Evaluation of oxidative DNA damage in blood lymphocytes during the transition period in dairy cows

Abstract

The present study was undertaken to evaluate 8-hydroxy-2′-deoxyguanosine (8-OHdG) level in peripheral lymphocytes during the transition period in dairy cows. Twenty cows that belong to the Iwate Livestock Breeding Center (Morioka city, Iwate prefecture, Japan) were subjected to study. Whole blood samples were collected from the same animals at two weeks perpartum and two weeks postpartum. Oxidative DNA damage marker (8-OHdG) was measured in peripheral lymphocytes using commercial ELISA kits. The results revealed that the 8-OHdG level was significantly higher (P < 0.01) during the prepartum period than its level during early lactation. It could be concluded that oxidative DNA damage in peripheral lymphocytes increased during the prepartum period in dairy cows.

Keywords:

• 8-OHdG
• cows
• DNA damage
• lactation
• transition period

1. Introduction

Free radicals are highly reactive substances produced continuously during metabolic processes. It is well known that reactive oxygen metabolites are produced continuously by normal metabolic processes, but the rate of production may be increased markedly under diverse conditions of increased metabolic demand. In the last few years, the detection of free radical damage and protection against it has become increasingly important in clinical medicine as a complementary tool in the evaluation of the metabolic status (Halliwell 1991; Stohs 1995; Castillo et al. 2003).

Under physiological conditions, the body usually has sufficient antioxidant reserves to cope with the production of free radicals (Miller et al. 1993; Castillo et al. 2001). However, when free radical generation exceeds the body’s antioxidant production capacity, oxidative stress develops. Oxidative stress has been suggested to contribute to dairy cattle under physiological conditions, including pregnancy, lactation, estrus cycle, transition period and also associating cattle diseases, including mastitis, retained placenta and udder edoema (Miller et al. 1993; Goff & Horst 1997; Abd Ellah 2010, 2013).

The role of oxidative stress in health and disease was studied extensively in both human and animal medicine (Valko et al. 2007; Abd Ellah et al. 2009). Free radical excess may result in impairment of DNA, enzymes and membranes, and induces changes in the activity of the immune system and in the structure of basic biopolymers which, in turn, may be related to mutagenesis and ageing processes (Poli 1993; Halliwell 1994; Schreck & Baeuerle 1994). Oxidative damage of DNA results in the oxidation of nucleosides. 8-hydroxy-2′-deoxyguanosine is an oxidized nucleoside that is excreted in the body fluids with DNA repair. Several studies have demonstrated that 8-OHdG in body fluids can act as a biomarker of oxidative stress (Chiou et al. 2003; Liu et al. 2004; Wu et al. 2004).

According to our knowledge, the present study constitutes the first that investigated the level of 8-OHdG in peripheral lymphocytes of dairy cows, as a biomarker for the oxidative DNA damage during the transition period of dairy cows. Although many studies were established the increased oxidative stress during lactation and pregnancy, none of these studies were concerned with the studying of oxidative DNA damage in blood lymphocytes of dairy cows. Consequently, the present study was aimed to evaluate the 8-OHdG level in peripheral lymphocytes during the transition period in dairy cows.

2. Materials and methods

2.1. Animals

This study was carried out using 20 healthy pregnant Holstein cows maintained at the National Livestock Breeding Center (Iwate Branch, Morioka, Iwate Prefecture, Japan). The Animals were examined during the transition period, which extended from two weeks before parturition to two weeks postpartum. During the study period all animals were kept under identical conditions.

2.2. Sample collection and analytical procedures

Whole blood samples were obtained from the jugular vein in evacuated (Vacutainer) tubes containing heparin. Samples were collected from the same animals at two weeks prepartum and two weeks postpartum. Tubes were kept in ice tank and transported directly after collection to the research laboratory at the Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Japan.

2.3. Separation of lymphocytes

Peripheral blood lymphocytes were separated using lymphocyte separation medium (LSM, MP Biomedical, Tokyo, Japan). Briefly, 6 ml of LSM was transferred to a 15-ml centrifuge tube, then 6 ml of whole blood was layered over the LSM. The tube was centrifuged at 400 × g at room temperature for one hour. The top layer of clear plasma was aspirated to 2–3 mm above the lymphocyte layer. The lymphocyte layer was aspirated plus about half of the LSM layer below it and transferred to a centrifuge tube. An equal volume of normal saline solution was added to the lymphocyte layer in the centrifuge tube and centrifuged at 250 × g for 10 minutes at room temperature. The cells were washed again with normal saline solution. The sediment containing the cells was processed directly for DNA extraction using a DNA extractor TIS kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

2.4. Extraction and digestion of DNA

Extraction of DNA was done using a DNA extractor TIS kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The extracted DNA was digested by using 8-OHdG assay preparation reagent set (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The prepared DNA is transferred to a filter tube of centrifugal ultrafiltration device (10,000 M W, Microcon, Millipore Corporation, Tokyo, Japan). The filter cup was centrifuged at 15,000 × g at 4° C for 20 minutes. The filtrate was collected and then proceeded to 8-OHdg assay.

2.5. Measurement of 8-OHdG in peripheral lymphocytes’ DNA

8-OHdG was quantitatively measured in DNA filtrate using enzyme-linked immunosorbent assay kit (Highly sensitive 8-OHdG Check ELISA kit, JaiCa, Nikken SEIL Co., Shizuoka, Japan). The absorbance of the standard and samples was measured at 450 nm using Microplate reader (MPR-A4i Tosoh, Tokyo, Japan); a standard curve was generated and used to determine the concentration of 8-OHdG (ng/ml) present in samples. Assay range was from 0.125 to 10 ng/ml.

2.6. Statistical analysis

Statistical significance was carried out using the Statistical Package for the Social Sciences for Windows (SPSS, version 10.0, Chicago, IL, USA). Groups were tested for difference using paired samples T test. Statistically significant differences were determined at P ≤ 0.05.

3. Results and discussion

Results presented in Table 1 revealed that 8-OHdG level in peripheral lymphocytes showed a significant increase (P < 0.05) during the prepartum (0.41 ± 0.06), when compared with its level during the postpartum (0.22 ± 0.02), which agreed with the result from a previous study by Abd Ellah et al. (2014), who reported increased 8-OHdG in serum of dairy cows during the dry period. Dairy cows undergo massive metabolic changes during lactation and pregnancy, which may have a negative impact on the health and productive performance of the dairy cows (Sordillo et al. 2009). It is well known that reactive oxygen metabolites are produced continuously by normal metabolic processes, but the rate of production may be increased markedly under diverse conditions of increased metabolic demands. Metabolic adaptations to lactation are initiated in late pregnancy, especially during the dry period (Bell 1995), which is associated with a variable tissue consumption of O₂, alteration of oxidative status and hence variable production of lipoperoxides (Miller et al. 1993; Formigoni et al. 1997; Ronchi et al. 2000; Bernabucci et al. 2002). The results of the present study declared increased oxidative DNA damage in the peripheral lymphocytes during the prepartum period compared with the early postpartum, which may be attributed to the increased oxidative stress due to the high metabolic demands. It could be concluded that 8-OHdG level in peripheral lymphocytes increased during the prepartum period compared with the early postpartum in dairy cows.

Table 1. Lymphocytes’ 8-OHdG during the transition period in dairy cows.

	Dairy cows (n = 20)
	Prepartum	Postpartum
8-OHdG (ng/ml)	0.41 ± 0.06	0.22 ± 0.02*

Note: Data were expressed as mean ± SE.

8 -OHdG, 8-hydroxy-2′-deoxyguanosine.

*P < 0.01.

Acknowledgements

The authors would like to thank all members at Department of Animal Medicine, Faculty of Agriculture, Iwate University, Japan for providing the financial support and the required facilities for research. Many thanks for Assiut University, Egypt, for supporting the collaborative research between Assiut University, Egypt and Iwate University, Japan. Many thanks for members at the National Livestock Breeding Center, Iwate branch for facilitating the collection of samples and for their help.

Disclosure statement

No potential conflict of interest was reported by the authors.