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Comparative Study
. 2009 Dec;158(7):1743-52.
doi: 10.1111/j.1476-5381.2009.00436.x.

Effects of 7,8-dihydro-8-oxo-deoxyguanosine on antigen challenge in ovalbumin-sensitized mice may be mediated by suppression of Rac

J Y Ro  1 , D Y Kim, S-H Lee, J W Park, M-H Chung
Affiliations
Comparative Study

Effects of 7,8-dihydro-8-oxo-deoxyguanosine on antigen challenge in ovalbumin-sensitized mice may be mediated by suppression of Rac

J Y Ro et al. Br J Pharmacol. 2009 Dec.

Abstract

Background and purpose: Earlier we reported that 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), an oxidatively modified guanine nucleoside, exerted anti-inflammatory activity through inactivation of the GTP binding protein, Rac. In the present study, the effects of 8-oxo-dG were investigated on responses to antigen challenge in sensitized mice, as Rac is also involved at several steps of the immune process including antigen-induced release of mediators from mast cells.

Experimental approach: Mice were sensitized and challenged with ovalbumin without or with oral administration of 8-oxo-dG during the challenge. Effects of 8-oxo-dG were assessed by measuring lung function, cells and cytokines in broncho-alveolar lavage fluid (BALF) and serum levels of antigen-specific IgE. Rac activity in BALF cells was also measured.

Key results: 8-oxo-dG inhibited the increased airway resistance and decreased lung compliance of sensitized and challenged mice to the levels of non-sensitized control mice and lowered the increased leukocytes particularly, eosinophils, in BALF. Furthermore, 8-oxo-dG suppressed allergy-associated immune responses, such as raised anti- ovalbumin IgE antibody in serum, increased expression of CD40 and CD40 ligand in lung, increased interleukin-4, -5, -13, interferon-gamma and tumour necrosis factor-alpha in BALF and mRNA levels of these cytokines in BALF cells, dose-dependently. The corresponding purine, 8-oxo-guanine, showed no effects in the same experiments. Finally, 8-oxo-dG, but not 8-oxo-guanine, inhibited the increased Rac activity in sensitized and challenged mice.

Conclusion and implications: 8-Oxo-dG had anti-allergic actions that might be mediated by Rac inactivation. This compound merits further evaluation of its therapeutic potential in allergic asthma.

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Figures

Figure 1
Figure 1
Effects of 8-oxo-dG on AHR of mice sensitized to ovalbumin. Mice were sensitized and then challenged with ovalbumin and AHR was assessed by measuring changes of airway resistance (RL) (upper panel) and lung compliance (lower panel) using the Fexivent system. After each MCh nebulization, lung resistance (cmH2O·L−1·s−1) and compliance (mL·cmH2O−1) were measured and expressed as cmH2O L−1·s−1 and % of the saline-nebulized group respectively. Sham represents mice sensitized and challenged with PBS (group A); OVA represents mice sensitized and challenged with ovalbumin (group C); 8-oxo-dG 30 mg and 8-oxo-dG 60 mg·kg−1, represent mice sensitized and challenged with ovalbumin as in group C but treated orally with 8-oxo-dG at these doses 6 h before every ovalbumin challenge (groups D and E); 8-oxo-Gua 60 mg·kg−1 represents mice as in group C but treated orally with 60 mg·kg−1 8-oxo-Gua 6 h before every ovalbumin challenge (group G). Data are mean ± SE of n= 8. * and +; P < 0.05 and P < 0.01 versus ovalbumin, respectively.
Figure 2
Figure 2
Effects of 8-oxo-dG on recruitment of leukocytes into BAL fluid in mice sensitized to ovalbumin. Sensitization, 8-oxo-dG treatment and cell counts were as described in Methods. The letters below the bars refer to treatment groups. Group A is mice sensitized and challenged with PBS; B, mice sensitized with ovalbumin and challenged with PBS; C, mice sensitized and challenged with ovalbumin; D, E and F, mice as in group C but treated orally with 6, 30 or 60 mg·kg−1 8-oxo-dG, respectively; G, mice as in group C but treated orally with 60 mg·kg−1 8-oxo-Gua. Data expressed are mean ± SE (n= 8). ** and ***; P < 0.01 and P < 0.001, respectively versus group A or group B. +, ++ and +++; P < 0.05, P < 0.01 and P < 0.001, respectively versus group C.
Figure 3
Figure 3
Effects of 8-oxo-dG on serum ovalbumin-specific IgE levels in mice sensitized to ovalbumin. Experimental details are described in Methods. The letters below the bars refer to treatment groups. Group A is mice sensitized and challenged with PBS; B, mice sensitized with ovalbumin and challenged with PBS; C, mice sensitized and challenged with ovalbumin; D, E and F, mice as in group C but treated orally with 6, 30 or 60 mg·kg−1 8-oxo-dG, respectively; G, mice as in group C but treated orally with 60 mg·kg−1 8-oxo-Gua. Data are expressed as mean ± SE (n= 8). ***; P < 0.001 versus group A +, ++ and +++; P < 0.05, P < 0.01 and P < 0.001, respectively versus group C (OVA).
Figure 4
Figure 4
Effect of 8-oxo-dG on CD40 or CD40L expression and inflammatory reaction in the lung tissues of mice sensitized to ovalbumin. Lung tissues were removed from mice and immunohistochemistry for CD40 and CD40L and H & E staining were carried out as described in Materials and Methods. Upper panel, CD40; middle panel, CD40L and lower panel, H & E staining. The stained tissues were observed microscopically under 200× magnification; bars in group A indicate 100 µm. The intensity of IHC colour developed in peribronchial and perivascular areas and peribronchial infiltration of inflammatory cells in H &E stained tissues were scored into four grades, which were 0, normal; 1, slight; 2, mild; 3, moderate; and 4, severe. Summary data are presented as histograms to the right of the histological sections. The letters below the bars refer to treatment groups. Group A is mice sensitized and challenged with PBS; B, mice sensitized with ovalbumin and challenged with PBS; C, mice sensitized and challenged with ovalbumin; D, E and F, mice as in group C but treated orally with 6, 30 or 60 mg·kg−1 8-oxo-dG, respectively; G, mice as in group C but treated orally with 60 mg·kg−1 8-oxo-Gua. The data obtained from group G (8-oxo-Gua) is shown only in the histogram. Data in the histograms are mean ± SE of n= 8. ***, P < 0.001 versus group A or group B. ++ and +++; P < 0.01 and P < 0.001, respectively versus group C.
Figure 5
Figure 5
Effects of 8-oxo-dG on cytokine proteins in BAL fluid and lung tissues of mice sensitized to ovalbumin. Cytokine levels in BAL fluids (BALF) and lung tissues (Lung Tissue) were determined by ELISA as described in Methods. The letters below the bars refer to treatment groups. Group A is mice sensitized and challenged with PBS; B, mice sensitized with ovalbumin and challenged with PBS; C, mice sensitized and challenged with ovalbumin; D, E and F, mice as in group C but treated orally with 6, 30 or 60 mg·kg−1 8-oxo-dG, respectively; G, mice as in group C but treated orally with 60 mg·kg−1 8-oxo-Gua. Data are expressed as mean ± SE (n= 8). ***; P < 0.001 versus group A (sham) or group B (OVA/PBS). +, ++ and +++; P < 0.05, P < 0.01 and P < 0.001, respectively versus group C (OVA).
Figure 6
Figure 6
Effects of 8-oxo-dG on activation of Rac and Rac-linked kinases in BAL cells of mice sensitized to ovalbumin. Collection of cells from BALF and activity assays of Rac1 and JNK were performed as described in Methods. The letters above the record refer to treatment groups. Group A is mice sensitized and challenged with PBS; B, mice sensitized with ovalbumin and challenged with PBS; C, mice sensitized and challenged with ovalbumin; D, E and F, mice as in group C but treated orally with 6, 30 or 60 mg·kg−1 8-oxo-dG, respectively; G, mice as in group C but treated orally with 60 mg·kg−1 8-oxo-Gua. The experiments were repeated four times. In each experiment, eight mice were used, each of which was randomly chosen out of each group of A∼G (n= 8) and a representative result of the four experiments was presented. Numerical values below the record with * are the ratio of band density of each group versus that of group A (sham), normalized to the band density of total Rac1.
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