The Impact of Oxidative Stress in Primary Congenital Glaucoma

 

Salwa H. N. Al-Rubaeꞌi¹, Suzanne Jubair², Ali N. M. Al-Sharifi³

¹Department of Chemistry, College of Science, Mustansiriyah University, Baghdad, Iraq.

²Department of Pharmaceutical Chemistry, College of Pharmacy, University of Kerbala, Kerbala, Iraq.

³Ibn Al-Haitham Teaching Eye Hospital, Baghdad, Iraq.

 

ABSTRACT:

Primary congenital glaucoma (PCG) is an aggressive type of glaucoma occurs early in life. Cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) enzyme, which is considered as phase I xenobiotic metabolizing enzyme, controls the eye oxidative homeostasis and correspondingly the normal development of the eye. The concentrations of CYP1B1 enzyme, and total antioxidant capacity and malondialdehyde as parameters reflecting the oxidative stress were determined using enzyme-linked immunosorbent assay and spectrophotometric methods. The study sample was included 100 patients diagnosed with primary congenital glaucoma and other 100 healthy subjects served as controls. The age of all the patients and the controls ranged between one month and three years. CYP1B1enzyme concentration was higher in PCG patients than in controls (0.867±0.11 ng/ml versus 0.524±0.06 ng/ml, p=0.01), malondialdehyde was also higher in PCG patients than in controls (0.968±0.06 µmol/L versus 0.359±0.02 µmol/L, p=0.0001), while the levels of total antioxidant capacity were lower in PCG patients (2.790±0.48 U/ml for patients versus 7.031±0.65 U/ml for controls, p=0.0001). Our findings indicate that high oxidative stress is related to PCG disease. Oxidative stress is one of the factors involved in the pathogenesis of primary congenital glaucoma and high CYP1B1enzyme concentrations are associated with high oxidative stress.

 

 

 

INTRODUCTION:

Glaucoma is a mixed group of ocular disorder includes optic nerve degeneration¹. Almost 15% of blindness worldwide due to glaucoma^2,3. Primary congenital glaucoma (PCG) is an aggressive type of glaucoma occurs early in life (from birth up to 3 years in age)⁴. PCG is caused by developing anomalies in the trabecular meshwork (TM) and the anterior chamber angle of the eye^1,5. These anomalies cause impairment in the aqueous humor (AH) outflow leading to elevated intraocular pressure (IOP) which causes optic nerve damage and when left without treatment, permanent blindness⁴.

 

Cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) gene was reported to be PCG associated gene ⁶, CYP1B1 gene encodes for a monooxygenase, that considered as phase I metabolizing enzyme, able to metabolize xenobiotics⁷, and also endogenous compounds such as retinoids, estrogen and testosterone ^8,9. The development of TM, which is the most important tissue in the case of PCG, depends critically on the CYP1B1 enzyme, therefore, the improper function of CYP1B1 can disrupt the proper development of TM ^10,11.

 

Under H₂O₂ treatment (high oxidative stress), the TM cells are less viable indicating deficiency in antioxidant capacity to reduce reactive oxygen species (ROS)¹². CYP1B1 enzyme activity straightly determines the oxidative status of the TM cells, in other word, CYP1B1 deficiency directly leads to increased oxidative stress in glaucomatous eye^13,14. On the other hand it was reported that higher CYP1B1 enzymatic activity causes 4-OH-estradiol accumulation leading to generation of quinones, which encourage the formation of ROS^15,16. Increased oxidative stress may cause cell death in TM and retinal ganglion cells¹⁷.

 

The imbalance between oxidants and antioxidants in preference of the oxidants is defined as oxidative stress, it is a potential reason for tissue damage¹⁸. The AH has antioxidant defense systems to reduce the damaging effects of oxidants. They include non-enzymatic, ions or compounds with low molecular weight such as ferritin, alpha tocopherol, ascorbate in addition to several enzymes such as glutathione peroxidase,catalase and superoxide dismutase, all these defense systems can be determined as total antioxidant capacity, in contrast malondialdehyde is formed as a result for lipid peroxidation by ROS¹⁹, the imbalance between total antioxidant capacity and malondialdehyde can represent the oxidative status²⁰. The AH in human glaucoma patients has a significant reduction in total antioxidant capacity because of the increased oxidative stress in the glaucomatous eye²¹.

 

This study was designed to examine the role of oxidative stress on the pathogenesis of PCG and to explore the relationship between CYP1B1enzyme concentration and the oxidative stress represented by total antioxidant capacity (TAC) and malondialdehyde (MDA) in the serum of PCG patients.

 

MATERIAL AND METHODS:

Study Subjects:

This study followed the principles of Helsinki Declaration, it was approved by the Ethics Committee, Department of Chemistry, College of Science, Mustansiriyah University, Baghdad, Iraq and the Iraqi Ministry of Health approved this work as well. The blood samples were taken after informed consent of the participants prior to their inclusion in the study. The patient children were recruited from Ibn Al-Haitham Teaching Eye Hospital, Baghdad, Iraq, while the healthy children were volunteers. All the patients were diagnosed by Dr. Ali N.M.Al-Sharifi, C.A.B.Opth, the standers that were adopted to diagnose PCG are as follows: Age of onset runs between first days and three years, enlarged cornea so the parameter is bigger than 11 mm, elevated IOP (over 21 mmHg) and increased cup to disk ratio. Patients have other ocular anomalies, like anterior segment dysgenesis, aniridia, neurofibromatosis, Sturge–Weber syndrome and congenital hereditary endothelial dystrophy were excluded.

One hundred unrelated Iraqi PCG patient children (58 male and 42 female) were included in this study and 100 unrelated healthy children (60 male and 40 female) from the same ethnicity without any systemic or ocular disease were served as controls. All the patients and controls aged between one month and three years. Three ml of venous blood was taken from the enrolled children, put into gel tubes and left at room temperature (25 °C) for 15 minutes, then it was centrifuged at 2000 ×g for 10 minutes in order to collect sera. Sera aliquots were placed in eppendorf tubes and stored at-40 Cº until used.

 

Biochemical Analysis:

The concentration of CYP1B1 enzyme was measured in the sera for the all patients and controls using enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer’s instructions (Cat#MBS760698, Mybiosource/US). TAC was also measured using the protocol of ELISA kit (Cat#MBS9304157, Mybiosource/ US). The concentration of serum MDA was determined by a spectroscopic method established on the reactivity of thiobarbituric acid ²². MDA reacts with thiobarbituric acid and produces a colored complex which has maximum absorbance at 532 nm.

 

Statistical Analysis:

Statistical Analysis System- 2012 (SAS) program was used for the statistical analysis of the study parameters (CYP1B1 enzyme, TAC and MDA). T-Test was used to compare between means, p value of 0.01 was considered to be statistically significant. Odds ratios and the corresponding 95% confidence intervals were calculated to determine potential associations.

 

RESULTS:

The concentration of CYP1B1 enzyme was measured to follow its effect on the oxidative status as a phase I xenobiotic metabolizing enzyme (XME), TAC and MDA concentrations were also measured as sensitive biochemical parameters reflecting the oxidative status. Table 1 shows a significant increasing of CYP1B1 enzyme concentration in patients compared to controls (0.867±0.11 ng/ml for patients versus 0.524±0.06 ng/ml for controls with p=0.01), it also shows a significant differences of TAC and MDA in patients compared to the controls. TAC range between 2.790±0.48 U/ml for patients versus 7.031±0.65 U/ml for controls with p=0.0001, while MDA run between 0.968±0.06 µmol/L for patients versus 0.359 ± 0.02 µmol/L for controls with p=0.0001.

 

 

Table 1: Comparison between PCG patients and controls groups concerning CYP1B1 enzyme, TAC and MDA concentrations.

Parameter	Group	Mean±SE	95% C.I. for Mean		P-Value	O.R.
			L.b.	U.b.
CYP1B1 Enzyme (ng/ml)	Controls	0.568± 0.06	0.432	0.703	0.0100**	1.037
	Patients	0.867 ± 0.11	0.668	0.910
TAC (U/ml)	Controls	7.031±0.648	5.724	8.336	0.0001**	1.882
	Patients	2.790 ± 0.48	1.867	2.986
MDA (µmol/L)	Controls	0.359± 0.021	0.313	0.405	0.0001**	1.263
	Patients	0.968 ± 0.06	0.909	1.001

SE: Standard error, C.I.: Confidence interval, O.R.: Odds ratio, L.b.: Lower bound, U.b.: Upper bound. ** Significant at p≤ 0.01.

 

 

DISCUSSION:

CYP1B1enzyme is a monooxygenase that catalyzes several reactions involved in the metabolism of endogenous compounds include retinals, 17β-estradiol, melatonin and arachidonic acid¹⁸. CYP1B1 was reported to be included in the synthesis pathway of retinoic acid ^23,24. CYP family of enzymes includes 70–80% of the total phase I xenobiotics metabolizing enzymes (XMEs) ²⁵. Phase I XMEs are known for their ability of both metabolic activation and detoxification. The metabolic activation of xenobiotics regularly leads to create electrophilic intermediates. Phase II XMEs help the electrophilic intermediates to conjugate with compounds such as acetate, sulphate, mercapturic acid, glutamine, glycine, and glucoside, yielding very hydrophilic compounds that can be easily expelled to complete the cycle of detoxification²⁶. Even though the result of the united work of phase I and phase II XMEs is detoxification of destructive chemical compounds, in the bath way taken by xenobiotic compounds from the beginning to the expelled product, there is a chance for reactive oxygenated intermediates to be formed through the CYP-mediated reactions, therefor increasing the oxidative stress of the tissues and body fluids^{27, 28}.

 

The statistical comparison for CYP1B1 enzyme, TAC and MDA between patients and controls groups showed a significant increasing for CYP1B1 enzyme in patients compared to controls, MDA is significantly increased in patients compared to controls and correspondingly TAC is significantly decreased in patients compared to controls (Table 1). It is very obvious that the decreased TAC is associated with increased MDA, in the same time the results indicate the relationship between increased CYP1B1 enzyme and the increased oxidative stress in the body fluids. An earlier report demonstrated that a higher CYP1B1enzymatic activity causes 4-OH-estradiol accumulation, leading to quinones formation and quinones can produce oxidative stress by encouraging ROS generation, that may cause cell death in retinal ganglion cells and TM, therefore could be involved in primary open angle glaucoma pathogenesis ¹⁸. We think that our results may be parallel to the demonstration in the mentioned report.

 

On the other hand, the higher enzymatic concentration does not necessarily mean higher enzymatic activity.

 

The increased concentration of the enzyme could has a genetic cause or it could be a result for environmental factors. Increased oxidative stress caused by generation of ROS from endogenous and exogenous sources could be an inducer for the elevation in the enzyme concentration as demonstrated by Verma and co-workers²⁹. We think that the effect between CYP1B1enzyme and the oxidative stress is interchanged, in the time that CYP1B1enzyme is expected to reduce the high oxidative stress in the tissues and body fluids especially those of eyes by metabolizing the xenobiotic compounds, its concentration is being influenced and increased to overcome the high oxidative stress. Increased oxidative stress could make unfavorable environment for CYP1B1 enzyme to perfume its optimum function in PCG patients, and it also could be an inducer for increasing CYP1B1 enzyme concentration.

 

Oxidative stress was reported as an influencing factor in many pathological conditions in case of adults and children, MDA is a result for lipid peroxidation and it is considered as an indicator of oxidative stress both in adult and children. When antioxidant defense systems are unsatisfactory, lipid peroxidation occurs and MDA increases leading to oxidative damage³⁰. There is a substantial diversity of free radicals that are produced as by-products from molecular oxygen reaction with endogenous and exogenous compounds in all organisms and that are able to exert extensive damage. Free radicals have the ability to destroy any molecule they confront.

 

The retina is like the TM, is a PCG related tissue. They both highly exposed to the light and composed of polyunsaturated fatty acids that make them highly susceptible to oxidative stress³¹. They both have their own enzymatic defense system of antioxidants, however they are still very susceptible to lipid peroxidation that is observed in many pathogenic conditions³².

 

 

TAC is a measurement for the total antioxidant power in the tissues and body fluids ³³. The significant decreasing of TAC in PCG patients is an expected result for the increased oxidative stress and it indicates its important role in the incidence and progression of PCG. It must be expected that a decreased TAC in tissues and body fluids could be the consequence of high oxidative reactions³⁴. Increased oxidative stress early in development may cause trabecular dysgenesis and result in PCG ³⁵.

 

In conclusion, this study revealed that high oxidative stress is involved PCG disease. High CYP1B1 concentrations are associated with high MDA concentrations and low levels of TAC. PCG patients have a significantly high CYP1B1enzyme concentration accompanied with a significantly high oxidative stress compared to healthy controls.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 14.09.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(11): 5013-5016.