2.3 Synchronization

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]. 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

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,

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],

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

The biological model used in this study was the wild strain (Bristol N2) nematode C. elegans, fed with the bacteria, uracil auxotroph, Escherichia coli OP50 in Luria-Bertani (LB) medium (10 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract) [27]. Both organisms were obtained through the Department of Chemical and Biological

Nematodes were maintained at 22 2°C in nematode growth medium (NGM) plates [3 g/L NaCl, 2.5 g/L peptone, 24 g/L bacteriological agar, 1 mL/L 1 M CaCl2,

an electron to the free radical to stabilize it [9]. Trolox (6-hydroxy-2,5,7,8-

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].

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2.2 Maintenance and growth conditions

among others.

properties.

2. Methodology

2.1 Materials

74

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.

### 2.4 Oxidative stress resistance

For oxidative stress resistance assays, synchronized nematodes were divided into the following groups: control group (without antioxidant) and two antioxidant groups (900 μM Trolox).

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

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.
