OXIDATIVE DEOXYRIBONUCLEIC ACID (DNA) DAMAGE from reactive oxygen species (ROS) is apparent in inflammation, malignant tumors, the aging process, and autoimmune diseases. (1) Often, urinary 8-hydroxydeoxyguanosine (8-OHdG) is the indicator of such oxidative DNA damage. An important target for ROS within a cell is DNA, the result of which is a broad range of alterations--including base and sugar modifications, covalent crosslinks with proteins, and single- and double-strand breaks. (1) The hypothesis that ROS modification of DNA is involved in the development of autoantibodies in systemic lupus erythematosus (SLE) has been supported by enhanced reactivity of SLE anti-DNA antibodies to ROS-denatured DNA. (2-5) In SLE, anti-double--stranded DNA (anti-ds-DNA) antibody--an autoantibody specific for double-stranded DNA--is a marker for the presence of disease and is an indicator of disease activity. (6) Anti-ds-DNA antibodies exist at high concentrations during active periods of SLE. The effect of exposure to sunlight (i.e., high-intensity vs. low-intensity sunlight), a well-established environmental factor in the induction and exacerbation of SLE, (7) on urinary excretion of 8-OHdG has not been considered in previous reports of oxidative DNA damage in SLE patients.
In the current study we measured urinary 8-OHdG levels to determine the effects of exposure to low-intensity and high-intensity sunlight on oxidative DNA damage in individuals with SLE.
Materials and Method
Study design and subjects. We first measured urinary 8-OHdG in consenting SLE patients and volunteers without any past diseases and present illnesses during 2 time periods: (1) late May to early September, a period of high-intensity sunlight in Japan; or (2) late November to early March, a period of low-intensity sunlight. We compared results obtained during both time periods for patients and controls. All patients with SLE fulfilled the American Rheumatism Association's 1987 revised criteria for the diagnosis of SLE. (8)
Strong sunlight period group. A total of 22 patients with SLE and 11 volunteers agreed to participate in this study. The ages for the SLE patients ranged from 23 yr to 63 yr (mean [[bar]x] [+ or -] standard deviation [SD] = 31.1 [+ or -] 11.7 yr). The volunteers' ages ranged from 23 yr to 57 yr ([bar]x [+ or -] SD = 29.8 [+ or -] 10.1 yr). In this group, age did not differ significantly between the volunteers and the SLE patients. All patients with SLE and volunteers in this group were female.
Weak sunlight period group. We enrolled 19 patients with SLE; their ages ranged from 23.0 yr to 63.0 yr ([bar]x [+ or -] SD = 37.8 [+ or -] 12.6 yr). Twelve volunteers who were 23.0-58.0 yr of age ([bar]x [+ or -] SD = 29.4 [+ or -] 10.0 yr) were also enrolled. Again, no significant difference in age was noted between the 2 groups. All patients with SLE and volunteers in this group were female.
Measurement of urinary 8-OHdG levels and anti-ds-DNA antibodies. We measured urinary 8-OHdG and anti-ds-DNA antibodies in a group of 39 patients with SLE during both high-intensity and low-intensity sunlight periods to evaluate seasonal changes. Seasonal changes in 8-OHdG levels among SLE patients were compared with levels in healthy volunteers who agreed to being studied for more than 1 yr. Among the SLE patients, 3 were male and 36 were female; their ages ranged from 1 6.0 yr to 64.0 yr ([bar]x [+ or -] SD = 39.4 [+ or -] 13.2 yr). Healthy volunteers were 8 females between the ages of 23.0 yr and 57.0 yr ([bar]x [+ or -] SD = 31.1 [+ or -] 11.7 yr). No significant age differences were noted between the low-intensity and high-intensity sunlight groups.
Urine samples. Participants with SLE provided urine samples during follow-up at an outpatient clinic; urine samples were provided by healthy controls at the time they reported to our laboratory. Urine samples were centrifuged at 3,000 g for 5 min, during which time suspended cell debris was removed. Subsequently, the supernatant was stored at -80[degrees]C until time of analysis.
Determination of 8-OHdG. Prior to examination, we again centrifuged urine samples at 3,000 g for 5 min to remove any suspended cell debris. We assayed the supernatant with a kit that included competitive enzyme-linked immunosorbent assay (ELISA; 8-OHdG Check, Japan institute for the Control of Aging [Fukuroi, Japan]). The specificity of monoclonal antibody N45.1 used in the competitive ELISA kit has been established previously. (9) At the conclusion of the assay, absorbance was measured at 450 nm with a computer-controlled ELISA reader (MPR Model 550; Bio-Rad [Tokyo, Japan]). The 8-OHdG standards used for the assay ranged between 0.5 and 200.0 ng/ml. The concentration of 8-OHdG in the test samples was interpolated from a standard curve drawn with the assistance of logarithmic transformation. Urinary creatinine was determined by the alkaline picrate method. (10) Data herein are expressed as the urinary 8-OHdG (ng/ml)-to-creatinine (mg/dl) ratio (ng/mg). (11)
Determination of anti-ds-DNA antibodies. We evaluated the activity or inactivity of SLE among patients by measuring serum anti-ds-DNA antibodies. We used an ELISA kit (Mesacup DNA-II test-ds [Nagoya, Japan]) to determine anti-ds-DNA antibodies.
Patients with SLE vs. controls, by season. Urinary 8-OHdG levels in the SLE patients and healthy controls were compared for the high-intensity and low-intensity sunlight periods. Among the patients with SLE, levels of 8-OHdG (31.0 [+ or -] 20.6 ng/mg [[bar]x [+ or -] SD]) were significantly higher than levels in volunteers (15.4 [+ or -] 7.2 ng/mg [p < .05]) during the high-intensity sunlight period. Nonetheless, no significant difference was observed between the SLE patients (15.4 [+ or -] 5.5 ng/mg [[bar]x [+ or -] SD]) and the volunteers (16.3 [+ or -] 4.6 ng/mg) during the low-intensity sunlight period (Fig. 1).
[FIGURE 1 OMITTED]
Season, 8-OHdG levels, and anti-ds-DNA antibodies. Seasonal changes in urinary 8-OHdG and serum anti-ds-DNA antibodies were examined in the SLE patients. Urinary 8-OHdG levels and serum anti-ds--DNA antibodies were 21.3 [+ or -] 20.6 ng/mg (values are [bar]x [+ or -] SD, unless noted otherwise) and 50.5 [+ or -] 65.8 IU/I, respectively, during the high-intensity sunlight period. During the low-intensity sunlight period, the respective values were only 12.6 [+ or -] 6.7 ng/mg and 42.1 [+ or -] 51.6 IU/I. Urinary 8-OHdG levels and anti-ds--DNA antibodies were significantly higher during the high-intensity sunlight period than during the low-intensity sunlight period (p < .01 and p < .05, respectively). In healthy control subjects, however, urinary 8-OHdG was 16.2 [+ or -] 8.0 ng/mg and 15.7 [+ or -] 5.1 ng/mg for high-intensity and low-intensity sunlight periods, respectively, thus providing no evidence of a seasonal change (Fig. 2).
[FIGURE 2 OMITTED]
In only a few studies have investigators compared urinary 8-OHdG between SLE patients and healthy persons. In 1994, Lunec et al. (12) used high-performance liquid chromatography in an attempt to measure urinary 8-OHdG in patients with SLE. Lunec et al. (12) did not detect 8-OHdG in the SLE patients. Recently, Evans et al. (13) used ELISA to reexamine urinary 8-OHdG levels in SLE patients; no difference between SLE patients and controls was detected. (13) It should be noted that the effect of sunlight intensity on urinary 8-OHdG levels was not considered in the study by Evans et al. (13) In our study, in addition to the influence of sunlight, fragments of DNA in urine that contained 8-OHdG may have gone undetected had we used chromatography, but use of ELISA permitted detection. Additional reasons for the differences between our study and others may involve disease activity or steroid therapy. In our study, urinary 8-OHdG levels in patients with SLE were significantly higher than in healthy persons during the period of high-intensity sunlight, although no significant difference was found for the period of low-intensity sunlight. This difference indicated that urinary 8-OHdG varied with seasonal changes in sunlight intensity. Environmental factors, especially the effect of sunlight, may be important in the evaluation of oxidative DNA damage in patients with SLE. In addition, oxidative DNA damage in our study was greater during high-intensity sunlight, which was associated with disease activity. Both urinary 8-OHdG levels and anti-ds-DNA antibodies were significantly higher in patients with SLE during periods of high-intensity sunlight than during periods of low-intensity sunlight. These results suggest that oxidative DNA damage from sunlight plays an important role in development and exacerbation of SLE.
Requests for reprints should be sent to Maeshima Etsuko, M.D., Ph.D., Third Department of Internal Medicine, Wakayama Medical University, Wakayama City, Wakayama 640-0012, Japan.
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