Abstract

The aim of this work was to evaluate the antioxidant activity of Trolox (6 hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble vitamin E analog, on the biological model Caenorhabditis elegans, and determine if the resistance provided to the nematode against oxidative stress has an inherited component. For this purpose, nematodes previously exposed to Trolox were transferred to medium plates with juglone (5-hydroxy-1,4-naphthalenedione), generating a prooxidant environment that induces lethal oxidative stress, and then nematode survival was evaluated every hour. Additionally, nematodes were synchronized and placed in new medium plates in the presence of Trolox, until the oxidative stress resistance of four different generations was evaluated. Trolox-treated C. elegans increased their oxidative stress resistance in comparison to those without treatment. Moreover, protection was potentialized through each generation, suggesting that Trolox not only neutralizes the oxidative damage but also induces molecular changes that extend nematode survival.

Keywords: Trolox, antioxidant, oxidative stress resistance, Caenorhabditis elegans

## 1. Introduction

During the cellular respiration process of aerobic organisms, there is a transfer of electrons toward oxygen that generates reactive oxygen species (ROS), since oxygen acts as the final acceptor of electrons in the electron transport chain for energy production. However, when there are leaks in the flow of electrons, as part of oxidative metabolism or as a response of the immune system, free radicals are generated [1, 2]. Despite the above, the organisms have developed an antioxidant defense system capable of seizing or neutralizing free radicals, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione transferase and glutathione reductase, and endogenous (glutathione) and exogenous (ascorbic acid, tocopherol, phenolic compounds, flavonoids, and carotenoids) antioxidants. When the production of reactive species increases or antioxidant levels decrease, the prooxidant-antioxidant balance that exists in organisms is broken, leading to the so-called oxidative stress [3].

ROS, generated as by-products of chemical cell reactions as well as by several stress factors (ultraviolet light, metallic ions, drugs, chemical modifications, heat, and ionizing radiation), are responsible for the oxidative damage of lipids, proteins, and DNA, mutations, processes involved in cellular aging, and onset or development of degenerative diseases such as diabetes, cataracts, hypertension, inflammation, cancer, rheumatoid arthritis, neuropathies, and cardiopathies, among others [4–8].

1 mL/L 1 M MgSO4∙7H2O, 20 mL/L pH 6.0 phosphate buffer, 1 mL/L cholesterol (0.005 g in 1 mL of ethanol)] supplemented with 200 μL of E. coli OP50. Nematodes

Synchronization was based on the methodology used by Surco-Laos et al. [25] with some modifications. Nematodes were taken in the adult stage (third day), washed with M9 solution to eliminate bacteria, and centrifuged at 4600 rpm and 4°C for 1 min. The supernatant was removed, and 1 mL of M9 solution was added and centrifuged again under the same conditions. The supernatant was removed, and 1 mL of 1 M NaOH was added, then vortexed (Vortex-Genie 2 G560, Scientific Industries, USA) for 30 s, and centrifuged under the same conditions. The supernatant was removed, and 500 μL of 1 M NaOH and 500 μL of NaOH:5% sodium hypochlorite (3 mL 1 M NaOH + 2 mL Cloralex®) were added, then vortexed for 60 s, and centrifuged under the same conditions. The supernatant was removed and washed two times with 1 mL of M9 solution, increasing the centrifugation speed to 5600 rpm. Lastly, the supernatant was removed, and the residue was placed on new

NGM plates with E. coli OP50. The plates were incubated at 22 2°C.

failed to respond at the stimulation with a platinum wire [7].

For oxidative stress resistance assays, synchronized nematodes were divided into the following groups: control group (without antioxidant) and two antioxidant

For oxidative stress resistance assays, the methodology suggested by Sangha et al. [28] was used with some modifications. It consisted of selecting 60 5 nematodes in L4 larval phase (2 to 2½ days), previously exposed to antioxidants, which were transferred to NGM plates with 400 μM of the prooxidant juglone (5-hydroxy-1,4-naphthoquinone; Sigma-Aldrich, Mexico), which induces lethal oxidative stress. Nematode survival was evaluated every hour until 8 h, scored as dead if they

Simultaneously, nematodes in L4 stage were synchronized, and eggs obtained were placed in new NGM plates with bacteria and antioxidants and incubated at 22 2°C until the L4 stage was reached again. At this point, one of the antioxidant groups continued with Trolox treatment (AO1), while in the other group, the treatment was discontinued after the first generation (AO2). Nematode survival was evaluated following the above-described methodology. This procedure was repeated until the oxidative stress resistance of four different generations was evaluated for each condition. Experiments were carried out in duplicate.

Survival assays of C. elegans were analyzed using the Kaplan-Meier methodology and log-rank test, using Minitab Statistical Software (18th version, Minitab Inc.,

were taken from NGM plates and washed with 2 mL of M9 solution (6 g/L Na2HPO4, 3 g/L KH2PO4, 5 g/L NaCl, 0.215 g/L MgSO4∙7H2O). They were centrifuged at 4600 rpm and 4°C for 1 min (centrifuge Z 366 K, HERMLE Labortechnik, Germany). Two washes were performed by removing the supernatant, adding 1 mL of M9 solution, and centrifuging under the same conditions. The supernatant was removed, and the residue was placed in new NGM plates with E.

coli OP50. The plates were incubated at 22 2°C [27].

Trolox Protection against Oxidative Stress in Caenorhabditis elegans

DOI: http://dx.doi.org/10.5772/intechopen.90099

2.3 Synchronization

2.4 Oxidative stress resistance

groups (900 μM Trolox).

2.5 Statistical analysis

USA).

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An antioxidant molecule is any substance that delays or prevents deterioration, damage, or destruction caused by oxidation. Antioxidants are compounds capable of slowing, inhibiting, or preventing the oxidation of molecules due to their ability to quench free radicals through electron transfer mechanisms. The antioxidant transfers an electron to the free radical to stabilize it [9]. Trolox (6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic acid) is a water-soluble vitamin E analog with antioxidant properties [10]. In this sense, several authors have demonstrated the ability of Trolox to reduce hydrogen peroxide (H2O2) levels, inhibit cell membrane damage and DNA fragmentation, and protect against damage by lipid peroxidation [11–14].

Caenorhabditis elegans is an organism frequently found in soils, feeding on bacteria and other microorganisms, and used as a biological model due to its small size, simple anatomy, short life span, transparent structure, easy reproduction, completely sequenced genome, and abundance of mutant strains [7, 15, 16]. The nematode genome has been completely sequenced; it contains 19,000 genes, and 65% of the genes is associated with human diseases, making clear its importance as a research model to understand the biological, metabolic, pathological, and molecular processes associated with the development of diseases, functioning, and toxicity of bioactive compounds and antioxidant substances [17, 18].

C. elegans can undergo experimental oxidative stress conditions upon being exposed to certain prooxidant compounds such as H2O2, tert-butylhydroperoxide, arsenite, paraquat, and juglone. This leads to increased levels of O2 and ROS, shortening the nematode life span and survival [19, 20], which has been advantageous to studies of antioxidant compounds using C. elegans as a research model organism. Several authors have established that its life span and/or resistance against oxidative stress increased after exposure to vitamin C [17]; vitamin E [21]; spinach extracts [22]; cocoa [23]; phenolic compounds such as quercetin [24, 25], epicatechin [26], and resveratrol [7]; and carotenoids such as β-carotene [17], among others.

The aim of this work was to evaluate the antioxidant effect of Trolox on the resistance of the nematode C. elegans against oxidative stress. Moreover, the effect of the antioxidant on continuous generations was studied with the purpose of determining if that resistance obeys to an inherited effect or solely to its antioxidant properties.
