Is the oxidative DNA damage level of human lymphocyte correlated with the antioxidant capacity of serum or the base excision repair activity of lymphocyte?

Abstract

A random screening of human blood samples from 24 individuals of nonsmoker was conducted to examine the correlation between the oxidative DNA damage level of lymphocytes and the antioxidant capacity of serum or the base excision repair (BER) activity of lymphocytes. The oxidative DNA damage level was measured with comet assay containing Fpg/Endo III cleavage, and the BER activity was estimated with a modified comet assay including nuclear extract of lymphocytes for enzymatic cleavage. Antioxidant capacity was determined with trolox equivalent antioxidant capacity assay. We found that though the endogenous DNA oxidation levels varied among the individuals, each individual level appeared to be steady for at least 1 month. Our results indicate that the oxidative DNA damage level is insignificantly or weakly correlated with antioxidant capacity or BER activity, respectively. However, lymphocytes from carriers of Helicobacter pylori (HP) or Hepatitis B virus (HBV) tend to give higher levels of oxidative DNA damage (P < 0.05). Though sera of this group of individuals show no particular tendency with reduced antioxidant capacity, the respective BER activities of lymphocytes are lower in average (P < 0.05). Thus, reduction of repair activity may be associated with the genotoxic effect of HP or HBV infection.

Figure 1

Oxidative DNA damage level of lymphocyte is steady. Lymphocyte samples of four volunteers collected at the indicated period of time were analyzed for the oxidative DNA damage with comet-Fpg/Endo III assay. 1–4: individual labels of the four volunteer; A: human AGS cells treated with 5 mM amoxicillin for 1 h, a positive control for enzyme (Fpg/Endo III) activity.

Figure 2

Variation of oxidative DNA damage levels of lymphocytes from 24 blood samples. Oxidative DNA damage levels of the lymphocytes isolated from 24 blood samples obtained from individuals who participated in health checkup. The oxidative DNA damage levels, represented as % DNA in tail, were aligned in the order from low to high, and each sample was coded (1–24) based on the respective DNA damage level. Carriers and noncarriers of HP or HBV are indicated with + or −, respectively.

Figure 3

Variation of antioxidant capacities of sera from 24 blood samples. (a) Antioxidant capacities of sera isolated from 24 blood samples as described in Figure 2. Top part: trolox standard curve. Bottom part: The sample code was given according to those described in Figure 2, and the carriers and noncarriers of HP or HBV are indicated with + or −, respectively. (b) Correlation between the oxidative DNA damage levels described in Figure 2 and the antioxidant capacities of Figure 3(a). The r value (= −0.085), the correlation coefficient, was obtained from statistical analysis.

Figure 4

Variation of excision activities in nuclear extracts of lymphocytes from 24 blood samples. (a) To test the lymphocyte's nuclear etract's (NE's) activity, the AGS cells were served as substrate. AGS cells were treated with 5 mM amoxicillin to induce oxidative DNA damage and examined by comet-NE assay with lymphocyte's NE. The sample code was given according to those described in Figure 2, and the carriers and noncarriers of HP or HBV are indicated with + or −, respectively. (b) Correlation between the oxidative DNA damage levels of Figure 2 and the excision activities of Figure 4(a) (note: samples coded 2, 13 and 18 were excluded from the correlation because the concern of artifact due to sample preparation; their respective NER activities shown in Figure 5(a) are also low). The r value (= −0.30), the correlation coefficient, was obtained from statistical analysis.

Figure 5

Control experiments for excision activities measured by the experiment described in Figure 4(a). (a) Nucleotide excision activities in some nuclear extracts of lymphocytes from the 24 blood sample. Nuclear extracts which failed to excise amoxicillin-induced DNA lesions were selected and used for examining their activities to excise UV-induced lesions. To determine the nucleotide excision activity, AGS cells treated with 5 J/m² UV were used as substrates in comet-NE assay. (b) Complementation test. Nuclear extracts samples from Helicobacter pylori carrier (HP) and HP-HBV (HP-HB) double carrier were mixed with the NE from p53 deficient cell line H1299 and used as in comet-NE assay. The NE from H1299 cell and healthy (H) people were severed as negative and positive control.