Detection of 8-Hydroxydeoxyguanosine (8-OHdG)

Enzyme in Recurrent Pterygium Raising a Question on its Role on

Recurrence

¹Omar M S Ismaeel, ² Hasnan Jaafar, ¹ Mohtar Ibrahim¹Department of Ophthalmology, School of Medical Sciences, University

Science Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

²Department of Pathology, School of Medical Sciences, Universiti Science Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

Introduction

• Pterygium is a common external ocular disease affecting people around the world, it is most common in tropical regions of the world due to exposure to sun light as it is considered a major factor in the development of

pterygium • The exact cause of pterygium pathogenesis is not fully established yet,

beside this the cause of recurrence is not identified.

• One of the proposed causes is the 8-OHdGenzyme associated mechanism.

• The sun light contains UVB component which has a photo oxidative damaging effects on ocular surface either by direct or indirect effect by damaging the DNA of the epithelial cells which is mediated by reactive oxygen species (ROS), that leads to the production of

8-Hydroxydeoxyguanosine ( 8-OHdG) enzyme.

• This enzyme is sensitive and stable biomarker for evaluating the degree of DNA oxidative damage.

• 8-OHdG enzyme frequently mispairs with adenine during DNA replication, ultimately giving rise to the G-C to T-A transversion mutations.

• This mutation may result in increase cellular proliferation and angiogenesis that lead to pterygium development attributed to high mutagenic effect of 8-OHdG enzyme.

• The role of 8-OHdG enzyme in the formation of primary pterygium is established.

• On the other hand no study supported the association of 8-OHdG enzyme with recurrent pterygium.

• In this study we aimed to find out if there is any positive association between 8-OHdG enzyme and recurrent pterygium.

Patients and Methods

• Inclusion criteria

– Primary pterygia that need surgical excision

– Recurrent pterygia within one year of surgical excision of the primary one

– Nasal pterygia only were included in the study. Temporal pterygia may have different pathology

• Exclusion criteria

– Secondary pterygia.

– Previous ocular surface surgery.

– Conjunctival inflammation due to other causes.

Methods

• Cross sectional study was performed to determine the expression of 8-OHdG enzyme in primary and recurrent pterygia and its relation to pterygia development.

• Samples of primary, recurrent pterygia and normal conjunctival (control) tissues were harvested from the patients who were eligible for surgery.

• The samples were collected from our indicated and consented patients whom had been selected from eye clinic and were planned for surgical excision of pterygium and conjunctival graft.

• Immunohistochemistry test was used to detect 8-OHdG enzyme by using Goat Anti 8-OHdG primary antibody.

• All the sections were pretreated with proteinase K (pretreatment reagent) for 40 minutes. Positive results were indicated by brownish staining of the epithelial cells under light microscope. (Figure. 1)

• Negative control slides were used to control non specific binding of 8-

OHdG.

Fig. 1 Positive staining for 8-OHdG enzyme, cells cytoplasm stained with brown color

Results

• Ninety two patients participated in this study and divided into three groups:

– Fifty five patients with primary pterygium participated in the first group. The average age of participants in this group was (55.3) year. Twenty seven participants were male (49%) and twenty eight participants were female (51%).

– Twelve patients with recurrent pterygium participated in the second group. The average age of participants in this group was (53.5) year. Nine participants were male (75%) and three participants were female (25%) of this group

Results

– Twenty five patients with normal conjunctiva participated in the third group. The average age of participants in this group was (64.3) year. Twelve participants were male (48%), and thirteen participants were female (52%)..

• Positive results in primary pterygia group were higher than positive results in both normal (control) and recurrent pterygium groups Table 1.

Table 1 The distribution of 8- OHdG enzyme in the three Groups of the study (n=92)

Groups Primary

N

(%)

Recurrent

N

(%)

Control

N

(%)

Total

N

(%)

Positive enzyme

35

63.6

2

16.7

4

16

41

44.6

Negative enzyme

20

36.4

10

83.3

21

84

51

55.4

Total 55

100

12

100

25

100

92

100

Results

Statistical analysis was done by Chi square test and showed:

• There is significant association of 8-OHdG enzyme with primary pterygium (P<0.001) as compared to control group.

• No significant association of 8-OHdG enzyme with recurrent pterygium (P= 0.959).

• To compare the association of 8-OHdGenzyme with primary pterygium and recurrent pterygium results show significant association with primary pterygium (P= 0.003).

Discussion

• Exposure to sun light is high in the tropical regions and the resultant UVB irradiation may play a role in the pathogenesis of the primary pterygium by its oxidative damaging effect on the cellular DNA.

• Kau et al, 2004 found abundant immunoreactivity of 8-OHdG enzyme in primary pterygium tissues (100 % of samples were positive).

• Tsai et al, 2005 found in his study 23.1 % of the specimens (primary pterygium) were positive for 8-OHdG enzyme.

Discussion

• Perra et al, 2006 detected the 8-OHdG enzyme in 67.74 % of the primary pterygium samples.

• These findings accord with our findings in the primary pterygium. The results of these studies support the hypothesis that oxidative damage of cellular DNA which might be brought by UVB exposure plays a role in the pathogenesis of primary pterygium indicated by detection of 8-OHdG

enzyme.

• Kau et al, 2004 detected the 8-OHdG enzyme in 100 % of normal conjunctival samples. The average level of 8-OHdG enzyme in primary pterygium was 4.7 fold higher than that in normal conjunctiva. All the normal conjunctiva samples were harvested from areas adjacent to the primary pterygium.

• In our study we found that 16 % of normal conjunctiva tissues were positive to 8-OHdG enzyme, three positive conjunctival samples were obtained from normal conjunctiva adjacent to primary pterygium while the

forth sample obtained from inferior conjunctiva.

Discussion

• Presence of 8-OHdG enzyme in normal conjunctiva indicates oxidative stress on epithelial cells of the normal conjunctiva which is not fully expressed to produce its mutagenic effect. This may be due to repair mechanism of the mutant gene conducted by hOGG 1 enzyme. The presence of 8-OHdG enzyme may precede the development by certain period of time.

• No previous study was done to detect the 8-OHdG enzyme in recurrent pterygium. In our study we got two positive samples out of twelve recurrent pterygium samples (16.7 %).

Discussion

• A “nonsignificant” result does not mean that there is no association in the population (Hulley et al, 2007).

• 8-OHdG enzyme may still play a role in the pathogenesis of recurrent pterygium.

• Recurrence may be due to other mechanisms like cytokines (interleukin-6 & 8) which induce angiogenesis and cellular proliferation and their expression increased after exposure to UVB light.

• Cyclooxygenase-2 may play a role in the pathogenesis of recurrent pterygium as high level was detected in the fibroblast of recurrent pterygium.

Conclusion

• There is significant association between the presence of 8-OHdG enzyme and primary pterygium.

• In spite of (no significant) association between the presence of 8-OHdG enzyme and recurrent pterygium, it is the first time that

8- OHdG enzyme has been detected in recurrent pterygium, this may reveal a role of 8-OHdG enzyme in recurrence of pterygium.

• Di Girolamo, N., Wakefield, D. & Coroneo, M. T. (2006). UVB-mediated induction of cytokines and growth factors in pterygium epithelial cells involves cell surface receptors and intracellular signaling. Invest Ophthalmol Vis Sci, 47(6), 2430-7.

• Karahan, N., Baspinar, S., Ciris, M., Baydar, C. L. & Kapucuoglu, N. (2008). Cycooxygenase-2 expression in primary and recurrent pterygium. Indian J ophthalmol, 56(4), 279-83.

• Kau, H. C., Tsai, C. C., Lee, C. F., Kao, S. C., Hsu, W. M., Liu, J. H. & Wei, Y. H. (2006). Increased oxidative DNA damage, 8-hydroxydeoxy- guanosine, in human pterygium. Eye, 20(7), 826-31.

• Perra, M. T., Maxia, C., Corbu, A., Minerba, L. Demurtas, P., Colombari, R., Martas, D., Bravo, S., Piras, F. & Sirigu, P. (2006). Oxidative stress in pterygia. Relation ship between P 53 and 8-OHdG. Mol Vis, 12, 1136-42.

• Tsai, Y. Y., Cheng, Y. W., Lee, H., Tsai, F. J., Tseng, S. H., Lin, C. L. & Chang, K. C. (2005). Oxidative DNA damage in pterygium. Mol Vis, 1171-5.