest dose tested. These results further support no toxicity associated with oral consumption of 2-HOBA acetate in rats and the continued development of 2-HOBA as a dietary supplement or functional food. Highlights • Ninety days of daily oral administration of 2-hydroxybenzylamine (2-HOBA) acetate was tested in rats. • 2-HOBA doses up to 1000 mg·kg BW−1·d−1 did not induce toxicological effects. • These results provide additional support for the safety of 2-HOBA as a functional food agent or dietary supplement. [ABSTRACT FROM AUTHOR]

Abstract 2-hydroxybenzylamine (2-HOBA), a compound naturally found in buckwheat, has been shown to protect cells and tissues from the damaging effects of oxidative stress. The purpose of this report was to evaluate 2-HOBA in preclinical oral rodent toxicity studies. This report includes the results from three oral toxicity studies in rodents: a preliminary 28-day feeding study in mice, a 14-day acute oral toxicity study in rats, and a 28-day repeated dose oral toxicity study in rats. The preliminary mouse feeding study showed no adverse effects of 2-HOBA at concentrations up to 0.456% by weight in feed, but decreased food intake and weight loss were observed at 1.56% 2-HOBA in the diet, likely due to poor palatability. In the acute dosing study, 2000 mg/kg BW 2-HOBA resulted in mortality in one of the six tested female rats, indicating a median lethal dose of 2500 mg/kg BW. In the 28-day repeated oral dose study, small differences were observed between 2-HOBA treated and control group rats, but none of these differences were determined to be of toxicological significance. Together, these studies support the lack of toxicity of oral administration of 2-HOBA acetate at doses up to 1000 mg/kg BW d−1 in rodents. Highlights • 2-HOBA (up to 0.456% of feed) over 28 days did not cause adverse effects in mice. • 2-HOBA is a low-toxicity compound when given as an acute oral dose in rats. • Daily 2-HOBA administration did not induce toxic effects in a 28-day rat study. • These results support the safety of 2-HOBA for use as a dietary supplement. [ABSTRACT FROM AUTHOR]

Posttranslational modification by isolevuglandins (isoLGs), arachidonate oxidation products, is an important yet understudied process associated with altered protein properties. This type of modification is detected in cytochrome P450 27A1 (CYP27A1), a multifunction enzyme expressed in almost every cell and involved in the metabolism of cholesterol and other sterols. Previously, the CYP27A1 Lys358-isoLG adduct was found in human retina afflicted with age-related macular degeneration. Yet, the effect of Lys358 modification on enzyme activity was not investigated. Herein, we characterized catalytic properties of Lys358 as well as Lys476 CYP27A1 mutants before and after isoLG treatment and quantified the extent of modification by multiple reaction monitoring. The K358R mutant was less susceptible to isoLG-induced loss of catalytic activity than the wild type (WT), whereas the K476R mutant was nearly as vulnerable as the WT. Both mutants showed less isoLG modification than WT. Thus, modification of Lys358, a residue involved in redox partner interactions, is the major contributor to isoLG-associated loss of CYP27A1 activity. Our data show the specificity of isoLG modification, provide direct evidence that isoLG adduction impairs enzyme activity, and support our hypothesis that isoLG modification in the retina is detrimental to CYP27A1 enzyme activity, potentially disrupting cholesterol homeostasis.

Abstract: Isoketals (IsoKs) are γ-ketoaldehydes formed via the isoprostane pathway of arachidonic acid peroxidation and are among the most reactive by-products of lipid peroxidation. IsoKs selectively adduct to protein lysine residues and are highly cytotoxic, but the targets and molecular events involved in IsoK-induced cell death are poorly defined. Our previous work established that physiologically relevant aldehydes induce mitochondrial dysfunction (Kristal et al., J. Biol. Chem. 271:6033–6038; 1996). We therefore examined whether IsoKs induced mitochondrial dysfunction. Incubation of mitochondria with synthetic IsoKs in the presence or absence of Ca2+ was associated with alterations in mitochondrial respiration, membrane potential (ΔΨ), and pyridine nucleotide redox state. IsoKs dose dependently (0.5–4µM) accelerated liver mitochondria swelling induced by low concentrations of Ca2+ and Zn2+ or by the prooxidant tert-butylhydroperoxide, and release of cytochrome c, with similar observations in heart/brain mitochondria. The mitochondrial permeability transition (mPT) inhibitor cyclosporine A delayed IsoK-induced mitochondria dysfunction. The actions of IsoKs are consistent with interactions with cytochrome c, a protein rich in lysine residues. Direct reaction of IsoKs with select lysines in cytochrome c was demonstrated using high-resolution mass spectrometry. Overall, these results suggest that IsoKs may, in part, mediate their cytotoxic effects through induction of the mPT and subsequent activation of downstream cell death cascades. [Copyright &y& Elsevier]