Responses to oxidative stress and antioxidant capacity in rats at different growth stages

Abstract

[Purpose] Oxidative stress is regulated by antioxidant capacity in vivo. However, its impact on aging characteristics remains debatable. This study is first to report oxidative stress, antioxidant capacity, and their ratio in five age groups of rats, and aimed to provide basic data useful for disease prevention. [Materials and Methods] Sixty male Wistar rats of different ages were used as experimental animals, grouped as follows: weaned (three weeks), growth (eight weeks), adulthood (six months), middle-age (12 months), and old-age (24 months). To assess oxidative stress and antioxidant capacity, derivatives of reactive oxygen metabolites and biological antioxidant potential were measured. [Results] The lowest level of oxidative stress and the highest level of antioxidant capacity were observed during the weaning stage, and remarkable dynamic changes were observed until adulthood. The highest oxidative stress and lowest antioxidant capacity were observed in the old-age group. [Conclusion] In vivo oxidative stress and antioxidant capacity are largely reflected in the characteristics of aging, and this ratio is greatly influenced by the dynamics of oxidative stress and antioxidant capacity with age.

INTRODUCTION

Reactive oxygen species (ROS) play an important role in in vivo immune functions¹⁾. However, when ROS accumulate in the body, they cause highly toxic oxidative stress and damage normal cells²⁾. Thus, oxidative stress is constantly regulated in vivo. The balance between ROS levels and antioxidant capacity is important for regulating oxidative stress and maintaining normal biological functions³⁾. Approximately 2% of the oxygen taken up by the body becomes ROS, which increases significantly owing to the decrease in hormone levels and strong anaerobic physical activity⁴⁾. In vivo oxidative stress is regulated by the antioxidant activities of super oxide dismutase (SOD) and glutathione peroxidase (GPx), which are influenced by antioxidants and hormones²⁾. Therefore, since aging affects hormonal and immune functions, it is conceivable that different age groups would have different responses to oxidative stress and antioxidant capacity. The purpose of this study was to clarify the dynamics of oxidative stress and antioxidant capacity in different age groups, and to gain knowledge on preventive medicine against oxidative stress.

MATERIALS AND METHODS

Sixty male Wistar rats (CLEA Japan, Inc., Tokyo, Japan) were divided according to their age into five groups of 12 individuals each, and their blood was collected during a resting state. The five groups were as follows: weaned (W; three weeks, 53.0 ± 3.3 g), growth (G; eight weeks, 254.3 ± 6.3 g), adulthood (A-1; six months, 441.5 ± 14.4 g), middle-age (A-2; 12 months, 546.3 ± 14.0 g), and old-age (A-3; 24 months, 621.5 ± 26.7 g). In all groups, two animals of the same groups were housed in one cage. The environmental conditions were set at 22 ± 2 °C, humidity of 55 ± 5%, and a 12-h light–dark cycle. The experimental animals had free access to food CE-2 (CLEA Japan, Inc.) and water.

This study was approved by the Animal Care and Use Committee of Aomori University of Health and Welfare and conducted in accordance with the guidelines for animal experiments at Aomori University of Health and Welfare.

To assess oxidative stress and antioxidant capacity, derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) were measured. Both d-ROMs and BAP were measured using a REDOXLIBRA (WISMERLL Co, Ltd., Tokyo, Japan). The d-ROMs are expressed in CARR U (1 CARR U=0.08 mg hydrogen peroxide/dL) and BAP are expressed in μmol/L as biological antioxidant capacity.

For analysis, Pearson’s correlation coefficients were calculated between d-ROMs and BAP, oxidative stress index (OSI) was determined using d-ROMs/BAP×100. d-ROMs, BAP and OSI were used as dependent factors and age was used as an independent factor to indicate the coefficient of determination by linear regression analysis. One-way analysis of variance was performed for comparisons between groups. Scheffé’s test was performed for multiple comparisons. Statistical analyses were performed using SPSS version 27 (IBM Japan, Ltd., Tokyo, Japan) and the statistical significance level was set at less than 5%.

RESULTS

A significant negative correlation was observed between d-ROMs and BAP in all experimental animals (r=−0.603; p<0.001; Table 1). In contrast, a significant positive correlation was noted between d-ROMs and BAP in Groups G (r=0.841; p<0.001) and A-3 (r=0.707; p<0.01) (Table 1). No significant correlation was noted between d-ROMs and BAP in Groups W (r=0.467; p=0.126), A-1 (r=0.075; p=0.816), or A-2 (r=−0.405; p=0.192; Table 1).

Table 1. Correlation coefficient between d-ROMs and BAP by age group.

Group					All animals
Weanling	Growth	Adult	Middle-age	Old-age
0.467	0.841***	0.075	−0.405	0.707**	−0.603***

**p<0.01, ***p<0.001.

d-ROMs: derivatives of reactive oxygen metabolites; BAP: biological antioxidant potential.

The d-ROMs ranged from 152 to 510 CARR U with a coefficient of determination (R2) of 0.705. Compared with the Group W, which had the lowest d-ROM value, the d-ROM values of Groups G, A-1, A-2, and A-3 were higher by 48.6%, 93.9%, 87.4%, and 114.7%, respectively (all p<0.001; Table 2). Compared with Group G, the d-ROM values of Groups A-1, A-2, and A-3 were 30.5% (p<0.001), 26.1% (p<0.01), and 44.5% (p<0.001) higher, respectively (Table 2). Notably, Group A-3 was significantly higher by 14.6% (p<0.05; Table 2).

Table 2. Comparison of d-ROMs, BAP, and OSI by age group.

	Group
	Weanling	Growth	Adult	Middle-age	Old-age	p-value1)
d-ROMs	188.1 ± 16.1***a,b,c,d	279.4 ± 21.1***a,e,g; **f	364.7 ± 29.9***b,e	352.4 ± 26.5***c; **f; *j	403.8 ± 73.0***d,g; *j	0.001
(CARR U)
BAP	3,323.6 ± 210.0***a,b,c,d	2,870.0 ± 116.0***a,g; *f	2,766.2 ± 105.4***b; *i	2,574.4 ± 178.8***c; *f	2,470.3 ± 328.6***d,g; *i	0.001
(μmol/L)
OSI	5.7 ± 0.5***a,b,c,d	9.7 ± 0.5***a,e,f,g	13.2 ± 1.7***b,e,i	13.8 ± 1.7***c,f; **j	16.3 ± 2.1***d,g,i; **j	0.001
(d-ROMs/BAP×100)

Mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001,¹⁾: one-way analysis of variance.

a: weanling vs. growth, b: weanling vs. adult, c: weanling vs. Middle-age, d: weanling vs. old-age, e: growth vs. adult, f: growth vs. middle-age, g: growth vs. old-age, h: adult vs. middle-age, i: adult vs. old-age, j: middle-age vs. old-age.

d-ROMs: derivatives of reactive oxygen metabolites; BAP: biological antioxidant potential; OSI: oxidative stress index.

The BAP was in the range of 2,075–3,714 μmol/L and the coefficient of determination (R2) was 0.635. Compared with Group W, which had the highest BAP value, the BAP values of Groups G, A-1, A-2, and A-3 were significantly lower by 13.7%, 16.8%, 22.6%, and 25.7%, respectively (p<0.001; Table 2). Compared with Group G, the BAP values of Groups A-2 and A-3 were significantly lower by 10.3% (p<0.05) and 13.9% (p<0.001), respectively (Table 2). The BAP value of Group A-3 was 10.7% lower than that of Group A-1 (p<0.05; Table 2).

The OSI, which is the ratio of d-ROMs to BAP, was within the range of 5.0–20.3, and the coefficient of determination (R2) was 0.839. The OSI was significantly higher in Groups G (71.7%), A-1 (133.0%), A-2 (143.2%), and A-3 (188.3%) (all p<0.001), as compared to that of Group W, which had the lowest OSI (Table 2). Compared with Group G, the OSI of Groups A-1, A-2, and A-3 were higher by 35.7%, 41.7%, and 68.0% (p<0.001), respectively (Table 2). Group A-3 had 23.8% higher OSI than Group A-1 (p<0.001; Table 2).

DISCUSSION

In this study, we investigated the dynamics of resting oxidative stress and antioxidant capacity in rats of different age groups. From the weaning period to adulthood, oxidative stress as expressed by OSI (Table 2), increased and antioxidant capacity, as expressed by BAP (Table 2), decreased more markedly than at other ages. Thereafter, the rate of change between ages decreased. The maximum value of oxidative stress and the minimum value of antioxidant capacity were observed in old-age, and both showed characteristics with large deviations.

Increased oxidative stress in healthy organisms activates antioxidant capacity and regulates oxidative stress. Approximately 90% of ROS are derived from mitochondria⁵⁾. An increase in oxygen uptake due to vigorous activity⁶⁾ and a decrease in antioxidant function increases oxidative stress⁷⁾. In this study, the rate of change in oxidative stress from weaning to adulthood was thought to be due to the growth-associated increase in muscle mass and activity-related increase in oxygen uptake. Moreover, the strong positive correlation between oxidative stress and antioxidant capacity during the growth stage was thought to be due to the transient enhancement of antioxidant capacity against oxidative stress. In contrast, age-related changes in the skeletal muscles are associated with a decrease in maximal oxygen uptake⁸⁾. Thus, the positive correlation between high oxidative stress and antioxidant capacity in old age is unlikely to be due to an increase in muscle mass and activity observed during the growth stage. This was considered to be the result of functional deterioration. Therefore, changes in oxidative power exceed those in antioxidant power, and the aging-induced enhancement of the ROS production system and deterioration of the antioxidant system leads to changes in the OSI.

The longer the lifespan in the natural world, the higher the response to oxidative stress⁹⁾. Antioxidant activity also plays an important role in pathology¹⁰⁾ and is highly relevant to health promotion and the prevention of chronic diseases¹¹⁾. In other words, the dynamics of oxidative stress are important factors related to health and longevity, and aging affects the balance between oxidative stress and antioxidant capacity. Consequently, it is necessary to consider the characteristics of the growth period and individual differences in old age with respect to the dynamics of oxidative stress and antioxidant activities in diseases that affect health.

Funding and Conflicts of interest

None.