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CYP1A1 gene polymorphism in inflammatory and non inflammatory

pterygium

Article · January 2017

DOI: 10.3923/jms.2017.26.30

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 Research Paper
J. Med. Sci., 17 (1): 26-30
JMS (ISSN 1682-4474) is an January-March, 2017
International, peer-reviewed DOI: 10.3923/jms.2017.26.30
scientific journal that publishes
original article in experimental CYP1A1 Gene Polymorphism in Inflammatory and
& clinical medicine and related non Inflammatory Pterygium
disciplines such as molecular
1
biology, biochemistry, genetics, Hendriati, 2Yanwirasti, 3Tjahjono Darminto Gondhowiardjo and 4Djong Hon Tjong
biophysics, bio-and medical
Background and Objective: Pathogenesis of pterygium is not fully understood.
technology. JMS is issued four
CYP1A1 gene found to have role in carcinogen metabolisms and also detected in
times per year on paper and pterygium tissue. The purpose of this study was to assess the CYP1A1 gene
in electronic format. polymorphisms in patients with inflammatory and non-inflammatory pterygium.
Methods: The PCR was conducted on blood samples of 80 patients with pterygium
For further information about which consisted of 40 inflammatory and 40 non-inflammatory pterygium. The
this article or if you need rs 4646903 SNP genotyping T>C (m1) in the CYP1A1 gene was conducted using
reprints, please contact: Restriction Fragment Length Polymorphisms-PCR (RFLP-PCR). The PCR products
were cut with restriction enzyme MspI. Results of restriction were then electrophorised
and then observed with GelDoc. Results: In inflammatory pterygium, 13 patients
Hendriati,
Department of Ophthalmology, obtained TT alleles (wild type), 19 TC allele (heterozygous mutants) and 8 CC alleles
Faculty of Medicine, (homozygous mutant). In non-inflammatory pterygium, 14 patients obtained TT allele
Andalas University, (wild type), 18 TC allele (heterozygous mutant) and 8 CC alleles (homozygous mutant).
Rawang Timur V No. 19, Padang, Conclusion: The CYP1A1 gene polymorphisms may occur in pterygium patients either
West Sumatra, Indonesia inflammatory or non-inflammatory. Polymorphisms occur in the form of heterozygous
and homozygous mutant.

Key words: CYP1A1 polymorphism, inflammatory, non-inflammatory pterygium

1
Department of Ophthalmology, Faculty of Medicine, Andalas University, Padang,
Indonesia

ANSI
2
Laboratory of Biomedical Sciences, Andalas University, Padang, Indonesia
3
Department of Ophthalmology, Faculty of Medicine, University of Indonesia, Jakarta,
Indonesia
4
Asian Network for Scientific Information Faculty of Biological Sciences, Andalas University, Padang, Indonesia

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 J. Med. Sci., 17 (1): 26-30, 2017

INTRODUCTION during the metabolic process, various unstable and reactive

metabolites of PAHs can be formed and these metabolites
Pterygium is epithelial hyperplasia characterized by attack to DNA, causing toxicity and cell transformation. The
fibrovascular tissue growth on the ocular surface, which grew polymorphism of CYP1A1 was previously found in pterygium
out of conjunctival bulbar and proliferates all over the cornea1. tissue. Thus, it is stated that carcinogenic process was also
The disease is closely associated with exposure to ultraviolet involved in the pathogenesis of pterygium10,12.
(UV)-B radiations, primarily affect the stem cells in the The CYP1A1 is a sub-family 1 of CYP gene superfamily,
limbus. Clinically, pterygium is associated with inflammation one of the important genes that encodes enzymes metabolizing
and neovascularization. Pterygium either primary or recurrent carcinogens and is involved in the metabolism of carcinogenic
can cause changes in normal conditions such as astigmatism PAHs. This gene has an important role in catalyzing the
and changes in ocular surface, which eventually can lead to oxidative reaction of benzo(a)pyrene, which is one of the
malfunctioning of vision due to worsen visual acuity2-4. most important class of PAHs, into carcinogenic reactive
The pathogenesis of pterygium is still not fully metabolites. The CYP1 enzymes are responsible for the
understood. According to the researchers there is a relationship activation and detoxification of various metabolites of PAHs12.
between pterygium with the involvement of environmental The aim of the present study was to determine CYP1A1 gene
factors, such as viral infections or oxidative stress, polymorphisms found in pterygium tissue and its correlation
anti-apoptotic mechanisms, mechanisms of immunology, with inflammatory and non inflammatory pterygium.
cytokines, growth factors, extracellular matrix modulators,
genetic factors and possibly other factors3-6. MATERIALS AND METHODS
Clinical symptoms of pterygium may be mild and often
with no complaints at all (asymptomatic). However, some Sample collection: Blood samples of 1 mL were obtained
patients may also have complaints such as red eye, from 80 patients with pterygium (40 inflammatory and
inflammation or irritation, burning sensation and foreign body 40 non-inflammatory) from Central Eye Care (Balai Kesehatan
sensation. Clinically, Donald Tan classified pterygium as Indra Masyarakat, BKIM) and private hospitals in Padang.
atrophic, intermediate and fleshy7. In practice, complaints and
Genomic DNA was isolated from blood samples of 300 mL
symptoms of pterygium are composed of inflammatory and
using Genomic DNA mini kit (Blood/cultured cell) GB100,
non-inflammatory.
Geneaid. The DNA isolation kit was carried out according to
Phototoxic ultraviolet radiation reacts directly or
procedures that outline sample preparation stage, the stage of
indirectly causing free radicals. According to previous studies
cell lysis, DNA binding phase, washing steps and DNA elution
free radicals are atoms or molecules that do not have a outer
stage.
electrons partner, highly reactive and can react with molecules
around them as discussed by Balci et al.8. Oxidative damage
Genotyping of rs4646903 (T>C): Genotyping of SNP
to cell membranes primarily experienced by unsaturated fatty
rs4646903 T>C (m1) in the CYP1A1 gene was done using
acids and the DNA will cause damage in the form of DNA
chain rupture, damage to deoxyribose and modified purine Restriction Fragment Length Polymorphism PCR
and pyrimidine. This damage will subsequently form (RFLP-PCR). The PCR reaction consisted of a total volume of
DNA-adducts8-10. 25 mL consisting of 0.2 μM deoxynucleoside triphosphates
Pterygium has long been regarded as a chronic (dNTPs), 1 μg of genomic DNA, 1.25 U HotStarTaq bufernya
degenerative condition, but after the discovery of abnormal DNA polymerase and its solution and 1.5 μM forward primer
expression of p53 protein in the epithelium of pterygium, the P79 (5'-AAGAGGTGTAGCCGCTGCACT-3') and 1.5 μM
pterygium is now regarded as an uncontrolled cell proliferation reverse primer P80 (5'-TAGGAGTCTTGTCTCATGCCT-3').
associated with ultraviolet, radiation like tumors. The P53 The PCR was performed with the following conditions:
mutant has a direct or indirect effect to the increase in gene initial denaturation at a temperature of 95EC for 5 min,
expression of growth by stimulating growth factors or growth followed by 35 cycles of a series of processes consisting of
receptor4,8. denaturation at 94EC for 1 min, annealing at 55EC for 30 sec
Environmental pollutants such as Polycyclic Aromatic and elongation at 72EC for 30 sec. The PCR process ended
Hydrocarbons (PAHs) are the result of incomplete combustion with a final elongation step at 72EC for 10 min and PCR
of organic materials derived from environmental carcinogens product size was 338 bp (Fig. 1).
and are metabolized by different xenobiotic-metabolizing The PCR products were cut with 1 U of restriction
enzymes, one of which is cytochrome P450 (P450 or CYP)11. enzyme MspI (the restriction = C^CGG) at 37EC for 16 h.
This enzyme is mainly instrumental in the change of PAHs Then the restriction results were analyzed by electrophoresis
into more polar metabolites and soluble in water, so the on 2% agarose gel dyed with GelRed DNA and then observed
metabolites formed can be excreted from the body. However, with GelDoc.

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 J. Med. Sci., 17 (1): 26-30, 2017

Fig. 1: Position of primer and the restriction enzyme on the gene CYP1A1 Mspl allele m1

RESULTS Table 1: Frequency distribution of clinical pterygium by age groups

Clinical manifestation
Groups of -------------------------------------------------
This study has been carried out an assessment of the age (years) Inflammatory Non-inflammatory Total
CYP1A1 gene polymorphisms in patients with pterygium. 30-39 4 2 6
Polymorphism is found in both groups of inflammatory and 40-49 9 9 18
non-inflammatory, either heterozygous or homozygous mutant, 50-59 13 12 25
in nearly equal numbers. The CYP1A1 gene is a sub-family 1 60-69 11 7 18
70-79 3 6 9
of CYP gene superfamily, that is involved in the metabolism 80-89 0 4 4
of carcinogenic PAHs.
Blood samples from 80 patients with pterygium were Table 2: Frequency distribution of clinical pterygium by occupation
examined, consisting of 40 inflammatory and 40 non- Clinical manifestation
inflammatory. Clinical pterygium when viewed based on ages -------------------------------------------------
can be seen in Table 1. Occupation Inflammatory Non-inflammatory Total
Outdoor 24 13 37
Most people with inflammatory pterygium had more Indoor 16 27 43
outdoor activities and otherwise people with non-inflammatory
pterygium were more active indoors (indoor), as shown in Table 3: Distribution of duration and complaints of pterygium
Table 2. People with pterygium have a variety of symptoms, Symptoms
including a red eye due to irritation and inflammation, Duration of ------------------------------
stinging, watering and foreign body sensation, but can also be pterygium (year) Yes No Total
found with no complaints at all. The correlation between <1 4 8 12
>1-2 11 16 27
duration of pterygium and patients complaints is shown in >2-3 9 5 14
Table 3. >3-4 0 0 0
After PCR the product was cut with restriction enzyme >4-5 0 0 0
MspI, the results were analyzed by electrophoresis restriction. >5 18 9 27
Individuals who have wild-type genotype rs4646903
(TT alleles) produced a 338 bp DNA size. Individuals who 8 non-inflammatory). Polymorphisms that occur in both
have a heterozygous genotype rs4646903 (TC alleles) groups of inflammatory and non-inflammatory were almost the
produced a 338 bp DNA size; 209 and 129 bp. Individuals same in mutant heterozygote, whereas mutant homozygote
who have a homozygous mutant genotype rs4646903 were equal in numbers (Table 4).
(CC allele) will produced a 209 bp DNA size and 129 bp, as
shown in Fig. 2. A total of 27 individuals (13 inflammatory DISCUSSION
and 14 non-inflammatory) have a wild type genotype
rs4646903 (TT alleles). Individuals who have a mutant Pterygium is a disease closely associated with exposure to
heterozygous genotype rs4646903 (TC alleles) of 37 ultraviolet (UV)-B radiation, primarily affect the stem cells in
(19 inflammatory and 18 non-inflammatory) and the corneoscleral limbus. Di Girolamo et al.13 found that
individuals who have mutant homozygous genotype UV-B radiation was to be relevant with pterygium
rs4646903 (CC alleles) were 16 (8 inflammatory and development. A stress-induced intracellular pathway and

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 J. Med. Sci., 17 (1): 26-30, 2017

Fig. 2: Result of 2% agarose gel electrophoresis

Wild type genotype produced 338 bp DNA size, while heterozygous genotype produced 338, 209 and 129 bp DNA size

Table 4: Distribution of CYP1A1 gene polymorphism frequency according to clinical pterygium

Polymorphism of gene CYP1A1
--------------------------------------------------------------------------------------------------------------
Clinical manifestation Wild type (TT) Mutant heterozygote (TC) Mutant homozygote (CC) Total
Inflammatory 13 19 8 40
Non-inflammatory 14 18 8 40
Total 27 37 16 80

potential cell-surface transmitters activated by UV radiation Young also analyzed the CYP1A1 gene polymorphisms
were identified in pterygium. Moreover, inflammatory and GSTM1 using RFLP-PCR and PCR on 205 pterygium
cytokines and angiogenic factors were also identified in specimens and 206 control to understand how the CYP1A1
pterygium samples. and GSTM1 polymorphisms increase the risk of pterygium. In
Clinically pterygium is associated with inflammation and the previous study, they found that CYP1A1 with genotype
neovascularization. Aspiotis et al.14 evaluated microvessel m1/m2 and m2/m2 was at risk of pterygium 1,553-fold greater
density (MVD) and expression of Vascular Endothelial than with genotype m1/m18. Young et al.17 formulated the
Growth Factor (VEGF) and thrombospondin-1 (TSP-1) in hypothesis that after exposure to environmental PAH, genetic
pterygium samples. The study found especially high polymorphisms of CYP1A1 contribute to the risk of the
expression levels for VEGF compared to normal conjunctiva pterygium formation thus CYP1A1 MspI polymorphisms can
and it was concluded that the angiogenesis-related factors were be used as a risk factor for pterygium18.
proved to be highly expressed in pterygium tissue. Meanwhile, Peng et al.19 found that CYP1A1 expression in pterygium
TSP expression level was low, allowing inducers of correlates with allelic variation and can be used as an
angiogenesis to act uninhibited which explained pathogenic independent risk marker. The study collected 150 pterygium
basis of pterygium formation14.
samples and 50 normal conjunctiva samples, which served as
The inflammation usually cause complaints including red
control. Forty eight (33.3%) pterygium specimens tested
eyes, pain, frequent watering and no foreign body sensation.
positive for CYP1A1 protein expression. The CYP1A1 protein
Non-inflammatory type of pterygium can present with no
expression was significantly greater in the pterygium group
complaints and all can be seen the white membrane that covers
than in the control group (p<0.0001). In addition, CYP1A1
the cornea. Age involved can be started from second decades
protein expression was associated with allelic variation.
until old age. The prevalence generally increased in older age.
Prevalence also tended to increase in outdoor workers, as they The CYP1A1 protein expression was significantly greater
gain more exposure to ultraviolet B radiations15,16. in the m2/m2 group than in the m1/m1 and m1/m2 groups
The association of CYP1A1 protein and genetic (p = 0.006)19.
polymorphism have been reported in several types of cancer, Tung et al.20 found that the BPDE-like DNA adduct
but less report was discussed about this in pterygium, formation in pterygium samples was associated with CYP1A1
especially in comparison between inflammatory and non polymorphisms. Benzo[a]pyrene 7,8-diol 9,10-epoxide
inflammatory pterygium. Therefore, in this study, the (BPDE), an ultimate metabolite of benzo[a]pyrene found to
association of CYP1A1 gene polymorphism was analyzed in have relationship with CYP1A1. In this study, the pterygium
inflammatory and non inflammatory pterygium. Young et al.17 was categorized into inflammatory and non inflammatory.
found the correlation between CYP1A1 polymorphism with The results showed that CYP1A1 gene polymorphism was
pterygium and stated that it might become a marker for the found in pterygium sample and it happened to be equal in both
prediction of pterygium susceptibility. inflammatory and non inflammatory pterygium.

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 J. Med. Sci., 17 (1): 26-30, 2017

CONCLUSION 9. De Almeida Junior, G.C., F.B. Frederico, K.P. Watanabe,

T.V. Garcia and A.Y. Iquejiri et al., 2008. Evaluation of
The CYP1A1 gene polymorphisms may occur in the both epithelial cell proliferating activity and fibroblast nuclear
types of pterygium, that is the inflammatory and non- kariometry in recurrent pterygium treated with mitomycin
inflammatory. Polymorphisms occur in form of mutant C. Arquivos Brasileiros de Oftalmologia, 71: 568-575.
heterozygote and homozygote. In both groups, polymorphism 10. Valko, M., D. Leibfritz, J. Moncol, M.T.D. Cronin,
of mutant heterozygote was found nearly equal in numbers M. Mazur and J. Telser, 2007. Free radicals and
while polymorphism of mutant homozygote was found just as antioxidants in normal physiological functions and human
much in both groups. disease. Int. J. Biochem. Cell Biol., 39: 44-84.
11. Shimada, T., 2006. Xenobiotic-metabolizing enzymes
SIGNIFICANT STATEMENT involved in activation and detoxification of carcinogenic
polycyclic aromatic hydrocarbons. Drug Metab.
The pathogenesis of pterygium has long been hypnotized Pharmacok., 21: 257-276.
by many studies. The CYP1A1 gene is one of the genes known 12. Ucakhan, O.O., A. Kanpolat, S. Elgun and I. Durak, 2009.
to have role in pathogenesis of pterygium. In this study, the The role of oxidative mechanisms in the etiopathogenesis
CYP1A1 gene polymorphisms was determined in of pterygium: A preliminary study. Ophthalmologica,
inflammatory and non inflammatory pterygium. To our 223: 41-46.
knowledge, this study is the first study to prove gene mutation 13. Di Girolamo, N., D. Wakefield and M.T. Coroneo, 2006.
in inflammatory and non inflammatory pterygium. UVB-mediated induction of cytokines and growth factors
in pterygium epithelial cells involves cell surface
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