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Ocular biomarkers in diseases and toxicities
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19 Ocular biomarkers in diseases and toxicities George A. Kontadakis, Argyro Plaka, Domniki Fragou, George D. Kymionis, and Aristides M. Tsatsakis
INTRODUCTION
where the light meets the retina. The crystalline lens is responsible for 30% of the refractive power of the eye in relaxed status and adds the needed power for accommodation in near vision in pre-presbyopic individuals. Behind the crystalline lens, the eye is filled by the vitreous body. In contact with the vitreous body is the retina (Figure 19.1). The retina contains the photoreceptors that transform light to neural signal, which, after a complex path of intraretinal transformations, travels to the visual cortex through the optic nerve and the rest of the optic pathway. This chapter describes the biomarkers of ocular diseases and toxicities.
The human eye serves as the receptor of light, giving rise to the sense of seeing after the cortical processing of neuronal signals originating from ocular reception. In order to achieve reception of light and transform it into the signal that is sent to the cortex, a combination of fine-tuned and organized tissues is utilized, tissues that combine to form the structure of the human eye. Thus, ocular histology includes all three types of tissue muscle, nerve, and epithelial at a very high level of organization. The light enters the eye through the cornea, which is a transparent tissue responsible for the 70% of the refractive power of the eye. The cornea is composed of epithelium externally, the corneal stroma (the basic part of its structure, consisting of organized collagen fibrils and proteoglycans) and the endothelium, which maintains the corneal humidity and transparency. The cornea is the front part of the bulbar wall; the rest of it is comprised of the sclera, a nontransparent collagen tissue. The cornea is covered by the tear film, which is composed of three layers: the lipid layer externally, the aqueous layer and the mucus layer internally. Each is produced by different types of glands. Behind the cornea the anterior chamber is filled by the aqueous humor, which preserves the intraocular pressure. It is produced by the ciliary body, and it is removed mainly through the trabecular meshwork in the angle of the anterior chamber. The anterior chamber is separated from the posterior by the iris and the ciliary body. The pupil is the iris diaphragm that serves to regulate the quantity of light that passes through to the crystalline lens and then to the posterior segment of the eye,
R. Gupta (Ed): Biomarkers in Toxicology. ISBN: 978-0-12-404630-6
OCULAR BIOMARKERS A large number of diseases with acute or chronic course and variable involvement of different ocular tissues may be present in ocular pathology. Symptoms of ocular disease vary from subjects’ discomfort to deterioration of ocular refractive function, or permanent visual loss due to damage in the retinal-neuronal pathway. Lately, several new methods for the identification and quantification of molecules in biological tissues have brought about the potential to identify molecular entities that may serve as potentially useful biomarkers in ophthalmology. Biomarkers may be helpful in early detection of a disease; may aid to predict severity of a disease; may predict the rate of disease progression; and may serve as predictors of response to treatment. Use of these molecular markers in the clinical setting seems to be a very fascinating option. However, there
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© 2014 Elsevier Inc. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-404630-6.00019-1
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Ciliary body Posterior chamber Anterior chamber
Retina Vitreous body
Cornea Pupil
Lens Optic disc
Iris Zonules
Optic nerve
Sclera
FIGURE
19.1
Overview
of
ocular
anatomy.
still are many limitations, such as lack of common procedures for proper banking of biological tissues and standardized methods and criteria in different studies. New studies are continually identifying biomarkers and seeking to standardize their values in health and disease. In this chapter, we focus on molecular biomarkers that are involved in the diagnosis and management of several ocular pathologies, as well as in ocular toxicity deriving from agents prescribed for other systemic conditions.
Molecular biomarkers in ocular surface disease Dry eye disease (DED) is an inflammatory disorder of the lacrimal functional unit of multifactorial origin leading to chronic ocular surface discomfort (Dry Eye Workshop, 2007). It is significant burden for ophthalmic healthcare, due to both its high prevalence and to its capacity of affecting patients’ quality of life. Because of its multifactorial origin and of the variability of patients’ subjective response to clinically observed disease parameters, it is not easy to determine objective parameters for the evaluation of DED. The pathophysiology of DED includes two distinct main pathways, each with several possible initial causes that result in the same disease entity. One of the two is the aqueous deficient dry eye, where the production of tear is less than needed, and the other is evaporative dry eye, where there is an increased evaporation of tears. In both conditions, there is increased osmolarity of tear film and increased inflammation of the ocular surface and patient discomfort. Proteomic analysis of the tear film of such patients has helped to identify
molecular parameters that may serve as biomarkers for disease severity or for predisposition to the disease. Immune processes have been demonstrated to play a key role in dry eye. Cytokines, growth factors and their receptors have been extensively studied in tears, corneal tissue, and conjunctival tissue of patients. TNF-a, IL-1, and IL-6 are found increased in dry eye samples, and also correlated with clinical parameters. Boehm et al. (2011) have published a cytokine profile of dry eye patients’ tears. They found increased tear levels of IL-1b, IL-6, IL-8, TNF-a, and IFN-g and increased expression of lipocalin, cystatin SN, and a-1 antitrypsin in dry eye patients, and also found correlations with clinical parameters of DED. In this study the findings were not significant in patients with evaporative dry eye, opposite to other reports that found increased levels in such patients (Enriquez-de-Salamanca et al., 2010). These differences may be attributed to differences in ways of material collection and differences in ways of patient and controls classification between studies. In a study by VanDerMeid et al. (2012) the tear levels of IL-1a, IL-1b, IL-6, IL-8, and TNF-a correlated inversely with Schirmer values; IL-1a, IL-6, and TNF-a also correlated directly with tear osmolarity. VanDerMeid et al. (2012) have studied the correlations of dry eye clinical parameters (Schirmer’s test, TBUT, tear osmolarity and ocular surface disease index) with tear MMP-9, MMP-1, MMP-2, MMP-7, and MMP-10 levels in a healthy volunteer group and found positive correlations of MMP levels with increasing disease severity according to each parameter. Chotikavanich et al. (2009) showed that tear MMP-9 activity was significantly higher in dry eye patients and proposed MMP-9 as a useful biomarker for dry eye. In addition, tear MMP-9 activity correlated strongly with clinical parameters, such as symptom severity score, topographic surface regularity index
OCULAR BIOMARKERS
scores, and corneal and conjunctival fluorescein staining scores. Diurnal variation of these molecules and age should always be taken into account if these parameters are to be considered as biomarkers. Another agent that has been studied in dry eye patients is ocular mucin (MUC) levels. Recently, Corrales et al. (2011) have shown differences in MUC-1, MUC-2, MUC-4, MUC-5ACn, and MUC-16 conjunctival levels in dry eye patients compared with controls. Another category of potential biomarkers for ocular surface disease is neuromediators associated with the functionality of ocular surface innervation. Corneal and ocular surface innervation is a part of the lacrimal functional unit and its function is impaired in dry eye, as shown by studies of corneal sensitivity in dry eye. Tear levels of some neuromediators have been proposed as potential useful markers of dry eye severity. Substance P, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and nerve growth factor (NGF) tear levels have been correlated with clinical parameters of dry eye (Lambiase et al., 2011).
Molecular biomarkers in keratoconus Keratoconus is a noninflammatory disorder that leads to corneal thinning, protrusion, and irregular astigmatism (corneal ectasia) (Figure 19.2). In end stages corneal scarring occurs and vision is significantly impaired. It affects young individuals, mainly during puberty, and progression occurs until 30 to 40 years of age. Currently, diagnosis of the disease is done clinically with specific imaging examinations. Timely diagnosis is very important because of the possibility for stabilization treatment
FIGURE 19.2
Slit lamp image of a patient with keratoconus.
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before the disease becomes visually impairing. In addition diagnosis of subclinical cases or cases predisposed to the disease is also very important, because these patients are poor candidates for refractive surgery and may develop corneal ectasia after being treated with laser surgery for their refractive error. Although it is considered a noninflammatory disorder, a chronic low grade inflammation may take part in the pathophysiology of keratoconus. Recent studies indicate that certain inflammatory molecules are elevated in tears from patients with keratoconus. Lema and Duran (2005) found that levels of IL-6, TNF-a and MMP-9 are higher in keratoconus in comparison to controls. In addition, in patients with unilateral keratoconus (clinically evident keratoconus in one eye and subclinical in the fellow eye), IL-6 and TNF-a were increased in the tears of both keratoconus eyes and fellow eyes (Lema et al., 2009). Pannebaker et al. (2010) showed that the level of MMP-1was increased in keratoconus. Other molecules found to be differently expressed in keratoconus corneas are keratins and mammaglobin B (Pannebaker et al., 2010), zinc-a2-glycoprotein, lactoferrin and immunoglobulin kappa chain (IGKC) (Lema et al., 2009), α-enolase and β-actin (Srivastava et al., 2006).
Molecular biomarkers in glaucoma Glaucoma is a vision-threatening disease affecting a large percent of the population worldwide. Glaucoma progression leads to loss of peripheral vision (visual field) and eventually blindness. One in 40 adults older than 40 years has glaucoma with loss of visual function, which equates to 60 million people worldwide being affected and 8.4 million being bilaterally blind. Increased intraocular pressure is the main cause of glaucoma, and it is caused mainly by obstruction in the outflow of aqueous humor. Timely diagnosis and initiation of IOP lowering medication is of great importance for prevention of visual loss. Follow-up of glaucoma patients is done mainly by IOP monitoring, visual fields sensitivity testing, and imaging of the optic nerve. The use of molecular biomarkers for the early diagnosis and for the follow-up of patients with glaucoma is a field of great scientific interest and research. Biomarkers are identified in blood or ocular fluids such as aqueous humor and tears and show up- or down regulation in patients with glaucoma. The development of clinically useful biomarkers in glaucoma is currently an area of active investigation. Weinstein et al. (1996) showed an association of decreased peripheral blood lymphocyte 3α-hydroxysteroid dehydrogenase (3α-HSD) activity with glaucoma. The authors concluded that the reduced levels of
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3α-HSD activity in the peripheral blood lymphocytes may serve as a marker for POAG or those at risk for developing the disease. Maruyama et al. (2002) suggested that a serum autoantibody against neuron-specific enolase (NSE) may be clinically useful for diagnosing early stages of glaucoma, and for monitoring glaucoma progression of NTG. According to the authors the anti-NSE antibody titers were relatively higher in patients with visual field deterioration than in those without it. Another molecule that may serve as a biomarker for early detection of glaucoma is brain-derived neurotrophic factor (BDNF), which contributes in building up and preserving neurons. BDNF has been found significantly decreased in the tears of normal tension glaucoma patients and open angle glaucoma patients in comparison to controls (Ghaffariyeh et al., 2011). Other studies suggest that CD44 content in ocular tissue may represent a biomarker for glaucoma (Mokbel et al., 2010). CD44 is transmembrane protein, the principal receptor of the glycosaminoglycan, hyaluronan. In the same study, erythropoietin was also recognized as a biomarker for glaucoma. Recent studies have revealed that the levels of homocysteine and hydroxyproline are significantly higher in the aqueous humor of patients with glaucoma than in controls (Ghanem et al., 2012). Currently, research is demonstrating several molecular biomarkers identified as possible indicators for the severity of glaucoma, for the monitoring of the response to treatment, and also for the identification of patients at high risk to develop glaucoma. Future research may provide valuable tools for clinical use in the treatment of glaucoma.
Molecular biomarkers in retinal disease Retinal pathology is one of the most intriguing parts of ophthalmology. Fundus disease may lead to irreversible visual impairment in several cases and early diagnosis may help in the prevention or the protection of the patients from disease progression. Diagnosis of retinal disorders is mainly from fundoscopic examination clinically, and also from specialized imaging examinations such as optical coherence tomography, and fluorescent angiography and indocyanin angiography. Most common disorders are age-related macular degeneration (ARMD) and diabetic retinopathy, which are included in the leading causes of blindness in the developed world.
Molecular biomarkers in age-related macular degeneration Currently research is targeted on identifying molecular agents that may contribute to the pathogenesis or the
progression of ARMD. A recent study by Kim et al. (2012) identified that in the aqueous humor of patients with ARMD the protein composition in the aqueous humor was different than in controls. Recently, elevated levels of CXCL10 were reported in the sera and choroid of individuals with ARMD, and elevated intraocular CCL2 levels were observed in neovascular ARMD (Mo et al., 2010; Jonas et al., 2010). Newman et al. (2012) showed that all ARMD phenotypes in the RPE-choroid are associated with elevated expression of all, or a subset, of the following chemokines: CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CCL2, and CCL8. In addition, the upregulation of immunoglobulin genes supports an adaptive, autoimmune response in ARMD that is consistent with previous reports of immunoglobulins in drusen and drusen-associated RPE as well as anticarboxyethylpyrrole adduct antibodies and antiretinal antigen autoantibodies in ARMD sera.
Molecular biomarkers in ocular oncology There are several studies that attempt to connect specific biomarkers with ocular cancers. Retinoblastoma (RB) is a malignant tumor of the retina that affects children. In the study of Beta et al. (2013), the miRNAs in the serum of children with RB were compared with those in normal age-matched serum. Expression of the oncogenic miRNAs, miR-17, miR-18a, and miR-20a by qRT-PCR was significant in the serum of the RB samples exploring the potential of serum miRNAs identification as noninvasive diagnosis. The researchers concluded that the identified miRNAs and their corresponding target genes could give insights on potential biomarkers and key events involved in the RB pathway. Uveal and conjunctival melanoma (Figure 19.3) has also been related to potential biomarkers. The Collaborative Ocular Oncology group (Onken et al., 2012) evaluated in 459 patients the prognostic performance of a 15-gene expression profiling (GEP) assay that assigns primary posterior uveal melanomas to prognostic subgroups: class 1 (low metastatic risk) and class 2 (high metastatic risk). They assumed that the GEP assay had a high technical success rate providing a highly significant improvement in prognostic accuracy over clinical TNM classification. Zoroquiain et al. (2012) analyzed in an immunohistochemical study the expression of p16ink4a (p16) in conjunctival melanocytic lesions and concluded that it seems to be a good marker to differentiate nevi and primary acquired melanoses (PAMs) from melanomas. Errington et al. (2012) found that uveal and conjunctival melanomas consistently expressed high levels of gp100, Melan-A/MART1, and tyrosinase, which are differentiation antigens. The
SYSTEMIC AGENTS AND OCULAR TOXICITY
FIGURE 19.3
Slit lamp image of a patient with uveal
melanoma.
relation of a stem cell marker, CD133, with uveal melanoma was explored by Thill et al. (2011). Differential expression of CD133 splice variants was found in iris, ciliary body, retina, and retinal pigment epithelium/ choroid as well as in uveal melanoma cell lines. Noninvasive biomarkers may potentially be used for the early diagnosis of ocular tumors and to follow up treatment. More experimental studies should be carried out to isolate more biomarkers, to evaluate their functional properties and to explore possible therapeutic approaches.
SYSTEMIC AGENTS AND OCULAR TOXICITY Many antidepressants, antipsychotics, anti-Parkinson drugs, antihistamines, anticonvulsants, decongestants, and beta-blockers, along with hormone replacement therapy, have been shown to cause dry eye symptom as an adverse toxic effect. Herbal products with the same effect include niacin, kava, echinacea, and anticholinergic alkaloids (Askeroglu et al., 2013). Moreover, corneal toxicity can be caused in a number of individuals by fluoroquinolones, nonsteroidal anti-inflammatory eye drops, glaucoma eye drops, preservatives in eye drops, aminoglycosides, chemotherapeutic medications, topical anesthetics, cyclooxygenase-2 inhibitors, bisphosphonates, retinoids, topical steroids, topical iodine, and some herbal medications such as black mustard, chamomile, cypress spurge, goa powder, and psyllium (Fraunfelder, 2006). Aminoglycosides, in particular, have been shown to cause visual loss, optic atrophy, glaucoma, and pigmentary degeneration (Hancock et al., 2005).
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Dexamethasone, fluocinolone, and triamcinolone are well-known corticosteroids that are very toxic to the retina at high doses and can even cause cataract and glaucoma (Penha et al., 2010). Retinal toxicity may also be caused by intravitreal use of the antibiotic amikacin (Widmer and Helbig, 2006). Hydroxychloroquine and chloroquine have been used as antimalarial agents and for the treatment of dermatological and rheumatological diseases. The administration of these drugs can lead to a wide range of toxic ocular effects such as retinal damage, pigmentary retinopathy, keratopathy, corneal deposits, cataract, photophobia, ocular muscle imbalance, and loss of peripheral and night vision (Tzekov, 2005; Tehrani et al., 2008; Tailor et al., 2012). Ethambutol hydrochloride has been known to cause dose and duration dependent ocular toxicity and, in particular, optic neuritis, since the 1960s. The drug is used for the treatment of tuberculosis and its most common ocular adverse effect is retrobulbar neuritis (Chan and Kwok, 2006). Low-dose fludarabine has been reported to cause hallucinations, visual changes, blurred vision, and even blindness. On the other hand, the ocular effects of highdose fludarabine include hallucinations, blurred vision, amaurosis, bilateral papillitis, and, in some cases, cortical blindness (Ding et al., 2008). Oral and inhaled steroids can cause cataract or glaucoma after chronic use or in individuals with high susceptibility. Oral antihistamine drugs used for the treatment of allergies can cause tear-film dysfunction (dry-eye syndrome) or conjunctival hyperreactivity (Bielory, 2006). Antineoplastic agents used in chemotherapies have been proven to cause tear-film changes and mucositis with involvement of the conjunctival mucosa (Chaves et al., 2007). Cisplatin, tamoxifen, and interferons, all chemotherapeutic agents, can cause visual loss and cisplatin can also cause retinal neovascularization (Kwan et al., 2006; Omoti and Omoti, 2006). In high concentrations ornithine can cause retinal toxicity when administered as a supplement in patients with gyrate atrophy of the retina and choroid (Hayasaka et al., 2011). Topiramate toxicity was noticed in two women who received the drug for the treatment of recurrent headaches and migraines. Both women presented with macular folds associated with angle-closure glaucoma. The symptoms disappeared after discontinuation of the drug (Kumar et al., 2006). Deferoxamine, a chelating agent of iron and aluminum ions, can cause ocular retinal toxicity by damaging the retinal pigment epithelium. Ophthalmologic monitoring may be required when treating hematological or kidney conditions with deferoxamine (Szwarcberg et al., 1997).
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Eye drops have been known to cause ocular toxicity after chronic use, sometimes caused by the preservatives included in the formulation. Therefore, symptoms such as cell loss and structural changes in the conjunctival epithelium and the corneal endothelium and epithelium, fibrosis and chronic inflammation of the subconjunctiva, and dry-eye syndrome may occur (Huber-van der Velden et al., 2012). For instance, in experiments carried out in rabbits it was shown that when benzalkonium chloride, the most common preservative used in ocular drugs, was applied topically, the whole cornea was impaired and at high doses the barrier integrity of the corneal endothelium was disrupted (Chen et al., 2011). Indocyanine green, a dye used in medical diagnostics, can cause retinal toxicity in high doses by degeneration of retinal layers and Mu¨ller cell dysfunction (Sato et al., 2002). Dry-eye disease has also been reported after ciguatera fish poisoning in a 47-year-old woman. The patient had consumed Spanish mackerel that contained the ciguatera toxin (Sheck and Wilson, 2010). Lead poisoning can also cause retinal toxicity as shown in a 35-year-old woman who suffered from loss of vision in the right eye (Gilhotra et al., 2007). Another heavy metal, iron, may play a role in retinal and macular degeneration, glaucoma, and cataract (He et al., 2007). In vitro experiments carried out in retinal pigment epithelium cells have shown that cadmium can cause disruption of the membrane integrity, alter the cell morphology, and decrease the survival of the cells and could therefore play a role in age-related retinal disease in smokers (Wills et al., 2008). Cobalt toxicity following hip implant has also been reported involving degenerative alterations of the photoreceptor-retinal pigment epithelium complex with coroidal infarction and paracentral scotomas (Ng et al., 2013). The toxic effects of crack-cocaine abuse include corneal disturbances. For instance, a crack-cocaine abuse case has been reported in which the patient had stromal ulceration and corneal epithelial disruption (Pilon and Scheiffle, 2006). Mustard gas can affect the eyes, especially in cases of chronic involvement. Symptoms include dry eye, limbal stem cell deficiency, limbal ischemia, chronic blepharitis, aberrant conjunctival vessels, meibomian gland dysfunction, corneal neovascularization, corneal irregularity, thinning and scarring, and lipid and amyloid deposition (Baradaran-Rafii et al., 2011). Ocular toxicity of hydrogen peroxide after habitual use as an eye wash was reported in a patient who was hospitalized for inflammation and scarring of the cornea and the conjunctiva (Memarzadeh et al., 1993). Paraquat, a common herbicide, can cause ocular surface destruction via topical exposure (Vlahos et al., 1993).
Light or electromagnetic radiation can cause retinal damage although the eye has adaptive mechanisms to avoid the possible damage (Youssef et al., 2011). Methanol poisoning by ingestion has led to ocular damage in two patient cases. Retinal and cystoid macular edema was observed with paracentral scotomas, decreased retinal sensitivity and enlargement of one blind spot (McKellar et al., 1992).
CONCLUDING REMARKS AND FUTURE DIRECTIONS Several molecules have been identified that may be helpful in the diagnosis of ocular disease, and also several systemic agents that may cause significant damage in the ocular tissue. Research in the field of biomarkers for ocular pathology is expanding and it is possible that soon those molecules may serve as biomarkers for early diagnosis, monitoring the treatment, and follow-up of the patient.
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Vlahos, K., Goggin, M., and Coster, D. (1993) Paraquat causes chronic ocular surface toxicity. Aus NZ J Ophthalmol 21: 187 190. Weinstein, B.I., Iyer, R.B., Binstock, J.M. et al. (1996) Decreased 3 alpha-hydroxysteroid dehydrogenase activity in peripheral blood lymphocytes from patients with primary open angle glaucoma. Exp Eye Res 62: 39 45. Widmer, S., and Helbig, H. (2006) Presumed macular toxicity of intravitreal antibiotics. Ophthalmologica 220: 153 158.
Wills, N.K., Ramanujam, V.M., Chang, J. et al. (2008) Cadmium accumulation in the human retina: effects of age, gender, and cellular toxicity. Exp Eye Res 86: 41 51. Youssef, P.N., Sheibani, N., and Albert, D.M. (2011) Retinal light toxicity. Eye (Lond) 25: 1 14, Doi: 10.1038/eye.2010.149. Epub 2010 Oct 29. Zoroquiain, P., Fernandes, B.F., Gonzalez, S. et al. (2012) p16ink4a expression in benign and malignant melanocytic conjunctival lesions. Int J Surg Pathol 20: 240 245.
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19 Ocular biomarkers in diseases and toxicities George A. Kontadakis, Argyro Plaka, Domniki Fragou, George D. Kymionis, and Aristides M. Tsatsakis
INTRODUCTION
where the light meets the retina. The crystalline lens is responsible for 30% of the refractive power of the eye in relaxed status and adds the needed power for accommodation in near vision in pre-presbyopic individuals. Behind the crystalline lens, the eye is filled by the vitreous body. In contact with the vitreous body is the retina (Figure 19.1). The retina contains the photoreceptors that transform light to neural signal, which, after a complex path of intraretinal transformations, travels to the visual cortex through the optic nerve and the rest of the optic pathway. This chapter describes the biomarkers of ocular diseases and toxicities.
The human eye serves as the receptor of light, giving rise to the sense of seeing after the cortical processing of neuronal signals originating from ocular reception. In order to achieve reception of light and transform it into the signal that is sent to the cortex, a combination of fine-tuned and organized tissues is utilized, tissues that combine to form the structure of the human eye. Thus, ocular histology includes all three types of tissue muscle, nerve, and epithelial at a very high level of organization. The light enters the eye through the cornea, which is a transparent tissue responsible for the 70% of the refractive power of the eye. The cornea is composed of epithelium externally, the corneal stroma (the basic part of its structure, consisting of organized collagen fibrils and proteoglycans) and the endothelium, which maintains the corneal humidity and transparency. The cornea is the front part of the bulbar wall; the rest of it is comprised of the sclera, a nontransparent collagen tissue. The cornea is covered by the tear film, which is composed of three layers: the lipid layer externally, the aqueous layer and the mucus layer internally. Each is produced by different types of glands. Behind the cornea the anterior chamber is filled by the aqueous humor, which preserves the intraocular pressure. It is produced by the ciliary body, and it is removed mainly through the trabecular meshwork in the angle of the anterior chamber. The anterior chamber is separated from the posterior by the iris and the ciliary body. The pupil is the iris diaphragm that serves to regulate the quantity of light that passes through to the crystalline lens and then to the posterior segment of the eye,
R. Gupta (Ed): Biomarkers in Toxicology. ISBN: 978-0-12-404630-6
OCULAR BIOMARKERS A large number of diseases with acute or chronic course and variable involvement of different ocular tissues may be present in ocular pathology. Symptoms of ocular disease vary from subjects’ discomfort to deterioration of ocular refractive function, or permanent visual loss due to damage in the retinal-neuronal pathway. Lately, several new methods for the identification and quantification of molecules in biological tissues have brought about the potential to identify molecular entities that may serve as potentially useful biomarkers in ophthalmology. Biomarkers may be helpful in early detection of a disease; may aid to predict severity of a disease; may predict the rate of disease progression; and may serve as predictors of response to treatment. Use of these molecular markers in the clinical setting seems to be a very fascinating option. However, there
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© 2014 Elsevier Inc. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-404630-6.00019-1
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Ciliary body Posterior chamber Anterior chamber
Retina Vitreous body
Cornea Pupil
Lens Optic disc
Iris Zonules
Optic nerve
Sclera
FIGURE
19.1
Overview
of
ocular
anatomy.
still are many limitations, such as lack of common procedures for proper banking of biological tissues and standardized methods and criteria in different studies. New studies are continually identifying biomarkers and seeking to standardize their values in health and disease. In this chapter, we focus on molecular biomarkers that are involved in the diagnosis and management of several ocular pathologies, as well as in ocular toxicity deriving from agents prescribed for other systemic conditions.
Molecular biomarkers in ocular surface disease Dry eye disease (DED) is an inflammatory disorder of the lacrimal functional unit of multifactorial origin leading to chronic ocular surface discomfort (Dry Eye Workshop, 2007). It is significant burden for ophthalmic healthcare, due to both its high prevalence and to its capacity of affecting patients’ quality of life. Because of its multifactorial origin and of the variability of patients’ subjective response to clinically observed disease parameters, it is not easy to determine objective parameters for the evaluation of DED. The pathophysiology of DED includes two distinct main pathways, each with several possible initial causes that result in the same disease entity. One of the two is the aqueous deficient dry eye, where the production of tear is less than needed, and the other is evaporative dry eye, where there is an increased evaporation of tears. In both conditions, there is increased osmolarity of tear film and increased inflammation of the ocular surface and patient discomfort. Proteomic analysis of the tear film of such patients has helped to identify
molecular parameters that may serve as biomarkers for disease severity or for predisposition to the disease. Immune processes have been demonstrated to play a key role in dry eye. Cytokines, growth factors and their receptors have been extensively studied in tears, corneal tissue, and conjunctival tissue of patients. TNF-a, IL-1, and IL-6 are found increased in dry eye samples, and also correlated with clinical parameters. Boehm et al. (2011) have published a cytokine profile of dry eye patients’ tears. They found increased tear levels of IL-1b, IL-6, IL-8, TNF-a, and IFN-g and increased expression of lipocalin, cystatin SN, and a-1 antitrypsin in dry eye patients, and also found correlations with clinical parameters of DED. In this study the findings were not significant in patients with evaporative dry eye, opposite to other reports that found increased levels in such patients (Enriquez-de-Salamanca et al., 2010). These differences may be attributed to differences in ways of material collection and differences in ways of patient and controls classification between studies. In a study by VanDerMeid et al. (2012) the tear levels of IL-1a, IL-1b, IL-6, IL-8, and TNF-a correlated inversely with Schirmer values; IL-1a, IL-6, and TNF-a also correlated directly with tear osmolarity. VanDerMeid et al. (2012) have studied the correlations of dry eye clinical parameters (Schirmer’s test, TBUT, tear osmolarity and ocular surface disease index) with tear MMP-9, MMP-1, MMP-2, MMP-7, and MMP-10 levels in a healthy volunteer group and found positive correlations of MMP levels with increasing disease severity according to each parameter. Chotikavanich et al. (2009) showed that tear MMP-9 activity was significantly higher in dry eye patients and proposed MMP-9 as a useful biomarker for dry eye. In addition, tear MMP-9 activity correlated strongly with clinical parameters, such as symptom severity score, topographic surface regularity index
OCULAR BIOMARKERS
scores, and corneal and conjunctival fluorescein staining scores. Diurnal variation of these molecules and age should always be taken into account if these parameters are to be considered as biomarkers. Another agent that has been studied in dry eye patients is ocular mucin (MUC) levels. Recently, Corrales et al. (2011) have shown differences in MUC-1, MUC-2, MUC-4, MUC-5ACn, and MUC-16 conjunctival levels in dry eye patients compared with controls. Another category of potential biomarkers for ocular surface disease is neuromediators associated with the functionality of ocular surface innervation. Corneal and ocular surface innervation is a part of the lacrimal functional unit and its function is impaired in dry eye, as shown by studies of corneal sensitivity in dry eye. Tear levels of some neuromediators have been proposed as potential useful markers of dry eye severity. Substance P, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and nerve growth factor (NGF) tear levels have been correlated with clinical parameters of dry eye (Lambiase et al., 2011).
Molecular biomarkers in keratoconus Keratoconus is a noninflammatory disorder that leads to corneal thinning, protrusion, and irregular astigmatism (corneal ectasia) (Figure 19.2). In end stages corneal scarring occurs and vision is significantly impaired. It affects young individuals, mainly during puberty, and progression occurs until 30 to 40 years of age. Currently, diagnosis of the disease is done clinically with specific imaging examinations. Timely diagnosis is very important because of the possibility for stabilization treatment
FIGURE 19.2
Slit lamp image of a patient with keratoconus.
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before the disease becomes visually impairing. In addition diagnosis of subclinical cases or cases predisposed to the disease is also very important, because these patients are poor candidates for refractive surgery and may develop corneal ectasia after being treated with laser surgery for their refractive error. Although it is considered a noninflammatory disorder, a chronic low grade inflammation may take part in the pathophysiology of keratoconus. Recent studies indicate that certain inflammatory molecules are elevated in tears from patients with keratoconus. Lema and Duran (2005) found that levels of IL-6, TNF-a and MMP-9 are higher in keratoconus in comparison to controls. In addition, in patients with unilateral keratoconus (clinically evident keratoconus in one eye and subclinical in the fellow eye), IL-6 and TNF-a were increased in the tears of both keratoconus eyes and fellow eyes (Lema et al., 2009). Pannebaker et al. (2010) showed that the level of MMP-1was increased in keratoconus. Other molecules found to be differently expressed in keratoconus corneas are keratins and mammaglobin B (Pannebaker et al., 2010), zinc-a2-glycoprotein, lactoferrin and immunoglobulin kappa chain (IGKC) (Lema et al., 2009), α-enolase and β-actin (Srivastava et al., 2006).
Molecular biomarkers in glaucoma Glaucoma is a vision-threatening disease affecting a large percent of the population worldwide. Glaucoma progression leads to loss of peripheral vision (visual field) and eventually blindness. One in 40 adults older than 40 years has glaucoma with loss of visual function, which equates to 60 million people worldwide being affected and 8.4 million being bilaterally blind. Increased intraocular pressure is the main cause of glaucoma, and it is caused mainly by obstruction in the outflow of aqueous humor. Timely diagnosis and initiation of IOP lowering medication is of great importance for prevention of visual loss. Follow-up of glaucoma patients is done mainly by IOP monitoring, visual fields sensitivity testing, and imaging of the optic nerve. The use of molecular biomarkers for the early diagnosis and for the follow-up of patients with glaucoma is a field of great scientific interest and research. Biomarkers are identified in blood or ocular fluids such as aqueous humor and tears and show up- or down regulation in patients with glaucoma. The development of clinically useful biomarkers in glaucoma is currently an area of active investigation. Weinstein et al. (1996) showed an association of decreased peripheral blood lymphocyte 3α-hydroxysteroid dehydrogenase (3α-HSD) activity with glaucoma. The authors concluded that the reduced levels of
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3α-HSD activity in the peripheral blood lymphocytes may serve as a marker for POAG or those at risk for developing the disease. Maruyama et al. (2002) suggested that a serum autoantibody against neuron-specific enolase (NSE) may be clinically useful for diagnosing early stages of glaucoma, and for monitoring glaucoma progression of NTG. According to the authors the anti-NSE antibody titers were relatively higher in patients with visual field deterioration than in those without it. Another molecule that may serve as a biomarker for early detection of glaucoma is brain-derived neurotrophic factor (BDNF), which contributes in building up and preserving neurons. BDNF has been found significantly decreased in the tears of normal tension glaucoma patients and open angle glaucoma patients in comparison to controls (Ghaffariyeh et al., 2011). Other studies suggest that CD44 content in ocular tissue may represent a biomarker for glaucoma (Mokbel et al., 2010). CD44 is transmembrane protein, the principal receptor of the glycosaminoglycan, hyaluronan. In the same study, erythropoietin was also recognized as a biomarker for glaucoma. Recent studies have revealed that the levels of homocysteine and hydroxyproline are significantly higher in the aqueous humor of patients with glaucoma than in controls (Ghanem et al., 2012). Currently, research is demonstrating several molecular biomarkers identified as possible indicators for the severity of glaucoma, for the monitoring of the response to treatment, and also for the identification of patients at high risk to develop glaucoma. Future research may provide valuable tools for clinical use in the treatment of glaucoma.
Molecular biomarkers in retinal disease Retinal pathology is one of the most intriguing parts of ophthalmology. Fundus disease may lead to irreversible visual impairment in several cases and early diagnosis may help in the prevention or the protection of the patients from disease progression. Diagnosis of retinal disorders is mainly from fundoscopic examination clinically, and also from specialized imaging examinations such as optical coherence tomography, and fluorescent angiography and indocyanin angiography. Most common disorders are age-related macular degeneration (ARMD) and diabetic retinopathy, which are included in the leading causes of blindness in the developed world.
Molecular biomarkers in age-related macular degeneration Currently research is targeted on identifying molecular agents that may contribute to the pathogenesis or the
progression of ARMD. A recent study by Kim et al. (2012) identified that in the aqueous humor of patients with ARMD the protein composition in the aqueous humor was different than in controls. Recently, elevated levels of CXCL10 were reported in the sera and choroid of individuals with ARMD, and elevated intraocular CCL2 levels were observed in neovascular ARMD (Mo et al., 2010; Jonas et al., 2010). Newman et al. (2012) showed that all ARMD phenotypes in the RPE-choroid are associated with elevated expression of all, or a subset, of the following chemokines: CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CCL2, and CCL8. In addition, the upregulation of immunoglobulin genes supports an adaptive, autoimmune response in ARMD that is consistent with previous reports of immunoglobulins in drusen and drusen-associated RPE as well as anticarboxyethylpyrrole adduct antibodies and antiretinal antigen autoantibodies in ARMD sera.
Molecular biomarkers in ocular oncology There are several studies that attempt to connect specific biomarkers with ocular cancers. Retinoblastoma (RB) is a malignant tumor of the retina that affects children. In the study of Beta et al. (2013), the miRNAs in the serum of children with RB were compared with those in normal age-matched serum. Expression of the oncogenic miRNAs, miR-17, miR-18a, and miR-20a by qRT-PCR was significant in the serum of the RB samples exploring the potential of serum miRNAs identification as noninvasive diagnosis. The researchers concluded that the identified miRNAs and their corresponding target genes could give insights on potential biomarkers and key events involved in the RB pathway. Uveal and conjunctival melanoma (Figure 19.3) has also been related to potential biomarkers. The Collaborative Ocular Oncology group (Onken et al., 2012) evaluated in 459 patients the prognostic performance of a 15-gene expression profiling (GEP) assay that assigns primary posterior uveal melanomas to prognostic subgroups: class 1 (low metastatic risk) and class 2 (high metastatic risk). They assumed that the GEP assay had a high technical success rate providing a highly significant improvement in prognostic accuracy over clinical TNM classification. Zoroquiain et al. (2012) analyzed in an immunohistochemical study the expression of p16ink4a (p16) in conjunctival melanocytic lesions and concluded that it seems to be a good marker to differentiate nevi and primary acquired melanoses (PAMs) from melanomas. Errington et al. (2012) found that uveal and conjunctival melanomas consistently expressed high levels of gp100, Melan-A/MART1, and tyrosinase, which are differentiation antigens. The
SYSTEMIC AGENTS AND OCULAR TOXICITY
FIGURE 19.3
Slit lamp image of a patient with uveal
melanoma.
relation of a stem cell marker, CD133, with uveal melanoma was explored by Thill et al. (2011). Differential expression of CD133 splice variants was found in iris, ciliary body, retina, and retinal pigment epithelium/ choroid as well as in uveal melanoma cell lines. Noninvasive biomarkers may potentially be used for the early diagnosis of ocular tumors and to follow up treatment. More experimental studies should be carried out to isolate more biomarkers, to evaluate their functional properties and to explore possible therapeutic approaches.
SYSTEMIC AGENTS AND OCULAR TOXICITY Many antidepressants, antipsychotics, anti-Parkinson drugs, antihistamines, anticonvulsants, decongestants, and beta-blockers, along with hormone replacement therapy, have been shown to cause dry eye symptom as an adverse toxic effect. Herbal products with the same effect include niacin, kava, echinacea, and anticholinergic alkaloids (Askeroglu et al., 2013). Moreover, corneal toxicity can be caused in a number of individuals by fluoroquinolones, nonsteroidal anti-inflammatory eye drops, glaucoma eye drops, preservatives in eye drops, aminoglycosides, chemotherapeutic medications, topical anesthetics, cyclooxygenase-2 inhibitors, bisphosphonates, retinoids, topical steroids, topical iodine, and some herbal medications such as black mustard, chamomile, cypress spurge, goa powder, and psyllium (Fraunfelder, 2006). Aminoglycosides, in particular, have been shown to cause visual loss, optic atrophy, glaucoma, and pigmentary degeneration (Hancock et al., 2005).
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Dexamethasone, fluocinolone, and triamcinolone are well-known corticosteroids that are very toxic to the retina at high doses and can even cause cataract and glaucoma (Penha et al., 2010). Retinal toxicity may also be caused by intravitreal use of the antibiotic amikacin (Widmer and Helbig, 2006). Hydroxychloroquine and chloroquine have been used as antimalarial agents and for the treatment of dermatological and rheumatological diseases. The administration of these drugs can lead to a wide range of toxic ocular effects such as retinal damage, pigmentary retinopathy, keratopathy, corneal deposits, cataract, photophobia, ocular muscle imbalance, and loss of peripheral and night vision (Tzekov, 2005; Tehrani et al., 2008; Tailor et al., 2012). Ethambutol hydrochloride has been known to cause dose and duration dependent ocular toxicity and, in particular, optic neuritis, since the 1960s. The drug is used for the treatment of tuberculosis and its most common ocular adverse effect is retrobulbar neuritis (Chan and Kwok, 2006). Low-dose fludarabine has been reported to cause hallucinations, visual changes, blurred vision, and even blindness. On the other hand, the ocular effects of highdose fludarabine include hallucinations, blurred vision, amaurosis, bilateral papillitis, and, in some cases, cortical blindness (Ding et al., 2008). Oral and inhaled steroids can cause cataract or glaucoma after chronic use or in individuals with high susceptibility. Oral antihistamine drugs used for the treatment of allergies can cause tear-film dysfunction (dry-eye syndrome) or conjunctival hyperreactivity (Bielory, 2006). Antineoplastic agents used in chemotherapies have been proven to cause tear-film changes and mucositis with involvement of the conjunctival mucosa (Chaves et al., 2007). Cisplatin, tamoxifen, and interferons, all chemotherapeutic agents, can cause visual loss and cisplatin can also cause retinal neovascularization (Kwan et al., 2006; Omoti and Omoti, 2006). In high concentrations ornithine can cause retinal toxicity when administered as a supplement in patients with gyrate atrophy of the retina and choroid (Hayasaka et al., 2011). Topiramate toxicity was noticed in two women who received the drug for the treatment of recurrent headaches and migraines. Both women presented with macular folds associated with angle-closure glaucoma. The symptoms disappeared after discontinuation of the drug (Kumar et al., 2006). Deferoxamine, a chelating agent of iron and aluminum ions, can cause ocular retinal toxicity by damaging the retinal pigment epithelium. Ophthalmologic monitoring may be required when treating hematological or kidney conditions with deferoxamine (Szwarcberg et al., 1997).
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Eye drops have been known to cause ocular toxicity after chronic use, sometimes caused by the preservatives included in the formulation. Therefore, symptoms such as cell loss and structural changes in the conjunctival epithelium and the corneal endothelium and epithelium, fibrosis and chronic inflammation of the subconjunctiva, and dry-eye syndrome may occur (Huber-van der Velden et al., 2012). For instance, in experiments carried out in rabbits it was shown that when benzalkonium chloride, the most common preservative used in ocular drugs, was applied topically, the whole cornea was impaired and at high doses the barrier integrity of the corneal endothelium was disrupted (Chen et al., 2011). Indocyanine green, a dye used in medical diagnostics, can cause retinal toxicity in high doses by degeneration of retinal layers and Mu¨ller cell dysfunction (Sato et al., 2002). Dry-eye disease has also been reported after ciguatera fish poisoning in a 47-year-old woman. The patient had consumed Spanish mackerel that contained the ciguatera toxin (Sheck and Wilson, 2010). Lead poisoning can also cause retinal toxicity as shown in a 35-year-old woman who suffered from loss of vision in the right eye (Gilhotra et al., 2007). Another heavy metal, iron, may play a role in retinal and macular degeneration, glaucoma, and cataract (He et al., 2007). In vitro experiments carried out in retinal pigment epithelium cells have shown that cadmium can cause disruption of the membrane integrity, alter the cell morphology, and decrease the survival of the cells and could therefore play a role in age-related retinal disease in smokers (Wills et al., 2008). Cobalt toxicity following hip implant has also been reported involving degenerative alterations of the photoreceptor-retinal pigment epithelium complex with coroidal infarction and paracentral scotomas (Ng et al., 2013). The toxic effects of crack-cocaine abuse include corneal disturbances. For instance, a crack-cocaine abuse case has been reported in which the patient had stromal ulceration and corneal epithelial disruption (Pilon and Scheiffle, 2006). Mustard gas can affect the eyes, especially in cases of chronic involvement. Symptoms include dry eye, limbal stem cell deficiency, limbal ischemia, chronic blepharitis, aberrant conjunctival vessels, meibomian gland dysfunction, corneal neovascularization, corneal irregularity, thinning and scarring, and lipid and amyloid deposition (Baradaran-Rafii et al., 2011). Ocular toxicity of hydrogen peroxide after habitual use as an eye wash was reported in a patient who was hospitalized for inflammation and scarring of the cornea and the conjunctiva (Memarzadeh et al., 1993). Paraquat, a common herbicide, can cause ocular surface destruction via topical exposure (Vlahos et al., 1993).
Light or electromagnetic radiation can cause retinal damage although the eye has adaptive mechanisms to avoid the possible damage (Youssef et al., 2011). Methanol poisoning by ingestion has led to ocular damage in two patient cases. Retinal and cystoid macular edema was observed with paracentral scotomas, decreased retinal sensitivity and enlargement of one blind spot (McKellar et al., 1992).
CONCLUDING REMARKS AND FUTURE DIRECTIONS Several molecules have been identified that may be helpful in the diagnosis of ocular disease, and also several systemic agents that may cause significant damage in the ocular tissue. Research in the field of biomarkers for ocular pathology is expanding and it is possible that soon those molecules may serve as biomarkers for early diagnosis, monitoring the treatment, and follow-up of the patient.
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