3. Results and discussion

Trolox-treated nematodes (900 μM) significantly increased their resistance against oxidative stress (p < 0.05) compared to untreated nematodes in all four generations evaluated (Figure 1 and Table 1). AO1 nematodes were exposed to Trolox over all four generations. On the other hand, only the first generation of AO2 nematodes was exposed to Trolox, and later generations were not exposed to the antioxidant (like control nematodes). The purpose of this was to evaluate whether nematode survival increases due to the antioxidant presence or through an inherited effect. In both treatments (AO1 and AO2), resistance increased after each generation since the antioxidant effect was potentialized, and this indicates the existence of an inherited effect. groups, although AO1 nematodes were slightly more resistant than those from group AO2. However, in later generations significant differences (p < 0.05) were detected between both groups. The resistance of AO1 nematodes was higher than that of AO2 nematodes. In the third and fourth generations, 28 and 39% of AO1 nematodes survived in comparison to 2 and 10% of AO2 nematodes, respectively. Oxidative stress resistance was higher in nematodes with continuous exposure to Trolox. Nevertheless, AO2 resistance was superior to control (p < 0.05) in all generations, proving once again the existence of an inherited effect that protected nematodes following exposure to the antioxidant (Trolox). These results are consistent with those of Yazaki et al. [29], who suggest that nematode resistance and survival are influenced by environmental factors (nutrients, oxygen, toxic substances, and pathogens) as well as heritage (20–50%). Additionally, Harrington and Harley [30] found that continuous exposure to vitamin E promotes nematode survival, which is consistent with the results of AO1 nematodes. On the other hand, Pietsch et al. [24] showed that quercetin (100 and 200 μM) increased the life span of three different generations of nematodes. However, survival was not shown to improve over the next generation, opposed to that observed in this work. In the same way, Zuckerman and Geist [31] noticed that vitamin E increased the life span and survival of C. elegans, although the effect of is not inherited from parents to

Trolox Protection against Oxidative Stress in Caenorhabditis elegans

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

Increased resistance against oxidative stress in nematodes treated with Trolox agrees with the results reported by several authors. In this sense, Chen et al. [7] observed that resveratrol (50 μM) increased nematode resistance by 27.6%. Also, researchers found that blue corn anthocyanin extract provided protection against oxidative stress by reducing ROS production and increasing C. elegans life span. On the other hand, Abbas and Wink [1] demonstrated that epicatechin gallate (EGCG) (200 μM) decreased H2O2 levels in C. elegans nematodes and increased its life span and resistance against oxidative stress. In the same way, González-Manzano et al. [26] reported that epicatechin (EG) (200 μM) increased nematode resistance against thermal stress by decreasing ROS levels and increasing CAT activity. Additionally, Kampkötter et al. [5] concluded that kaempferol extends the life span of nematodes through increased resistance to thermal stress. Thermal stress leads to an accumulation of ROS; therefore, the treatment reduces ROS levels and gives protection to the nematodes. In addition, Surco-Laos et al. [25] found that quercetin (200 μM) increased breeding, survival, and resistance against thermal and oxidative stress of C. elegans. Similarly, Lee et al. [16] showed that vitexin (50 and 100 μM) increased life span, decreased ROS levels, and induced thermotolerance, leading to

Trolox has been shown to increase the life span of nematodes as well as their thermotolerance, and it has been proposed that longevity is enhanced by inducing stress response [19, 20]. In this sense, Zhang et al. [32] showed that Trolox was capable of neutralizing ROS levels and reversing the oxidative damage induced by calycosin-7-O-β-D-glucoside, findings that are consistent with the observations of this work. In contrast, Schulz et al. [33] observed that treatment with Trolox (100 μM) did not improve the life span of nematodes. It is likely that the researchers have not witnessed any effect due to the low concentration of Trolox, since in the current study, the concentration of Trolox was higher (900 μM) and its protective effect was exhibited. This theory is particularly true as mentioned by Gems and Doonan [19] who explained that antioxidant dosage affects oxidative stress

Vitamin E administration has been shown to increase life span, survival, and growth, protect against oxidative stress during gametogenesis, decrease toxicity and oxidative stress, and reverse damage caused by UV radiation in C. elegans [19, 20, 34].

progeny.

increased resistance against oxidative stress.

response and survival of nematodes.

77

In the first generation, no significant differences (p > 0.05) were observed between groups AO1 and AO2 (as they had the same treatment). In the second generation, no significant differences (p > 0.05) were observed between both

#### Figure 1.

Resistance against oxidative stress of nematodes treated with Trolox. Data shown correspond to mean (n = 2). AO1 nematodes were exposed to Trolox through all generations. AO2 nematodes were exposed to Trolox only in the first generation. Control nematodes were not exposed to any antioxidant through all generations.


Data shown correspond to mean (n = 2). Superscript letters indicate significant differences (p < 0.05) between treatments within each generation.

#### Table 1.

Half-life time of Trolox-treated nematodes subjected to oxidative stress.

groups, although AO1 nematodes were slightly more resistant than those from group AO2. However, in later generations significant differences (p < 0.05) were detected between both groups. The resistance of AO1 nematodes was higher than that of AO2 nematodes. In the third and fourth generations, 28 and 39% of AO1 nematodes survived in comparison to 2 and 10% of AO2 nematodes, respectively. Oxidative stress resistance was higher in nematodes with continuous exposure to Trolox. Nevertheless, AO2 resistance was superior to control (p < 0.05) in all generations, proving once again the existence of an inherited effect that protected nematodes following exposure to the antioxidant (Trolox). These results are consistent with those of Yazaki et al. [29], who suggest that nematode resistance and survival are influenced by environmental factors (nutrients, oxygen, toxic substances, and pathogens) as well as heritage (20–50%). Additionally, Harrington and Harley [30] found that continuous exposure to vitamin E promotes nematode survival, which is consistent with the results of AO1 nematodes. On the other hand, Pietsch et al. [24] showed that quercetin (100 and 200 μM) increased the life span of three different generations of nematodes. However, survival was not shown to improve over the next generation, opposed to that observed in this work. In the same way, Zuckerman and Geist [31] noticed that vitamin E increased the life span and survival of C. elegans, although the effect of is not inherited from parents to progeny.

Increased resistance against oxidative stress in nematodes treated with Trolox agrees with the results reported by several authors. In this sense, Chen et al. [7] observed that resveratrol (50 μM) increased nematode resistance by 27.6%. Also, researchers found that blue corn anthocyanin extract provided protection against oxidative stress by reducing ROS production and increasing C. elegans life span. On the other hand, Abbas and Wink [1] demonstrated that epicatechin gallate (EGCG) (200 μM) decreased H2O2 levels in C. elegans nematodes and increased its life span and resistance against oxidative stress. In the same way, González-Manzano et al. [26] reported that epicatechin (EG) (200 μM) increased nematode resistance against thermal stress by decreasing ROS levels and increasing CAT activity. Additionally, Kampkötter et al. [5] concluded that kaempferol extends the life span of nematodes through increased resistance to thermal stress. Thermal stress leads to an accumulation of ROS; therefore, the treatment reduces ROS levels and gives protection to the nematodes. In addition, Surco-Laos et al. [25] found that quercetin (200 μM) increased breeding, survival, and resistance against thermal and oxidative stress of C. elegans. Similarly, Lee et al. [16] showed that vitexin (50 and 100 μM) increased life span, decreased ROS levels, and induced thermotolerance, leading to increased resistance against oxidative stress.

Trolox has been shown to increase the life span of nematodes as well as their thermotolerance, and it has been proposed that longevity is enhanced by inducing stress response [19, 20]. In this sense, Zhang et al. [32] showed that Trolox was capable of neutralizing ROS levels and reversing the oxidative damage induced by calycosin-7-O-β-D-glucoside, findings that are consistent with the observations of this work. In contrast, Schulz et al. [33] observed that treatment with Trolox (100 μM) did not improve the life span of nematodes. It is likely that the researchers have not witnessed any effect due to the low concentration of Trolox, since in the current study, the concentration of Trolox was higher (900 μM) and its protective effect was exhibited. This theory is particularly true as mentioned by Gems and Doonan [19] who explained that antioxidant dosage affects oxidative stress response and survival of nematodes.

Vitamin E administration has been shown to increase life span, survival, and growth, protect against oxidative stress during gametogenesis, decrease toxicity and oxidative stress, and reverse damage caused by UV radiation in C. elegans [19, 20, 34].

3. Results and discussion

Technology, Science and Culture - A Global Vision, Volume II

Figure 1.

generation.

Table 1.

76

Trolox-treated nematodes (900 μM) significantly increased their resistance against oxidative stress (p < 0.05) compared to untreated nematodes in all four generations evaluated (Figure 1 and Table 1). AO1 nematodes were exposed to Trolox over all four generations. On the other hand, only the first generation of AO2 nematodes was exposed to Trolox, and later generations were not exposed to the antioxidant (like control nematodes). The purpose of this was to evaluate whether nematode survival increases due to the antioxidant presence or through an inherited effect. In both treatments (AO1 and AO2), resistance increased after each generation since the antioxidant effect was potentialized, and this indicates the existence of an inherited effect. In the first generation, no significant differences (p > 0.05) were observed between groups AO1 and AO2 (as they had the same treatment). In the second generation, no significant differences (p > 0.05) were observed between both

Resistance against oxidative stress of nematodes treated with Trolox. Data shown correspond to mean (n = 2). AO1 nematodes were exposed to Trolox through all generations. AO2 nematodes were exposed to Trolox only in the first generation. Control nematodes were not exposed to any antioxidant through all generations.

Control 3.5<sup>b</sup> 3.7<sup>b</sup> 3.6<sup>c</sup> 3.7<sup>c</sup> AO1 4.1<sup>a</sup> 5.1a 5.9<sup>a</sup> 6.6a AO2 4.2<sup>a</sup> 4.6a 4.7<sup>b</sup> 4.8b Data shown correspond to mean (n = 2). Superscript letters indicate significant differences (p < 0.05) between treatments within each

First generation Second generation Third generation Fourth generation

Trolox treatment Half-life time (h)

Half-life time of Trolox-treated nematodes subjected to oxidative stress.

In this sense, Harrington and Harley [30] and Zuckerman and Geist [31] showed that vitamin E (200 μg/mL) prolonged the life span and survival of C. elegans. Meanwhile, Kim et al. [35] mentioned that vitamin E improved longevity of nematodes by increasing their resistance against oxidative stress. In contrast, higher concentration of vitamin E (400 μg/mL) proved to have a toxic effect on nematode survival, exhibiting adverse effects on nematode reproduction and growth delay [30]. This is consistent with findings of Li et al. [34] who showed that high concentrations of vitamin E had neurotoxic effects on C. elegans. They found that elevated levels of vitamin E induce abnormal neuronal development that disrupts nematode thermosensation and thermotaxis. Similarly, nematodes supplemented with high doses of antioxidants, such as EUK-8 and EUK-134 (SOD mimetics), display shorter life span due to increased ROS production [19, 21]. In addition, Chen et al. [7], Yazaki et al. [29], and Desjardins et al. [36] have suggested that high levels of antioxidants exhibit a prooxidant and toxic effect on the organism, while lower concentrations display a protective effect.

Antioxidant compounds achieve their protective effect through at least two mechanisms, direct suppression of free radicals and oxidants and potentiation of the synthesis and activity of metabolites and enzymes in the body [37]. Numerous studies have shown that resistance against oxidative stress of nematodes fed with antioxidants is not solely caused by the ability to scavenge free radicals and reverse oxidative damage. Resistance also involves the regulation of antioxidant enzymes and defenses (SOD, CAT, and GPx) [8, 29]. In this sense, it has been noted that mutant strains age-1 and daf-2 have higher expression and activity of CAT and SOD enzymes than the wild strain, thus increasing their life span and tolerance to oxidative and thermal stress. On the other hand, mutant strain mev-1 has lower life span due to paraquat hypersensitivity and decreased SOD levels [20]. Other mechanisms involved in the resistance are modulation of transcriptional factors, signaling pathways, and reduction of ROS production, mechanisms that influence development, growth, metabolism, and survival of C. elegans [8, 24, 26, 29, 32, 35].

## 4. Conclusions

Continuous exposure of nematodes to Trolox increased their resistance against oxidative stress and survival in comparison to those without treatment. This suggests that Trolox is not only capable of neutralizing oxidative damage but also triggers changes at physiological and molecular levels that enhanced its antioxidant activity and the organism's antioxidant defenses, allowing them to cope with the oxidative stress and increasing their survival.

Author details

Puebla, Mexico

79

Marco Antonio González-Peña<sup>1</sup>

and Ana Eugenia Ortega-Regules<sup>3</sup>

Puebla, Cholula, Puebla, Mexico

Puebla, Cholula, Puebla, Mexico

, José Daniel Lozada-Ramírez<sup>2</sup>

\*

Trolox Protection against Oxidative Stress in Caenorhabditis elegans

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

\*Address all correspondence to: ana.ortega@udlap.mx

provided the original work is properly cited.

1 Department of Chemical and Food Engineering, Universidad de las Américas

2 Department of Chemical and Biological Sciences, Universidad de las Américas

3 Department of Health Sciences, Universidad de las Américas Puebla, Cholula,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
