Biological effects of an oxyphytosterol generated by β-Sitosterol ozonization

Highlights

• βSec reduced HepG2 cell viability, with lower potency but higher efficacy than βSito.

• βSec induced a cell cycle arrest in the G0/G1 phase and reduced the mitotic index.

• βSec caused alteration in cell and nuclear morphologies.

• βSec disrupted the organization of the microtubules network.

• N-acetyl l-cysteine was not able to inhibit βSec biological effects.

Abstract

β-Sitosterol (βSito) is the most abundant phytosterol found in vegetable oils, grains such as wheat, beans, and corn, and in many phytosterol-enriched foods. It is prone to oxidation by reactive oxygen species, such as ozone, leading to the formation of oxyphytosterols. A better understanding regarding the biological effects and mechanism of action of oxyphytosterols is required since the beneficial and adverse side effects of these compounds on human health remain highly controversial. In this work, we investigated the biological effects of β-Secosterol (βSec), a new oxyphytosterol generated by the reaction of βSito with ozone. Treatment of HepG2 cells with βSito or βSec (0.1–100 μM) for 24, 48, and 72 h induced a dose-dependent reduction of cell viability in the MTT assay, with βSec showing higher efficacy than βSito. However, βSec presented a lower potency than βSito, showing IC₅₀ = 37.32 μM, higher than βSito (IC₅₀ = 0.23 μM) at 48 h. Cell cycle analyses by flow cytometry showed a slight decrease of G0/G1 phase with βSito 0.5 μM, but a significant cell cycle arrest at the G0/G1 phase in the treatment for 48 h with βSec 20 μM (62.69 ± 2.15%, p < 0.05) and βSec 40 μM (66.96 ± 5.39%, p < 0.0001) when compared to control (56.97 ± 2.60%). No suggestion of apoptosis was indicated by flow cytometry data. Also, βSec (20 and 40 μM) reduced the mitotic index. In the laser scanning confocal microscopy analysis no alterations in cell morphology were observed with βSito (0.5 μM). Nevertheless, round-shaped cells, abnormal nuclear morphology with shrinkage, and formation of microtubules clusters were observed in the treatment with βSec, indicating a disruption in the microtubules network organization. N-acetyl-l-cysteine was not able to inhibit any of these cellular effects, indicating a lack of involvement of oxidative stress in the mechanism of action of βSec. Although not further investigated in this study, it was discussed the hypothesis that covalent adduct formation with lysine residues of proteins, could play an important role in the biological effects elicited by βSec. Elucidation of the primary cellular processes induced by βSec provides the essential knowledge to be aware of its potential adverse side effects or therapeutic use of this oxyphytosterol.

Introduction

Phytosterols are plant sterols found in vegetable oils, seeds, nuts, grains, fruits, and phytosterol-enriched functional foods and supplements, which are widely consumed [1], primarily due to their ability to reduce the risks of atherosclerosis by inhibiting cholesterol absorption [2]. Although this benefit, there is a concern in terms of health and food safety, particularly due to the potential biological effects of their oxidized products, named oxyphytosterols or phytosterol oxidation products, which are generated by autoxidation during heating at high temperatures or prolonged storage of foods [3]. The generation of oxyphytosterols in vivo is still unclear and controversial [4].

Oxyphytosterols have already been detected in the plasma of healthy human subjects [5], but studies conducted by Baumgartner et al. related that intake of 3.0 g/day of plant sterols in humans causes no increase in the concentration of oxyphytosterols [6,7]. Conversely, other studies indicate elevated concentration in the plasma of phytosterolemic patients with low plant sterol intake, suggesting endogenous oxidation of phytosterols [8].

Although less potent than oxysterols (oxidation products of cholesterol), oxyphytosterols are capable of causing similar biological effects to a great variety of cells [4], which include cytotoxicity, apoptosis [[9], [10], [11]], disruption of mitochondrial membrane potential [12], modulation of intestinal cholesterol metabolism [13], and modulation of inflammation and immunity [14,15]. These properties have been associated not only with pathological processes such as atherosclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis [16] but also with antitumoral [17] and antiviral activities [18].

Less attention has been paid to the effects on cell cycle, microtubules network, and centrosomes. These cell components have important roles in many fundamental biological processes. Microtubules, filamentous polymers of tubulins, are the major component of the cytoskeleton and are implicated in intracellular transport, driving of chromosome separation during cell division, regulation of cell polarity, and morphogenesis [19]. Moreover, microtubule mitotic spindle and parallel microtubule arrays in neurons are major targets for several compounds used in the treatment of cancer and neurological disorders [20]. The centrosome is the major microtubules-organizing center composed of centrioles and pericentriolar material (PCM), which comprises several proteins, including nucleating factors, such as γ-tubulin and scaffolding proteins, such as pericentrin [21]. While centrioles are essential to centrosome duplication in every cell cycle, PCM is responsible for most of the centrosome microtubule-organizing activity, including microtubules nucleation and control of microtubules number, polarity, distribution, and flux [22].

Ozone (O₃) is a gas with a high oxidation potential, used in food processing and as a sanitizing agent [23]. Besides, O₃ has been detected in elevated concentrations in the atmosphere of urban areas due to the increase of human activities related to economic development [24].

The growing consumption of phytosterol-enriched products and the elevated concentration of ozone in the atmosphere [25] potentially enhance the feasibility of oxyphytosterol formation in foods from the O₃ reaction with unsaturated molecules. Thus, taking into account the structural similarities of cholesterol and β-Sitosterol (βSito), recently Martins et al. (2020) reported the synthesis, chemical characterization, and mechanism of formation of oxyphytosterols generated by reaction with ozone. The main product, βSec, a β-Sitosterol secoaldehyde (2-[(7aR)-5-[(1R,4S)-4-hydroxy-1-methyl-2-oxocyclohexyl]-1,7a-dimethyl-1,2,3,3a, 4,5,6,7- octahydroinden-4-yl]acetaldehyde) was able to cause a decrease in HepG2 cell viability [26].

Therefore, in the light of the availability of βSec and considering the potential effects by oxyphytosterols in several cellular processes and the lack of studies on cell morphology and distribution of the microtubule network, we sought to evaluate the biological effects of this new oxyphytosterol. In the present work, we compared the potency and efficacy of βSito and βSec in reducing the viability of HepG2 tumor liver cells and investigated the biological effects of βSec by measuring cell cycle phase distribution by flow cytometry, analyzing cell and nuclear morphologies and effects in microtubules network by immunofluorescence. Also, we considered the involvement of oxidative stress in the mechanism of action of βSec, using N-acetyl-l-cysteine (NAC) as an antioxidant. Although not further investigated in this study, it was discussed the hypothesis that covalent adduct formation with lysine residues of proteins, could play an important role in the biological effects elicited by βSec.