Increased 8-Hydroxy-2′-Deoxyguanosine in Leukocyte DNA in Leber’s Hereditary Optic Neuropathy

Abstract

purpose. This study was conducted to test the hypothesis that oxidative stress is involved in the pathogenesis of Leber’s hereditary optic neuropathy (LHON). The level of 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidized DNA base common in cells undergoing oxidative stress, was measured in leukocyte DNA from patients with LHON and normal control subjects.

methods. The 8-OHdG and deoxyguanosine (dG) content in leukocyte DNA from 25 patients with LHON with an 11778 mitochondrial (mt)DNA mutation, 14 asymptomatic maternal relatives, and 27 unrelated normal control subjects were measured using high-performance liquid chromatography and electrochemical detection methods.

results. The mean 8-OHdG/10⁵ dG ratio from leukocyte DNA was 1.34 ± 0.99 in patients with LHON, 1.00 ± 0.91 in their asymptomatic maternal relatives, and 0.31 ± 0.20 in normal control subjects, respectively. There was a statistically significant difference in the mean 8-OHdG/10⁵ dG ratio between patients with LHON and normal control subjects and between asymptomatic maternal relatives and normal control subjects. The difference between patients with LHON and asymptomatic maternal relatives did not reach statistical significance.

conclusions. Patients with LHON with an 11778 mtDNA mutation had higher oxidative DNA damage. Oxidative stress has a key role in the pathogenesis of LHON.

Leber’s hereditary optic neuropathy (LHON) is a maternally transmitted disease associated with acute visual loss that occurs mostly in young adult males. The first LHON-associated mitochondrial (mt)DNA 11778 mutation was reported by Wallace et al. ¹ in 1988. There are 25 point mutations in mtDNA reported to be associated with LHON. ² Based on genetic, clinical, and biochemical parameters, mutations at nucleotide positions 3460, 11778, and 14484 are regarded as primary mutations in that each alone can cause LHON and they are not present in normal control subjects. There remain several confusing aspects of the pathophysiology of LHON, however, that cannot be explained by mitochondrial inheritance alone, including male predominance, reduced penetrance, late age of onset, and expression limited to the optic nerve. 

The pathogenesis of LHON remains largely unknown. The bioenergetic defect has little consistent support for a common enzymatic defect in complex I activity in LHON with the three pathogenic mutations. ^{3 4} Heteroplasmy does not explain the clinical variations of patients. ^{5 6} Whereas the possibility of nuclear gene involvement has been strongly suggested, ^{7 8} linkage and X-inactivation analyses to demonstrate such a locus have been unsuccessful. ^{9 10}  

Deficiencies in respiratory chain function and reactive oxygen species (ROS) are believed to have pivotal roles in the pathogenesis of LHON. ^{11 12 13} Human cells depend on mitochondrial oxidative phosphorylation to generate energy. Through defective respiration, mitochondria produce a large number of ROS, which may cause oxidative damage to cellular constituents, including membrane lipids, proteins, and DNA. Among the many types of modifications induced by ROS, 8-hydroxy-2′-deoxyguanosine (8-OHdG) is one of the most abundant oxidative products of DNA. The 8-OHdG content in leukocyte DNA and in urine is a specific biomarker of DNA damage. ^{14 15} In the present study, we measured the 8-OHdG content in leukocyte DNA from patients with LHON to determine whether oxidative stress is involved in LHON. 

Materials and Methods

Subjects

Twenty-five patients (22 male and 3 female) with LHON, 14 asymptomatic maternal relatives (3 male and 11 female), and 27 unrelated normal control subjects (21 male and 6 female) were included in this study. The mean age of patients with LHON was 30 years (range, 12–67 years). The mean age of onset of patients with LHON was 20 years (range, 6–40 years). All patients with LHON had the 11778 mtDNA mutation. The mean age of asymptomatic maternal relatives was 37 years (range, 9–50 years). The mean age of normal control subjects was 32 years (range, 12–68 years). All control subjects tested negative for the 11778 mtDNA mutation, and none had systemic disease such as diabetes mellitus. 

Blood Sampling

The study was performed according to the tenets of the Declaration of Helsinki for research involving human subjects. The protocol was approved by the Institutional Review Board of Taipei Veterans General Hospital. Informed consent was obtained from all subjects. Whole blood was withdrawn from patients with LHON, asymptomatic maternal relatives, and normal control subjects and stored in EDTA-containing glass tubes. 

DNA Isolation

Total DNA was extracted from the blood cells and purified using a DNA purification kit (Puregene; Gentra System, Inc., Minneapolis, MN). 

Enzymatic Hydrolysis and Determination of 8-OHdG

The specific content of 8-OHdG in total DNA was measured by high-performance liquid chromatography. ¹⁶ All procedures were performed by the same person (S-HK) in the same laboratory. Briefly, a 100-μg aliquot of DNA dissolved in 100 μL of 10 mM Tris-HCl (pH 7.4) was digested by incubation with 1 μL DNase I (20 U/μL) and 11 μL 0.1 M MgCl₂ solution at 37°C for 30 minutes. After adjusting the pH to 5.0 by adding 4.8 μL of 1 M sodium acetate (pH 5.3) and 1.2 μL of 0.1 M ZnSO₄, the DNA sample was digested with 5 μL nuclease P1 (1 U/3 μL in 20 mM sodium acetate, pH 5.3) at 65°C for 10 minutes. The DNA was hydrolyzed to the corresponding nucleosides by incubation with 5 μL alkaline phosphatase (1 U/μL) for 30 minutes at 37°C. Processed DNA samples were separated with a C-18 column (particle size 5 μm, 200 × 4.6 mm; JT Baker, Inc., Phillipsburg, NJ) on a high-performance liquid chromatography system (Jasco, Tokyo, Japan) connected in series with an electrochemical detector (Bioanalytical Systems, West Lafayette, IN) and a UV detector (at 254 nm). Elution was performed at a flow rate of 0.8 mL/min for 40 minutes with a mobile phase consisting of 12.5 mM citric acid, 25 mM sodium acetate, and 1 mM acetic acid containing 6% methanol (pH 5.8). The amount of deoxyguanosine (dG) in the sample was calculated from the peak area of dG in the chromatogram recorded through a UV monitor. The amount of 8-OHdG in the sample was expressed as the ratio to the amount of total dG. 

Statistical Analysis

The mean 8-OHdG/10⁵ dG ratios in total DNA from the blood of patients with LHON, asymptomatic maternal relatives, and normal control subjects were compared by one-way ANOVA and the Kruskal-Wallis test. P < 0.05 was considered to indicate statistical significance. 

Results

Tables 1 2 and 3 show the demographic, clinical information and the 8-OHdG results of each patient in three groups. The scattergram of 8-OHdG results of each group was shown on Figure 1 . The mean 8-OHdG/10⁵ dG ratios of blood cells were 1.34 ± 0.99 in patients with LHON, 1.00 ± 0.91 in asymptomatic maternal relatives, and 0.31 ± 0.20 in normal control subjects (Fig. 2) . There was a statistically significant different 8-OHdG/10⁵ dG ratio between patients, asymptomatic maternal relatives and normal control subjects (one-way ANOVA, P < 0.001). The mean 8-OHdG/10⁵ dG ratios in patients with LHON and in asymptomatic maternal relatives were significantly elevated compared with normal control subjects (Kruskal-Wallis test, P = 0.000 and 0.004, respectively). The difference between patients with LHON and asymptomatic maternal relatives did not reach statistical significance. 

Discussion

Oxidative DNA damage is thought to contribute to aging ^{17 18} and to a host of degenerative diseases of aging, including cancer. ^{19 20} DNA damage can occur intrinsically as a consequence of normal metabolism. The rate of oxidative DNA damage is directly related to the metabolic rate and is inversely related to the lifespan of the organism. ²¹ Leukocyte DNA 8-OHdG levels are increased in cigarette smokers, ²² patients with diabetes, ²³ and patients on chronic hemodialysis. ²⁴ The 8-OHdG content in cybrids increases as the proportion of mtDNA with the 4977 deletion increases. ²⁵ The present study indicated that the leukocyte DNA 8-OHdG content was significantly increased in patients with LHON. This is powerful evidence supporting the presence of oxidative stress in the pathogenesis of LHON. 

Others have implicated oxidative stress in LHON. ^{13 26 27 28 29 30} In in vitro studies, cybrid cell lines bearing the pathogenic LHON 11778 mutation were much more sensitive than the parental cell line to oxidative stress, which causes cell death in a Ca²⁺-dependent manner. ²⁶ Biochemical studies of LHON suggest that the cytotoxicity induced by the loss of complex-I activity was not from reductions in oxidative phosphorylation, but was due to increased production of ROS. Chronic overproduction of ROS may be an important consequence of the pathogenic mtDNA mutations. ²⁷ ROS production is increased in cybrids carrying the three primary mutations associated with LHON and different mutations in mtDNA result in a modified pattern of the antioxidant machinery. ²⁸ In animal studies, optic neuropathy induced by reductions in mitochondrial superoxide dismutase, the essential antioxidant that catalyzes the dismutation of superoxide radicals, was strikingly similar to the histopathology of LHON. ²⁹ Patients with LHON and asymptomatic carriers have a reduced α-tocopherol/lipid ratio in their plasma, which most probably reflects increased free radical generation and α-tocopherol consumption. ³⁰ In the present study, mtDNA damage was increased in patients with the 11778 mtDNA mutation, which may reflect the increased ROS production. 

How does optic neuropathy in LHON occur? The pathogenic mtDNA mutations of complex-I genes result in defective respiration, inhibiting the electron transport chain, thereby generating ROS to levels beyond the capability of endogenous antioxidants present within the mitochondria. The selective vulnerability of the optic nerve in LHON, however, remains a mystery. Using a neuronal precursor cell line NT2 containing mitochondria bearing the 11778 and 3460 mutations, differentiation significantly reduced LHON cells (by 30%) compared with control subjects, indicating either a decreased proliferative potential or increased cell death of the LHON-NT2 cells. ³¹ There are increased mitochondrial ROS observed in differentiated LHON-NT2 cells. The findings suggest that the LHON phenotype might be the result of an increase in mitochondrial ROS, which is caused by LHON mutations, possibly mediated through neuron-specific alterations in the complex-I structure. 

In summary, the present study indicated that the 8-OHdG content in leukocyte DNA was significantly increased in patients with LHON. This provides further evidence linking oxidative stress to the pathogenesis of LHON. 

 Supported by Grant VGH 91-316 from Taipei Veterans General Hospital (M-YY).

 Submitted for publication June 6, 2003; revised February 4, 2004; accepted February 5, 2004.

 Disclosure: M.-Y. Yen, None; S.-H. Kao, None; A.-G. Wang, None; Y.-H. Wei, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Demographic, Clinical Information and 8-OHdG/10⁵dG Ratio of LHON Patients

Case	Sex	Age	Age of Onset	8-OHdG/105dG Ratio
1	M	12	11	1.300
2	M	17	17	0.781
3	M	17	15	2.767
4	M	18	12	0.329
5	M	19	19	2.191
6	M	19	6	0.889
7	M	19	17	0.765
8*	M	20	20	3.618
9	M	23	17	2.099
10	M	24	16	1.349
11	M	24	23	0.257
12	M	26	16	0.912
13	M	30	28	2.939
14	M	32	17	0.342
15	M	33	9	0.854
16	M	34	31	0.316
17	M	35	30	1.360
18	M	35	22	0.865
19	M	38	38	1.286
20	M	39	10	2.087
21	M	41	40	0.447
22	M	58	34	3.255
23	F	51	38	0.097
24	F	21	20	1.281
25	F	67	17	1.099

*  A case of recent onset of visual loss.

Table 2.

 

Demography and 8-OHdG/10⁵dG Ratio of Asymptomatic Maternal Relatives

Case	Sex	Age	8-OHdG/105dG Ratio
1	M	9	1.299
2	M	19	0.556
3	M	43	0.711
4	F	26	0.275
5	F	28	2.435
6	F	35	0.634
7	F	36	1.757
8	F	42	0.452
9	F	44	1.563
10	F	46	0.629
11*	F	48	3.076
12	F	48	0.390
13	F	50	0.074
14	F	50	0.159

*  Mother of case 8 in Table 1 . Her son’s visual loss may cause her stress, associated with a high level of 8-OHdG.

Table 3.

 

Demography and 8-OHdG/10⁵dG Ratio of Unrelated Normal Control Subjects

Case	Sex	Age	8-OHdG/105dG Ratio
1	M	12	0.090
2	M	21	0.419
3	M	22	0.094
4	M	22	0.319
5	M	22	0.436
6	M	24	0.090
7	M	24	0.091
8	M	25	0.435
9	M	25	0.430
10	M	26	0.157
11	M	26	0.235
12	M	26	0.216
13	M	27	0.399
14	M	31	0.625
15	M	32	0.435
16	M	37	0.116
17	M	40	0.294
18	M	45	0.090
19	M	55	0.474
20	M	67	0.762
21	M	68	0.529
22	F	20	0.116
23	F	23	0.080
24	F	28	0.268
25	F	32	0.467
26	F	44	0.656
27	F	50	0.094

Figure 1.

 

The 8-OHdG/10⁵ dG ratio in each group.

Figure 2.

 

The mean 8-OHdG/10⁵ dG ratio in each group.